Creatine Deficiency Syndromes: A Clinical - VU

Creatine Deficiency Syndromes: A Clinical,
Molecular and Functional Approach
Joseph Dingbobga Tanyi Ndika
The studies described in this thesis were carried out at the Metabolic Unit, Department of Clinical Chemistry,
VU University Medical Center in Amsterdam, The Netherlands.
This research was carried out under the tutelage of the Graduate School Neurosciences Amsterdam Rotterdam
(ONWAR) as part of the Brain Mechanisms in Health and Disease research program of the Neuroscience
Campus Amsterdam.
This work was financed by the Neuroscience Campus Amsterdam and the Metabolic Laboratory, Department
of Clinical Chemistry, VU University Medical Center in Amsterdam, The Netherlands.
The publication of this thesis was financially supported by:
-
Department of Clinical Chemistry, VU University Medical Center and VU University, Amsterdam
VU University, College van Decanen, Amsterdam
Reading committee:
prof.dr. C.B.M. Oudejans
prof.dr. T.J. de Grauw
dr. A. Thijs
dr. S. Mahmutoglu
dr. O. Braissant
ISBN: 978-90-5383-093-2
Cover picture: Creatine monohydrate powder. Source: www.relentlessgains.com
Printed by DPC/Huisdrukkerij, VU University Medical Center, Amsterdam, The Netherlands
Copyright © Joseph Dingbobga Tanyi Ndika. All rights reserved
VRIJE UNIVERSITEIT
Creatine Deficiency Syndromes: A Clinical,
Molecular and Functional Approach
ACADEMISCH PROEFSCHRIFT
ter verkrijging van de graad Doctor aan
de Vrije Universiteit Amsterdam,
op gezag van de rector magnificus
prof.dr. F.A. van der Duyn Schouten,
in het openbaar te verdedigen
ten overstaan van de promotiecommissie
van de Faculteit der Geneeskunde
op donderdag 19 juni om 11.45 uur
in de aula van de universiteit,
De Boelelaan 1105
door
Joseph Dingbobga Tanyi Ndika
geboren te Bamenda, Kameroen
promotoren:
prof.dr. G.S. Salomons
prof.dr.ir. C.A.J.M. Jakobs
copromotor:
dr. C. Martinez-Muñoz
The ability of the genes to vary and, when they vary (mutate), to reproduce themselves
in their new form, confers on these cell elements, as Muller has so convincingly pointed
out, the properties of the building blocks required by the process of evolution. Thus, the
cell, robbed of its noblest prerogative, was no longer the ultimate unit of life. This title
was now conferred on the genes, subcellular elements, of which the cell nucleus
contained many thousands and, more precisely, like Noah's ark, two of each kind.
Boris Ephrussi1
Dedicated to my parents;
Henry Tanyi (in loving memory) and Beatrice Tanyi
1
Nucleo-cytoplasmic Relations in Micro-Organisms: Their Bearing on Cell Heredity and Differentiation (1953), p.2-3
TABLE OF CONTENTS
PAGE
Chapter 1
General Introduction and Outline of the Thesis.
Chapter 2
Developmental progress and creatine restoration upon long-term creatine 29
supplementation of a patient with arginine:glycine amidinotransferase
deficiency.
Thirteen new patients with guanidinoacetate methyltransferase
39
deficiency and functional characterization of nineteen novel missense
variants in the GAMT gene.
Post-transcriptional Regulation of the Creatine Transporter Gene:
49
Functional Relevance of Alternative Splicing.
Chapter 3
Chapter 4
9
Chapter 5
Cloning and characterization of the promoter regions from the parent and
paralogous creatine transporter genes.
61
Chapter 6
RNA sequencing of creatine transporter (SLC6A8) deficient fibroblasts
reveals impairment of the extracellular matrix.
69
Chapter 7
Summary, concluding remarks and perspectives
99
Appendices Nederlandse samenvatting
107
Acknowledgements
110
Curriculum vitae
113
List of publications
114
9
Chapter 1
General Introduction and Outline of the Thesis
"Creatine Kinase is located near sites where the real action occurs"
Theo Wallimann
10
11
Introduction
Creatine, Phosphocreatine and Creatine Kinase system
Creatine (methyl guanidine-acetic acid) – which is the Greek word for meat, was first identified in 1832
as a component of skeletal muscle by the French scientist, Michel Eugène Chevreul. Phosphocreatine
was discovered about a hundred years later (1927) with the observation that its levels changed
between resting and contracting muscle [1,2]. Phosphorylation of creatine (Cr) to phosphocreatine
(PCr) and back catalyzed by the creatine kinase enzymes (CK, EC 2.7.3.2) is crucial for the supply of
high energy phosphates (Fig. 1) in tissues and cells with high and fluctuating energy demands like
skeletal and cardiac muscle, brain, spermatozoa and photoreceptor cells [3]. The ATP/ADP ratio, local
concentrations of ATP, ADP and AMP are amongst key regulators influencing numerous metabolic
processes, reviewed in [4], as such it doesn’t suffice to merely increase the levels of ATP in these
tissues. An example of this can be found in neurons, where the rate of ATP hydrolysis can be increased
by several orders of magnitude within seconds for their energy needs, however their intracellular ATP
levels remain surprisingly constant. This led to the concept of the stability paradox [5] which purports
that there are ... “immediately available, fast and efficiently working energy supporting and back-up
systems that connect sites of energy consumption to those of energy production via phosphoryl
transfer networks” (reviewed in [6]). In this regard, it is believed that the Cr/PCr/CK system has
evolved to play a major role in the regulation of intracellular energy metabolism. CK is the only existing
phosphagen kinase in vertebrates, with at least five subunit isoforms that are expressed in a tissueand cell- specific manner with defined subcellular locations; a brain type (CK-B), a muscle type (CK-M),
a hetero-dimeric heart type (MB-CK) and two mitochondrial creatine kinases (mt-CK) that exist as
homo-octamers (a sarcomeric mt-CK and a ubiquitously expressed mt-CK) [4,7]. It has long been
recognized that efficient energy metabolism requires the close spatial organization of the enzymes
generating ATP, co-localization of sites of ATP synthesis with sites of ATP consumption, and the
sustained delivery of high energy phosphates to ATPases in cases where ATP generation is separated
from consumption [8]. The primary mode of regulation of the CK isoenzymes is by subcellular
compartmentation (enabling the functional coupling of the CK reaction to various cellular ATPases)
and Cr/PCr due to their relatively smaller size when compared to ATP/ADP as well as their superior
concentrations in cytosol (ATP, 3-5mM; ADP, 20-40µM; Cr, 5-10mM; PCr, 20-35mM) [4] by definition
(Fick’s law) have a higher diffusion coefficient. Taken together, this renders the Cr/PCr/CK system very
effective in providing sudden and sufficiently high concentrations of high energy phosphates during
conditions of rapid ATP hydrolysis. In summary; Cr/PCr are most suited to act as shuttle molecules
between sites of ATP hydrolysis (ATPases) and sites of ATP production (glycolysis and mitochondrial
oxidative phosphorylation) or the CK/PCr system can be directly coupled to ATPases to regenerate
ATP from ADP (Fig. 1).
12
Figure 1: Creatine/Phosphocreatine/Creatine Kinase system (obtained with permission from Wallimann et al. [9]): Creatine
(Cr) is transported into cells expressing creatine kinase (CK) via the creatine transporter (CRT) and is utilized in a number of
ways; Cr can be coupled to mitochondrial oxydative phosphorylation (via mtCK) or glycolysis (via CK-g) to yield
phosphocreatine (PCr). In the cytosol, interconversion between Cr and PCr by CK-c serves to replenish ATP from ADP. And
finally, at sites of ATP consumption (for example by ATPases), PCr is recruited by CK-a to regenerate ATP from ADP.
Creatine synthesis and transport:
Creatine and phosphocreatine are spontaneously broken down to creatinine which is then excreted
into the urine. The rate of loss has been estimated to be about 1.7% of the total body pool per day
[10]. This loss of creatine requires continuous replacement which occurs through a combination of
endogenous synthesis and uptake from the diet.
Creatine synthesis is carried out by two enzymes using arginine, glycine and methionine. The entire
glycine molecule is incorporated into creatine whereas arginine and methionine provide their amidino
and methyl groups respectively. Creatine synthesis (Fig. 2) takes place in two steps; in a first step Larginine glycine amidino transferase (AGAT/GATM, EC 2.1.4.1) catalyzes the transfer of the amidino
group from arginine to glycine to yield ornithine and guanidinoacetate (GAA). The next step involves
the transfer of a methyl group from S-adenosylmethionine (SAM) to GAA to produce creatine and Sadenosylhomocysteine (SAH) catalyzed by the enzyme guanidinoacetate N-methyl transferase
(GAMT, EC 2.1.1.2) [11].
Synthesized creatine or that which is taken in with the diet is distributed via the bloodstream and is
able to cross lipid-rich biological membranes mainly via the action of a specialized carrier - the creatine
transporter (SLC6A8) [12]. Tissues and cells with high and fluctuating energy requirements like the
skeletal muscle, brain, kidneys, and retina, express high levels of SLC6A8, which by virtue of its
homology to the GABA family of transporters has been classified as a Na+/Cl- -dependent
neurotransmitter transporter (also known as solute carrier transporter of family number 6 - SLC6),
that are responsible for transport of solutes (GABA, serotonin, dopamine, glutamate, etc) against a
high concentration gradient [13].
13
Figure 2: Creatine synthesis and transport (adapted with permission from Wada et al. [14]): AGAT catalyzes the synthesis of
guanidinoacetic acid (GAA) using equimolar amounts of arginine and glycine. GAMT subsequently transfers a methyl group
from S-adenosylmethionine to GAA forming creatine. Highest expression levels of AGAT and GAMT have been detected in the
kidney and liver respectively, suggesting these organs are primarily responsible for creatine synthesis. Synthesized creatine
is secreted into the blood from where it can be taken up into tissues, across the plasma membrane by the creatine transporter.
Creatine is spontaneously broken down and excreted into urine as creatinine.
Inborn errors of creatine metabolism
Creatine is distributed in the entire organism, with highest levels detected in skeletal muscle (30mM)
and brain (10mM) [10]. Accordingly, one of the most striking effects of creatine supplementation
(coupled to an exercise program) is an increase in muscle mass [15–18], indicating that the increase
in energy demands as a result of exercise stimulates an increase in muscle creatine metabolism. This
so called ergogenic property of creatine has been studied in detail [19,20] and five mechanisms have
been proposed for this effect: increase in phosphocreatine and glycogen levels, rapid regeneration of
phosphocreatine, increase in expression of growth factors, decrease inflammation and muscular
damage as well as an increase in muscle volume due to the osmotic effects of creatine. Thus it can be
expected that “defective muscle functions” will be the main symptom of creatine deficiency. However
following the identification of the first patients with creatine deficiency [21–23] encompassing more
than 200 clinically diagnosed cases to date (www.lovd.nl/GATM, www.lovd.nl/GAMT,
www.lovd.nl/SLC6A8), it has become evident that impaired muscle function is not the major
consequence of altered creatine metabolism. These disorders drastically affect brain function with
intellectual disability being the most prevalent phenotypic outcome [24] . It is worth noting that
although the brain constitutes only about 2% of the body’s mass, it may spend up to 20% of the body’s
energy consumption [25]. Meaning that a very high turnover of ATP is necessary to sustain cerebral
function and consequently the brain will be severely affected by creatine deficiency.
14
Creatine synthesis defects
AGAT deficiency (OMIM: 602360) and GAMT deficiency (OMIM: 601240) are autosomal recessivelyinherited errors of creatine synthesis with the main clinical manifestations of defective creatine
synthesis being; intellectual disability, speech and language delay, autistic-like behavior and epileptic
seizures. However clinical presentation can be quite non-specific and include other symptoms like
hypotonia, growth delays, self-mutilating behavior and movement disorders. Affected individuals can
be diagnosed by analysis of creatine, guanidinoacetate, and creatinine in urine, plasma and
cerebrospinal fluid [26]. It is of note that, irrespective of the presence of a functional creatine
transporter in these patients, the biochemical hallmark of AGAT and GAMT deficiency is a decrease or
absence of the creatine peak as revealed by cranial proton magnetic resonance spectroscopy (1HMRS). The main metabolic difference between AGAT and GAMT deficiency is the elevated GAA levels
in GAMT deficiency patients. Patients with GAMT deficiency may also suffer from a predominantly
extrapyramidal movement disorder and more severe epilepsy compared to AGAT deficient patients
[27,28].
Creatine transport defect
To date four creatine transporters have been described; SLC6A8 (CT1, CRT1, CRTR or CrT) located on
chromosome Xq28, SLC6A10pA and SLC6A10pB – two duplicated unprocessed pseudogenes of SLC6A8
located on chromosome 16p11.2 [29], and more recently the monocarboxylate transporter (MCT12)
was also shown to be capable of creatine transport [30].
A defect in the SLC6A8 gene was the first inborn error of creatine transport identified (Salomons,
2001). Creatine transport deficiency as a result of a mutation in the SLC6A8 gene mapped at the Xchromosome (OMIM: 300036) is X-linked, and as expected the symptoms are most severe in males,
with female carriers presenting with a milder phenotype. The most common symptoms overlap with
those due to creatine biosynthesis deficiency and these include; intellectual disability, autism-like
behaviour, movement disorders and epilepsy. Individuals with SLC6A8 deficiency may also have, mild
generalized muscular hypotrophy, dysmorphic facial features, microcephaly and gastrointestinal
disturbances [31–33]. Female carriers (heterozygotes) usually present with learning disabilities [34].
Current estimates indicate that mutations in the SLC6A8 gene could be responsible for around 2% of
cases of X-Linked intellectual disability [35], and about 1% of cases with intellectual disability of
unknown aetiology [36].
Although the two SLC6A8 paralogues (SLC6A10pA and SLC6A10pB) at chromosome 16p11.2 share
95.8% sequence similarity with SLC6A8, their predicted amino acid sequence harbours a nonsense
mutation in “exon 4” (with respect to CTR1), indicating that a creatine transporter protein cannot be
translated from the SLC6A10 mRNA and they are most likely pseudogenes [37,38]. However, a
translocation breakpoint on chromosome 16p11.2 was mapped to disrupt the 5' flanking sequence of
SLC6A10pA in a patient presenting with autism (developmental as well as severe language delay with
no evident dysmorphic features) [39,40]. No data on brain/plasma/urinary creatine levels was
provided for this patient.
Another case of creatine transport-related deficiency has been recently reported in a patient
harboring a non-synonymous p.Gly407Ser mutation in the SLC16A12 gene. SLC16A12 encodes a solute
carrier of the monocarboxylate transporter family (MCT12). Mutations in SLC16A12 were initially
identified as the underlying defect in age-related cataract and renal glucosuria [41,42]. MCT12mediated creatine transport was found to be independent of Na+/Cl- and pH dependent, but the most
interesting difference with creatine transport by CTR1 was the bidirectional nature of MCT12 creatine
transport [30] because so far the nature of creatine efflux from cells was unknown. Taken together,
the fact that a mutation identified in SLC16A12 decreases creatine uptake in vitro, and that Slc16a12
15
knockout rats have elevated urinary creatine, as well as the tissue-/cell-specific differences in
expression patterns between SLC16A12 and SLC6A8, led the authors to conclude that MCT12 and CTR1
may have distinct, but possibly synergistic functions in terms of creatine homeostasis. Not surprisingly,
due to the presence of functional creatine synthesis and transport in patients SLC16A12 deficiency
(OMIM: 611910), the classical neurological phenotype of creatine deficiency is not seen in these
patients. Incidentally, the Km of the MCT12 creatine transport was found to be 567.4µM, which is
higher than that of CTR1 creatine transport, determined to be around 17-77 µM in human and rat
(reviewed in [43]). As such MCT12 has a correspondingly lower affinity for creatine than CTR1, and the
latter is most likely the primary transporter responsible for intracellular uptake of creatine. However
this does not minimize the importance of MCT12 as it is the first transporter identified that is capable
of creatine efflux from cells.
Diagnosis of creatine deficiency syndromes
In addition to biochemical screening of creatine, creatinine and guanidinoacetate in body fluids,
molecular genetic tests are also performed for sequence variants in AGAT (GATM), GAMT or SLC6A8
genes. Moreover, functional assays have been developed for AGAT and GAMT enzyme activities
[44,45] as well as creatine uptake [22,46] in primary fibroblasts and lymphoblasts. Table 1 summarizes
the current approaches used in diagnoses of creatine deficiency syndromes. Reference values for the
investigated metabolites and details of the diagnostic methodologies can be found in the references
cited therein.
16
Table 1: Diagnostic criteria for creatine deficiency syndromes
Diagnosis
SLC6A8 deficiency
GAMT deficiency
AGAT deficiency
Brain Creatine
Low to absent
Low to absent
Low to absent
Plasma Creatine
Increased
Decreased
Decreased
Urinary Creatine
Increased
Decreased
Decreased
CSF Creatine
Normal to Increased
Decreased
Normal
Plasma GAA
Normal
Increased
Decreased
Urinary GAA
Normal
Increased
Decreased
CSF GAA
Not done
Increased
Not done
Plasma Creatinine
Normal
Normal
Decreased
Urinary
Creatinine
Low
Low
Low
CSF creatinine
Decreased
Not done
Not done
presence of sequence
Not applicable
Not applicable
presence of sequence
Not applicable
Metabolic
Creatine1, Creatinine2
Guanidinoacetate3 (GAA)
Sequencing
(DNA/RNA)
SLC6A8
variants/deletions/insertions
GAMT
Not applicable
variants/deletions/insertions
GATM
Not applicable
Not applicable
presence of sequence
variants/deletions/insertions
Functional
SLC6A8
Creatine uptake assay in
Not applicable
Not applicable
GAMT enzyme activity in
Not applicable
primary fibroblasts
GAMT
Not applicable
lymphoblasts/fibroblasts
GATM
Not applicable
Not applicable
AGAT enzyme activity in
lymphoblasts/fibroblasts
1_[47–50]
2_[48,50]
3_[47–52]
Understanding the pathophysiology of creatine deficiency
The dawn of this millennium has seen tremendous progress being made in the areas of genomics,
transcriptomics, proteomics and metabolomics. This is evident by the increasing number of
17
individuals, and the different genetic diseases being diagnosed using next generation sequencing
technologies. It has been no different for the creatine deficiencies and there is an ever growing list of
individuals in the literature diagnosed with an inborn error of creatine metabolism. In this post
genomic era, the need to understand the pathophysiological processes common amongst otherwise
unrelated genes is now stronger, because it is indeed the first step towards developing therapeutic
strategies.
As mentioned above, creatine synthesis deficiency occurs following a defect in either AGAT or GAMT
genes. The key difference between patients with AGAT or GAMT deficiency is the prevalence of
intractable epilepsies with GAMT deficiency. Elevated GAA levels in GAMT patients are responsible for
its more severe phenotype [53,54]. Further clarifications on the pathogenicity of defective creatine
synthesis to the brain emerged, when it was revealed via in situ hybridization that SLC6A8 is only
expressed by microcapillary endothelial cells at the blood–brain barrier (BBB). Suggesting that the BBB
has a limited permeability for peripheral creatine and this limitation has to be compensated for by
endogenous creatine synthesis in the brain [55–57].
In the context of the pathogenicity of a creatine transporter defect – despite the presence of
functional creatine synthesis enzymes, three models have been proposed. The first model derives
from the fact that although on a global scale the brain is capable of creatine synthesis the cellular
expression of AGAT and GAMT can be dissociated [58]. Specifically, in cortical grey matter only 12%
of cells were found to express both AGAT and GAMT, and a further 43% expressing either AGAT (22%)
or GAMT (21%) [58,59]. Due to the expression patterns of AGAT + SLC6A8 (6.7%) and GAMT + SLC6A8
(7.9%) in these cells and the previously established strong decrease of creatine in the brain of SLC6A8
deficient patients, the authors propose that the creatine transporter could be required for the
provision of GAA synthesized in AGAT-expressing cells to GAMT-expressing cells. This model is
supported by the finding of elevated GAA levels in one SLC6A8 deficient patient [60] and more recent
experiments that show SLC6A8-mediated efflux of GAA into the CSF as well as an SLC6A8mediated/Na+- and Cl-- dependent uptake of GAA by choroid plexus epithelial cells [61]. In a second
model, based on in vitro creatine uptake experiments using primary cultures of rat type I astrocytes
and cerebellar granule cells the creatine transporter was apparently more effective in building up
intracellular creatine than endogenous synthesis [62]. Hypothetically this is in line with experiments
done by Tachikawa et al., [63] that show that the Km for GAA transport by CTR1 (269µM-oocytes,
412µM-HEK293) was at least 10 fold higher than that for creatine. It follows that with such a relatively
higher affinity to take up creatine, cells could potentially accumulate exogenous creatine faster than
they can get rid of GAA, and the ensuing high levels of accumulating creatine will then act as a negative
feedback for GAA synthesis by downregulating AGAT expression [64]. Finally, taking into account; the
evidence that creatine is released at the synapse in an action-potential dependent manner [65], the
homology of CTR1 to other SLC6 transporters (for example the dopamine, glycine, noerepinephrine,
serotonin and transporters) that are known to facilitate neurotransmitter reuptake at the synaptic
cleft [66] as well as the presence of normal creatine levels in CSF of SLC6A8 deficient patients, the
most recent model purports to explain the cerebral creatine deficiency in SLC6A8 deficient patients as
arising from the failure to recycle creatine at the synapse following its release [67].
Supplemental and therapeutic use of creatine
Due to its known role in muscle physiology, creatine has been predominantly used as a nutritional
supplement to improve muscle performance in healthy individuals [68,69]. The most popular use of
creatine has been as a performance-enhancing substance by athletes. Over the last two decades, the
discovery of patients with primary creatine disorders has played a major role in understanding the
18
physiological relevance of creatine. The predominantly neurological symptomatology of creatine
deficiency, as well as co-occurring muscular deficits established the basis for its increased use (with
varying degrees of success) as a supplement not only in the treatment of primary creatine deficiency
disorders but also as a neuroprotective agent (in Parkinson’s disease, Huntington’s disease, Ischemia,
Amyotrophic lateral sclerosis, etc.), an anti-inflammatory agent, as well as an in mitochondrial
cytopathies. A more extensive overview of the benefits of creatine supplementation is covered by
[6,10,70–72]. For the purpose of this thesis only the relevance of creatine supplementation to creatine
synthesis and transport deficiency will be highlighted. In the literature the total dose of creatine
administered to patients with creatine deficiency ranges from 100 to 800 mg/kg/bw/day.
AGAT deficiency
Replenishment of brain creatine in AGAT and GAMT patients as measured with 1H-MRS is slow, usually
requiring a high dose (which is on average about 20 times the daily requirement) of creatine and/or
long-term supplementation to achieve brain creatine levels greater than or equal to 60% of controls
[48]. Nonetheless a positive clinical outcome almost always ensues creatine supplementation.
Observations made on patients suggest that, compared to GAMT deficiency, AGAT deficiency is
associated with a milder phenotype, and/or with a better response to creatine supplementation. In
response to creatine supplementation, AGAT patients undergo a general improvement of cognitive
functions (improved speech, fading autism) which also includes progress in psychomotor
development, however mild intellectual disability still persists [73]. Even more remarkable, was the
abrogated manifestation of classical creatine deficiency symptoms in a patient diagnosed with AGAT
deficiency at birth and treated pre-symptomatically [74], suggesting that onset of treatment is crucial
in determining phenotypic response to creatine therapy.
GAMT deficiency
Similarly, onset of symptoms in GAMT deficiency can also be prevented by pre-symptomatic
treatment of neonatal GAMT deficiency [75]. Creatine supplementation in the earliest GAMT patients
quickly revealed that brain creatine replenishment without targeted decrease of GAA levels is not as
potent in improving clinical symptoms in patients as combining creatine supplementation with
ornithine supplementation and dietary arginine (protein) restriction; which confer a significant
decrease in urinary and plasma GAA concentrations and a significant improvement of epilepsy and
EEG findings [54,76,77]. Pre-symptomatic treatment suggests treatment onset could determine
phenotypic response to treatment. This is emphasized in a recent study involving analysis of treatment
outcome in 48 patients diagnosed with GAMT deficiency [78], wherein 4 patients treated before the
age of 9 months had normal or almost normal developmental outcomes. Moreover there was a
positive correlation between treatment onset and severity of intellectual disability/developmental
delay in the entire patient group.
SLC6A8 deficiency
Creatine supplementation has been largely inefficient in treating SLC6A8 deficient patients [27]. When
combined with L-arginine and L-glycine to enhance creatine biosynthesis results have been far more
encouraging, especially in females since they tend to have mild SLC6A8 deficiency (heterozygous) [79].
The only positive effects reported in males has been an increased muscle mass and improved gross
motor skills [80]. Consequently the use of alternatives like lipophilic creatine derivatives that could
cross the blood brain barrier by passive diffusion has been gaining popularity in recent years. These
studies remain promising with especially positive results in vitro [81–84] and in an SLC6A8 deficient
19
mouse model supplemented with cyclocreatine [85]. Nonetheless no breakthroughs have yet been
attained in human subjects due to a number of issues amongst which are (but not limited to); the
highly spontaneous conversion/enzymatic degradation of some derivatives to creatinine [86–88],
inability to maintain sufficiently high brain concentrations of these compounds [89] and of course
some toxicity issues have been noticed as well [81].
20
Objectives and Outline of Thesis
In view of the clinical relevance of creatine, especially within the context of patients affected with
cerebral creatine deficiency, the objective of this thesis was centered on two themes; 1– to explore
potential avenues which can be used to improve diagnosis of patients and, 2– unraveling aspects on
creatine transport regulation with a view towards improved treatment outcome in patients with a
defect in creatine biosynthesis.
The absence of pathologic manifestations in AGAT or GAMT deficient individuals treated presymptomatically underscores the importance of early diagnosis. This illustrates the urgency of a
proper diagnosis of patients early on in life. Hereto we thought to enlarge the awareness of these
disorders by describing the clinical phenotype and long term treatment results in AGAT deficiency
(Chapter 2). GAMT deficiency had been described already more thoroughly, but studies on missense
mutations were still lacking. Since missense mutations are usually difficult to interpret we
implemented a functional study for this type of mutations and characterized all current known
missense variants. In addition we investigated 13 new GAMT patients with missense mutations
(Chapter 3).
In both AGAT and GAMT deficiencies, treatment aims are in replenishment of the cerebral creatine
pool by dietary creatine supplementation. In case of GAMT deficiency additional supplements are
being used aiming at the decrease of the cerebral accumulation of guanidinoacetate. By exploring the
regulation of the creatine transporter, this treatment regimens can potentially be improved.
Therefore we investigated the potential presence and function of yet unidentified creatine transporter
isoforms or alternative transporters capable of creatine transport. Two creatine transporter isoforms
were identified with a similar expression profile to that of the creatine transporter. Via overexpression
assays, we investigated if they are capable of (regulation of) creatine transport or if they influence
SLC6A8-mediated creatine uptake (Chapter 4).
Defects in the creatine transporter are fairly common amongst individuals with X-linked intellectual
disability. However screening for potentially pathogenic variants on the SLC6A8 gene has so far not
included its yet unknown promoter region. Because it is now evident from whole genome sequencing
data that disease variants are also being found within such non-coding regions we sought to identify
the promoter region of SLC6A8 (Chapter 5). The sequence similarity between SLC6A8 and its
paralogous loci on chromosome 16 (SLC6A10P) is approximately 95%. However as opposed to SLC6A8,
expression of the SLC6A10P pseudogenes had only been reported in brain and testis. We hypothesized
that the sequence variants in the corresponding pseudogene promoters could account for their
limited expression. Consequently we also cloned and investigated the transcriptional promoter of the
SLC6A10P pseudogene (Chapter 5).
So far attempts to treat patients with SLC6A8 deficiency have been unsuccessful, largely as a result of
the impermeability of the blood brain barrier to creatine. By gaining insight into the molecular
pathophysiology of creatine deficiency, alternative treatment strategies that bypass the need for
creatine supplementation should be developed. To further improve our understanding of altered
pathways in the background of creatine deficiency we carried out genome–wide differential RNA
expression in SLC6A8 deficient fibroblast cells. The applied technique (RNAseq) and its outcome is
described in Chapter 6.
21
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28
29
Chapter 2
Developmental progress and creatine restoration upon long-term
creatine supplementation of a patient with arginine:glycine
amidinotransferase deficiency
"It’s not that I’m so smart, it’s just that I stay with problems longer"
Albert Einstein
30
31
Molecular Genetics and Metabolism
Developmental progress and creatine restoration upon long-term creatine
supplementation of a patient with arginine:glycine amidinotransferase deﬁciency
Joseph D.T. Ndika a, 1, Kathreen Johnston b, 1, James A. Barkovich c, Michael D. Wirt d, Patricia O'Neill e,
Oﬁr T. Betsalel a, Cornelis Jakobs a,⁎, Gajja S. Salomons a
a
Metabolic Unit, Department of Clinical Chemistry, VU University Medical Center, Amsterdam, The Netherlands
Genetics Department, Kaiser Permanente, San Francisco, CA, USA
Departments of Radiology and Biomedical Imaging, Neurology, and Pediatrics, University of California, San Francisco, CA, USA
d
Clinical Services Blanchﬁeld Army Community Hospital, Fort Campbell, KY, USA
e
Rehabilitation Services Department, Kaiser Permanente, San Francisco, CA, USA
b
c
a r t i c l e
i n f o
Article history:
Received 22 December 2011
Received in revised form 19 January 2012
Accepted 19 January 2012
Available online 27 January 2012
Keywords:
AGAT
Creatine deﬁciency
Developmental delay
Creatine supplementation
a b s t r a c t
Background: Arginine:glycineamidinotransferase (AGAT/GATM) deﬁciency has been described in 9 patients
across 4 families. Here we describe the clinical outcome and response to creatine supplementation in a patient of the second family affected with AGAT deﬁciency—a 9-year-old girl.
Patient and methods: Delayed motor milestones were noticed from 4 months of age and at 14 months moderate
hypotonia, developmental delay and failure to thrive. Laboratory studies revealed low plasma creatine as well as
extremely low levels of guanidinoacetic acid in urine and plasma. Proton magnetic resonance spectroscopy
(MRS) of the brain showed absence of creatine. DNA sequence analysis revealed a homozygous mutation
(c.484+ 1 G > T) in the AGAT/GATM gene. AGAT activity was not detectable in lymphoblasts and RNA analysis
revealed a truncated mRNA (r.289_484del196) that is degraded via Nonsense Mediated Decay. At 16 months, Bayley's Infant Development Scale (BIDS) showed functioning at 43% of chronologic age. Oral creatine supplementation (up to 800 mg/kg/day) was begun.
Results: At age 9 years she demonstrated advanced academic performance. Partial recovery of cerebral creatine
levels was demonstrated on MRS at 25 months of age. Brain MRS at 40 months of age revealed a creatine/NAA
ratio of about 80% of that in age-matched controls.
Conclusions: 8 years post initiation of oral creatine supplementation, patient demonstrates superior nonverbal and
academic abilities, with average verbal skills. We emphasize that early diagnosis combined with early treatment
onset of AGAT deﬁciency may lead to improvement of developmental outcome.
© 2012 Elsevier Inc. All rights reserved.
1. Introduction
Creatine deﬁciency syndromes (CDS) are a set of inborn errors of
metabolism whose common biochemical feature is the absence of
creatine/phosphocreatine in the brain. Clinical manifestations are
often characterized by intellectual disability, speech and language
delay, autistic-like behavior and epilepsy [1]. Biosynthesis of creatine
begins with the synthesis of guanidinoacetic acid (GAA) from arginine and glycine by L-argine:glycineamidinotransferase (AGAT;
OMIM 602360). Creatine is then synthesized by the transfer of a
methyl group from S-adenosyl methionine to GAA by guanidinoacetate methyl transferase (GAMT; OMIM 601240). Biosynthesized or dietary creatine is taken up via the creatine transporter (CTR), encoded
⁎ Corresponding author at: Department of Clinical Chemistry, VU University Medical
Center Amsterdam, De Boelelaan 1117, PK 1X 014, 1081 HV Amsterdam, The Netherlands.
Fax: +31 20 4440305.
E-mail address: [email protected] (C. Jakobs).
1
Contributed equally.
1096-7192/$ – see front matter © 2012 Elsevier Inc. All rights reserved.
doi:10.1016/j.ymgme.2012.01.017
for by the SLC6A8 gene (OMIM 300036). Genetic deﬁciencies in either
the creatine biosynthesis enzymes or transporter protein result in
CDS [2–4].
Treatment of creatine biosynthesis defects leads to improvement
in movement disorder, epilepsy and developmental progress. The
treatment of GAMT deﬁciency consists of creatine and ornithine supplementation and an arginine restricted diet; and AGAT deﬁciency is
treated with creatine supplementation alone, whereas no successful
treatment strategy yet exists for SLC6A8 deﬁciency.
AGAT (ofﬁcial HGNC symbol is GATM, however to avoid confusion
we refer to it as AGAT—as used in all previously reported patients) and
GAMT deﬁciencies are autosomal recessive disorders, and SLC6A8 deﬁciency is an X-linked disorder. AGAT deﬁciency is the least frequently reported of the three disorders, with a total of nine patients from
four families [3,5–11]. We report here the diagnostic work-up and
long-term outcome of the creatine supplementation therapy in the
second family diagnosed with AGAT deﬁciency [8,9]. Additionally
we review all patients with AGAT deﬁciency reported in the literature
and developed a novel database—the Leiden Open Variation Database
32
Table 1
RT-PCR primer sequences.
No. Forward/reverse sequence
1
2
mRNA
AGAT location
Amplicon size
(bp)
5′TACATCGGATCTCGGCTTGG 3′
AGAT
Exon 1/Exon 2 Wild type: 716
5′TCAGCAGCATCAAAGCATGGC 3′
Exon 5
ΔExon 3: 520
5′GCTGGTGATGTGAGCTGAGT 3′
GAPDH –
Wild type: 819
5′TCAAGGCTGAGAACGGGAAGC 3′
(LOVD), to include genetically relevant data of all patients (http://
www.LOVD.nl/GATM).
2. Case report: diagnosis and treatment
This 9-year-old girl was born at term after an uneventful pregnancy to non-consanguineous parents of Chinese ancestry. Gross motor
delay was ﬁrst noted at 4 months of age when she was not pushing
up when placed in prone position. She started sitting with support
at 6 months and sat independently at 9 months.
Upon evaluation at 10 months of age, she had moderate central
hypotonia. She sat independently but did not have appropriate righting responses when she began to fall, and bore weight when held vertically by locking her knees. Poor weight gain was also noted.
4 months later she was evaluated for developmental delay. She
stood with support but did not cruise, and could not independently
get to a sitting position. She transferred objects hand to hand and
said consonant sounds such as “mama” non-speciﬁcally. Physical examination revealed weight 7.76 kg (below 5th percentile), length
69.5 cm (below 5th percentile) and head circumference at 5th–10th
percentiles. She had moderate diffuse hypotonia. Her deep tendon reﬂexes were normal. She had a unilateral single palmar ﬂexion crease
and unilateral preauricular skin tag.
2.1. Laboratory diagnosis
Laboratory analysis revealed moderate generalized organic aciduria
(2–5 times the upper limits of normal), a hallmark of low creatinine
excretion; thus a creatine synthesis defect was considered. Urinary
and plasma guanidinoacetate (GAA) were markedly reduced
(0.22 mmol/mol creatinine; ref 53.9 ± 26.9 SD and 0.07 μM; control
1.34 ± 0.64 SD respectively) with very low creatine in plasma
(2.3 μM; control 75.6±21.6 SD); all measured as previously described
[12]. This result suggested AGAT deﬁciency. DNA sequence analysis of
the AGAT gene revealed a novel homozygous mutation—c.484 +
1 G > T. This mutation as predicted by Alamut™ software causes the
A
1
RT -
2
+
-
-
2.2. Creatine supplementation: dosage, cerebral replenishment, side
effects
The initial creatine dose was 400 mg/kg/day, corresponding to
15–20 times the daily creatine requirement. Throughout therapy,
the oral creatine dose has varied from 400 to 800 mg/kg/day
(Fig. 2). Dosing adjustments were based on evaluation of cerebral creatine content, serum creatine levels, urine exams and clinical status
including developmental progress.
Proton MR spectroscopy data were acquired from a 2 × 2 × 2 cm
voxel that included frontal white matter and basal ganglia in the left
cerebral hemisphere. It revealed an increase in cerebral creatine content when measured prior to and post treatment onset (Fig. 2). However, the brain creatine content was essentially unchanged at 55–60%
of normal between 8.5 and 17 months of therapy (25 to 34 months of
age). In an effort to improve brain creatine content and developmental outcome, the creatine dose was gradually increased from 400 to
800 mg/kg/day over 4 weeks beginning at 34 months of age. Repeat
brain MRS 5 months later showed improvement in brain creatine/
NAA from approximately 60% to 80% of normal. She was noted however to have 4+ creatinine crystals for the ﬁrst time in her urine. To
avoid possible renal toxicity, the creatine dose was decreased to
400 mg/kg/day for 1 month and then increased back to 500 mg/kg/
day. The family was advised to increase ﬂuid intake. There has been
B
3
+
erroneous splicing of exon 3. AGAT enzyme activity was not detectable
in cultured lymphoblasts [13]. Both parents are heterozygous for this
same mutation. In order to conﬁrm erroneous splicing in patient's
mRNA, RT-PCR was performed (see Table 1 for primer sequences).
RNA was isolated from the patient's lymphoblasts using the SV Total
RNA Isolation System (PROMEGA). Following cDNA synthesis the general
PCR conditions were as follows: initial denaturation cycle of 15 min at
95 °C; followed by 38 cycles of 45 s at 94 °C, 45 s at 61 °C, and 1 min at
72 °C; and a ﬁnal extension of 10 min at 72 °C. Direct sequencing was
performed on puriﬁed PCR products by use of BigDye Terminator v3.1
and an ABI 3100 sequence machine (PE Applied Biosystems). The
obtained electropherograms were analyzed by use of the Mutation Surveyor™ software package (SoftGenetics). This conﬁrms a truncated
mRNA lacking exon 3—r.289_484del196, which predicts the substitution of alanine at position 97 by valine and an alternative reading
frame that ends in the 11th codon of exon 4 (p.Ala97ValfsX11). The
truncated mRNA is subjected to nonsense mediated decay (NMD) as
it is only faintly detectable on RNA samples obtained from AGAT deﬁcient lymphoblasts cultured with cycloheximide (250 μg/mL). This is
in contrast to control mRNA isolated from lymphoblasts cultured without cycloheximide (Figs. 1A/B).
+
blank
p.= (GATM)
716 bp
1
2
3
1
2
4
4
5-8
9
AGAT
819 bp
GAPDH
5-8
9
p.Ala97ValfsX11
Fig. 1. Identiﬁcation of truncated AGAT transcript as well as conﬁrmation of nonsense mediated decay (NMD) in AGAT deﬁcient lymphoblasts by RT-PCR. (A) Prior to RNA isolation,
patient's lymphoblast cells were either treated without (1) or with (2) cycloheximide. Lymphoblasts of control (3) were not treated with cycloheximide. The control cell line shows
expected fragment size (716 bp), while the cycloheximide-treated cells show low expression of the truncated AGAT mRNA (control Δ exon 3: 520 bp). In the absence of cycloheximide treatment no AGAT mRNA could be detected (1). In PCR reactions that lacked Reverse Transcriptase (RT) (−) no GAPDH or AGAT amplicons were present indicating that the
RNA was not contaminated with gDNA. (B) Fig. 1B shows a scheme of the consequences of the splice site mutation (c.484 + 1 G > T) as revealed by RT-PCR and sequence analysis.
Wild type AGAT mRNA is translated from exon 1–9 (upper transcript). Due to the canonical splice site mutation exon 3 is skipped resulting in a frameshift with Ala 97 being the ﬁrst
replaced amino acid by Val and a new reading frame which leads to a premature stop codon in exon 4 (lower transcript).
33
A
B
NAA
Cho
Oral creatine dose vd Cr/NAA ratio in WM
1
Oral creatine dose
(mg/kg/d)
800
Cr/NAA ratio
0.25
700
2
8 months Cr treatment
3
17 months Cr treatment
4
23 months Cr treatment
0.2
600
500
0.15
400
Cr/NAA ratio
Oral Cr Dose (mg/kg/d)
Prior to treatment
Cr
0.3
900
0.1
300
200
0.05
100
0
0
2
3
3.5 8.5
9
9.5 11 13 17 17.5 18.5 19.5 22 23.5
Months of therapy
0
4
3
2
1
0
Fig. 2. Creatine Restoration in AGAT Deﬁcient Patient. (A) Oral Creatine Dose vs Cr/NAA Ratio. Treatment efﬁcacy was monitored by measuring brain creatine before treatment and
at intermittent creatine doses following creatine supplementation. (B) Single voxel long echo (TE = 288 ms) frontal white matter proton magnetic resonance spectra were acquired
at 1.5 T. (1) Initial MR spectrum collected prior to oral creatine supplementation at age 16.5 months. Note the absence of the creatine resonance at 3.02 ppm consistent with the
diagnosis of AGAT deﬁciency. (2–4) The creatine resonance at 3.02 ppm demonstrates a progressive increase in peak area over 23 months of oral creatine supplementation, but
remains below normal levels.
no evidence of gastrointestinal disease or malabsorption. As urine
specimens continued to be negative for crystals, 31 months after initiating therapy the dose was increased to 600 mg/kg/day and then to
700 mg/kg/day 2.5 months later. She has remained on this dose since
then (50 months chronologic age and at 33.5 months of therapy).
Urinalysis has been monitored every 3–6 months, and has been normal except for intermittent appearance of crystals on rare occasions.
Kidney function tests and renal ultrasounds performed every 6 months
showed no abnormalities, except for one occasion—6 years and 9 months
into therapy, when blood creatinine was 0.81 mg/dL (reference range
0.3–0.7). Repeat creatinine measurements 1, 2, and 9 months later
were normal—most recently 0.62 mg/dL.
2.3. Occupational, physical and speech-language therapy
From age 16 months she received occupational and physical therapy for global developmental delay. But because of improvements in
her developmental milestones following creatine supplementation,
both services were discontinued at age 24 months (7.2 months into
creatine therapy).
At 20 months of age, 3 months into creatine therapy, she babbled
but did not say mama or dada speciﬁcally. A further evaluation at age
26 months revealed a delay in comprehensive and expressive language skills of 2 to 6 months. A month later speech therapy was introduced in conjunction with continued creatine supplementation. She
received speech-language therapy throughout (except for a long
break from age 54 to 63 months) until discontinued at age 9.25 years.
2.4. Developmental progress (Table 2)
Neuropsychological assessment showed greatly improved functioning on the Bayley Scales of Infant Development, second edition
(BSID II) from 43% at diagnosis to 100% 23 months into creatine therapy. BSID-II scores of 100 ± 15 represent the mean ± 1SD; score ≤70
is 2SDs below the mean. Patient continued to demonstrate strengths
in her cognitive and academic development even though there was
a signiﬁcant discrepancy between verbal and nonverbal abilities
(WPPSI-III test). She scored in the bottom of the average range on
verbal subtests (Verbal IQ-91), and in the high average to superior
range on nonverbal tests of intellectual ability (Performance IQ-112).
She also scored in the very superior range on tests of purely nonverbal
skills (Toni-3 and Leiter Tests). Formal IQ testing has not been repeated
since age 5 years. At age 9 years (7.5 years of therapy) educational
assessments according to School District of California Standardized
Testing (State target for all students is 350–600) revealed the following
results: English-Language Arts score of 424 (advanced is 402–600),
Mathematics score of 506 (advanced is 414–600). She has been in regular preschool and elementary school classes. There have been no behavioral difﬁculties, and no evidence of attention deﬁcit hyperactivity
disorder or autistic spectrum disorder. The child is cooperative, friendly,
and delightful.
2.5. Language development (Table 2)
Upon diagnosis at age 16 months patient demonstrated comprehensive and expressive language skills in the 6–12 months range.
She indicated needs with vocalization and used vowel and consonant
sounds non-speciﬁcally. Contribution of creatine supplementation
and speech therapy to patient's language development were evaluated using a number of tests from age 36 to 104 months.
In terms of expressive language the patient has shown an overall
improvement in her rate of single word recall from long-term memory (EOWPVT test [14]), despite an unusual variable pattern of progression. Her pragmatic use of language as noted by observation is
improved for initiation of conversation, answering and inquiring.
Concerning her receptive language, she showed consistent average
performance on the basic concept subtest (CELF tests [15]), important
to the follow-through of verbal instructions, from the 49th percentile
at age 75 months to the 58th percentile at age 104 months. Despite
the patient's tendency to lose skill development in one area (reasoning and thinking) of receptive language, as she gained in another
(recognition of the main idea, story comprehension), her collective
scores on the Listening Test [16] did reﬂect consistent growth from
>7 months delay at age 75 months down to 6 months delay at age
34
Table 2
Summary of developmental and speech tests carried out during the course of therapy.
Symptom assessed
Developmental progress
Chronologic age
Duration of creatine
Test
Scores (and Percentiles)
(months)
suppl. (months)
and age equivalents (months)
16
0
BSID-II
Mental scale
N/A(1)
7
Motor scale
N/A(1)
29
12.2
BSID-II
67(1)
23
80(9)
25
40
23.2
BSID-II
96(39)
40−42
101(53)
40-42
19.2
WPPSI-III
Full scale IQ
94(34)
Performance IQ
100(50)
Verbal IQ
36
47
30.2
WPPSI-III
107(34)
107(25)
60
43.2
WPPSI-III
104(61)
105(50)
112(79)
47
30.2
Leiter
140
60
43.2
Leiter
145
60
43.2
Toni-3
135
Age equiv.
Age equiv.
Uncorrected Nonverbal Ratio IQ**
Age equiv.
7
90(25)
91(27)
(Mean=100, SD=16)
Nonverbal Ratio IQ (Mean =100, SD = 16)
Expressive language
41
24.2
EOWPVT
Standard score
98(45)
development
47
30.2
EOWPVT
90(25)
39
60
43.2
EOWPVT
112(79)
73
92
75.2
EOWPVT
89(23)
78
99
82.2
EOWPVT
98(45)
95
104
87.2
EOWPVT
103(58)
110
39
Comprehensive language
61
44.2
CELF-P2
Standard score: Core language*
96(36)
development
69
52.2
CELF-P2
102(55)
77
60.2
CELF-4
96(39)
85
68.2
CELF-4
99(47)
75
58.2
LT
Standard score
89(23)
<68
83
66.2
LT
99(49)
77
104
87.2
LT
102(46)
100
Score
Age equiv.
th
5 grade
104
87.2
LAC
93
Age equiv.
Sub-Listening Test scores (and Percentiles)
Chronologic age (months):
75
83
104
Main idea
Details
83(12)
90(27)
106(58)
87(49)
117(84)
Concepts
97(49)
107(66)
92(29)
105(58)
Reasoning
96(43)
90(28)
91(26)
Story comprehension
81(12)
90(28)
110(70)
BSID-II: Bayley's Scale of Infant Development - II
CELF-4: Clinical Evaluation of Language Fundamentals -4
WPPSI-III: Wechsler Preschool and Primary Scale of Intelligence -III
LT: Listening Test
EOWPVT: Expressive One Word Picture Vocabulary Test
LAC : Lindamood Auditory Conceptualization Test
CELF-P2: Clinical Evaluation of Language Fundamentals - Preschool 2
+/− : with or without speech language therapy
*Combined result of both expressive and receptive language subtests
**Uncorrected Nonverbal Ratio IQ is derived using norms that were published in 1968 and overestimate Nonverbal IQ when used in the year 2007
83 months and 4 months delay at age 104 months. Finally her phonemic processing was quantiﬁed using the Lindamood Auditory Conceptualization test (LAC)—a criterion referenced tool giving only
grade level equivalencies. The patient did well, scoring at a 5th
grade equivalency which was 2 years advanced beyond her actual
3rd grade level at the time.
Speech-language therapy continued once a week until 9.25 years
of age notwithstanding her low average to average performance on
speech-language evaluations, because of the relative weakness of
her verbal skills compared to her nonverbal skills.
3. Discussion
Oral creatine supplementation in creatine biosynthesis deﬁciencies (AGAT and GAMT deﬁciencies), as opposed to creatine uptake deﬁciency (SLC6A8 deﬁciency), results in a better developmental
outcome. AGAT deﬁciency patients have a less severe phenotype
and a more favorable response to treatment compared to GAMT deﬁciency patients, possibly due to the neurotoxic effect(s) of elevated
guanidinoacetate in GAMT deﬁciency patients [17]. At the time of
treatment onset in our patient little was known about the optimum
35
creatine dose, so the goal was to titrate creatine supplementation to
maximize the cerebral creatine level and developmental progress
while avoiding side effects. Although creatine is generally considered
safe, there is theoretic concern that long-term high-dose creatine
treatment may be harmful to kidney function. No renal impairment
has been observed in treated patients, however. Reported pharmacologic creatine dosage ranges from 350 to 2000 mg/kg/day [5,18–20].
A dose of 15–20 times the daily creatine requirement (corresponding
to 350–400 mg/kg/day in children) does not induce side effects in
healthy adult volunteers [18]. Renal toxicity has been reported occasionally in individuals taking creatine as a supplement to enhance
athletic performance. A gradual nephrotoxicity, usually tubular,
from excess crystal excretion might rarely occur. If oral ﬂuid intake
is not adequate, renal damage from crystals could occur. However direct glomerular toxicity from creatine is unproven.
Lack of signiﬁcant improvement in cerebral creatine content between 8 and 17 months of treatment on the 400 mg/kg/day creatine
dose was concerning. After doubling the creatine dose to 800 mg/
kg/day, brain Cr/NAA by 23 months of treatment increased from
60% to 80%, and developmental scores increased from 80% (after
13 months of treatment) to 100% of that expected for chronologic
age. Although the high dose of 800 mg/kg/day creatine appeared to
be effective in improving brain creatine content and developmental
outcome, it was reduced to 500 mg/kg/day and then gradually increased, because of presence of urinary creatinine crystals for a
short period. Since 50 months of age the patient has been treated
with a creatine dose of 700 mg/kg/day. Despite possible nephrotoxicity, the high creatine dose has been continued as the child has done
very well developmentally. She has exhibited no side effects on this
creatine dose, and creatinine crystals have not recurred (except on
rare occasions). The brain MRS has not been repeated since
40 months of age for two reasons. First, the main goal of treatment
is developmental outcome, which has generally been excellent. It is
not clear that increasing cerebral creatine content above 80% would
further improve developmental outcome. Secondly, there is a paucity
of data on normal brain creatine values in age-matched controls.
The direct effect of speech-language therapy on language development in creatine deﬁcient patients has not been previously assessed.
Given the patient's overall average performance on recent speech
and language evaluations, and that review of the data suggests that
the previous break (from age 54 to 63 months) in speech therapy
was not associated with any signiﬁcant deterioration in her progress,
the decision has been made recently to discontinue speech therapy
for 6 months and reassess her progress after this period.
Table 3
Key features of all described AGAT patients: diagnosis, treatment and response.
Features
Family 1
Family 2
Family 3
Family 4
Reference
Bianchi et al. (2000); Item et al. (2001);
Present study.
Edvardson et al. (2010).
A Verma (2010).
2
c.1111dup ;
p.(Met371AsnfsX6)
♂
Depleted
18 years
♀
Depleted
12 years
2
c.505C > T ;
p.(R169X)
♀
N
18 years
♂
N
23 years
Battini et al. (2002, 2006)
General
Number of patients
Mutation
Sex
Brain creatine prior to treatment
Age at diagnosis
4
c.446G > A ;
p.(W149X)
♀
Depleted
4 years
♀
Depleted
6 years
♂
Depleted
3 weeks
♂
Depleted
2 years
1
c.484 + 1G > T;
p.Ala97ValfsX11
♀
Depleted
1.4 years
Symptoms at diagnosis
Cognitive disability or delay
Seizures
Expressive speech/Language delay
Behavioral problems
Muscle weakness
+
*+
+
+
−
+
−
+
+
−
NA
−
NA
−
−
+
−
+
+
Mild
+
−
+
−
Moderate
+
−
+
−
+
+
−
+
−
+
+
−
+
−
+
+
−
+
−
+
Treatment (creatine suppl.)
Start of treatment
Dose (mg/kg/day)
4.3 years
400
6.4 years
400
4 months
100
2 years
400
1.4 years
400-800
18 years
100
12 years
100
18 years
400 1
20 years
5 g/day 2
Clinical response (Treatment Eff.)
Duration (months)
Brain creatine after treatment
16
>95%
16
>95%
8
60%
15
90%
102
~ 80%3
11
18%
11
70%
6
65%
~ 36
66%
42↗68
57↗62%
c
77
↗
N
N
N
a
b
c
60↗77%
↗
d
65↔65
N
N
a
105
NA
NA
Normal
NA
NA
NA
↗
N
N
N
normal
104
91
112
↔
f
47↔49
f
53↔55
f
50↔50
↗
g
60↗70
g
66↗71
g
59↗72
N
N
N
↗
N
N
N
N
N
N
↗
N
N
N
↗
normal
↗
normal
N
normal
↗
N
normal
normal
N
↗
N
↗
↗
↗
N
↗
Clinical response (Symptoms)
Cognitive function
Performance (Developmental)
Eye-hand coordination
Visual-perceptual abilities
Expressive speech
Full scale IQ scores
Verbal IQ scores
Performance IQ scores
General
Behavior or speech
Muscle strength
a
a
+: present
−: absent
*: single episode at 18 months
NA: not applicable
N: not determined/available
1: initial dose of 5g/day
2: not measured prior to treatment
3: 23 months post creatine supplementation
b50↗63
100%
a: Griffith Developmental Scales
b: Bayley Infant Development Scale (at 40 months)
c: Visual Motor Integration Test
d: Leiter International Performance Scale
e: Wechsler Preschool and Primary Scale of Intelligence
f: Wechsler Adult Intelligence Scale
g: Wechsler Intelligence Scale for Children
>95%: almost complete
↗: improved
↔: unchanged
%: with respect to normal
36
A
LOVD - Leiden Open Variation Database
L-arginine:glycine amidinotransferase, nuclear gene encoding mitochondrial (GATM)
Curator: Gajja Salomons
Home
Variants
V iew unique variants
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Submit
Documentation
Searc h unique variants
V iew all c ontents
Full databas e s earc h
V ariant lis ting bas ed on patient origin
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LOVD - Variant listings
Unhide all columns
| Hide Specif ic Columns
| Hide all columns
Exon
DNA change
RNA change
Protein change
03
c .4 4 6 G > A
(Reported 2 times)
r .( ? )
p .( T r p 1 4 9 X )
-
Bianchi 2000
04
c .5 0 5 C >T
(Reported 2 times)
r .( ? )
p .( A r g 1 6 9 X )
-
Verma 2010
08
c .1 1 1 1 dup
(Reported 2 times)
r .( ? )
p . ( Met371AsnfsX6)
-
Edvardson 2010
c .4 8 4 +1 G >T
(Reported 2 times)
r.289_484del196
p.A la9 7 V alfs X1 1
Ref erence
T his variant is a proven
pathogenic mutation s inc e
erroneous s plic ing was
demons trated
5’UTR 1
2
3
4
5
6
c.
11
11
du
p;
p.
(M
c.
44
c. 6G>
48 A
c. 4+ ; p
50 1G .( T
5C > rp
>T T; p 14
; p .A 9X
.(A la9 )
rg 7V
16 a
9X lfsX
)
11
B
Salomons 2005
et
37
1A
sn
fs
X6
)
03i
Variant remarks
7
8 9
3’ UTR
400 bp
100 bp
Fig. 3. Screenshot of the newly developed LOVD/GATM database (A), and a schematic representation of the AGAT/GATM gene and its pathogenic variants (B). References for each
mutation as well as key clinical descriptions for every patient as a result of the said mutation are summarized in Table 3.
Thus far, 9 patients affected with AGAT deﬁciency from 4 unrelated families have been described. In the present paper clinical and
treatment data on these patients are summarized (Table 3). The age
at diagnosis varies between 3 weeks and 23 years. In a male sibling
of the ﬁrst AGAT deﬁcient patients the disease onset was prevented
by initiating creatine supplementation a few months after birth [7].
However, in all the other patients seen so far cognitive disability or
delay and speech/language delay are the most common phenotypic
manifestations of AGAT deﬁciency and as such can be used to monitor
the disease (in case of a relapse) or treatment progress. Based on the
dose of creatine used in all the other AGAT deﬁciency patients
(Table 3), it is possible that developmental outcome in the present
case would have been as successful on a lower dose of creatine, and
that her cerebral creatine content would have gradually increased
to the same level on the lower creatine dose. Recently, long-term follow up (7 to 10 years) has been reported in patients of the ﬁrst family
wherein an initial dose of 400 mg/kg/day and a gradual decrease to
100 mg/kg/day has been sufﬁcient to enable clinical improvement
in adaptive behavior and communicative skills [21]. The time course
of 23 months required to achieve a Cr/NAA of 80% of normal in our
case is similar to what has been observed in patients with GAMT deﬁciency, but longer than observed for the AGAT deﬁciency patients in
the ﬁrst reported family [5,6]. It is not clear whether the excellent
outcome in the present case with respect to the other symptomatic patients (Table 3) is due to the high dose of creatine, the early implementation of treatment, the long duration of treatment, or a combination
thereof. Moreover it is of note that all reported mutations are severe
mutations that result in (or predict) truncated proteins (Fig. 3) therefore the more favorable response in our patient is unlikely to be genotype speciﬁc. More research will be required to further determine the
optimum creatine dose required to treat AGAT deﬁciency. For now it
seems an initial dose of 400 mg/kg/day is appropriate. Dose increase
up to 700 mg/kg/day may be considered to optimize cerebral creatine
content and developmental outcome, and appears to be well tolerated
in the present case. Alternatively, dose decrease can also be considered.
The creatine dose of 100 mg/kg/day has also been associated with good
clinical outcome and cerebral creatine replenishment in one patient
treated since early infancy [22]. The genetic data of all patients have
been included in the newly developed LOVD database (http://www.
LOVD.nl/GATM) to provide a ﬂexible and freely available tool for collection and display of DNA variations (Fig. 3).
4. Conclusion
We describe here outcome in a patient with AGAT deﬁciency treated for 8 years. The initial developmental outcome of our patient is
better than the older sibling in the ﬁrst family reported [3,5–7], in
whom treatment did not begin until 5 years of age, even though the
brain creatine recovery in this patient is better than that of our patient.
Our initial experience in treating this patient with creatine deﬁciency
secondary to AGAT deﬁciency adds important information to the experience from the previously reported cases. Initial treatment from
16 months of age conﬁrms that early diagnosis and treatment may be
associated with excellent neurodevelopmental outcome. The longterm follow-up of the patient described in this report adds further evidence to the importance of early diagnosis/treatment onset of patients
with AGAT deﬁciency. Efﬁcient cataloguing of all identiﬁed variants as
well as long-term follow-ups of all patients will be very useful in unraveling the pathophysiology of the disease. The creatine dose of
700 mg/kg/day used in this patient over 5 years has been effective
with no apparent side effects.
Acknowledgements
The authors would like to thank Drs. Lauren Plawner, Sheldon Orloff,
Annette Finkel and Peter Kim for their excellent clinical care, Linda
37
Cooper MS for her compassionate skillful genetic counseling and assistance with the manuscript, and the parents for their extraordinary
diligence and devotion in the raising of this talented and remarkable
child.
References
[1] S. Stöckler-Ipsiroglu, G.S. Salomons, Inborn metabolic diseases: diagnosis and
treatment, Mol. Cell. Biochem. 244 (2006) 211–217.
[2] S. Stöckler, D. Isbrandt, F. Hanefeld, B. Schmidt, K. von Figura, Guanidinoacetate
methyltransferase deﬁciency: the ﬁrst inborn error of creatine metabolism in
man, Am. J. Hum. Genet. 58 (1996) 914–922.
[3] C.B. Item, S. Stöckler-Ipsiroglu, C. Stromberger, A. Mühl, M.G. Alessandrì, M.C.
Bianchi, et al., Arginine:glycine amidinotransferase deﬁciency: the third inborn
error of creatine metabolism in humans, Am. J. Hum. Genet. 69 (2001) 1127–1133.
[4] G.S. Salomons, S.J. van Dooren, N.M. Verhoeven, K.M. Cecil, W.S. Ball, T.J. Degrauw,
et al., X-linked creatine-transporter gene (SLC6A8) defect: a new creatinedeﬁciency syndrome, Am. J. Hum. Genet. 68 (2001) 1497–1500.
[5] M.C. Bianchi, M. Tosetti, F. Fornai, M.G. Alessandri’, P. Cipriani, G. De Vito, et al.,
Reversible brain creatine deﬁciency in two sisters with normal blood creatine
level, Ann. Neurol. 47 (2000) 511–513.
[6] R. Battini, V. Leuzzi, C. Carducci, M. Tosetti, M.C. Bianchi, C.B. Item, et al., Creatine
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Salomons, et al., Diagnostic enzyme assay that uses stable-isotope-labeled substrates to detect L-arginine:glycine amidinotransferase deﬁciency, Clin. Chem.
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[17] F. Nasrallah, M. Feki, N. Kaabachi, Creatine and creatine deﬁciency syndromes:
biochemical and clinical aspects, Pediatr. Neurol. 42 (2010) 163–171.
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[19] M. Wyss, Health implications of creatine: can oral creatine supplementation protect against neurological and atherosclerotic disease? Neuroscience 112 (2002)
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neuropsyscological follow-up of AGAT-D patients after ten years from the diagnosis. J. Inherit. Metab. Dis. 34(3):P-143 SSIEM Annual Symposium (2011)
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[22] M.C. Bianchi, M. Tosetti, R. Battini, V. Leuzzi, M.G. Alessandri’, C. Carducci, et al.,
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38
39
Chapter 3
Thirteen new patients with guanidinoacetate methyltransferase
deficiency and functional characterization of nineteen novel
missense variants in the GAMT gene
"99 percent of all statistics only tell 49 percent of the story"
Ron DeLegge II
40
41
RESEARCH ARTICLE
OFFICIAL JOURNAL
Thirteen New Patients with Guanidinoacetate
Methyltransferase Deficiency and Functional
Characterization of Nineteen Novel Missense Variants in
the GAMT Gene
www.hgvs.org
Saadet Mercimek-Mahmutoglu,1,2∗ † Joseph Ndika,2 † Warsha Kanhai,2 Thierry Billette de Villemeur,3 David Cheillan,4
Ernst Christensen,5 Nathalie Dorison,6 Vickie Hannig,7 Yvonne Hendriks,8 Floris C. Hofstede,9 Laurence Lion-Francois,10
Allan M. Lund,11 Helen Mundy,12 Gaele Pitelet,13 Miquel Raspall-Chaure,14 Jessica A. Scott-Schwoerer,15
Katalin Szakszon,16 Vassili Valayannopoulos,17 Monique Williams,18 and Gajja S. Salomons2∗
1
Division of Clinical and Metabolic Genetics, Department of Pediatrics, The Hospital for Sick Children, University of Toronto, Toronto, Canada;
Metabolic Laboratory, Department of Clinical Chemistry, VU University Medical Center, Amsterdam, The Netherlands; 3 AP-HP Service de
´
´
ˆ
´ editaires
´
´
Neuropediatrie,
Pathologie du Developpement,
Hopital
Trousseau, Paris, France; 4 Service Maladies Her
du Metabolisme,
Groupement
5
Hospitalier Est, Hospices Civils de Lyon, France; Department of Clinical Genetics, Juliane Marie Center, Copenhagen, Denmark; 6 AP-HP Service
´
´
ˆ
de Neuropediatrie,
Pathologie du Developpement,
Hopital
Trousseau, Paris, France; 7 Division of Medical Genetics and Genomic Medicine,
Vanderbilt University Medical Center, Nashville, Tennessee; 8 Department of Clinical Genetics, Free University Medical Center, Amsterdam, The
´
Netherlands; 9 Wilhelmina Children’s Hospital, Utrecht, The Netherlands; 10 Service de Neuropediatrie,
Groupement Hospitalier Est, Hospices Civils
de Lyon, Bron, France; 11 Department of Clinical Genetics, Centre for Inherited Metabolic Diseases, Copenhagen, Denmark; 12 Evelina Centre for
Inherited Metabolic Disease, Goys and St Thomas NHS Foundation Trust, Evelina Children’s Hospital, London, England; 13 Department of
Pediatrics, Chulenval, Nice, France; 14 Department of Paediatric Neurology, Hospital Universitari Vall d’Hebron, Barcelona, Spain; 15 Department of
Pediatrics, Division of Genetics and Metobolism, University of Wisconsin, Madison, Wisconsin; 16 Institute Pediatrics, Clinical Genetics Center,
University of Debrecen, Hungary; 17 Reference Center for Inherited Metabolic Disease, Paris, France; 18 Department of Pediatrics, Sophia Childrens
Hospital, Erasmus Medical Center, The Netherlands
2
´ Sobrido
Communicated by Mar´ıa-Jesus
Received 16 September 2013; accepted revised manuscript 6 January 2014.
Published online 10 January 2014 in Wiley Online Library (www.wiley.com/humanmutation). DOI: 10.1002/humu.22511
ABSTRACT: Guanidinoacetate methyltransferase deficiency (GAMT-D) is an autosomal recessively inherited disorder of creatine biosynthesis. Creatine deficiency
on cranial proton magnetic resonance spectroscopy, and
elevated guanidinoacetate levels in body fluids are the
biomarkers of GAMT-D. In 74 patients, 50 different
mutations in the GAMT gene have been identified with
missense variants being the most common. Clinical and
biochemical features of the patients with missense variants were obtained from their physicians using a questionnaire. In 20 patients, 17 missense variants, 25% had
a severe, 55% a moderate, and 20% a mild phenotype.
The effect of these variants on GAMT enzyme activity
was overexpressed using primary GAMT-D fibroblasts: 17
variants retained no significant activity and are therefore
considered pathogenic. Two additional variants, c.22C>A
(p.Pro8Thr) and c.79T>C (p.Tyr27His) (the latter detected in control cohorts) are in fact not pathogenic as
†
∗
These authors share first authorship.
Correspondence to: Saadet Mercimek-Mahmutoglu, FCCMG, Division of Clinical
and Metabolic Genetics, Department of Pediatrics, University of Toronto, The Hospital for Sick Children, Toronto, Canada. E-mail: [email protected]; Gajja
S. Salomons, Metabolic Unit, Department of Clinical Chemistry, Neuroscience Campus Amsterdam, VU University Medical Center, Amsterdam, The Netherlands. E-mail:
[email protected]
these alleles restored GAMT enzyme activity, although
both were predicted to be possibly damaging by in silico
analysis. We report 13 new patients with GAMT-D, six
novel mutations and functional analysis of 19 missense
variants, all being included in our public LOVD database.
Our functional assay is important for the confirmation
of the pathogenicity of identified missense variants in the
GAMT gene.
C 2014 Wiley Periodicals, Inc.
Hum Mutat 35:462–469, 2014. KEY WORDS: GAMT; GAMT-D; site-directed mutagenesis; missense variants
Introduction
Guanidinoacetate methyltransferase deficiency (GAMT-D; MIM
#612736) is an autosomal recessively inherited disorder of creatine biosynthesis [Stockler et al., 1996]. Its estimated incidence is
1:114,072 in Utah [Viau et al., 2013] and its carrier frequency was
1/1475 in a small cohort of newborns [Mercimek-Mahmutoglu et al.,
2012b]. Creatine has a buffering and transport function of highenergy phosphates in brain and muscle, and is essential for growth
cone migration, dendritic and axonal elongation, neurotransmitter
release, and cotransmission on GABA postsynaptic receptors in the
central nervous system (CNS) [Wallimann et al., 1992; Wyss and
Kaddurah-Daouk, 2000; Almeida et al., 2006a].
42
Creatine deficiency in brain proton magnetic resonance spectroscopy (1 H-MRS), and elevated guanidinoacetate (GAA) levels in
urine, blood, and cerebral spinal fluid) are the biochemical hallmarks of GAMT-D [Stockler et al., 1996; Wyss and KaddurahDaouk, 2000]. Clinical features are global developmental delay
(GDD) and seizures in infants and intellectual disability, movement disorder, epilepsy, and behavioral problems in children. Hypotonia and gross motor delay together with choreiform movements have been recently reported in a 10-month-old patient with
GAMT-D as early presenting symptoms [Viau et al., 2013]. Treatment consists of high-dose creatine and ornithine supplementation,
arginine-restricted diet, and sodium benzoate therapy to replenish
cerebral creatine deficiency and decreased neurotoxic GAA accumulation in CNS [Schulze et al., 1997; Schulze et al., 2003, MercimekMahmutoglu et al., 2009]. Normal neurodevelopmental outcome
have been reported in three patients who were diagnosed and treated
in the neonatal period [Schulze et al., 2006; El-Gharbawy et al., 2013;
Viau et al., 2013]. Given the effectiveness of early intervention and
severe neurodevelopmental outcome in untreated patients, GAMT
deficiency is an excellent candidate for newborn screening.
So far, 74 patients with GAMT-D have been reported [Cheillan
et al., 2012, Dhar et al., 2009; Mercimek-Mahmutoglu et al., 2009,
Mercimek-Mahmutoglu et al., 2012a; Nasrallah et al., 2012; Akiyama
et al., 2014; Comeaux et al., 2013; El-Gharbawy et al., 2013; Viau
et al., 2013]. In these patients, 50 different mutations have been
identified in the GAMT gene (MIM #601240) and 64% of them are
missense variants. Deletions, splice errors, frame shift, nonsense,
and (other) truncating mutations are usually classified as pathogenic
mutations. Missense variants pose problems for the interpretation of
the pathogenicity. Currently used in silico analyses [e.g., using Sorting Intolerant From Tolerant (SIFT, http://sift.jcvi.org), Polymorphism Phenotyping (PolyPhen, http://coot.embl.de/PolyPhen)] are
important for predicting damaging effects of missense mutations,
but do not confirm the pathogenicity. Functional characterization
of gene variants via targeted amino-acid substitutions and enzyme
assays still remain vital in order to establish the effect (if any) of
an existing variant on the catalytic activity of a protein. All patients
with GAMT-D had novel homozygous or compound heterozygous
variants or known disease-causing mutations in the GAMT gene
and heterozygous carrier status were confirmed in parents. Here,
we report 19 missense variants in the GAMT gene and their functional characterization. We also report 20 patients with 17 of these
variants (either homozygous or compound heterozygous) for clinical and biochemical phenotype: 13 not previously published; two
partially published; and five previously published patients.
Materials and Methods
This study was approved by the Institutional Research Ethics
Board (REB#1000033694) at The Hospital for Sick Children.
Clinical and Biochemical Data of the Patients
Patients with GAMT-D with one of the missense variants not
previously studied for functional characterization in our laboratory
were included into this study. Physicians following these patients,
who had not published, or partially published their cases, were
invited to participate to this study. Physicians were provided a questionnaire including following questions: age of onset, clinical features (GDD, intellectual disability, seizure, movement disorder, behavioral problems), and biochemical features (urinary GAA levels,
cerebral creatine by 1 H-MRS or 31 P-MRS, brain magnetic resonance
imaging (MRI), GAMT enzyme activity in the cultured skin fibroblasts). The questionnaire was completed for previously reported
patients (by authors) according to information acquired from the
literature. We applied a clinical severity scoring system based on the
information from the questionnaire for the clinical phenotype using
previously reported scoring system [Mercimek-Mahmutoglu et al.,
2006]. The details of the clinical severity scoring system are summarized under Table 1 legends. Sum of all clinical features according to
clinical severity scoring were given as phenotype.
Molecular Genetic Studies and Functional Characterization
of Missense Variants
Sequencing of the GAMT gene (NM 000156.5) was performed as
´ et al.,
previously described for mutation analysis [Caldeira Araujo
2005]. Nucleotide numbering of variants reflects cDNA numbering
with +1 corresponding to the A of the ATG translation initiation
codon in the reference sequence, according to journal guidelines
(www.hgvs.org/mutnomen).
The open reading frame of the GAMT gene (NP 000147.1) was
cloned as a fusion protein with EGFP in a pEGFPN1 vector. Recombinant pGAMT-EGFPN1 plasmids for each missense mutation were generated by standard molecular biology techniques [see
Almeida et al., 2006b for details]. A primary GAMT-deficient human fibroblast cell line (homozygous for a frameshift mutation),
cultured according to standard procedures to 70% confluence, was
used for transient expression of generated constructs. For the transfections, 45 μg of each plasmid was incubated at room temperature for 10 min with polyethyleneimine (Polysciences, Omnilabo,
Eppelheim, Germany) in serum-free medium, and subsequently the
complex was applied to the cells. Cells transfected with either the
wild-type pGAMT-EGFPN1 construct or with the pEGFPN1 empty
vector as well as untransfected cells were taken along as controls. All
transfections were done in triplicate, and green fluorescence of the
EGFP tag was used to estimate the transfection efficiency. Cells were
harvested 48 h after transfection, flash-frozen, and stored at –80°C
for Western blot analysis and GAMT enzyme activity measurement.
Immunodetection of the GAMT–EGFP fusion protein was carried
out as described previously [Almeida et al., 2006b] using an EGFP
antibody (Abcam, Cambridge, UK). The GAMT enzyme assay was
performed with the supernatants from lysed cells as described by
Verhoeven et al. [2004].
Results
Clinical and Biochemical Data of the Patients
We received a completed questionnaire from 13 physicians. Two
physicians diagnosed GAMT-D in a sibling and they were both included (P2 siblings of P1 and P8 siblings of P7) into this study.
Clinical, biochemical, and molecular genetic results were summarized in Table 1.
Thirteen patients were not reported previously (P1, P2, P3, P7,
P8, P10, P11, P13, P14, P15, P16, P17, P19, P21); two patients
were reported partially (P4, P9) and five patients were reported
previously [P5 in Mercimek-Mahmutoglu et al., 2006 P9; P6 in
Mercimek-Mahmutoglu et al., 2006 P12; P12 in Engelke et al., 2009
P4; P18 Verbruggen et al., 2007; P20 in Leuzzi et al., 2006] (Table 1).
Median age of the patients was 12 ± 7.41 SD (range 5–35 years),
median age of diagnosis was 6.5 ± 7.22 SD (range 10 months to
20 years) of 21 patients with GAMT-D. Two patients (P8, P12)
were adults at the time of the diagnosis. According to available
HUMAN MUTATION, Vol. 35, No. 4, 462–469, 2014
463
6–12 mo/6 yrs
2.5 yrs/10 yrs
3–6 yrs/10 yrs
2b /M/8 yrs
3/F/16 yrs
4/M/19 yrs [P5 in Lion-Francois et al.,
2006]
5/M/13yrs [P9 in Mercimek-Mahmutoglu
et al., 2006]
6/M/14 yrs [P12 in
Mercimek-Mahmutoglu et al., 2006]
7b /M/8 yrs
b
8 /F/26 yrs
9/M/11 yrs [Scott-Schwoerer et al., 2011]
10/F/5 yrs
11/F/11 yrs
12 mo/7 yrs
N/A
15/M/10 yrs
16/M/16 yrs N/A
17/F/5 yrs
18/M/10 yrs [Verbruggen et al., 2007]
19/M/8 yrs
20/F/24 yrs [Leuzzi et al., 2006]
21/F/14 yrs
c.476T>C (p.Leu159Pro)c
c.497T>C (p.Leu166Pro)
c.506G>A (p.Cys169Tyr)
[Caldeiro et al., 2005]
c.590T>C (p.Leu197Pro)
c.623G>C (p.Arg208Pro)c
18–24 mo/6 yrs
NA/12yrs
1–2 yrs/3 yrs
12–18 mo/4 yrs
12–18 mo/2 yrs 9 mo
Severe ID, AED responsive seizures,
hyperactivity
Severe GDD, severe ID, (IQ < 30), intractable
epilepsy, myoclonus bradikinesia, ADHD
Severe GDD, occasional seizures, Ataxia,
autism ADHD, aggressive behavior
Mild ID
Moderate GDD, mild ID, ADHD
Severe GDD, movement disorder, AED
responsive seizures, autistic
Moderate GDD, ataxia, ADHD
Severe ID, ataxia, autism, aggressive behavior
Moderate GDD, intractable epilepsy, hypotonia
Moderate-severe GDD, autistic like features
Mild ID, AED responsive seizures, stereotypic
movements
Severe GDD, movement disorder, AED
nonresponsive seizures, aggressive behavior
Borderline ID, seizures nonresponsive to AED,
ADHD, autistic-like features
Severe GDD, seizures nonresponsive to AED,
spasticity, aggressive behavior
Moderate GDD, AED responsive seizures,
ataxia, tremor, ADHD, autistic-like features
N/A
Severe ID, AED responsive seizures, aggressive
behavior
Mild GDD, AED responsive seizures, autistic
Severe GDD, drug responsive seizures,
aggressive behavior
Severe GDD, occasional seizures, hyperactive,
self injurious, aggressive behavior
Moderate GDD, AED responsive seizures, ADD
Clinical features
Severe
Moderate
Mild
Mild
N/A GAMT enzyme
activity deficient
Severe
Severe
Severe
Moderate
Moderate
Moderate
Moderate
Moderate
Mild
Moderate
Severe
Mild
Moderate
Moderate,
Moderate
Moderate
Phenotypea
Normal/partially absent/absent
Bilateral dorsal pons increased
signal/absent/absent
NA/absent/present
Mild cortical atrophy, white/gray
matter signal
changes/absent/absent
Normal/absent/N/A
N/A
Bilateral globus pallidi increased
signal/absent/present
Normal/absent/present
Normal/partially absent/absent
Delayed
myelination/absent/absent
Normal/absent/absent
NP
Normal/NP
Normal/absent/absent
Bilateral thalami increased
signal/absent/absent
N/A
Normal/partially absent/absent
Normal/partially absent/absent
Normal/partially absent/present
Normal/partially absent/present
Normal/absent/present
MRI/creatine peak/GAA peak on
1
H-MRS
c.623G>C/c.623G>C
c.590T>C/c.590T>C
c.497T>C/c.497T>C
c.506G>A/c.506G>A
c.439C>T; c.11 36dup26bp
(p.Gly13ProfsX38)
c.476T>C/c.476T>C
c.407C>T/c.316C>T
c.403G>A; c.327G>A
c.328G>T; c.327G>A
c.274A>G/c.274A>G
c.202G>T/c.202G>T
c.202G>T/c.202G>T
c.220G>C/c.327G>A
c.220G>C/c.220G>C
c.224C>T/c.224C>T
c.160G>C/c.160G>C
c.152A>C/c.526dupG
c.148A>C/c.148A>C
c.133T>A/c.11 36dup26bp
c.133T>A/c.327G>A
c.133T>A/c.327G>A
Genotype
c
b
Clinical severity scoring system.
Siblings.
Novel mutations.
Clinical severity scoring system: (A) intellectual disability/global developmental delay was scored: 0, normal; 1, mild; 2, moderate; 3, severe; (B) seizures were scored 0, none; 1, occasional seizures; 2, antiepileptic drug-responsive seizures; 3,
antiepileptic drug-resistant seizures. (C) Movement disorder was scored 0, no movement disorder; 1, nonspecific movement abnormalities; 2, extrapyramidal movement disorder; 3, pyramidal and extrapyramidal movement disorder. Sum of all
clinical features according to clinical severity scoring were given as phenotype.
F, female; M, male; yrs, years; mo, months; GDD, global developmental delay; AED, antiepileptic drugs; ADD, attention deficit disorder; ID, intellectual disability; ADHD, attention deficit hyperactivity disorder; N/A, not available; MRI, magnetic
resonance imaging; GAA, guanidinoacetate; 1 H-MRS, proton magnetic resonance spectroscopy; NP, not performed.
a
9–12 mo/4 yrs
14/F/12 yrs
c.403G>A (p.Asp135Asn)
[O’Rourke et al., 2009]
c.407C>T (p.Thr136Met)
[Sempere et al., 2009]
c.439C>T (p.His147Try)c
18–24 mo/7 yrs
13/F/10 yrs
c.328G>T (p.Val110Phe)c
1–2 yrs/27 yrs
12/M/35 yrs [P4 in Engelke et al., 2009]
6–9 mo/10 mo
12–18 mo/20 yrs
6–12 mo/1 yr
6–12 mo/3 yrs
18–24 mo/7 yrs
1–2 yrs/2 yrs
6–12 mo/3 yrs
6–12 mo/11 years
Age at onset/Age at
diagnosis
1b /F/16 yrs
Patient/sex/age (reference for previously
published patients)
c.274A>G (p.Asn92Asp)
c.202G>T (p.Gly68Cys)c
c.202G>T (p.Gly68Cys)c
c.220G>C (p.Ala74Pro)
c.220G>C (p.Ala74Pro)
c.224C>T (p.Ala75Val)c
c.160G>C (p.Ala54Pro)
c.152A>C (p.His51Pro)
c.133T>A (p.Trp45Arg)
[Comeaux et al., 2013]
c.133T>A (p.Trp45Arg)
[Comeaux et al., 2013]
c.133T>A (p.Trp45Arg)
[Comeaux et al., 2013]
c.148A>C (p.Met50Leu)
Missense variant (reference
for previously published
mutations)
Table 1. Clinical, Biochemical, and Molecular Genetic Results of the Patients with a Missense Variant and Confirmed GAMT-D
43
44
clinical information of the 20 patients, four patients (20%) had
mild; 11 patients (55%) had moderate and five patients (25%) had
severe phenotype (Table 1). Fifteen patients (P1–6, P9, P11–17,
P20–21) had seizures (75%). Age of onset for the first seizure was
between 9 months (P9) and 7 years (P11). Eight patients (P1, P6, P7,
P8, P12, P15, P17, P21) (40%) had movement disorder. Ataxia was
the most common movement disorder. Behavioral problems were
present in 19 patients (95%) ranging from hyperactivity, attention
deficit hyperactivity disorder (ADHD) to self-injurious aggressive
behavior. The most common behavioral problems were autism or
autistic-like features (35%) and aggressive behavior (35%). The age
of the patients with aggressive behavior was from 5 to 35 years.
Six patients (30%) presented with ADHD. Bilateral increased signal
intensity in the globus pallidi in one patient (P14) and in bilateral
thalami in one patient was present (P11). In both patients, these
MRI changes were associated with movement disorder.
Urine GAA levels were elevated in all patients and were between
1.1 and 12 times above the upper limit of reference range. Seventeen
patients underwent 1 H-MRS. In none of these patients, neither
creatine nor GAA were quantified. In 11 patients, creatine was totally
absent (P1, P6, P7, P10, P11, P14, P15, P17, P18, P19, P20) and in six
partially absent. GAA peak was present in six patients (P1, P2, P3,
P14, P15, P18) (four with total absent and two with partial absent
creatine peak) (Table 1). GAMT enzyme activity was measured in
four patients (P2, P9, P12, P21) and was nondetectable or strongly
decreased in all patients.
We had no clinical information for P16 (compound heterozygous for c.439C>T (p.His147Try), but GAMT-D was confirmed
biochemically by deficient GAMT enzyme activity in the cultured
skin fibroblasts.
One patient with unknown etiology was heterozygous for the
c.22C>A (p.Pro8Thr) variant with no identifiable second variant
in the GAMT gene who presented with severe GDD, hypotonia,
intractable epilepsy and died at the age of 11 months. Urine GAA was
elevated 2.5 times and plasma GAA was 1.8 times above the upper
limit of reference range. On 1 H-MRS, creatine peak was about 90%
of normal, marginally low, which was not suggestive of GAMT-D.
Another patient with unknown etiology was homozygous for the
c.79T>C (p.Tyr27His) variant in the GAMT gene identified by whole
exome sequencing suggestive of GAMT-D. GAMT enzyme activity
was within normal range in the cultured skin fibroblasts excluding
GAMT deficiency in this patient.
Molecular Genetic Studies and Functional Characterization
of Missense Variants
We detected six novel variants (Table 1). Twelve patients had
homozygous and nine patients had compound heterozygous mutations. The panethnic c.327G>A mutation was found in five patients
as second mutations.
We introduced 19 missense variants identified in the GAMT gene
via SDM of which 17 were detected in patients affected with GAMT
deficiency. Two additional variants, including one polymorphism as
described above were tested.
Homogenates from cells transfected with either one of the other
17 variants retained no significant activity approximately 0%–4%
of recombinant wild-type GAMT activity (Fig. 1). In all transfectants, a green fluorescent signal was detected indicating successful transfection (data not shown). On the Western blot (Fig. 1),
no detectable expression could be seen for the following variants: c.152A>C (p.His51Pro), c.160G>C (p.Ala54Pro), c.202G>T
(p.Gly68Cys), c.220G>C (p.Ala74Pro), c.506G>A (p.Cys169Tyr),
Figure 1. A: GAMT (NM_000156.5) assay showing effect of variants on catalytic activity. GAMT−/− is the untransfected GAMT-deficient
fibroblast cell line. Empty vector is a deficient cell line transfected
with a basic pEGFPN1 vector and, wild-type a deficient cell line transfected with a p.GAMT-EGFPN1 vector. GAMT+/+ represents a cell line
with wild-type endogenous GAMT activity. The error bars represent
the standard error of the mean from triplicate transfections. B: Western blot using an anti-GAMT antibody to detect expression of GAMT–
EGFP fusion proteins. A Western blot was carried out for all triplicate
transfections and one representative blot of each sample is shown.
No detectable expression of the fusion protein could be seen for
variants; p.His51Pro, p.Ala54Pro, p.Gly68Cys, p.Ala74Pro, p.Val110Phe,
p.His147Tyr, p.Cys169Tyr, and p.Leu197Pro. Twenty-four micrograms of
protein was loaded in each lane. The dashed line separates samples
that were run on the same gel.
and c.590T>C (p.Leu197Pro) variants (Fig. 2). GAMT enzyme
activity was undetectable in homogenates from cells overexpressing the c.133T>A (p.Trp45Arg) and c.220G>C (p.Ala74Pro) variants and was very low for the c.160G>C (p.Ala54Pro), c.274A>G
(p.Asn92Asp), c.403G>A (p.Asp135Asn), c.407C>T (p.Thr136Met),
c.497T>C (p.Leu166Pro), and c.590T>C (p.Leu197Pro). Two mutations, c.439C>T (p.His147Try) and c.623G>C (p.Arg208Pro), revealed highest residual GAMT enzyme activity (both 4% of the activity of GAMT wild-type transfectants). The c.623G>C (p.Arg208Pro)
variant proved pathogenic in our functional analysis although it was
conserved only across four species and SIFT and PolyPhen predicted
it was not pathogenic.
Homogenates from cells overexpressing GAMT variants c.22C>A
(p.Pro8Thr) and c.79T>C (p.Tyr27His) were found to restore GAMT
activity to similar levels as the wild-type GAMT transfectants. Surprisingly, the c.22C>A (p.Pro8Thr) and c.79T>C (p.Tyr27His) were
conserved across nine species and SIFT predicted these variants to
be nontolerated and PolyPhen predicted the c.22C>A (p.Pro8Thr)
to be benign and the c.79T>C (p.Tyr27His) probably damaging, respectively. Conservation of the substituted amino acids in GAMT
orthologs from 12 other species is depicted in Figure 2.
Discussion
We report 13 new patients with GAMT-D, six novel pathogenic
mutations in the GAMT gene and functional characterization of
19 missense variants in this study. All 13 patients had biochemical features of GAMT deficiency including elevated GAA in urine,
creatine deficiency on brain MRS, and/or deficient GAMT enzyme
activity in the cultured skin fibroblasts or lymphoblasts. In two
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45
Figure 2. Scheme showing conservation of amino acid residues in GAMT orthologs across 13 different species (source: ENSEMBL). Depicted are
the amino acids 8, 27, 45, and so on until Arg208. Functional analysis of GAMT activity in recombinant cells reveals the amino-acid substitutions at
the conserved Pro8 and Tyr27 residues (p.Pro8Thr and p.Tyr27His, respectively) are NOT pathogenic, whereas substitution of the poorly conserved
Arg208 residue by proline proves to be pathogenic.
sibling pairs (P1 and P2 and P7 and P8), clinical phenotype was
moderate, despite an age difference of 8 and 18 years, respectively.
In the first sibling pair, behavioral problems were more severe in
the younger sibling, self-injurious behavior compared with his 8
years older sister. Whereas in second sibling pair (P7 and P8),
younger sibling had ADHD; 18 years older sibling had aggressive
behavior. As an observation, behavior disorder might be progressive in GAMT-D. The prevalence of behavior disorder, aggressive
behavior, and autism or autistic-like features being most common,
was 95% in this study, which was reported as 78% in a previous
study including 27 patients [Mercimek-Mahmutoglu et al., 2006].
In our study, 40% of the patients had movement disorder, ataxia
being most common, which was similar to previous report (48%)
[Mercimek-Mahmutoglu et al., 2006]. Seizures were one of the clinical features in 75% of the patients in our study, which was reported
to be higher, (93%) in a previous study [Mercimek-Mahmutoglu
et al., 2006]. 30% of the patients had intractable epilepsy in both,
previous and this study. On qualitative 1 H-MRS, total absence of
creatine peak was associated with severe phenotype in four patients,
moderate phenotype in four patients, and mild phenotype in three
patients. Total absence of creatine peak together with presence of
GAA peak was associated with severe phenotype in two patients,
moderate phenotype in one patient, and mild phenotype in one
patient. Additionally, two patients with moderate phenotype had
partial creatine deficiency and present GAA peak on qualitative
1
H-MRS. These results show us that abnormal metabolite profile on
1
H-MRS is also not helpful to predict severity of disease phenotype.
All clinical data were based on single physician observations, either,
clinical geneticist, pediatric neurologist, or pediatrician with specific
training to recognize and categorize clinical features of GAMT-D.
No neuropsychological assessments for degree of intellectual disability, and no standardized developmental assessments for degree
of GDD, autism diagnostic tests were applied because of the retrospective nature of our study. However, in our clinical practice, even
with well-designed prospective case control studies, standardized
clinical follow-up assessments are difficult to apply due to various
interfering factors such as limited availability of clinical resources,
funding cuts and limitations of standardized tests. We still think that
single physician observations are valuable information resources to
assess clinical phenotype in very rare disorders like GAMT-D.
Three patients (P1, P2, and P3) with c.133T>A (p.Trp135Arg)
mutation had the moderate phenotype. Two nonrelated patients
were heterozygous for c.220G>C (p.Ala74Pro) mutation one with
moderate (P9) and one with mild phenotype (P10). The heterozygous c.403G>A (p.Asp135Asn) mutation detected in P14 with severe
phenotype and bilateral globus pallidi involvement on brain MRI
has been also reported previously in heterozygous state in another
non-related patient with severe phenotype and bilateral globus pallidi involvement on brain MRI [O’Rourke et al., 2009]. Compound
heterozygous c.407C>T (p.Thr136Met) and c.316C>T (p.Gln106)
mutations were detected in P15, a Spanish patient with severe phenotype and both mutations had been previously reported in another
Spanish patient with GAMT-D who had moderate phenotype and
was diagnosed at the age of 45 years [Sempere et al., 2009]. Homozygous c.506G>A (p.Cys169Tyr) mutation was detected in patient P19
with a mild phenotype; however, same homozygous mutation was
reported previously in a Portuguese patient with severe phenotype
[P27 in Mercimek-Mahmutoglu et al., 2006].
Recombinant c.133T>A (p.Trp45Arg) cells did not have any
GAMT enzyme activity with decreased amount of protein on the
Western blot. Three heterozygous patients (P1, P2, P3) for this mutation had a moderate phenotype, albeit the presence of a second
deleterious mutation. Recombinant c.220G>C (p.Ala74Pro) cells revealed no enzyme activity as well as no protein expression on the
Western blot. Surprisingly P10, homozygous for this mutation, presented with a mild phenotype and P9 was compound heterozygous
and had a moderate phenotype. Of all tested variants, recombinant c.439C>T (p.His147Try) (P16, no clinical information) and
c.623G>C (p.Arg208Pro) (P21) cells revealed relatively higher residual enzyme activity (4% compared with wild-type GAMT transfectants). P21 was homozygous for the p.Arg208Pro mutation and
had a severe phenotype. Because of the fact that the severity of the
symptoms varies amongst patients harboring the same mutation(s)
[Mercimek-Mahmutoglu et al., 2006] and that all the pathogenic
variants are devoid of significant residual GAMT activity, no genotype/phenotype correlation can be drawn from this cohort. In other
words, the level of (residual) GAMT activity does not explain the
differences between patients presenting with a moderate, mild, or
severe phenotype. It seems that GAMT activity aside; there are other
yet unknown factors that influence the pathophysiology of GAMT
deficiency.
Analyzing the conservation of gene sequences (DNA, RNA, or
amino acids) within a functional class or across species, amino-acid
residues or domains relevant for gene function can often be extrapolated because the amino-acid sequence determines the protein’s
3D structure and hence function. However, the relation of sequence
46
Figure 3. Graphical representation of the UniProtKB, PDB – ATOM and PDB – SEQRES sequences of rat GAMT (PDB ID: 1XCJ; Komoto et al.).
In this representation, an overview of the secondary structure (DSSP), author assigned annotations (Site Record), published amino-acid sequence
(UniProtKB) plus the amino-acid annotation as provided by author of solved structure (PDB) are shown. Amino-acid residues depicted above the
nascent GAMT sequence represent the amino-acid substitutions that were tested. Indicated by a yellow or purple circle (on the Site Record)
are the amino-acid residues that are directly involved in binding to GAA or SAH, respectively. The W20 amino-acid residue (amongst others) is
directly bound to SAH, and the importance of this residue is highlighted by the pathogenicity of the well-characterized W20S mutant (Almeida et al.,
2006a,b).
to structure is not a unique relationship, as different sequences,
sometimes totally unrelated, may have similar 3D structures. Thus,
the degree of conservation of the 3D structure is much higher than
the degree of conservation of the amino-acid sequence. Functional
characterization of gene variants via targeted amino-acid substitutions and enzyme assays still remains vital in order to establish the
effect (if any) of an existing variant on the catalytic activity of a
protein. The availability of solved 3D structures greatly facilitates
the interpretation of such functional studies. Two missense variants, (c.22C>A (p.Pro8Thr) and c.79T>C (p.Tyr27His)) were not
damaging to GAMT protein and activity.
The heterozygous c.22C>A (p.Pro8Thr) variant was present in
a patient with mildly elevated urine and plasma GAA, but almost
normal creatine on 1 H-MRS, unfortunately the patient passed away
before the completion of biochemical investigations to exclude diagnosis of GAMT-D. This variant was neither found in a cohort
of 6002 individuals (http://evs.gs.washington.edu/EVS/) nor in our
220 control alleles.
However, the c.79T>C (p.Tyr27His) variant is in fact a
polymorphism which is reported in the exome database
(http://evs.gs.washington.edu/EVS/) with a minor allele frequency
of 0.2% and at a frequency of 1.5% in a Portuguese newborn population (data not published) which suggest that this polymorphism is
not associated with GAMT-D, although software prediction mod-
els suggest that the variant could be pathogenic. Additionally, in
one individual this polymorphism was detected in homozygous
state who had normal GAMT enzyme activity in the cultured primary fibroblasts excluding the diagnosis of GAMT-D. This is in
line with the findings of normal GAMT enzyme activity in our
GAMT overexpression assay and confirms that this variant is not
associated with GAMT-D. Based on the solved 3D structure of the
intact GAMT ternary complex (Fig. 3) [GAMT – (SAH + GAA);
[Komoto et al., 2002, 2004], fusion protein expression, and GAMT
activity measurements, we attempt to provide an explanation for
the pathogenicity or lack thereof, of the missense variants tested
[www.pdb.org – DSSP program; Kabsch and Sander, 1983]. Seven
of the mutated amino-acid residues interact directly via H-bonds
with SAH (Met50, Gly68, Ala74, Asn92, Glu135, and Thr136) or
GAA (Cys169). The pathogenicity of the tested mutants at these positions is probably because of their inability to bind the substrate or
a loss of active site conformation/integrity. The secondary structure
of GAMT comprises alpha helices and beta strands as well as the
rare 310 helix motifs [Pauling et al, 1951]. Expression of the fusion
protein, but no GAMT activity, was detected in cell homogenates
of the pathogenic variants; p.Met50Leu (located within an alpha
helix), p.Trp45Arg (located within a 310 -helix) and p.Arg208Pro
(located within a beta strand). Substitutions of the residues at these
sites may have the effect of distorting the backbone conformation
HUMAN MUTATION, Vol. 35, No. 4, 462–469, 2014
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47
(orientation), interfering with hydrogen bonding (via steric hindrance) and eventually lead to a misfolded protein. Misfolding of the
GAMT protein could also affect its EGFP fusion protein (by disrupting its anti-EGFP epitope) preventing its detection by an anti-EGFP
antibody. On the Western blot, no detectable expression for the
above-mentioned variants could be seen, all of which were located
either within an alpha helix (His51, Ala54, Ala74, Ala75, His147,
Leu159, and Leu197) or a beta strand (Gly68 and Val110). Residue
Tyr27 is incorporated into a beta strand, however, the p.Tyr27His
variant is not pathogenic (see above). A possible explanation could
be that Tyr and His have side chains of similar bulk and polarity,
thus there is no (significant) change in the torsion angle when His
substitutes Tyr at this position. Pro8 is not involved in secondary
structure formation—a possible reason why the p.Pro8Thr substitution is tolerated.
Physicians, trying to establish a diagnosis, are always challenged
with the finding of novel variants and their pathogenicity. Genetic
prenatal diagnosis can be offered in families with two pathogenic
GAMT alleles in the index case. To confirm that the mutations are
inherited in trans, the presence of heterozygous mutations needs to
be confirmed in parents. In a case with a heterozygous mutation and
biochemically confirmed GAMT-D, the second disease allele may
be identified by an informative polymorphism. In this case, prenatal
diagnosis can be still offered.
The diagnosis of GAMT-D can be based on metabolite measurements (i.e., increased levels of GAA) in body fluids and creatine
deficiency on 1H-MRS. However, for the interpretation of the missense variants additional studies are needed. These are the reasons
that functional studies are essential to prove pathogenicity of missense variants. The GAMT enzyme activity measurement in cultured
primary skin fibroblasts is the gold standard for the confirmation of
the diagnosis in GAMT-D, which is clinically available only in our
laboratory worldwide. The sample collection from the patients is an
invasive procedure and shipment of the cells as well as funding for
the clinical testing is not available in many countries. With the new
developments in molecular genetics technologies, such as, next generation genome and/or exome sequencing, we will find more and
more missense variants with unclear pathogenicity and functional
studies will be an essential part of clinical diagnostics.
Concluding Remarks
We report 13 new patients with GAMT-D, six novel mutations
in the GAMT gene and functional analysis of 19 missense variants.
We showed that using the described functional assay, two variants
(p.Pro8Thr and p.Tyr27His) predicted to be pathogenic by software modeling did not interfere with GAMT function, and one
(p.Arg208Pro) that was predicted not to be pathogenic is in fact
pathogenic. This illustrates that introducing mutations by SDM in
recombinant GAMT protein followed by a functional enzyme assay
is essential for the confirmation of the pathogenicity of the missense variants in the GAMT gene. Furthermore, our novel GAMT
mutation database http://www.LOVD.nl/GAMT for the information of pathogenic variants will help physicians to decide on the
pathogenicity of the missense variants.
As we know that standard treatment protocols are not widely
used in patients with GAMT-D because of restricted availability
of supplements (e.g., ornithine) and difficulties in maintaining an
arginine-restricted diet. Additionally, a patient with GAMT-D had a
limited effect on neurodevelopmental and biochemical outcome on
strict arginine-restricted diet [Mercimek-Mahmutoglu et al., 2012].
Because of various unknowns and no available clinical treatment
trials for GAMT-D for the last 20 years, we initiated an international database to help us for the evaluation of treatment outcome
and development of new treatment recommendations for GAMT
deficiency. We use an online questionnaire hosted by the Research
Electronic Data Capture (REDCap) online software. All physicians
who requested genetic testing for the confirmation of GAMT-D in
Amsterdam are being contacted via e-mail and invited for this study.
We are hoping to include all treated patients with GAMT-D into this
study.
Acknowledgment
We would like to thank the families allowing their physicians to share their
children’s clinical information.
Disclosure statement: The authors declare no conflict of interest.
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49
Chapter 4
Post-transcriptional Regulation of the Creatine Transporter Gene:
Functional Relevance of Alternative Splicing
"Real biologists who actually do the research will tell you that they almost never find
a phenomenon, no matter how odd or irrelevant it looks when they first see it, that doesn't
prove to serve a function. The outcome itself may be due to small accidents of evolution"
E. O. Wilson
50
51
Biochimica et Biophysica Acta
Post-transcriptional regulation of the creatine transporter gene:
Functional relevance of alternative splicing
Joseph D.T. Ndika a,b, Cristina Martinez-Munoz a, Nandaja Anand a, Silvy J.M. van Dooren a, Warsha Kanhai a,
Desiree E.C. Smith a, Cornelis Jakobs a, Gajja S. Salomons a,b,c,⁎
a
b
c
Department of Clinical Chemistry, Metabolic Unit, VU University Medical Center, Amsterdam, The Netherlands
Neuroscience Campus, VU University Medical Center, Amsterdam, The Netherlands
Department of Clinical Genetics, VU University Medical Center, Amsterdam, The Netherlands
a r t i c l e
i n f o
Article history:
Received 28 May 2013
Received in revised form 7 February 2014
Accepted 12 February 2014
Available online 20 February 2014
Keywords:
Na+/Cl− cotransporter
Creatine transporter
Alternative splicing
Creatine uptake upregulation
Intellectual disability
a b s t r a c t
Background: Aberrations in about 10–15% of X-chromosome genes account for intellectual disability (ID); with a
prevalence of 1–3% (Gécz et al., 2009 [1]). The SLC6A8 gene, mapped to Xq28, encodes the creatine transporter
(CTR1). Mutations in SLC6A8, and the ensuing decrease in brain creatine, lead to co-occurrence of speech/
language delay, autism-like behaviors and epilepsy with ID. A splice variant of SLC6A8–SLC6A8C, containing intron 4 and exons 5–13, was identiﬁed. Herein, we report the identiﬁcation of a novel variant — SLC6A8D, and
functional relevance of these isoforms.
Methods: Via (quantitative) RT-PCR, uptake assays, and confocal microscopy, we investigated their expression
and function vis-à-vis creatine transport.
Results: SLC6A8D is homologous to SLC6A8C except for a deletion of exon 9 (without occurrence of a frame shift).
Both contain an open reading frame encoding a truncated protein but otherwise identical to CTR1. Like SLC6A8,
both variants are predominantly expressed in tissues with high energy requirement. Our experiments reveal that
these truncated isoforms do not transport creatine. However, in SLC6A8 (CTR1)-overexpressing cells, a
subsequent infection (transduction) with viral constructs encoding either the SLC6A8C (CTR4) or SLC6A8D
(CTR5) isoform resulted in a signiﬁcant increase in creatine accumulation compared to CTR1 cells re-infected
with viral constructs containing the empty vector. Moreover, transient transfection of CTR4 or CTR5 into
HEK293 cells resulted in signiﬁcantly higher creatine uptake.
Conclusions: CTR4 and CTR5 are possible regulators of the creatine transporter since their overexpression results
in upregulated CTR1 protein and creatine uptake.
General signiﬁcance: Provides added insight into the mechanism(s) of creatine transport regulation.
© 2014 Elsevier B.V. All rights reserved.
1. Introduction
Genetic deﬁciencies involving AGAT (OMIM ID: 602360), GAMT
(OMIM ID: 601240) or SLC6A8 (OMIM ID: 300036), make up the creatine deﬁciency syndromes — a group of inborn errors of metabolism
with symptoms such as intellectual disability, autism-like behavior
and epilepsy [2–4]. Intellectual disability, with a prevalence of 1-3%
[1], is also the main symptomatic of defective creatine synthesis or
transport. Creatine supplementation is the main therapeutic approach,
with success (notably improved cognitive function) in treating AGAT
and GAMT deﬁciencies [5]. The poor permeability of the blood–brain
barrier to creatine (reviewed in [6]) renders creatine supplementation
ineffective for treating SLC6A8 deﬁciency. A recent comprehensive overview of creatine metabolism and transport in relation to CNS function is
⁎ Corresponding author at: Metabool Lab, PK 1X 009, De Boelelaan 1117, 1081HV
Amsterdam, The Netherlands. Tel.: +31 204443053.
E-mail address: [email protected] (G.S. Salomons).
http://dx.doi.org/10.1016/j.bbagen.2014.02.012
0304-4165/© 2014 Elsevier B.V. All rights reserved.
reviewed in Braissant et al. [45]. Congenital creatine deﬁciency aside,
creatine has also been used to treat mitochondrial and muscular diseases,
to alleviate brain and spinal cord injuries and to improve mental performance [7]. Creatine has also been employed as a neuroprotective agent
in animal models of Parkinson's and Huntington's diseases [8]. In a more
recent study [9], overexpression of the creatine transporter in mice resulted in protection against acute myocardial infarction. Consequently insight
into the mechanisms of how the transporter is regulated is valuable.
Alternative splicing is one of the most important mechanisms regulating gene expression. It enables diversiﬁcation of one gene into different
protein products and is thought to provide a molecular mechanism for
ﬁne-tuning the gene functions of a single locus [10,11]. Contrary to the expected minor role of alternative splicing in functional regulation, large scale
sequencing- and bioinformatics-based studies have reported that it occurs
in up to 94% of human genes [12,13]. The presence of a novel SLC6A8 splice
variant (SLC6A8C), was revealed in primary ﬁbroblasts of different individuals and in different human tissues [14]. The authors were however unable
to detect SLC6A8B (GenBank: U17986) — the ﬁrst identiﬁed splice variant
52
of SLC6A8. In the present study, we report the identiﬁcation in human and
mouse, of a new variant (SLC6A8D) of presumably the SLC6A8C mRNA,
with an in-frame deletion of exon 9. In order to investigate the functional
relevance of alternative splicing of SLC6A8 in terms of creatine uptake
and/or regulation of the full-length creatine transporter, we generated recombinant mouse 3T3 Swiss cells overexpressing each splice variant, the
full length transporter, as well as cells co-expressing the full length transporter with either one or the other splice isoform.
conditions as for the human samples. For screening of the mouse cells,
the primers were speciﬁcally designed to amplify mouse slc6a8derived sequences (Table 1, Nº. 6, Nº. 7, Nº. 8); consequently the PCR annealing temperature (Ta) was adjusted to 61 °C for slc6a8d. As control,
ampliﬁcation of Slc6a8c and Slc6a8 (Ta, 59 °C) was included. All PCR
products were gel-puriﬁed and sequenced.
2. Materials and methods
Investigation of differential splice variant expression was carried out
on RNA isolated from 20 different tissues (FirstChoice™ Human Total
RNA survey panel; Ambion). Gene-speciﬁc primers and probes used
for ampliﬁcation of each transcript are shown in Table 2.
To investigate if upregulation of creatine uptake in 3T3 Swiss cells coexpressing CTR1 with a splice isoform compared to cells co-expressing
CTR1 with an empty vector, was as a result of enhanced SLC6A8 transcription, standard Q-PCR was performed on an ABI7300 (Applied Biosystems)
using a probe (5′-FAM 3′-TAMRA labeled probe: 5′-TGGGTGCTGGTCTACT
TCTGTGTC-3′) and primers (5′-AAGTCTTGAGGCTGTCTGG-3′ and
5′-ACGATCTTTCCCGTGGAT-3′) speciﬁc for exon 4 of human SLC6A8.
All reactions were performed in the presence of 1 M Betaine and
ROX reference dye, and corrected for input by normalizing to GAPDH
(human tissues) or Gapdh (mouse 3T3 Swiss cells) gene expression
assay (PrimeTime™ Std qPCR Assay; IDT Technologies). Quantiﬁcation
(threshold cycle number, CT) of both target and reference genes was
carried out in triplicate and in independent wells using the 2−ΔCt method. Analysis was done using the Q-Gene™ software package [15]. The
mean normalized expression was obtained by averaging the CT values
of target and reference genes, respectively and subsequent calculation
of the standard error of the mean normalized expression [16].
2.1. Identiﬁcation of SLC6A8D transcript
Total RNA was extracted (RNA isolation kit; Promega) from HEK-293
cells (since SLC6A8 expression is high in kidney) and from primary
human ﬁbroblasts obtained from both controls and a patient with a genomic deletion encompassing the entire SLC6A8 gene. cDNA synthesis
(Fermentas) was performed (with and without reverse transcriptase,
to check for gDNA contamination) using 1 μg of RNA. A forward primer
complementary to intron 4 and a reverse primer complementary to the
3′UTR of the SLC6A8 locus were designed to amplify both SLC6A8C and
SLC6A8D from HEK293 cDNA (Table 1, Nº. 1). Subsequently nested
primers were used to detect presence of SLC6A8C and SLC6A8D transcripts
in the ampliﬁed PCR products. SLC6A8D-speciﬁc primers were designed
complementary to intron 4 and spanning exon 8/10 such that only messages lacking exon 9 will be ampliﬁed (Table 1, Nº. 2). For detection of speciﬁc SLC6A8C and SLC6A8 transcripts, previously designed primers [14]
were used (Table 1, Nº. 3 and Nº. 4). RT-PCR of all transcripts was performed using KAPA HiFi™ Hotstart DNA polymerase with GC buffer
(Kapa Biosystems). The general PCR conditions used were as speciﬁed
by the manufacturer, except for the inclusion of 0.2 M Betaine in each reaction. Thermocycling was as follows: initial denaturation for 5 min at
95 °C; followed by 38 cycles of 20 s at 98 °C, followed by 15 s at 66 °C
(SLC6A8, SLC6A8C, SLC6A8D) or 61 °C (GAPDH), and 2 min at 72 °C; with
a ﬁnal extension step of 10 min at 72 °C. Transcript identities were conﬁrmed by sequencing (ABI 3130XL Genetic Analyser; Applied Biosystems).
2.2. Detection of SLC6A8D expression in monkey and mouse
Slc6a8d expression in mouse was investigated in NIH-3T3 2.2 ﬁbroblasts and primary mouse embryonic ﬁbroblasts (MEFs) of FVB mice.
Two monkey cell lines (CP132 and Vero) were also included. RT-PCR
for the monkey cell lines was performed with same primers and PCR
2.3. Quantitative real-time PCR (Q-PCR)
2.4. Construction of pBABE-hygro-SLC6A8-EGFP, pBABE-puro-SLC6A8C-EGFP
and pBABE-puro-SLC6A8D-EGFP expression vectors
The open reading frame (ORF) of all isoforms was cloned in-frame to
the N-terminal of EGFP (enhanced green ﬂuorescent protein). In order to
obtain a pBP-SLC6A8D-EGFP construct; RNA was isolated from HEK293
cells followed by cDNA synthesis. The ORF of SLC6A8D (exons 7–13)
was ampliﬁed by RT-PCR using primers with HindIII and EcoRI restriction
site overhangs (Table 1, Nº. 9), and then cloned into pEGFPN1 by standard cloning techniques. Via PCR (Table 1, Nº. 11) and site directed mutagenesis, an EcoRI-SLC6A8D-EGFP-SalI construct was then shuttled from
its pEGFPN1 vector into a pBABE-puro (pBP) destination vector. The ORFs
Table 1
PCR and cloning primers.
No.
Species
Forward/reverse sequence
mRNA sequence
SLC6A8 location
Amplicon size (bp)
1
Human
SLC6A8C/SLC6A8D
Human/monkey
3
Human/monkey
4
Human/monkey
Intron 4
3′ UTR
Intron 4
Exon 8/10
Intron 4
Exon 9
Exon 1
3′ UTR
1969/1831
2
5
Human
6
Mouse
7
Mouse
8
Mouse
9
Human
10
Human
11
Recombinant
5′-GAGGTACTGAAAGCCAAGCAATGC-3′
5′-GCTGGTGATGTGAGCTGAGT-3′
5′-CTCCCACACCTGCACTGCCC-3′
5′-GACGTACATCCCGCCCTGGC-3′
5′-CTCCCACACCTGCACTGCCC-3′
5′-GGAGAGATCGATGACAAAGCAG-3′
5′-ATGGCGAAGAAGAGCGCCGAG-3′
5′-GCTGGTGATGTGAGCTGAGT-3′
5′-ATGGCGAAGAAGAGCGCCGAG-3′
5′-GCTGGTGATGTGAGCTGAGT-3′
5′-CAAGAATGATCTGGAGTTTGGG-3′
5′-GACGTACATTCCACCCTGGC-3′
5′-CAAGAATGATCTGGAGTTTGGG-3′
5′-GGAGAGATCGATGACAAAGCAG-3′
5′-GGTATCTATAGCGTGTCTGG-3′
5′-TTACATGACACTCTCCACCACG-3′
5′-CCCAAGCTTCCACCATGGCTGCAGAGCAGGGCGTGC-3′
5′-CGGAATTCGCATGACACTCTCCACCACG-3′
5′-CGGGATCCACCATGGCGAAGAAGAGC-3′
5′-CGGGATCCCATGACACTCTCCACCACG-3′
5′-CGCGAATTCCACCATGGCTGCAGAGCAGGGCGTGC-3′
5′-CGCGTCGACCTCTACAAATGTGGTATGGCTG-3′
SLC6A8D
SLC6A8C
SLC6A8
GAPDH
Slc6a8d
Slc6a8c
Slc6a8
SLC6A8C/SLC6A8D
SLC6A8
SLC6A8C-/SLC6A8D-(EGFP)
827
938
1930
819
Intron 4
Exon 8/10
Intron 4
Exon 9
Exon 2
Exon 13
Exon 7
Exon 13
Exon 1
Exon 13
Exon 7
944
1056
1884
809/671
1905
1650/1515
53
Table 2
Q-PCR primer pairs and probes. To quantify SLC6A8C transcripts, cDNA synthesis was carried out with a primer in exon 9 (to distinguish SLC6A8C from SLC6A8D) followed by PCR with
SLC6A8C-speciﬁc primers. To quantify SLC6A8 and SLC6A8D transcripts, cDNA synthesis was done with an oligo(dT) primer followed by qPCR with gene-speciﬁc primers.
Gene
cDNA synthesis
Forward primer (10 μM)
Reverse primer (10 μM)
Probe (5 μM) 5′Fam, 3′Tamra
SLC6A8
SLC6A8C
SLC6A8D
Oligo(dT)20
5′-CAGCATCAATGTCTGGAACA-3′
5′-GCCAGCACCATGATGTAGTA-3′
5′-TCAAAGGCCTGGGCTACGCCT-3′
5′-GGAGAGATCGATGACAAAGCAG-3′
5′-GGGACCTCTGAACATACCT-3′
5′-GCAGTGAAGTACACGATCTG-3′
5′-ACAGCCTCCGCTGAGCAGCCT-3′
Oligo(dT)20
5′-GACAGCCAGGGCGGGAT-3′
5′-ACCACGCACTCCCAAAAG-3′
5′-ACTACTCGGCCAGCGGCACCA-3′
of SLC6A8 and SLC6A8C were cloned previously in-frame with EGFP in a
pEGFPN1 plasmid [14,17]. A BamHI-EGFP-SalI restriction-enzymedigested fragment was isolated from pEGFPN1 and re-cloned into both
pBABE-hygro (pBH) and pBP vectors. Via PCR (Table 1, Nº. 10) a BamHISLC6A8-BamHI fragment was obtained from pEGFPN1-SLC6A8 and recloned in-frame to EGFP in pBH to produce pBH-SLC6A8-EGFP. A pBPSLC6A8C-EGFP construct was generated by shuttling a PCR-generated
EcoRI-SLC6A8C-EGFP-SalI fragment from SLC6A8C-pEGFPN1 into a pBP
vector. Integrity of all clones was veriﬁed by sequencing.
2.5. Generation of recombinant viruses
Viruses containing the empty vectors (pBP-EGFP or pBH-EGFP), the
truncated isoforms (pBP-SLC6A8C-EGFP or pBP-SLC6A8D-EGFP) and the
full-length transporter (pBH-SLC6A8-EGFP) constructs were generated.
pBP and pBH vectors have the viral packaging signal sequence (Psi)
while Phoenix cells (HEK293T) express viral GAG-POL and ENV genes.
Transfection of these Phoenix producer cell lines with pBP/pBH-based
vectors yields viral supernatants with an ecotropic host range. Phoenix
cells were cultured under standard mammalian cell culture conditions
to 70% conﬂuence in DMEM cell culture medium (GIBCO, Scotland) supplemented with 1% pen/strep, 1% L-glutamine and 10% FBS. They were
subsequently transfected (CaCl2/HBS method; Sigma) with 25 μg
endotoxin-free plasmid DNA (Nucleobond™ 500 EF Kit; Bioke). Transfection efﬁciency was monitored via EGFP ﬂuorescence. 48 and 72 h after
transfection, 10 ml of medium containing recombinant viruses was
tapped, ﬁltered, ﬂash-frozen in liquid nitrogen and stored at −80 °C.
2.6. Infection of 3T3 Swiss mouse ﬁbroblast cells with construct-containing
viruses
In a ﬁrst round of infections two “primary” cell lines were generated
expressing either the empty vector (pBH-EGFP) or CTR1 (pBH-SLC6A8EGFP) with hygromycin resistance as a selectable marker. Next, these
two cell lines were re-infected (in duplicate, here-in called Infection I
and Infection II) with pBP-EGFP-, pBP-SLC6A8C-EGFP- or pBP-SLC6A8DEGFP-containing viruses with puromycin resistance as the co-selectable
marker. Thus in total duplicates of 6 cell lines were generated coexpressing; the two empty vectors (1. pBH-EGFP/pBP-EGFP), one splice
variant with an empty vector (2. pBH-EGFP/pBP-SLC6A8C-EGFP or 3. pBHEGFP/pBP-SLC6A8D-EGFP), SLC6A8 with an empty vector (4. pBH-SLC6A8EGFP/pBP-EGFP), SLC6A8 with a splice variant (5. pBH-SLC6A8-EGFP/pBPSLC6A8C or 6. pBH-SLC6A8-EGFP/pBP-SLC6A8D-EGFP). The infections
were carried out as follows; 3T3 Swiss cells were cultured (same medium
and conditions as above) to 50% conﬂuence. Next, individual viral taps
were thawed to 37 °C and mixed. Cell growth medium was refreshed
with virus-containing supernatant (5 ml/10 cm dish, supplemented
with 4 μg/ml polybrene). A non-infected control plate was taken along.
24 h post infection; successful transformants were selected by addition
of 250 μg/ml hygromycin for 5 days (or 7 μg/ml puromycin +
125 μg/ml hygromycin for 3 days, 24 h after the second round of infections). After the hygromycin-based infection, stable transformed cells
were maintained for 7 days in 125 μg/ml hygromycin until the
puromycin-based infections. Following the second selection, cell populations were recovered in 3.5 μg/ml puromycin- and 125 μg/ml
hygromycin-supplemented medium. 24 h prior to creatine uptake assays,
recombinant 3T3 Swiss cells were seeded to 70% conﬂuence in medium
without antibiotics. 4 replicates per infected cell line were seeded — 3
for creatine uptake and 1 plate to make pellets for both Western blot
and Q-PCR. Cell pellets were ﬂash-frozen in liquid nitrogen, and stored
at −80 °C until further use.
2.7. Creatine uptake assay
Prior to incubation of the cells in creatine, intracellular amino acids
were depleted, based on a previously described method [18]. Brieﬂy; recombinant cells were incubated for 1 h at 37 °C in Earle's Balanced Salts
(EBSS; Sigma-Aldrich) supplemented with 0.1% D-glucose and 0.25%
BSA. Cell monolayers were washed with EBSS and incubated (5 min for
48 h; 95% air/5% CO2 at 37 °C) in DMEM (10% FBS, 1% Pen/Strep, 1%
L-Glu) with the desired amount [5 μM (5 μmol/l)–2000 μM
(2000 μmol/l)] of stable-isotope-labeled creatine-[methyl-13C]
monohydrate. Uptake was stopped by rapidly washing cells with cold
Hank's Balanced Salt Solution (HBSS). The cells were harvested (2 min
at 37 °C in 0.05% Trypsin + EDTA, GIBCO; Scotland), resuspended in
cold HBSS followed by centrifugation (5 min, 1400 rpm).
Cell pellets were resuspended in Milli-Q grade water and disrupted by
vortexing. After centrifugation (1 min, 12,000 rpm), the supernatant was
cleaned further on an Amicon Ultra — 10 K membrane. Measurement of
creatine-[methyl-13C] was performed on the protein-free eluate, using
creatine-[2H3] (Sigma-Aldrich) as internal standard. In parallel, protein
content was determined from a fraction of the disrupted cells using a
bicinchoninic acid protein assay kit, according to the instructions of the
manufacturer (Sigma-Aldrich). Creatine concentration is expressed as
pmol creatine/μg total protein. Measurements were performed on an
API 3000 triple quadruple tandem mass spectrometer (Applied
Biosystems) with a Perkin-Elmer Series 200 HPLC pump and a PerkinElmer Series 200 auto sampler (operated at 4 °C). Using a Symmetry
Shield RP18 analytical column (3.0 × 100 mm; 3.5 μm; Waters) 10 μl of
the sample was separated using 5 mM nonapentanoic acid as an ionpair reagent. In 5 min the acetonitrile content was linearly increased
from 5% to 15%, and subsequently in 5 min from 15% to 50%. The ﬂow
rate was 0.3 ml/min. The turbo ion electrospray was operated in positive
ion mode, the cone temperature was set to 450 °C and the cone voltage
was 5000 V. Nitrogen was used as the turbo ion gas at a ﬂow rate of
8 l/min. Collision induced dissociation was initiated using nitrogen as
the collision gas at a pressure of 0.06 kPa. Creatine was fragmented
with a collision energy of 35 V. The following MS/MS transitions were
measured (creatine; 132.0 → 90.0, creatine-[methyl-13C]; 133.0
→ 91.0 and creatine-[2H3]; 135.0 →93.0). The LC–MS/MS data were
acquired and processed using Analyst for Windows software (Applied
Biosystems). The limit of quantiﬁcation (S/N = 10) was estimated to
be 20 pmol.
2.8. Western blot
Cell pellets were lysed in urea lysis buffer (8 M urea/100 mM
NaCl/10 mM Tris–HCL, pH 8.0). Protein concentrations for each sample were determined as described above and normalized to equal
concentrations with additional lysis buffer and SDS sample buffer.
54
Cell lysates (40 μg) were size separated on a 12% Bis–Tris Gel
(NuPAGE™, Invitrogen), and transferred to a PVDF membrane (iBlot
™, Invitrogen). Immunodetection was performed using antibodies directed against the EGFP tag and against endogenous Actin as a loading
control (anti-EGFP, anti-Actin; Abcam). Immune complexes were detected by enhanced chemiluminescence (Lumi-LightPLUS; Roche Applied Science). For analysis of plasma membrane targeting of fusion
protein isoforms, the cell membrane component was ﬁrst extracted
(membrane protein extraction kit; BioVision) and then immunoblotted
as described above. Immunoblotting with antibodies to endogenous
Guanidinoacetate Methyl Transferase (GAMT) was used to conﬁrm
the purity of the extracted membrane fractions.
2.9. Fluorescence microscopy
Recombinant 3T3 Swiss cells seeded on glass slides (Menzel™
Superfrost) were ﬁxed for 15 min at room temperature (RT) in 4% paraformaldehyde plus 1% Triton X-100 dissolved in phosphate buffered saline (PBS). Slides were washed and mounted for microscopy with
Vectashield™ mounting medium (Vector Laboratories). Fluorescence
was visualized at 63 × magniﬁcation (Objective Plan-Apochromat
63×/ 1.40 oil) with a confocal microscope (LSM 510 Meta, Zeiss).
2.10. Transient transfection of HEK 293 cells
Kidney-derived HEK293 cells were cultured in 60 mm cell culture
dishes under standard mammalian cell culture conditions to 40% conﬂuence in DMEM cell culture medium (GIBCO, Scotland) supplemented
with 1% pen/strep, 1% L-glutamine and 10% FBS. They were subsequently transfected (Fugene™ HD transfection reagent; Promega) with 10 μg
of pEGFPN1-based SLC6A8C (CTR4), SLC6A8D (CTR5) or SLC19A3 (thiamine transporter) endotoxin-free plasmid DNA. All constructs were
transfected in triplicate and the transfection efﬁciency based on EGFP
ﬂuorescence was estimated to be N90% in all plates (data not shown).
24 h after transfections, cell monolayers were washed, harvested with
trypsin and seeded into 100 mm dishes to achieve a conﬂuence of
about 70% after 24 h in cell culture medium. 48 h after transfections, creatine uptake was performed as described in Section 2.7.
3. Results and discussion
3.1. Molecular identiﬁcation and characterization of SLC6A8D
While cloning the SLC6A8C variant [14] from a HeLa cell line, a novel
variant, which we named SLC6A8D (GenBank: KC800563), was discovered. This variant was the product of an in-frame deletion of exon 9,
resulting in a novel 138 bp shorter form of the SLC6A8C mRNA
(GenBank: EU280316). The rest of the sequence is 100% homologous
to the SLC6A8C mRNA and its coding region is identical to that of
SLC6A8C and SLC6A8. Thus at the protein level SLC6A8D (CTR5) is 100%
homologous to SLC6A8C (CTR4) with the exception of a 46 amino acid
in-frame deletion of residues 54 through 99. The deletion comprises
the ﬁrst cytosolic loop and the second transmembrane domain of the
CTR4 protein (based on the LeuT model [19], Fig. 1). A pseudogene of
SLC6A8–SLC6A10P, has been mapped to chr16 [20,21]. No SLC6A8 PCR
product was observed following a PCR on cDNA of a patient with a genomic deletion of the entire SLC6A8 locus, using a forward primer complementary to both SLC6A8 and SLC6A10P and a reverse primer speciﬁc to
the 3′ UTR of SLC6A8. Furthermore, nested fragments of both SLC6A8C
and SLC6A8D were ampliﬁed from PCR products of a forward primer
in intron 4 and the SLC6A8–speciﬁc reverse primer. Taken together,
these results conﬁrm that SLC6A8D is a chrX transcript, and possesses
identical 5′ and 3′ UTRs to those of SLC6A8C (Fig. 2).
3.2. Biological relevance of the SLC6A8D splice variant
In mouse we conﬁrmed the expression of an exon 9-deﬁcient variant
(Slc6a8d) of Slc6a8c. Slc6a8d (GenBank: KC994641) expression was detected in transformed mouse embryonic ﬁbroblasts (NIH-3T3 2.2) and,
albeit at lower levels, in a mouse mammary gland cell line (NMuMG).
For the ﬁrst time we also show that SLC6A8C is expressed in monkey
(Fig. 3).
3.3. Tissue-speciﬁc expression of SLC6A8, SLC6A8C and SLC6A8D
The expression levels of SLC6A8 and its splice variants are highest in
tissues with high energy requirement like the kidney, colon and small
intestine (Fig. 4). Suggesting that like SLC6A8, the splice variants could
Fig. 1. Schematic representations of SLC6A8, SLC6A8C and SLC6A8D and their putative transmembrane domains. Because the exons, open reading frame and hence amino acids from SLC6A8
are conserved with splicing, the last ﬁve (SLC6A8C) and last four (SLC6A8D) transmembrane domains are identical to those of the full length creatine transporter (CTR1). To be consistent
with previous nomenclature, we refer to the SLC6A8D protein as CTR5.
55
Fig. 2. Molecular characterization of SLC6A8D. (A) Shows speciﬁcity of reverse primer (Table 1, Nº. 4) to the SLC6A8 transcript. No amplicon could be detected in ﬁbroblasts from an individual with a genomic deletion encompassing the entire SLC6A8 and its ﬂanking genes (lanes 3 and 4) as opposed to control ﬁbroblasts (lanes 1 and 2). This reverse primer in combination
with an intron 4 forward primer was used to amplify both SLC6A8C and SLC6A8D messages (B) from HEK 293 cDNA (lane 6, primers; Table 1, Nº. 1). A nested PCR on the resulting amplicons
with the same intron 4 forward primer and a reverse primer speciﬁc for either SLC6A8C (lane 7, primers; Table 1, Nº. 3) or SLC6A8D (lane 8, primers; Table 1, Nº. 2) conﬁrms that the 5′UTR
of SLC6A8D is at least as big as that of SLC6A8C. The mRNA sequence of SLC6A8D, as conﬁrmed by sequencing of cloned PCR products from lane 6, is shown in (C). The underlined sequence
depicts intron 4 of SLC6A8, while the bold printed nucleotides represent the cloned open reading frame of the SLC6A8D (CTR5) splice isoform. In all cell lines, absence of gDNA contamination was conﬁrmed by PCR on cDNA synthesized without reverse transcriptase (data not shown).
also be involved in regulating creatine uptake. Across all 20 tissues tested, SLC6A8C is the most abundant splice variant of the two, with
expression levels noticeably higher than that of SLC6A8 in prostrate
and thymus.
Fig. 3. Biological relevance of SLC6A8D. Expression of SLC6A8D was analyzed in several mouse cell lines (NIH-3T3 2.2, Mefs, S49, L1210) and tissues (stomach, liver) as well as in two monkey cell lines (Vero, CP132). Ampliﬁcation of SLC6A8/Slc6a8 and SLC6A8C/Slc6a8c as included controls. The reverse transcriptase (RTO) containing reactions are depicted by a+ and the RTO
negative reactions by a− character. Experiments were repeated at least three times and similar results were obtained: representative pictures are shown. Slc6a8d expression is only observed for the transformed ﬁbroblasts (NIH-3T3 2.2) and the mammary epithelial cells (NMuMG).
56
Fig. 4. Expression levels of SLC6A8, SLC6A8C and SLC6A8D across 20 human tissues. All target gene expression values were normalized using GAPDH as reference gene. Ampliﬁcation efﬁciency of each primer pair (all between 98.22% and 99.91%) was determined by the standard curve method using serial dilutions of cDNA. The mean normalized expression is a function of
the average CT values of the target and reference genes respectively. Bars are mean ± SEM.
Fig. 5. Time course (A) and saturation kinetics (B) of creatine transport in recombinant 3T3 Swiss overexpressing SLC6A8-EGFP. Creatine uptake (on cells supplemented with 5, 25, 50, 100,
250, 500, 1000 and 2000 μM creatine) was performed as described in Section 2.7, in triplicate. Each bar represents the mean ± SEM. In the duplicate infections (I and II) uptake is linear
within the ﬁrst 4 h (A). Evaluation with a Michaelis–Menten model revealed a Vmax of 138 ± 3.8 and 113.3 ± 4.4 pmol/4 h/μg protein and a Km of 41.2 ± 5.5 and 34 ± 6.7 μM for Infections I and II respectively (B). Following overnight incubations, cells overexpressing the splice isoforms {SLC6A8C (CTR4) or SLC6A8D (CTR5)} accumulate similar levels of isotopelabeled creatine as those cells expressing only the empty vector or uninfected cells. On the other hand overexpression of SLC6A8 (CTR1) results in a signiﬁcantly higher creatine uptake
capacity at both non-saturating (25 μM) and saturating (500 μM) creatine concentrations (C). Panel (D) shows the expression of EGFP fusion constructs in 40 μg of cell lysate. All cells
were infected with two constructs using puromycin and hygromycin as co-selectable markers. “Empty vector” represents a combination of the pBPuro-EGFP/pBHygro-EGFP plasmids,
“SLC6A8C” — the pBHygroEGFP/pBPuro-SLC6A8C-EGFP plasmids, “SLC6A8” — the pBHygroEGFP/pBPuro-SLC6A8-EGFP plasmids and “SLC6A8D” — the pBHygroEGFP/pBPuro-SLC6A8CEGFP plasmids. To ensure that all lanes were loaded with equal amounts of protein, the blot was stripped and re-probed with an anti-actin antibody.
57
3.4. Splice variants SLC6A8C (CTR4) and SLC6A8D (CTR5) do not transport
creatine
In recombinant SLC6A8 cells (overexpressing CTR1) the time
course for creatine uptake was linear for up to 4 h in both sets of infections (Fig. 5A). The saturation kinetics of creatine transport was
determined following incubation in 5 μM–2 mM of creatine-[methyl-13C]-supplemented medium for 4 h (Fig. 5B). Creatine transport
followed Michaelis–Menten kinetics with Vmax values of 113.3 ±
4.4 and 138 ± 3.8 pmol/4 h/μg protein for infections I and II respectively, as well as Km values of 34 ± 6.7 μM (Infection I) and 41.2 ±
5.5 μM (Infection II). Following overnight incubations, cells overexpressing the splice isoforms accumulate similar levels of creatine-[methyl-13C] compared to both cells expressing only the empty vector and
uninfected cells. On the other hand overexpressing SLC6A8 (CTR1) results in a 10 fold and a 13 fold increase in creatine uptake capacity at
physiological (25 μM) and saturating (500 μM) creatine-[methyl-13C]
concentrations respectively (Fig. 5C). Expression of splice isoforms and
the full length creatine transporter as a fusion to EGFP is conﬁrmed on
Western blot (Fig. 5D). Thus, the splice variants by themselves are not
bonaﬁde creatine transporters.
3.5. Re-infecting CTR1-expressing cells with either CTR4 (SLC6A8C) or CTR5
(SLC6A8D) upregulates creatine uptake at physiological concentrations,
while increasing Vmax at saturating creatine concentrations
To explore whether the splice isoforms regulate SLC6A8, the transporter was expressed with or without additional expression of the
splice variants, followed by analysis of creatine transport (Section 2.7).
To rule out the possibility of passive creatine transport, a ﬁrst set of incubations was carried out in medium supplemented only with
creatine-[methyl-13C] and in parallel a second set of incubations was
carried out in a medium supplemented with equimolar amounts of
creatine-[methyl-13C] and guanidinopropionic acid (25, 500 and
1000 μM each). Guanidinopropionic acid (GPA) is a known competitive
inhibitor of the creatine transporter [22]. Measurement of cellular creatine levels in the absence and presence of GPA shows that co-expression
of the transporter with its splice variant enhances creatine uptake
(p b 0.05) at both physiological (25 μM) and saturating (500 μM and
1000 μM) creatine concentrations. Decreased intracellular accumulation of creatine in the presence of GPA conﬁrms that this enhanced uptake is not due to passive diffusion of creatine into the cells (Fig. 6 and
6B). We observe an increase in the maximum rate of creatine transport
Fig. 6. Upregulated creatine transport due to increased SLC6A8 protein (CTR1) levels in 3T3 Swiss-SLC6A8 cells re-infected with SLC6A8C or SLC6A8D. Infection I (A, upper panel) and Infection II (B, lower panel) represent independent secondary infections of each construct (empty vector, SLC6A8C or SLC6A8D) on cells already overexpressing SLC6A8-EGFP. Incubations
were carried out in triplicate and the measured intracellular creatine was normalized to the total protein content. Cells were incubated with only creatine (Cr) or with equimolar amounts
of creatine and guanidino propionic acid (Cr + GPA). Each bar represents the mean ± SEM, and * a p value b 0.05 (one-way ANOVA followed by Dunnett's post-test, comparing SLC6A8/
empty vector to either SLC6A8/SLC6A8C or SLC6A8/SLC6A8D using the GraphPad Prism software). At non-saturating creatine concentrations (25 μM), creatine transport is signiﬁcantly upregulated 2–3 fold in SLC6A8/SLC6A8C and SLC6A8/SLC6A8D cells when compared to cells expressing only SLC6A8 and the empty vector (SLC6A8/empty vector). At saturating creatine concentrations (500 and 1000 μM), the same holds true for the SLC6A8/SLC6A8C cell lines (up to 4 fold increase); however the upregulation (10–60% increase) is lower in the SLC6A8/SLC6A8D
cells when compared to the SLC6A8/empty vector cell lines. To determine if this enhanced creatine accumulating capacity following addition of a splice variant was due to increased SLC6A8
transcription/translation and hence the amount of transporter available at the plasma membrane, quantitative real time PCR was carried out on cell pellets from Infection I (C, upper
panel). The normalized expression is a function of the average CT values of SLC6A8-EGFP and mouse Gapdh respectively. Bars are mean ± SEM. There is no signiﬁcant difference in recombinant SLC6A8-EGFP mRNA expression between 3T3 Swiss-SLC6A8 cells co-expressing a splice variant and those co-expressing the empty vector. However on a Western blot (C, lower
panel) SLC6A8 protein levels (CTR1) are higher when either the SLC6A8C (CTR4) or SLC6A8D (CTR5) isoform was co-expressed in place of the empty vector. CTR4 protein levels are higher
than those of CTR45 (possibly resulting from higher transfection efﬁciencies and as such a higher viral titer) with a corresponding higher expression of CTR1. Re-probing the same blot with
an anti-actin antibody was carried out as a loading control.
58
(thiamine transporter) construct. Their effect on endogenous creatine
uptake was determined as described in Section 2. At both 25 μM and
500 μM creatine concentrations HEK293 cells overexpressing CTR4
and CTR5 had a signiﬁcant increase (p value b 0.005) in creatine transport compared to cells over-expressing SLC19A3. Though moderate
(around 25% increase), the increase of creatine uptake following overexpression of CTR4 and CTR5 in HEK293 cells is in line with what is observed for the heterologous mouse 3T3 Swiss expression system
(Fig. 7). This conﬁrms that overexpression of the splice variants has a
positive effect on the function of the endogenously expressed creatine
transporter.
Fig. 7. Physiological relevance of splice variants on endogenous CTR1 in HEK293 cells. Creatine transport is signiﬁcantly increased by 26–28% in HEK293 cells overexpressing the
CTR4 or CTR5 splice isoform compared to cells overexpression the thiamine transporter
(SLC19A3). * indicates p value b 0.001 (one-way ANOVA followed by Dunnett's posttest — GraphPad Prism software) between effect on endogenous creatine transport due
to CTR4/CTR5 compared to SLC19A3. The uptake of creatine in cells overexpressing
SLC19A3 (THTR2) was 8% reduced compared to the pEGFPN1 (empty vector)
transfectants (data not shown), which is most likely explained by the fact that the cells
have more difﬁculties in expressing a membrane protein than EGFP only.
following addition of SLC6A8C or SLC6A8D as opposed to addition of the
empty vector, in recombinant SLC6A8-expressing cells. At nonsaturating creatine concentrations (25 μM) the extent to which
SLC6A8D enhances creatine transport is comparable to that by
SLC6A8C (2–3 fold). On the other hand at saturating substrate concentrations (500/1000 μM) the maximum SLC6A8D-mediated increase in
creatine uptake was only about 1.5 fold as opposed to a 4 fold increase
in the case of SLC6A8C. This is most likely due to the higher expression of
CTR4 (SLC6A8C-EGFP) compared to CTR5 (SLC6A8D-EGFP) (Fig. 6C,
lower panel), resulting in higher CTR1 (SLC6A8-EGFP) protein levels
and a concomitant increase in creatine uptake capacity.
3.6. Physiological relevance ofCTR4 and CTR5
To conﬁrm that the splice variants increase the creatine uptake mediated by the endogenous creatine transporter we transiently
transfected the kidney-derived HEK293 cells with a pEGFPN1 based
SLC6A8C (CTR4) or SLC6A8D (CTR5) plasmid. As a control to exclude
that overexpression of an unrelated transporter would give a similar effect we also transiently transfected a pEGFPN1 based SLC19A3
3.7. Splice-variant-dependent upregulation of creatine transport by CTR1
occurs at post transcription
After depletion of intracellular creatine followed by incubation in 25,
500 and1000 μM creatine-[methyl-13C]-supplemented medium for 4 h,
SLC6A8-EGFP mRNA levels were evaluated in recombinant SLC6A8 cells
additionally expressing the empty vector or a splice isoform. At both
physiological and saturating creatine concentrations there is no signiﬁcant difference in SLC6A8 (SLC6A8-EGFP) transcription in the SLC6A8/
empty vector cell line compared to the SLC6A8/SLC6A8C or SLC6A8/
SLC6A8D cell lines (Fig. 6C, upper panel). A Western blot (Fig. 6C,
lower panel) shows bands at 100 kDa and 90 kDa corresponding to
the glycosylated and unglycosylated SLC6A8-EGFP fusion proteins.
There are also bands at 50 kDa (SLC6A8C-EGFP), 40 kDa (SLC6A8DEGFP) and 25 kDa (EGFP). Western blotting reveals that expression of
CTR1 (SLC6A8-EGFP) compared to Actin levels is lower in the absence
of a co-expressed splice isoform. Interestingly, expression of CTR4
(SLC6A8C-EGFP) is higher than CTR5 (SLC6A8D-EGFP) with a corresponding higher co-expression of CTR1. Thus, additional expression of
either CTR4 or CTR5 to CTR1-expressing cells results in an increase in
total CTR1 levels. This splice-variant-associated increase in CTR1 protein
levels was not accompanied by an increase in mRNA levels, suggesting
(post)translational rather than transcriptional regulation. This positive
regulation of a parent transporter by its otherwise non-functional variant is of great interest as previous reports investigating the functional
relevance of splicing among neurotransmitter transporters revealed a
dominant negative effect of the splice isoforms on the function of the
transporter. Co-expression of a non-functional rat norepinephrine
transporter splice isoform (rNETb) with a functional rNETa isoform in
COS cells, decreased the expression and hence activity of the rNETa
Fig. 8. Localization of EGFP fusion constructs. To investigate if fusion proteins are targeted to the plasma membrane, 3T3 Swiss-SLC6A8, -SLC6A8/empty vector, -SLC6A8/SLC6A8C and -SLC6A8/
SLC6A8D cells were incubated in 25 μM creatine overnight. A whole cell fraction (lanes 1–4) and a plasma membrane fraction (lanes 5–7) of each recombinant cell type were analyzed on a
Western blot. Whole cell fractions were loaded as follows: lane 1; SLC6A8, lane 2; SLC6A8/SLC6A8D, lane 3; SLC6A8/SLC6A8C, lane 4; SLC6A8/Empty vector. Lanes 5, 6 and 7 are plasma membrane fractions of the samples in lanes 2, 3 and 4 respectively. An antibody directed against cytosolic guanidinoacetate N-methyl transferase (GAMT) was used to conﬁrm the purity of the
plasma membrane fraction. The Western blots show that, of all fusion proteins, only SLC6A8-EGFP (CTR1) is targeted to the plasma membrane. Absence of the GAMT protein and the
unglycosylated SLC6A8-EGFP (glycosylation of the creatine transporter occurs at the Golgi before it is targeted to the plasma membrane, thus these unglycosylated moieties are most likely
its ER–Golgi intermediates [44]) conﬁrms the purity of the plasma membrane fractions.
59
Fig. 9. Co-localization of CTR1 with CTR4 and CTR5. Confocal image acquisition (Objective Plan-Apochromat 63×/ 1.40 oil; LSM 510 Meta, Zeiss) of cells co-expressing SLC6A8-EGFP (CTR1)
and SLC6A8C-mCherry (CTR4) or SLC6A8D-mCherry (CTR5) indicates that the full length transporter co-localizes with its truncated splice isoforms.
isoform [23]. A similar scenario has been described for the glutamatetransporting EAAT1 transporter and its EAAT1exon9skip splice variant
[24]. This is very interesting as it raises the possibility that although
the regulatory mechanisms in this family of transporters seem to be
conserved, the effect on protein function may be different.
3.8. Splice isoforms may facilitate endoplasmic reticulum exit and/or
trafﬁcking of full-length transporter
Puriﬁcation of the plasma membrane (PM) fraction of recombinant
3T3 Swiss cells shows that the splice variants are not localized to the
plasma membrane like CTR1 (Fig. 8). Thus the splice isoforms do not internalize together with the full-length transporter at the PM to facilitate
or enhance its afﬁnity for creatine. However, by replacing the EGFP tag
on CTR4 and CTR5 with mCherry (SLC6A8C-mCherry and SLC6A8DmCherry respectively), we show that both CTR4 and CTR5 co-localize
with CTR1 (Fig. 9). It is plausible that the splice variants do oligomerize
to CTR1 at the level of the ER to facilitate its trafﬁcking to the Golgi,
where it will undergo further modiﬁcation(s) before being targeted to
the plasma membrane. This is consistent with previous studies involving regulation of Na+/Cl− cotransporters and further emphasizes the
notion that oligomerization-dependent regulation seems to be common
among members of this transporter family [25,26].
4. Concluding remarks
In this study we identiﬁed a novel variant of SLC6A8–SLC6A8D; identical to SLC6A8C, but lacking exon 9. Differential expression between tissues and conservation across species suggested a functional relevance of
these variants. Functional creatine transport analyses reveal that
although these splice isoforms do not transport creatine, their coexpression with the full-length transporter results in an enhanced capacity for creatine uptake, reﬂected by an increase in protein expression
of the creatine transporter. Varying the cell surface levels of the transporter via altered trafﬁcking is a fairly common avenue for regulation
of neurotransmitter transporters. This results in the maximum rate of
transporter activity (Vmax) being altered with little to no change in
substrate afﬁnity (Km), reviewed in [27,28]. Studies investigating changes in creatine transporter levels in response to extracellular creatine have
shown that creatine saturation or depletion was associated with a reduced or increased Vmax respectively, of creatine transport. In these
studies, changes in plasma membrane rather than total CTR1 levels resulted in the altered Vmax [29–33]. The increase in total CTR1 levels in
our case, following addition of a splice isoform is an indication that besides being involved in CTR1 trafﬁcking, these splice isoforms may also
be involved in extending the half-life of the mature transporter. A possible avenue could be via the ubiquitin-protein ligase — Nedd4-2 (neuronal precursor cell expressed developmentally down-regulated protein),
which targets a number of membrane proteins including receptors and
ion channels for degradation by the proteasome. A Nedd-4-2-mediated
decrease in plasma membrane levels has been shown for the dopamine
[34], glucose [35], glutamate [36,37] and thiazide-sensitive [38] Na+/Cl−
cotransporters, suggesting that this mechanism could be relevant for
regulating the Na+/Cl− creatine transporter as well. In fact there is evidence that the serum/glucocorticoid-regulated kinases (SGK1 and
SGK3), which are inhibitors of Nedd4-2 [38–42] increase the maximal
transport rate of CTR1 [43]. For the moment we can only speculate that
by virtue of their homology to the full-length transporter, SLC6A8C
(CTR4) and SLC6A8D (CTR5) can act as alternative targets and thus insulate the transporter from ubiquitin ligases. The extent and nature of this
regulation of SLC6A8 by SLC6A8C and SLC6A8D is subject to future investigations. Their effect of enhancing creatine accumulation by SLC6A8 provides new avenues to explore the regulation of the creatine transporter,
which may also be relevant for other members of this transporter family.
Acknowledgements
The authors are grateful to Herman ten Brink for synthesis of the
creatine-[methyl-13C]. The anti-GAMT antibodies were a kind gift from
Theo Walliman's lab. The mCherry plasmid was obtained by courtesy
of Roger Tsien's lab.
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61
Chapter 5
Cloning and characterization of the promoter regions from the
parent and paralogous creatine transporter genes
"It appears unlikely that the role of the genes in development is to be understood so
long as the genes are considered as dictatorial elements in the cellular economy. It
is not enough to know what a gene does when it manifests itself. One must also
know the mechanisms determining which of the many gene-controlled potentialities will be realized"
David Ledbetter Nanney
62
63
Gene
Cloning and characterization of the promoter regions from
the parent and paralogous creatine transporter genes
Joseph D.T. Ndika a,b, Vera Lusink a, Claudine Beaubrun a, Warsha Kanhai a,
Cristina Martinez-Munoz a, Cornelis Jakobs a, Gajja S. Salomons a,b,c,⁎
a
b
c
Department of Clinical Chemistry, Metabolic Unit, VU University Medical Center, Amsterdam, The Netherlands
Neuroscience Campus, VU University Medical Center, Amsterdam, The Netherlands
Department of Clinical Genetics, VU University Medical Center, Amsterdam, The Netherlands
a r t i c l e
i n f o
Article history:
Accepted 2 October 2013
Available online 18 October 2013
Keywords:
Creatine transport
Transcriptional regulation
Promoter
Pseudogene
a b s t r a c t
Interconversion between phosphocreatine and creatine, catalyzed by creatine kinase is crucial in the supply of
ATP to tissues with high energy demand. Creatine's importance has been established by its use as an ergogenic
aid in sport, as well as the development of intellectual disability in patients with congenital creatine deﬁciency.
Creatine biosynthesis is complemented by dietary creatine uptake. Intracellular transport of creatine is carried
out by a creatine transporter protein (CT1/CRT/CRTR) encoded by the SLC6A8 gene. Most tissues express this
gene, with highest levels detected in skeletal muscle and kidney. There are lower levels of the gene detected in
colon, brain, heart, testis and prostate. The mechanism(s) by which this regulation occurs is still poorly
understood. A duplicated unprocessed pseudogene of SLC6A8–SLC6A10P has been mapped to chromosome
16p11.2 (contains the entire SLC6A8 gene, plus 2293 bp of 5′ﬂanking sequence and its entire 3′UTR). Expression
of SLC6A10P has so far only been shown in human testis and brain. It is still unclear as to what is the function of
SLC6A10P. In a patient with autism, a chromosomal breakpoint that intersects the 5′ﬂanking region of SLC6A10P
was identiﬁed; suggesting that SLC6A10P is a non-coding RNA involved in autism. Our aim was to investigate the
presence of cis-acting factor(s) that regulate expression of the creatine transporter, as well as to determine if
these factors are functionally conserved upstream of the creatine transporter pseudogene.
Via gene-speciﬁc PCR, cloning and functional luciferase assays we identiﬁed a 1104 bp sequence proximal to the
mRNA start site of the SLC6A8 gene with promoter activity in ﬁve cell types. The corresponding 5′ﬂanking
sequence (1050 bp) on the pseudogene also had promoter activity in all 5 cell lines. Surprisingly the pseudogene
promoter was stronger than that of its parent gene in 4 of the cell lines tested. To the best of our knowledge, this is
the ﬁrst experimental evidence of a pseudogene with stronger promoter activity than its parental gene.
© 2013 Elsevier B.V. All rights reserved.
1. Introduction
1.1. Creatine pathway
Creatine is a nitrogenous organic acid whose intracellular pool is
maintained by both endogenous synthesis and nutritional uptake. The
Abbreviations: ATP, adenosine triphosphate; bp, base pair; kb, kilo base pair; cDNA,
DNA complementary to RNA; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase;
gDNA, genomic DNA; EGFP, enhanced green ﬂuorescent protein; SV40, Simian virus 40;
chr, chromosome; MEFs, mouse embryo ﬁbroblasts; HEK293, human embryonic kidney
cell line; 3T3 Swiss, mouse ﬁbroblast cell line; SK-N-SH, human neuroblastoma cell line;
mRNA, messenger RNA; miRNA, microRNA; ORF, open reading frame; PCR, polymerase
chain reaction; RTPCR, reverse transcriptase-polymerase chain reaction; SLC6A8, solute
carrier family 6, member 8; SLC6A10P, solute carrier family 6, member 10 pseudogene;
UTR, untranslated region.
⁎ Corresponding author at: Department of Clinical Chemistry, Metabolic Unit,
VU University Medical Center, Amsterdam, The Netherlands. Tel.: + 31 204442880;
fax: + 31 204440305.
E-mail address: [email protected] (G.S. Salomons).
0378-1119/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.gene.2013.10.008
creatine transporter gene, also known as SLC6A8 (GeneID 6535) or
CT1, is located on the human chromosome Xq28 and consists of 13
exons (Gregor et al., 1995; Sandoval et al., 1995). Mutations in the
SLC6A8 gene lead to congenital creatine deﬁciency (Salomons et al.,
2001). The importance of creatine transport is highlighted by in vitro
studies in cerebellar granule cells which show that creatine transport
seems to be more efﬁcient in building up total intracellular creatine
than de novo synthesis (Carducci et al., 2012). The two enzymes
responsible for endogenous creatine synthesis are AGAT (from arginine
and glycine) and GAMT (via methylation of guanidinoacetate produced
by AGAT). Genetic deﬁciency of either creatine synthesis enzymes
results in depletion of brain creatine, similar to SLC6A8 deﬁciency
(OMIM: 300036). Key clinical features of creatine deﬁciency are intellectual disability, autism like behavior, movement disorders and
epilepsy (Mercimek-Mahmutoglu et al., 2009). Creatine supplementation therapies, especially when initiated in the newborns, have
been successful in preventing disease onset and in treating several
cases of creatine biosynthesis deﬁciency (Bianchi et al., 2000, 2007;
64
Mercimek-Mahmutoglu et al., 2006; Ndika et al., 2012; Schulze and
Battini, 2007). No treatment options yet exist for creatine transporter
deﬁcient patients. Thus unraveling aspects of creatine transporter
regulation remain crucial; both in terms of understanding the pathophysiology of creatine biosynthesis deﬁciencies — to improve existing
treatment regimens, and also to explore possible avenues for therapy
vis-a-vis SLC6A8 deﬁciency.
1.2. Intellectual disability
Intellectual disability (now the preferred term for mental retardation)
is the most common developmental disorder with a worldwide prevalence of 1.37% (Maulik and Mascarenhas, 2011). For the majority of
cases of inherited intellectual disability, the genetic cause has not yet
been elucidated. X-linked intellectual disability is estimated to account
for 5%–12% of all cases of intellectual disability (Herbst and Miller,
1980). The frequency of SLC6A8 mutations in an XLMR population of
288 patients was as high as 2.1% (Rosenberg et al., 2004). In another
study, a 1% prevalence of mutations in SLC6A8 was found in boys with
intellectual disability of unknown etiology (Clark et al., 2006). So far
patients are being diagnosed either via metabolic workup (i.e. creatine
and guanidinoacetic acid measurements in urine and plasma) via cranial
MRS or genetic testing. In the latter, so far only the coding exons and the
neighboring splice sites of the SLC6A8 gene are being analyzed. The
unknown promoter region has not yet been included in these analyses.
The functional relevance of such (promoter) regions is highlighted
by Dunham et al., based on their observation that disease-relevant
single nucleotide polymorphisms (SNPs) are enriched within noncoding functional elements — a majority of which reside within or
in the vicinity of regions that are outside of protein-coding genes
(Dunham et al, 2012).
1.3. Duplicated paralogues of SLC6A8 on chromosome 16
There is a poor understanding of why some genes (paralogous
genes) are ampliﬁed in our genome during evolution and whether
they have a function. Until recently it was believed that paralogous
genes were “faults” of nature and had no function. However, nowadays
several paralogue genes are known to be expressed and even functional
(Pei et al., 2012). A duplicated paralogue of SLC6A8–SLC6A10 (alias CT2)
with a transversion of the T in its initial termination codon to a G,
extending its open reading frame by 50 amino acids, was mapped
to chromosome 16p11.2 (Iyer et al., 1996; Xu et al., 1997). However
in chromosome 16 the predicted amino acid sequence was found to
harbor a nonsense mutation in “exon 4” (compared to CT1), indicating
that a creatine transporter protein cannot be translated from the
SLC6A10 mRNA and is most likely a pseudogene (Eichler et al., 1996;
Iyer et al., 1996). This established the basis for a change in nomenclature
from SLC6A10 to SLC6A10P (Gene ID: 386757). Further clariﬁcation on
the nature of the SLC6A8 duplication on chromosome 16 was provided
by Höglund and colleagues (Höglund et al., 2005). By searching the May
2004 assembly of the human genome on the UCSC genome browser,
they found out that there are two adjacent pseudogenes of SLC6A8 on
chromosome 16: one at 32797531–32799840 on the reverse strand
and the other at 33690486–33692794 on the forward strand. Both loci
share a 95.8% sequence similarity with SLC6A8. All published instances
of SLC6A10P referred to the pseudogene on the reverse strand, now
denoted as SLC6A10pA (Gene ID: 386757), while that on forward
strand, SLC6A10pB is listed in Entrez Gene as a predicted gene
(Gene ID: 653562). However, with a sequence similarity of 99.6%, it
is very likely that some of the cDNA identiﬁed as SLC6A10pA also
included transcripts from SLC6A10pB. SLC6A10P transcripts (most
likely SLC6A10pA and SLC6A10pB messages) have been reported in testis
(Iyer et al., 1996) and brain (Bayou et al., 2008). Moreover, according to
data from the Gene Expression Atlas — a database of publicly
available gene expression data obtained from functional genomics
experiments by the European Bioinformatics Institute (EMBL-EBI),
differential expression of SLC6A10P (SLC6A10pA and SLC6A10pB)
has been seen for some tissues (kidney, skeletal muscle, spinal
cord, etc) and cell lines (HT1080, Hela, A498, etc) (http://www.ebi.
ac.uk/gxa/gene/ENSG00000214617). This differential and tissuespeciﬁc expression pattern suggests that there is/are functional role(s)
associated with the SLC6A10P pseudogenes. Supportive of this hypothesis, a translocation breakpoint on chromosome 16p11.2 was mapped
to disrupt the 5′ﬂanking sequence of SLC6A10pA in a patient presenting
with autism (Bayou et al., 2008). Approximately 2.3 kb of the 5′ﬂanking
sequence of SLC6A10pA and SLC6A10pB shares 95% homology to the
same region on their parent gene, and 99.9% homology with one
another. Other than their differential expression across tissues and
experimental conditions, nothing else is known of the regulation and
even function of the SLC6A10P pseudogenes. As a ﬁrst step towards
understanding the physiological relevance of these pseudogenes we
investigated the presence of a functional promoter and its activation
across different cell types. Sequence analysis of the cloned promoter
reveals it to be the 5′ﬂanking sequence of the pseudogene on the
reverse strand (SLC6A10pA). For simplicity we will refer to both
SLC6A10pA and SLC6A10pB as SLC6A10P, except otherwise indicated.
2. Methodology
2.1. Sequence alignment and analysis
Using the genome browser feature on UCSC Genome Browser
(February 2009 version), we obtained a sequence of approximately
3 kb (2868 bp) upstream of the start of the 5′UTR of SLC6A8. Next we
performed a BLAT alignment (Kent et al., 2002) of this region (including
the entire 5′UTR [279 bp] and 33 bp into the SLC6A8 open reading frame
[ORF]). Restriction site analysis of the selected potential upstream
regulatory region (URR) — 3180 bp in total, was done in Vector NTI™
(Invitrogen, Carlsbad, CA, USA).
2.2. Isolation of 5′ﬂanking sequences
DNA isolation was carried out using a genomic DNA (gDNA) isolation
kit (Promega). Via PCR on isolated gDNA, 5′ﬂanking sequences of
SLC6A8 were ampliﬁed from whole blood of a healthy donor (male)
while 5′ﬂanking sequences of SLC6A10 were ampliﬁed from the ﬁbroblast cells of an anonymized individual (male) with a complete deletion
of the SLC6A8 locus. Primers were designed to amplify and clone
5′ﬂanking sequences of − 3146/+ 34 base pairs (bp) relative to
the ﬁrst methionine of SLC6A8 and −1016/+34 bp relative to the
same methionine on SLC6A10pA/SLC6A10pB. The primers used to obtain
the 5′ﬂanking sequence of SLC6A8 were gene-speciﬁc while those used
to obtain 5′ﬂanking sequences of the pseudogenes could also amplify
SLC6A8; however this is avoided by carrying out the PCR on gDNA of
the individual with a deletion of the SLC6A8 locus. PCR primers were
designed using Vector NTI™ and synthesized by IDTdna (Leuven,
Belgium).
All PCRs were carried out using a high ﬁdelity polymerase (KAPA
HiFi™ HotStart — KapaBiosystems, MA, USA) as speciﬁed by the
manufacturer. A gradient of different annealing temperatures (Ta)
was used to determine the most optimum Ta for each primer pair. In
order to enable cloning of PCR products into the reporter vector,
forward and reverse primers were designed to contain restriction site
sequences at their 5′ends. Potential 5′ﬂanking regulatory regions as
well as PCR primers are depicted in Table 1. Truncated 5′ﬂanking
sequences of 2345 bp (−2311/+34) and 1104 bp (−1070/+34)
were derived by making use of native XhoI and SacI restriction sites
within the −3146/+34 — SLC6A8 fragment and a created HindIII
restriction site at its 3′end.
66
32896789
−
chr16:
5′-GGAGAgagctcAAGGAAGGAGGGAGC-3′
5′-GGaagcttTATAGATGCCGTTCTCGGCGCTCTTCTTCG-3′
32897838
66
152952960
+
chrX:
Digestion
152954063
N/A
152952965
+
chrX:
152951749
68
152954063
152951751
+
chrX:
5′‐CGCaagcttCTCGAGCCTCTGCTTTCTCTCC‐3′
5′‐GGctcgagTATAGATGCCGTTCTCGGCGCTCTTCTTCG‐3′
Digestion
pGL4.20[luc2/puro].
A DNA sequence of 1.2 kb from the 5′‐most end of the −2279/+34 bp insert was obtained via XhoI/SacI digestion, linearized with Klenow Pol and religated into a linearized and blunted pGL4.20 vector.
2
1
gDNA from SLC6A8 — patient (male)
pGL4.20—2.3 kb (SLC6A8) plasmid
pGL4.20—2.3 kb (SLC6A8) plasmid
pGL4.20—3.2 kb (SLC6A8) plasmid
−3146/+34 (SLC6A8) pGL4.20—3.2 kb (SLC6A8) plasmid
+
chrX:
152951751
152954063
68
NheI
HindIII
XhoI
HindIII
HindIII
XhoI
XhoI
SacI
SacI
HindIII
SacI
HindIII
70
152954063
152950884
+
5′-gctagcAAACTCCAGCCTTACCAGCCTGAACTTCATGC-3′
5′-GGaagcttTATAGATGCCGTTCTCGGCGCTCTTCTTCG-3
Digestion
−3146/+34 (SLC6A8)
5′ → 3′
−2279/+34 (SLC6A8)
5′ → 3′
+34/−2279 (SLC6A8)
3′ → 5′
−2279/−1064 (SLC6A8)2
5′ → 3′
−1070/+34 (SLC6A8)
5′ → 3′
−1016/+34 (SLC6A10P)
5′ → 3′
chrX:
Primer pair/cloning
strategy
Chro
2.3. Vectors, transfections and promoter assay
5′Flanking region (bp) cloned
in pGL4.201
Table 1
Primer sequences and cloning strategy of potential upstream regulatory regions.
Locus
Strand
Start
End
Annealing
temp. (°C)
Restriction
sites
Template
gDNA from healthy subject (male)
65
The characterization of 5′ﬂanking sequences from SLC6A8 and
SLC6A10P was based on a pGL4.20-Basic promoterless plasmid containing the ﬁreﬂy luciferase gene (pGL4.20[luc2/puro]; Promega).
Varying lengths and orientation of the 5′ﬂanking sequences of both
SLC6A8 (−3146/+34, −2311/+34, −1070/+34, −2311/−1070,
+34/−2311) and SLC6A10P (−1016/+34) were introduced into
the MCS of pGL4.20 by standard molecular biology techniques.
Sequence integrity of cloned inserts was conﬁrmed by sequencing
(Big Dye Termination kit v3.1 — Applied Biosystems). A second
plasmid — pGL3-Promoter (pGL3-SV40; Promega), containing a
luciferase gene under the control of an SV40 promoter was used as
a control for stable expression of luciferase in the different cell
types (HeLa, HEK293, SK-N-SH, CEPH, human ﬁbroblast and mouse
3T3 Swiss). Prior to transfections, all cells had been cultured for at
least a week in suitable media (HeLa and CEPH — RPMI; HEK293,
SK-N-SH, 3T3 Swiss and human ﬁbroblast — DMEM), supplemented
with 1% pen/strep, 1% L-glutamine and 10% FBS under standard
mammalian cell culture conditions (humidiﬁed atmosphere of 95%
air/5% CO2 at 37 °C). HeLa, HEK293, SK-N-SH and 3T3 Swiss
cells were transfected with FugeneHD™ transfection reagent (Roche
Applied Science), using a Fugene to DNA ratio of 6.25 μl:1.25 μg (HeLa,
HEK293, 3T3 Swiss — for 24 h), and 16 μl:2 μg (SK-N-SH — for 72 h) in
12 well plates. Transfection of CEPH and human ﬁbroblasts was carried
out using the 4D-Nucleofector™ System; Lonza (600 ng DNA/well). To
circumvent the problem of crosstalk between promoters (Farr and
Roman, 1992) we did not use a second plasmid harboring a functional
promoter to normalize for transfection efﬁciencies. In order to confound
variations as a result of varying transfection efﬁciencies, all plasmid
transfections were done on the same day (same cell line passage),
in triplicate. Furthermore transfection efﬁciencies were estimated
based on EGFP ﬂuorescence from co-transfections (in adjacent wells)
of a pGL3-SV40 plasmid with a plasmid expressing enhanced green
ﬂuorescent protein (pEGFP-N1; Clonetech) in a 50:1 ratio (1.225 μg
pGL3-SV40 + 0.025 μg pEGFP-N1 for the Fugene transfections and
1.960 μg pGL3-SV40 + 0.04 μg pEGFP-N1 for the Nucleofector
transfections). Cells transfected with the pGL4.20-Basic plasmid are
used as a negative control. Analysis of promoter activity was carried
out by measuring luminescence signal intensity (Victor Wallac™
luminometer — PerkinElmer) generated from cell lysates of transfected
cells to which luciferase assay reagent had been added (Luciferase Assay
Kit; Promega). Luciferase activity (and consequently promoter activity)
is expressed as luminescence units (LU)/μg of total protein.
3. Results
3.1. Selection of potential upstream regulatory region
− 3146 bp to + 34 bp relative to the ﬁrst methionine (+ 1ATG) of
SLC6A8 (RefSeq Gene: NM_00569) contains a region − 2311/+ 34
that is 95% homologous to the two paralogous loci on chromosome 16.
Native XhoI and SacI restriction sites located at −2279 and −1070
respectively, were used to generate truncated fragments from the 5′end
of the −3146/+34 fragment. Sequence homology of all three fragments:
−3146/+34 [3180 bp], −2279/+34 [2313 bp] and −1070/+34
[1104 bp], to the paralogous chromosome 16 loci is shown in Fig. 1. The
entire Xq28 locus that has been duplicated on chr16 is shown in Fig. 2.
3.2. Functional identiﬁcation of 5′ﬂanking regulatory sequence of SLC6A8
Since SLC6A10P conserves a 5′ﬂanking sequence of 2311 bp as its
parent gene, we hypothesized that promoter features will be localized
in this upstream sequence. However, unlike SLC6A8, SLC6A10P expression has so far only been reported for testes and fetal brain. A possible
explanation for this could be that the sequence variants which
66
Fig. 1. Human BLAT search results (UCSC Genome Browser, version February 2009) depicting cloned 5′ﬂanking sequences. a: −3146/+34 b: −2279/+34 and c: −1070/+34 base pairs
relative to the ﬁrst methionine (+1ATG) of the SLC6A8 open reading frame. d: −1016/+34 base pairs relative to the ﬁrst methionine (+1ATG) of creatine transporter gene paralogous
at chromosome 16p11.2. The 5′regulatory regions of the two pseudogenes share 99.9% sequence similarity. Sequence ID can be obtained via genomic coordinates (from the left; columns
6, 7, 8 and 9).
have accumulated upstream of SLC6A10P (RefSeq Gene: NR_003083)
decreased transcription factor binding afﬁnity and/or created new
transcription factor binding sites. Alternatively, upstream of the
2311 bp region on SLC6A8 there could be enhancer-like elements
facilitating transcription of the creatine transporter gene — a region
which is not conserved upstream of SLC6A10P. Thus to identify
the proximal promoter and potential enhancer(s), we cloned the
− 3146/+ 34 and − 2279/+ 34 fragments from the SLC6A8 gene
into the pGl4.20 vector. Subsequent transfections and luciferase
assays in 3 different cell types conﬁrmed the presence of promoter
activity. However there was no signiﬁcant difference in promoter
strength between the − 3146/+ 34 and − 2279/+ 34 5′ﬂanking
regulatory sequences (Fig. 3).
3.3. Characterization of truncated promoter fragments
To further characterize the promoter region, we truncated the
−2279/+34 fragment into two: −2279/−1064 and −1070/+34.
The − 1070/+ 34 fragment gives the same degree of transcriptional activation as the − 3146/+ 34 and − 2279/+ 34. Fragment
− 2279/− 1064 was unable to drive transcription of the luciferase
gene. Cloning of fragment − 2279/+ 34 in reverse orientation to
the luciferase gene (+ 34/− 2279) led to a repression of promoter
activity (Fig. 4).
Fig. 2. A schematic of chromosome 16 showing two paralogous copies of a duplicated locus
from chromosome Xq28. Comparative sequence analysis between Xq28 and 16p11.2 using
the most recent assembly of the human genome (Feb 2009, GRCh37/hg19) at the
UCSC genome browser, reafﬁrms the duplication of a 26,542 bp segment of Xq28
(chrX:152,951,719-152,978,261) to two separate regions on chromosome 16p11.2
which are approximately 877 kb from each other {one on the + strand [chr16:
33,776,247 to 33,802,719] and the other on the — strand [chr16: 32,899,081 to
32,872,610]}. Both paralogous segments include 2311 bp of 5′sequence ﬂanking the ﬁrst
methionine of SLC6A8, the entire SLC6A8 gene as well as its entire 3′UTR and part of the
ﬂanking BCAP31 gene.
Fig. 3. Identiﬁcation of upstream regulatory region on SLC6A8. In all 3 cell lines,
− 3146/+ 34 bp and − 2279/+ 34 bp of 5′ﬂanking sequences signiﬁcantly induce
transcription of the luciferase gene compared to the empty vector.
67
Fig. 4. Characterization of promoter sequences in Hela cells. Arrow direction indicates the orientation of the cloned sequence. pGL4.20 refers to cells transfected with the empty vector.
Progressive truncation of the SLC6A8 5′ﬂanking sequence of 3180 bp (−3146/+34) to 2313 bp (−2279/+34) and 1104 bp (−1070/+34) results in no signiﬁcant change in promoter
activity (p-value: a = 0.11 and d = 0.15 respectively), thus the core promoter sequence is conserved within the 1104 bp sequence. This is furthermore conﬁrmed by the absence of
promoter activity in fragment −2279/−1064 consisting of 1241 bp of 5′sequence from the 2313 bp fragment (p-value: c = 0.0001). Analysis of a paralogous region on chromosome
16 (−1016/+34 of 5′sequence ﬂanking of SLC6A10pB) corresponding to the −1070/+34 fragment indicates that the pseudogene indeed has a functional and potentially stronger
promoter compared to its parent gene (p-value: e = 0.00035). Reversing the orientation of the −2279/+34 fragment represses promoter activity (p-value: b = 0.0001). Statistical
signiﬁcance was determined using an unpaired t-test.
3.4. 5′ﬂanking sequence on pseudogene conserves core promoter activity
Because the upstream regulatory region homologous between SLC6A8
and SLC6A10P (Fig. 3) has promoter activity, we wondered if promoter
function was conserved for the paralogue gene as well. A 5′ﬂanking
sequence (−1016/+34) that was 93% homologous to the −1070/+34
fragment, containing the core promoter of SLC6A8 was isolated from
SLC6A10P (Fig. 1c and Table 1). Sequence analysis conﬁrmed this
paralogous fragment to be from the reverse strand, which is 99.9%
homologous to the second duplicated locus on the forward strand
(Fig. 1d) of Chr 16.
Interestingly the −1016/+34 fragment ﬂanking the pseudogene
harbors a stronger promoter compared to the same region ﬂanking
SLC6A8. The characterized promoter activity of 5′ﬂanking sequences of
both SLC6A8 and its paralogue from chr16 is shown in Fig. 4.
binding sites within, and/or absence of enhancer elements further
upstream of this 2.3 kb region resulted in a lower and varying
expression of SLC6A10P. Functional analyses of 5′ﬂanking sequences
from SLC6A8 provide evidence of a promoter restricted within
1104 bp, spanning the entire 5′UTR and 34 bp into its ORF. This
1104 bp also entirely spans previously predicted transcription factor
binding sites (Sandoval et al., 1995). The observed promoter activity
3.5. Relative strength of the parent and pseudogene promoters
Promoter strength across ﬁve different cell lines was determined
relative to that of a constitutive SV40 promoter located within the
pGL3-Promoter plasmid (Section 2.3). The paralogous promoter is
signiﬁcantly stronger than its parent promoter in four of the ﬁve cell
lines tested (Fig. 5).
4. Discussion and conclusion
Transcriptional gene regulation is mediated by the presence of cisacting (promoters and/or enhancers), or trans-acting (DNA-binding
proteins) elements. DNA-binding proteins are targeted to their respective
binding sites on the promoter, and via their concerted action, RNA
polymerase is able to bind and synthesize mRNA from the DNA strand.
Sequence analysis of a 2313 bp sequence ﬂanking the SLC6A8 open
reading frame (ORF) reveals that it is 94% homologous to the same
region ﬂanking the two paralogous copies of the SLC6A8 gene on Chr
16. Because of the altered expression proﬁle of SLC6A10P relative to
SLC6A8 we hypothesized that loss of integrity of transcription factor
Fig. 5. Transcriptional potential of SLC6A8 (−1070/+34) and SLC6A10pB (−1016/+34)
core promoter across different cell lines. Promoter strength (transcriptional potential) is
described in terms of relative normalized luminescence units — luminescence signal as a
result of the 5′ﬂanking promoter sequence normalized to the mean luminescence signal
generated by transfecting the cell line in question with a promoterless pGL4.20 vector.
The promoter of SLC6A10pB is a stronger activator of transcription (2- to 7-fold greater)
than the promoter of SLC6A8 in 4 of the 5 cell lines tested (HEK293, CEPH, Fibroblast and
SK-N-SH). Statistical signiﬁcance was determined using an unpaired t-test. The p-values
for the difference in promoter activity between SLC6A8 and SLC6A10pB for each cell line
are HEK293—0.0300 (a), CEPH—0.0080 (b), Fibroblast—0.6300 (c), Hela—0.0007 (d) and
SK-N-SH—0.0060 (e).
68
in the mouse 3T3 Swiss cell suggests that transcription factor binding
sites have been conserved across species (Fig. 3). Transcription factor
activation of the SLC6A8 promoter is cell-type dependent (Fig. 5) and
differential expression of SLC6A8 has been seen across various cells
and tissue types (http://www.ebi.ac.uk/gxa/gene/ENSG00000130821).
Thus altered promoter activation, via differential transcription factor
expression, could very well be one of the key mechanisms by which
SLC6A8 mRNA levels are regulated.
SLC6A8 deﬁciency, an X-linked intellectual disability syndrome is
caused by mutations including large genomic deletions in the SLC6A8
gene. It could also be that mutations in the promoter region cause this
disorder. By having the knowledge on the promoter of SLC6A8 we
now can include this region in the diagnostic workup of patients
suspected to have a creatine transporter defect. Thus this gene region
can be tested for the presence of variants, as is currently being done
for the coding region and the splice sites by DNA sequence analysis.
The proximal promoter region of SLC6A8 (1070 bp from the ﬁrst
methionine) can now be included during DNA sequence analysis in
patients presenting with clinical and biochemical features of SLC6A8
deﬁciency or intellectual disability of unknown etiology.
Contrary to expectations, the 5′ﬂanking sequence of the paralogous
SLC6A10pA gene harbors a stronger promoter than that of its parent
gene. This SLC6A10pA promoter sequence has a 99.9% homology to
the 5′ﬂanking sequence of SLC6A10pB; as such the promoter upstream
of SLC6A10pB is most likely functional. Albeit the presence of a very
strong promoter, sequence analyses of the SLC6A10P pseudogenes
indicate the presence of a premature termination codon in exon 4
(with respect to SLC6A8). Prior to the resolution of the structure of
a bacterial homologue of the creatine transporter (bacterial LeuT
[Yamashita et al., 2005]), SLC6A10P was predicted to be a creatine
transporter. However transmembrane domains 1 and 6 (exons 1 and
6) are involved in substrate binding, thus it is very unlikely that the
SLC6A10pA or SLC6A10pB transcripts will encode a protein capable of
transporting creatine. Taken together all these ﬁndings suggest that,
creatine transport aside, the creatine transporter pseudogene may be
functional. This raises two (amongst others) possibilities; on the one
hand transcribed SLC6A10P could serve as a buffer of miRNAs that will
otherwise target and downregulate expression of the parent SLC6A8
gene. The 3′UTR of SLC6A8 mRNA shares about 97% sequence homology
with its paralogous loci on Chr 16, supporting the notion that transcribed SLC6A10P might act as a decoy for miRNA families that will
otherwise target SLC6A8. Such a miRNA-based regulation of a gene by
its pseudogene(s) has been demonstrated for OCT4 (Suo et al., 2005),
KRAS (Poliseno et al., 2010) and PTEN (Alimonti et al., 2010; Carracedo
et al., 2011) transcripts. On the other hand alternative splicing and
alternative promoter usage are known to contribute to mRNA
heterogeneity and thus the proteomic complexity of a single locus. It
is therefore conceivable that the identiﬁed proximal promoter could
represent the promoter of an alternative SLC6A10P variant (conserved
open reading frame) or a different gene (different open reading
frame) at the same locus. According to the autism chromosome
rearrangement database (http://projects.tcag.ca/autism/) about 53% of
autism-related copy number variants identiﬁed so far on chromosome
16 are at the 16p11.2 locus, and may account for approximately 1% of
all autism spectrum disorder cases (Kumar et al., 2008; Weiss et al.,
2008). Bearing in mind that a patient presenting with autism was
identiﬁed with a chromosomal deletion of a region immediately
upstream of SLC6A10pA (Bayou et al., 2008), the experimental evidence
provided herein of a potent promoter ﬂanking the SLC6A10pA locus
(and probably SLC6A10pB, by virtue of the 99.9% sequence homology
of its potential promoter to that of SLC6A10pA) should pave the way
for subsequent functional studies aimed at unraveling the functional
relevance of the SLC6A10P pseudogenes. Be it as possible regulators of
SLC6A8 or some other unanticipated role.
In summary, we have provided functional evidence of cell-type
dependent transcriptional activation of both the SLC6A8 and SLC6A10P
promoters. Further analysis will be required to evaluate the identities
of important transcription factor-binding sites and to identify elements
involved in tissue-speciﬁc expression of these genes.
Conﬂict of interest
The authors declare no conﬂict of interest.
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69
Chapter 6
RNA sequencing of creatine transporter (SLC6A8) deficient
fibroblasts reveals impairment of the extracellular matrix.
"If Louis Pasteur were to come out of his grave because he heard that the cure for cancer still had not been found, NIH would tell him, “Of course we'll give you assistance.
Now write up exactly what you will be doing during the three years of your grant.”
Pasteur would say, “Thank you very much,” and would go back to his grave. Why?
Because research means going into the unknown. If you know what you are going
to do in science, then you are stupid! This is like telling Michelangelo or Renoir that
he must tell you in advance how many reds and how many blues he will buy, and exactly how he will put those colors together"
Albert Szent-Gyorgyi
70
71
RNA sequencing of creatine transporter (SLC6A8) deficient fibroblasts reveals impairment of the
extracellular matrix.
Benjamin Nota PhD1, Joseph D. T. Ndika MSc1, Jiddeke M. van de Kamp MD2, Warsha A. Kanhai MSc1,
Silvy J. M. van Dooren MSc1, Mark A. van de Wiel PhD3, Gerard Pals PhD 2, and Gajja S. Salomons PhD1,*
1Metabolic
unit, Department of Clinical Chemistry, VU University Medical Center, Amsterdam, The Netherlands
of Clinical Genetics, VU University Medical Center, Amsterdam, The Netherlands
3Department of Epidemiology and Biostatistics, VU University Medical Center, Amsterdam, The Netherlands
2Department
*
Corresponding author, email: [email protected], telephone: +31204443053
Postal Address: Metabool Lab, PK 1X 009. De Boelelaan 1117. 1081HV. Amsterdam, The Netherlands.
72
Abstract
OBJECTIVE: Creatine transporter (SLC6A8) deficiency is the most common cause of cerebral creatine
deficiency syndromes, and is characterized by depletion of creatine in the brain. Manifestations of this
X-linked disorder include intellectual disability, speech/language impairment, behavior abnormalities,
and seizures. At the moment no effective treatment is available. The objective of this study was to
investigate the molecular pathophysiology of SLC6A8 deficiency.
METHODS: In order to investigate the molecular pathophysiology of this disorder we performed RNA
sequencing on fibroblasts derived from patients. The transcriptomes of fibroblast cells from eight
unrelated individuals with SLC6A8 deficiency and three wild type controls were sequenced. SLC6A8
mutations with different effects on the protein product resulted in different gene expression profiles.
RESULTS: Differential gene expression analysis followed by gene ontology term enrichment analysis
revealed that especially the expression of genes encoding components of the extracellular matrix and
cytoskeleton are changed in SLC6A8 deficiency, such as collagens, keratins, integrins, and cadherins.
INTERPRETATION: Our findings suggests an important novel role for creatine in the structural
development and maintenance of cells. It is likely that the (extracellular) structure of brain cells is also
impaired in SLC6A8 deficient patients, but further studies are necessary to confirm this and to reveal the
true functions of creatine in the brain.
73
Introduction
Inborn errors of creatine metabolism lead to cerebral creatine deficiency syndromes and are
characterized by low levels of creatine in the brain. Manifestations of these syndromes are intellectual
disability, speech and language impairment, behavior abnormalities, and often seizures 1,2. These
disorders are either autosomal recessive caused by defects in the genes encoding the creatine
biosynthesis enzymes guanidinoacetate methyltransferase (GAMT) 3 or arginine:glycine
amidinotransferase (AGAT) 4, or they are X-linked caused by defects in the creatine transporter SLC6A8 5.
Oral creatine supplementation is beneficial, especially when initiated at early age, for individuals with
GAMT or AGAT deficiency 4,6, however not effective for individuals with SLC6A8 deficiency 7. Currently
there is no appropriate treatment for SLC6A8 deficiency but the results of recent studies with
cyclocreatine treatment in creatine transporter deficient mice 8, or lipophilic creatine derivatives in
SLC6A8 deficient human fibroblasts 9, look promising.
Although the ensuing symptoms due to SLC6A8 deficiency in particular and creatine deficiency as a
whole, are similar across all patients, the severity of these symptoms does in fact differ between groups
of patients. Especially concerning the extent of intellectual disability as well as the presence/absence
and frequency of epileptic seizures. No concrete genotype-phenotype correlation has yet been
established, but there might be a correlation between residual transporter activity (and consequently
residual brain creatine) and severity of the symptoms10.
In order to develop successful treatment regimens for SLC6A8 deficiency it is crucial, in our opinion, to
unravel the pathophysiology (deregulated pathways) of SLC6A8 deficiency. The advent of novel
technologies, such as next-generation sequencing, now make it possible to elucidate the molecular
mechanisms behind the pathophysiology of disorders 11. To gain insight into the pathophysiology of
SLC6A8 deficiency, we performed RNA sequencing (RNA-seq) analysis on different fibroblast cultures
derived from patients or controls. RNA-seq is a very powerful tool for determining differences in
transcriptomes 12. The principle behind RNA-seq is to sequence the mRNA molecules derived from a
certain sample. This allows the determination of which transcripts and how many copies are present at a
certain time or condition. RNA-seq studies have been performed already to understand numerous
neurological disorders, such as autism 13, and schizophrenia 14. It can reveal for example which cellular
processes, molecular pathways, or transcriptional networks are disrupted or impaired in disorders.
We used fibroblasts as a model for this disorder, because it is easily accessible patient material.
Furthermore fibroblasts express SLC6A8, and are used to confirm/exclude the diagnosis SLC6A8
deficiency by so called creatine uptake assay 15. In addition, primary SLC6A8 deficient fibroblast are
being used to study the effect of novel missense variants in this gene by overexpression studies 15. We
investigated the transcriptomes of fibroblasts containing different mutations in SLC6A8 that had
different effects on the protein product. The first mutation: c.1631C>T; p.(Pro544Leu), a missense
mutation commonly found in different families, was associated with a 40% residual creatine uptake
activity compared to wild type (WT) 15. The second mutation: c.1222_1224delTTC; p.(Phe408del), a
common in frame deletion of three nucleotides resulting in the deletion of one amino acid, causes 0%
residual activity of the transporter compared to WT 15. To enable sound statistical analysis we took
74
biological variation into account; we assessed the transcriptomes of fibroblasts of three unrelated
individuals for each of these mutations, and compared it to three different WT fibroblasts. We also
included fibroblasts from two other individuals; one with a nonsense mutation and the other with a
mutation consisting of a large genomic deletion in which the entire SLC6A8 gene has been deleted as
well as a portion of the neighboring genes, which we herein refer to as SLC6A8 del1-13.
Material and Methods
Subjects, cell culturing, and RNA isolation
Eight anonymous male subjects diagnosed with SLC6A8 deficiency were included in this study. A short
summary of the most common clinical manifestations in mild (Pro544Leu) and severe (Phe408del)
SLC6A8 deficiency can be found in Supplemental Table S1. Fibroblasts taken from skin were cultured in
Gibco DMEM medium (Life technologies), supplemented with 10% FBS and 1% Pen/Strep, under
standard mammalian cell culture conditions. As control three (male) fibroblast cell lines with WT SLC6A8
were used. Cultures with 70% confluence were harvested by centrifugation (340 g ~ 1520 rpm), and cell
pellets were stored at –80°C. The study was approved by the ethics committee of the VUMC,
Amsterdam, The Netherlands.
RNA was isolated from the cell pellets with the SV Total RNA Isolation kit (Promega), according to
manufacturer’s guideline with a slight modification: the pellets were pre-treated with RNA BEE (Amsbio)
and chloroform, in order to remove genomic DNA. RNA quality was assessed with an Agilent Eukaryote
Total RNA Nano assay in a 2100 Bioanalyzer (Agilent). RNA integrity number (RIN) values between 9.50
and 10 were derived, which indicated intact RNA in the samples. Quantification and purity assessment
of the RNA samples were done on a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific), and
produced 260/280 nm ratios between 2.09–2.13 which indicated “pure” RNA.
Library preparation and next-generation sequencing
Low-throughput protocol of the TruSeq RNA Sample Preparation Kit (Illumina) was followed to generate
libraries suitable for sequencing, using 2 µg total RNA input per sample. Libraries were quantified with
an Agilent DNA 1000 assay in a 2100 Bioanalyzer. Twelve different libraries (including the eleven of this
study) were divided over two equimolar pools which contained six libraries each. Each pool was loaded
into four channels of a flow cell for cluster generation with a cBot system (illumina), and subsequent
sequencing by synthesis was performed with a paired-end 100 bp protocol on a HiSeq 2000 (Illumina).
Data analysis
The paired-end reads were aligned to the human reference genome (UCSC Genome Browser hg19) using
TopHat v2.0.6 16, which made use of Bowtie v0.12.7.0 17, SAMtools v0.1.18.0 18, and was run under
default settings (with phred33 quality scale). For each sample a BAM file was loaded into R (2.15.2) using
Rsamtools (1.10.2), the aligned reads were counted per known transcript (annotation from UCSC
Genome Browser hg19) using IRanges (1.16.4) and GenomicRanges (1.10.6). In total the aligned reads
were counted for 39,680 transcripts of 23,398 genes. The raw read counts per transcript, per sample (n
75
= 11), were used in edgeR v3.0.8 19 for differential expression assessment. In edgeR the read counts
were not adjusted for gene or transcript length since this is not recommended by the developers,
however for library size a correction factor was taken into account by the model. Only the transcripts
with at least > 1 reads in ≥ 3 samples were kept for analysis. Common dispersion was estimated with the
general linear models method in edgeR, and differential expression of transcripts were determined for
the following comparisons (contrasts): “Pro544Leu” vs. “WT”, “Phe408del” vs. “WT”, “Phe408del” vs.
“Pro544Leu”, “nonsense” vs. “WT”, and “SLC6A8 del1–13” vs. “WT”. False discovery rate (FDR) control
was used to correct for multiple testing 20, and FDR adjusted p-values < 0.05 were considered significant.
Genes, of which transcripts were found significantly differentially expressed in the edgeR analysis, were
further used for GO enrichment analysis using GOseq 21. Enrichment of GO terms was assessed in each
list of differential genes per contrast separately, taking length bias into account. The derived p-values
were corrected for multiple testing using FDR control, and adjusted p-values < 0.05 were considered
significant.
Quantitative reverse transcription PCR
For five genes (and one reference gene) qPCR assays were designed with ProbeFinder version 2.50 for
Human (Roche). In Supplemental Table S2 the primer sequences and the universal probe numbers are
given for each assay. The same RNA samples from the three patients with the Phe408del mutation and
from the three WT samples were used, cDNA was made from approximately 1 µg RNA (OmniscriptTM RT
kit - Qiagen). Each sample was assayed for each gene in triplicate on a light cycler type LC480 (Roche),
according to the manufacturer’s guidelines. A mean normalized expression (MNE) value was calculated
from the obtained Ct values with the Q-Gene module 22, as described previously 23. GAPDH was used as a
reference gene for normalization of input cDNA. For each target gene a mean Phe408del/WT ratio was
calculated which was log2 transformed. A Pearson product-moment correlation coefficient was
calculated in R v2.15.1, and a p-value < 0.05 was considered significant.
Results
RNA derived from different fibroblast cultures from individuals with SLC6A8 deficiency and controls was
assessed using RNA-seq analysis. A whole Illumina HiSeq2000 flow cell was run with 12 different RNAseq libraries, including all 11 libraries generated from the 11 samples of this study. Therefore 2/3 lane
was available per library. All together the run produced 2.5 × 10 9 100 bp long paired-end reads that
could be assigned to the 11 samples. On average 59% could be mapped to the human genome (hg19),
using TopHat with default settings. Further details about the reads are available in Supplementary Table
S3. The mapped reads for each known (hg19 USCS annotated) transcript were counted using Rsamtools,
to allow subsequent statistical analysis. In total these “raw counts” were determined for 39,680
transcripts, from 23,398 genes.
We used edgeR 19 to test which transcript levels were significantly different between groups (false
discovery rate; FDR < 0.05), and the number of differentially expressed transcripts for each group
comparison (contrast) are summarized in Table 1. The largest number of transcripts that showed
differential expression was found between mutation Phe408del and WT (n = 120), and the smallest
number was between Pro544Leu and WT (n = 29). Ten transcripts (from five genes) were significantly
76
differentially expressed in both Pro544Leu vs. WT and Phe408del vs. WT comparisons (Supplementary
Fig. 1). For each group comparison; all differentially expressed transcripts and their fold changes and pvalues are shown in Supplementary Table S4–8. In general, more significantly up- than downregulated
transcripts were identified in SLC6A8 deficient cells compared to WT. In total the expression of 132
genes was significantly altered in one or more comparisons. Twenty-six (19.6%) of these genes are
associated with brain and/or neurons (see Supplementary Table S4–8).
In our analysis, we identified significantly differentially expressed transcripts from almost all
chromosomes (except chromosome 22 and Y). From some regions, however, three or more genes were
differentially co-expressed suggesting a common regulatory mechanism within the locus. In the
Phe408del vs. WT comparison, we identified three of such loci, notably; 17q21.2 (KRT33A, KRT33B, and
KRT34), 17q25.3 (FSCN2, NPTX1, and TSPAN10), and 19p13.11–12 (C19orf44, CALR3, CHERP, EPS15L1,
CIB3, and HSH2D). In the nonsense mutation vs. WT comparison, the six aforementioned genes at
19p13.11–12 were also upregulated, and from the SLC6A8 del1–13 vs. WT contrast six genes at 15q25.2
(AGSK1, GOLGA6L9, LOC440297, LOC727849, UBE2Q2P2, and UBE2Q2P3) were upregulated. When
compared to WT, there was no locus with three or more differentially co-expressed genes in the cells
with 40% residual creatine transport (Pro544Leu), suggesting that the common regulatory mechanism(s)
at the identified loci could be linked to creatine transport. This is supported by the fact that in the
Phe408del vs. Pro544Leu contrast, four such regions were identified: 19p13.11-12, at which the same six
genes in the Phe408del vs. WT and the nonsense mutation vs. WT contrasts were upregulated, 17p13.1
(MYH1, MYH2, and MYH4 upregulated), 17q21.2 (KRT31, KRT33A, KRT33B, and KRT34 downregulated;
also identified in the Phe408del vs. WT comparison), and Xq28 (LOC100507404, F8, and BRCC3
upregulated).
To gain insight into the mechanisms involved in SLC6A8 deficiency, we performed gene ontology (GO)
enrichment analysis using GOseq 21. Assigning GO terms to gene products is an initiative of the GO
Consortium with the goal to develop a structured and unified way of gene annotation 24. These GO
terms were used in GOseq to identify terms significantly enriched in our lists of differentially expressed
genes. To distinguish the Pro544Leu missense mutation with residual activity from the Phe408del (a one
amino acid deletion) mutation with no activity we performed GOseq analysis for each of the Phe408del
vs. WT, Pro544Leu vs. WT, and Phe408del vs. Pro544Leu differentially expressed genes separately. After
Benjamini-Hochberg’s method to correct for multiple testing 20, we identified 18 GO terms significantly
enriched (FDR < 0.05) for the differentially expressed genes from the Phe408del vs. WT contrast (Figure
1A, Table 2), and two GO terms for the genes from the Phe408del vs. Pro544Leu contrast (Figure 1B,
Table 3). The genes differentially expressed due to the Pro544Leu vs. WT contrast, do not contain any
enriched GO terms. The differentially expressed genes from the nonsense mutation vs. WT and the
SLC6A8 del1–13 vs. WT contrasts did neither contain any enriched GO terms.
Intellectual disability (ID) is present in all SLC6A8 deficient patients diagnosed so far. Although more
than 450 genes have been implicated in ID, it has become clear over the years that these genes
converge onto common networks involving neurogenesis, neuronal differentiation/migration and a
much larger group involved in defects in synapse formation, maturation and plasticity 25,26. By searching
through SynaptomeDB 27, a database of synapse proteins with the differentially expressed transcripts
from our dataset, we identified one gene from the Pro544Leu vs. WT, four genes from the Phe408del vs.
Pro544Leu and eleven genes from the Phe408del vs. WT contrasts, that encode components of the
77
synaptome (Table 4). The SLC6A8 nonsense and del1–13 mutated transcriptomes contained respectively
two (EPB41L3 and NGEF) and one (AQP1), differentially expressed synapse genes compared to WT
fibroblasts.
We chose to validate the expression of five genes (ADAMST7, CDH10, COL4A5, COL5A3, and COL15A1)
that were found differentially expressed in the Phe408del vs. WT contrast, with quantitative reverse
transcription PCR (qPCR). Our aim was to assess whether we could get similar fold changes with this
alternative technique, and used Roche’s Universal ProbeLibrary System Technology. The same RNA
samples from all three patients with the Phe408del mutation and all three WT were used, and were
assayed in triplicate for each of the five genes. In Figure 2 the average (log2 transformed) fold changes
are given for both methods, and showed that they were in accordance with each other. Also a significant
Pearson correlation coefficient of 0.97 (p-value = 0.0063) was found between the two methods (Figure
2).
Discussion
In this study we have shown the effects on the transcriptome of SLC6A8 deficiency in fibroblast cell lines
derived from patients. To our knowledge this is the first transcriptomic study on human cerebral
creatine deficiency syndromes. We used RNA-seq analysis in our study, which has several advantages
above alternative methods, such as the large dynamic range, low or no background signals, and high
levels of reproducibility 12. Furthermore, we chose to investigate different types of SLC6A8 mutations
because they have different effects on the gene product and function. We investigated a mutation with
a milder effect on the protein (Pro544Leu; 40% residual transporter activity), with a more severe effect
(Phe408del; 0% residual activity), a nonsense mutation, and a large genomic deletion (del1–13) both
resulting in the absence of SLC6A8 accumulation. Although the expression of some genes is affected by
almost all SLC6A8 mutations (such as FTCD and ITIH5), it becomes clear that each mutation affects the
transcriptome in a unique way.
To account for biological variability, we used three biological replicates for both the Pro544Leu and
Phe408del mutations, as well as for the WT control. We did this by using fibroblasts from three
unrelated individuals for each mutation or control group. The results therefore derived from the
contrasts with these groups elucidate reliably what general effect each mutation has, on a
transcriptional level. We also included mutations of which no biological replication was available (i.e.,
the SLC6A8 nonsense and the del1–13 mutations). Many published RNA-seq studies do not contain any
biological replication in their experiments, which is not ideal, however can still be informative.
Furthermore we validated the expression of some genes with an alternative method; qPCR. Our study
showed a high and significant correlation between both methods.
The GO enrichment analysis revealed cellular processes and components which are possibly impaired in
SLC6A8 deficiency. The extracellular matrix was the most affected cellular component, as well as cellular
processes such as cell structure, junction assembly, and cell adhesion. Transcripts encoding components
of collagen (COL4A5, COL5A3, COL15A1), a fundamental part of the extracellular matrix 28, are all
upregulated compared to WT. Furthermore, transcripts encoding keratin (KRT8, KRT31, KRT33A,
KRT33B, KRT34) and cadherins (CDH2, CDH10, CDH13, CDH18, PCDH7, DCHS1) are differentially
expressed. Keratin is an important constituent of the cytoskeleton and is involved in the binding of cells
78
to the extracellular matrix 29, whereas cadherins also play a role in cell-matrix and cell-cell adhesion 30.
Many more transcripts associated with the extracellular matrix or cell structure are differentially
expressed such as integrins (ITGA4, ITGA8), myosins (MYH1, MYH2, MYH4), and many more (see Table
1–2). Moreover, the expression of transcripts associated with growth factors is affected by SLC6A8
deficiency, such as EPS15L1, GDF6, IGFBP2, and IGFBP7. This suggests again involvement of the
extracellular matrix, since the matrix is responsible for growth factor binding, activation, distribution,
and presentation to cells 31.
The extracellular matrix is of high importance in proper development and functioning of neurons 32–34,
and many components originally discovered in non-neuronal tissue are also present in neurons 32.
Especially structural support of neurons, elongation, migration, and conduction of communication
signals are critical properties provided by the extracellular matrix. Dysfunctional extracellular matrix
molecules have been linked to different neurodegenerative disorders 35, learning 36, and epilepsy 37. Our
results, from skin fibroblasts, showed many genes associated with the brain or with neurons (almost
20% of all the significantly differentially expressed genes). It is therefore likely that the extracellular
matrix would also be altered in SLC6A8 deficient neurons, however further research is needed to
confirm this. This could be established, for instance, with SLC6A8 deficient neuron cell cultures for
example derived from induced pluripotent stem cells 38,39, in creatine transporter deficient mouse
models 40, or with post mortem patient material. Restoration of the extracellular matrix could become a
novel therapeutic target for cerebral creatine deficiency syndromes.
In conclusion we present genome-wide expression data of SLC6A8 deficiency in human fibroblasts. We
identified significantly differentially expressed genes due to this disorder, of which many are involved in
cell structure and the extracellular matrix. Our results suggest an important role of creatine in
extracellular matrix maintenance. This lays a fundament for understanding the biological role of creatine
in cells, on which further neurological research for can be build.
Acknowledgment
We would like to thank Cees Oudejans, Marie van Dijk, Hari Thulluru, and Omar Michel (Clinical
Chemistry, VUmc Amsterdam) for the help with the RNA sequencing experiment. Furthermore we thank
Wessel van Wieringen (Epidemiology and Biostatistics, VUmc Amsterdam) for advice on the statistical
analyses.
Potential Conflicts of Interest
Nothing to report.
79
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Tables
Table 1. Number of differentially expressed transcripts with FDR < 0.05 in edgeR analysis. Total number of transcripts in analysis is 39,680
(and after filtering in edgeR 24,158).
Contrasta
Transcripts with FDR < Upregulated transcripts c
Downregulated
0.05b
transcriptsc
Pro544Leu (n = 3) vs. WT (n = 3)
29 (20)
23 (16)
6 (4)
Phe408del (n = 3) vs. WT (n = 3)
120 (69)
69 (44)
51 (25)
Phe408del (n = 3) vs. Pro544Leu (n = 3)
60 (42)
36 (25)
24 (17)
Nonsense (n = 1) vs. WT (n = 3)
44 (27)
25 (16)
19 (11)
SLC6A8 del1-13 (n = 1) vs. WT (n = 3)
34 (20)
22 (14)
12 (6)
aComparison between two groups. bNumber of differentially expressed transcripts, transcribed from number of genes given between
parentheses. cUp- or downregulated in first group of contrast compared to second group.
Table 2. Significantly enriched GO terms (Benjamini-Hochberg adjusted p-value < 0.05) in Phe408del vs. WT. Total genes in analysis: 23,347.
Total genes significant with edgeR: 69.
GO ID
P-value
Adjusted P
Description
Total1
Sig2
Gene symbol
GO:0005576
1.35 × 10-7
0.002232
extracellular region
2046
21
GO:0030246
4.89 × 10-7
0.002731
carbohydrate binding
402
10
GO:0005201
4.97 × 10-7
0.002731
78
6
GO:0005198
1.40 × 10-6
0.005778
599
11
GO:0007155
3.65 × 10-6
0.010252
extracellular matrix
structural constituent
structural molecule
activity
cell adhesion
925
14
GO:0022610
3.73 × 10-6
0.010252
biological adhesion
927
14
GO:0016337
7.70 × 10-6
0.018132
cell-cell adhesion
378
9
GO:0031012
1.05 × 10-5
0.022699
extracellular matrix
416
9
GO:0034332
1.37 × 10-5
0.022699
40
4
GO:0044421
1.38 × 10-5
0.022699
adherens
junction
organization
extracellular region
part
ABI3BP; ADA; ADAMTS7; CDH13; CHI3L1;
COL15A1; COL4A5; COL5A3; EMCN; F2R;
IGFBP2; ITIH5; LOXL3; LYNX1; MGP; NPTX2;
OLFM2; OLFML2B; RSPO4; SERPINB2;
SFRP1
ABI3BP; CALR3; CHI3L1; COL5A3; GALNT5;
GFPT2; LPHN2; NPTX2; RSPO4; SFRP1
CD4; CHI3L1; COL15A1; COL4A5; COL5A3;
MGP
CD4; CHI3L1; COL15A1; COL4A5; COL5A3;
KRT33A; KRT33B; KRT34; KRT8; MGP; RPL9
ABI3BP; ADA; CD4; CDH10; CDH13; CDH18;
CDH2; COL15A1; COL5A3; ITGA8; MGP;
PCDH7; SFRP1; SOX9
ABI3BP; ADA; CD4; CDH10; CDH13; CDH18;
CDH2; COL15A1; COL5A3; ITGA8; MGP;
PCDH7; SFRP1; SOX9
ADA; CDH10; CDH13; CDH18; CDH2; ITGA8;
MGP; PCDH7; SOX9
ABI3BP; ADAMTS7; CHI3L1; COL15A1;
COL4A5; COL5A3; MGP; OLFML2B; SFRP1
CDH10; CDH13; CDH18; CDH2
1066
13
GO:0009888
1.84 × 10-5
0.027616
tissue development
1152
14
GO:0045987
2.35 × 10-5
0.032313
18
3
GO:0005578
3.04 × 10-5
0.036559
352
8
GO:0034329
3.10 × 10-5
0.036559
positive regulation of
smooth
muscle
contraction
proteinaceous
extracellular matrix
cell junction assembly
166
6
ABI3BP; ADAMTS7; CHI3L1; COL15A1;
COL4A5; COL5A3; MGP; SFRP1
CDH10; CDH13; CDH18; CDH2; GJC1; SFRP1
GO:0032501
10-5
0.036559
multicellular
5277
33
ADA; ATF5; BDKRB2; CALR3; CAMK2A; CD4;
3.14 ×
ABI3BP; ADA; ADAMTS7; CDH13; CHI3L1;
COL15A1; COL4A5; COL5A3; IGFBP2;
LOXL3; MGP; SERPINB2; SFRP1
CHI3L1; COL5A3; F2R; GJC1; HAND2; ITGA8;
KRT34; LOXL3; MAMSTR; MEOX2; MGP;
NR4A3; SFRP1; SOX9
ADA; F2R; PTGS1
83
organismal process
GO:0003008
3.99 × 10-5
0.041131
system process
1587
16
GO:0009653
5.00 × 10-5
0.048503
anatomical structure
morphogenesis
1901
18
GO:0050850
5.30 × 10-5
0.04852
CDH13; CDH2; CHERP; CHI3L1; COL15A1;
COL4A5; COL5A3; F2R; FSCN2; FXYD1;
GJC1; HAND2; ITGA8; KRT34; LOXL3;
MAMSTR; MEOX2; MGP; NFATC2; NPTX1;
NPTX2; NR4A3; PTGS1; SERPINB2; SFRP1;
SGK1; SOX9
ADA; BDKRB2; CAMK2A; CDH2; F2R;
FSCN2; FXYD1; GJC1; ITGA8; MEOX2;
NPTX1; NPTX2; NR4A3; PTGS1; SGK1; SOX9
ADA; CDH13; CDH2; CHI3L1; COL15A1;
COL4A5; F2R; FSCN2; GJC1; HAND2; ITGA8;
LOXL3; MEOX2; MGP; NPTX1; NR4A3;
SFRP1; SOX9
ADA; CD4; CDH13
positive regulation of 24
3
calcium-mediated
signaling
1In total 17,414 genes of 23,347 could be assigned to one or more GO term. 267 of the 69 significant genes could be assigned to one or more
GO terms.
Table 3. Significantly enriched GO terms (Benjamini-Hochberg adjusted p-value < 0.05) in Phe408del vs. Pro544Leu. Total genes in analysis:
23,347. Total genes significant with edgeR: 42.
GO ID
GO:0005198
P-value
9.59 ×
10-7
Adjusted P
Description
Total1
Sig2
Gene symbol
0.015817
structural molecule 599
9
CD4; COL4A5; KRT31; KRT33A; KRT33B;
activity
KRT34; MYH2; MYH4; RPL9
GO:0005859
5.90 × 10-6
0.048637
muscle
myosin 18
3
MYH1; MYH2; MYH4
complex
1In total 17,414 genes of 23,347 could be assigned to one or more GO term. 239 of the 42 significant genes could be assigned to one or more GO
terms.
Table 4. Differentially expressed synapse-associated genes
Contrast
Pro544Leu vs. WT
Differentially
expressed
SynaptomeDB1
ATP2A1
Phe408del vs. WT
CAMK2A
transcript
found
in
Description
ATPase, Ca++ transporting, cardiac muscle, fast twitch 1
calcium/calmodulin-dependent protein kinase II alpha
cadherin 2, type 1, N-cadherin (neuronal)
CDH2
collagen, type V, alpha 3
COL5A3
PLEKHA6
pleckstrin homology domain containing, family A
member 6
ribosomal protein L9
RPL9
cadherin 10, type 2 (T2-cadherin)
CDH10
Ly6/neurotoxin 1
LYNX1
cadherin 13, H-cadherin (heart)
CDH13
neuronal pentraxin I
84
NPTX1
Neuronal Olfactomedin Related ER Localized Protein 2
OLFM2
epidermal growth factor receptor pathway substrate 15like 1
Phe408del
Pro544Leu
vs.
EPS15L1
RPL9
ribosomal protein L9
HAPLN1
hyaluronan and proteoglycan link protein 1
tuberous sclerosis 1
TSC1
cadherin 2, type 1, N-cadherin (neuronal)
CDH2
1An
integrated database to retrieve, compile, and annotate genes comprising the synaptome. These
genes encode components of the synapse including neurotransmitters and their receptors, adhesion/cytoskeletal proteins, scaffold proteins,
transporters, and others 27.
85
Figures
Figure 1. Significantly enriched GO terms in SLC6A8 deficiency. With GOseq analysis (A) 18 GO terms were
identified as significantly enriched (Benjamini-Hochberg adjusted p-value < 0.05) for Phe408del vs. WT, and (B) 2
GO terms for Phe408del vs. Pro544Leu. The GO term description is given with the –log10(adjusted P-value).
Between parentheses; first the number of genes assigned to the GO term that were significantly differentially
expressed in the Phe408del vs. WT or Phe408del vs. Pro544Leu contrast is given, and then the total number of
genes assigned to the GO term. Note –log10(0.05) = 1.30.
86
Figure 2. Comparison between gene expression values derived from RNA-seq and qPCR. (A)The expression of five
genes was assessed with qPCR and the log2 transformed fold changes (ratio Phe408del/WT) are given next the
RNA-seq derived values. (B) A significant Pearson correlation coefficient of 0.97 (p-value = 0.0063) was found
between the log2 fold change values derived with both methods.
87
Supplementary Table S1. Summary of clinical features of Pro544Leu and Phe408del mutations as a cause of SLC6A8 deficiency.
Number of
Phe408del
Pro544Leu
7
5
Intellectual disabilityb
(1) Degree unspecified
(1) Mild
(4) Severe
(1) Degree unspecified
(1) Mild
(3) Moderate
Speech delayb
(3) No speech (at 4-9.5 years)
(3) Single words (at 1-3.8 years)
(1) Sentences (at 20 years)
(1) No speech (at 2.4 years)
(1) Single words (at 5 years)
(3) Sentences (at 12.7-15.9 years)
Behavior abnormalities
All
All
Seizuresb
(4) No
(3) Yes
(4) No
(1) Severe
aNumber
patientsa
of patients known from literature. bNumber of patients between parentheses.
Supplementary Table S2. Primers and probes of the quantitative reverse transcription PCR assays.
Gene
COL15A1
(NM_001855)
ADAMTS7
(NM_014272)
COL5A3 (NM_015719)
CDH10 (NM_006727)
COL4A5 (NM_000495)
GAPDH (NM_002046)
Forward
ttccagcaacccacatca
Reverse
gttcagagcagccaaatgc
Probe
#16
Amplicon length
91 nt
tcctctatgatgtaagccaccag
caccagagtgtgtggcaga
#8
94 nt
gcagaatatccatctcggactc
tcaaaacctcttgaccgtga
agagcccacggtcaagact
agccacatcgctcagacac
ggctctccttttgctccttt
cgtgttgtctctttgggattg
catgaaaggcatggtactaaagc
gcccaatacgaccaaatcc
#67
#59
#8
#60
71 nt
86 nt
69 nt
66 nt
Supplementary Table S3. Number of RNA-seq derived (and mapped) reads.
Sample
WT rep. 1
WT rep. 2
WT rep. 3
Pro544Leu rep. 1
Pro544Leu rep. 2
Pro544Leu rep. 3
Phe408del rep. 1
Phe408del rep. 2
Phe408del rep. 3
Nonsense rep. 1
Del1–13 rep. 1
Total
Total # reads
215,415,784
272,175,472
165,117,286
197,528,518
260,597,912
232,520,430
271,099,194
265,099,500
219,378,006
234,958,444
170,502,478
2,504,393,024
Left reads (Phred33)
107,706,399
136,082,833
82,557,586
98,762,776
130,294,166
116,258,703
135,544,827
132,545,331
109,685,167
117,477,243
85,247,953
1,252,162,984
Right reads
(Phred33)
107,686,785
134,912,461
82,543,375
98,746,029
129,154,810
116,237,905
134,338,838
131,318,806
108,720,810
117,456,623
84,539,002
1,245,655,444
Mapped reads left
Mapped reads right
64,583,251 (59.96%)
81,578,128 (59.95%)
49,690,717 (60.19%)
57,716,577 (58.44%)
80,296,557 (61.63%)
69,313,165 (59.62%)
81,830,640 (60.37%)
76,919,746 (58.03%)
64,508,081 (58.81%)
71,606,626 (60.95%)
52,071,558 (61.08%)
750,115,046
(59.91%)
62,857,190 (58.37%)
77,234,037 (57.25%)
49,132,468 (59.52%)
57,159,720 (57.89%)
75,916,620 (58.78%)
68,219,732 (58.69%)
77,380,364 (57.60%)
72,382,448 (55.12%)
59,815,904 (55.02%)
70,631,271 (60.13%)
49,082,396 (58.06%)
719,812,150
(57.79%)
88
Supplementary Table S4. Differentially expressed transcripts with FDR < 0.05 in edgeR analysis for Pro544Leu vs. WT contrast. A total of 29
transcripts derived from 20 genes were significantly differential.
Gene / transcript name
Location
Description
logFC
PValue
FDR
COL15A1 NM_001855
9q22.33
collagen, type XV, alpha 1
3.725255
4.18E-13
1.01E-08
SLC26A3 NM_000111
7q31.1
solute carrier family 26, member 3
-10.1468
9.06E-12
1.09E-07
ERAP2 NM_001130140
5q15
-4.08825
3.21E-08
0.000214
ERAP2 NM_022350
5q15
-4.08278
3.54E-08
0.000214
FTCD NM_006657
21q22.3
7.491242
1.35E-07
0.000651
FTCD NM_206965
21q22.3
7.413364
2.22E-07
0.000895
F8 NM_000132
Xq28
-2.84042
8.87E-07
0.003063
GOLGA8B NR_027410
15q14
endoplasmic reticulum
aminopeptidase 2
endoplasmic reticulum
aminopeptidase 2
formiminotransferase
cyclodeaminase
formiminotransferase
cyclodeaminase
coagulation factor VIII,
procoagulant component
golgin A8 family, member B
2.198931
1.05E-06
0.003158
GOLGA8A NR_027409
15q14
golgin A8 family, member A
2.041493
1.85E-06
0.004972
GOLGA8B NM_001023567
15q14
golgin A8 family, member B
2.176148
2.15E-06
0.005197
SLC6A7a
5q32
9.840472
2.79E-06
0.005926
NM_014228
GOLGA8A NM_181077
15q14
solute carrier family 6
(neurotransmitter transporter, Lproline), member 7
golgin A8 family, member A
1.978223
3.04E-06
0.005926
LOC653075 NR_033933
15q13.2
golgin A8 family, member T
2.165785
3.24E-06
0.005926
ITIH5 NM_030569
10p14
2.739546
3.44E-06
0.005926
ITIH5 NM_032817
10p14
2.748934
3.68E-06
0.005926
SAMD14 NM_174920
17q21.33
2.840417
4.83E-06
0.007286
ITIH5 NM_001001851
10p14
2.706861
6.37E-06
0.008845
DCHS1 NM_003737
11p15.4
inter-alpha-trypsin inhibitor heavy
chain family, member 5
inter-alpha-trypsin inhibitor heavy
chain family, member 5
sterile alpha motif domain
containing 14
inter-alpha-trypsin inhibitor heavy
chain family, member 5
dachsous 1 (Drosophila)
2.302375
6.59E-06
0.008845
MALAT1 NR_002819
11q13.1
2.730747
1.32E-05
0.016778
SNORD36C NR_000016
9q34.2
metastasis associated lung
adenocarcinoma transcript 1 (nonprotein coding)
small nucleolar RNA, C/D box 36C
3.496889
1.91E-05
0.023082
LOC100507404 NR_039991
Xq28
TMLHE antisense RNA 1
-3.40604
2.06E-05
0.023726
SLC26A4 NM_000441
7q22.3
solute carrier family 26, member 4
-3.84293
2.21E-05
0.024216
LOXL4 NM_032211
10q24.2
lysyl oxidase-like 4
2.32251
2.83E-05
0.029697
HLA-DPA1 NM_033554_7
6p21.32
3.212388
4.26E-05
0.042927
2.947903
4.64E-05
0.044798
2.040369
5.50E-05
0.047526
2.932111
5.51E-05
0.047526
2.039148
5.51E-05
0.047526
2.091354
5.98E-05
0.049799
major histocompatibility complex,
class II, DP alpha 1
HLA-DPA1 NM_001242524_1
6p21.32
major histocompatibility complex,
class II, DP alpha 1
ATP2A1 NM_004320
16p11.2
ATPase, Ca++ transporting, cardiac
muscle, fast twitch 1
HLA-DPA1 NM_001242525_3
6p21.32
major histocompatibility complex,
class II, DP alpha 1
ATP2A1 NM_173201
16p11.2
ATPase, Ca++ transporting, cardiac
muscle, fast twitch 1
CHRFAM7A a NM_139320
15q13.2
CHRNA7 (cholinergic receptor,
nicotinic, alpha 7, exons 5-10) and
FAM7A (family with sequence
similarity 7A, exons A-E) fusion
aGene is associated with brain and/or neurons according to Entrez Gene or UniProtKB/Swiss-Prot.
89
Supplementary Table S5. Differentially expressed transcripts with FDR < 0.05 in edgeR analysis for Phe408del vs. WT contrast. A total of 120
transcripts derived from 69 genes were significantly differential.
Gene / transcript name
Location
Description
logFC
PValue
FDR
HSH2D NM_032855
19p13.12
8.026207
7.43E-15
1.79E-10
CALR3 NM_145046
19p13.11
hematopoietic SH2 domain
containing
calreticulin 3
10.19589
1.91E-13
2.31E-09
CIB3 NM_054113
19p13.12
11.21249
1.54E-10
1.24E-06
FSCN2 NM_001077182
17q25.3
6.59186
3.30E-10
1.63E-06
FSCN2 NM_012418
17q25.3
6.602358
3.38E-10
1.63E-06
COL15A1 NM_001855
9q22.33
3.056435
8.79E-10
3.54E-06
VAT1La
16q23.1
-6.92099
1.12E-09
3.86E-06
-5.61606
2.07E-09
6.25E-06
NM_020927
calcium and integrin binding family
member 3
fascin homolog 2, actin-bundling
protein, retinal
(Strongylocentrotus purpuratus)
fascin homolog 2, actin-bundling
protein, retinal
(Strongylocentrotus purpuratus)
collagen, type XV, alpha 1
EMCN NM_016242
4q24
vesicle amine transport protein 1
homolog (T. californica)-like
endomucin
EMCN NM_001159694
4q24
endomucin
-5.54346
3.91E-09
1.05E-05
C19orf44 NM_032207
19p13.11
3.117484
1.58E-08
3.81E-05
NPTX1a NM_002522
17q25.3
chromosome 19 open reading
frame 44
neuronal pentraxin I
5.052242
1.03E-07
0.000227
EPS15L1 NM_021235
19p13.11
2.423863
1.17E-07
0.000235
ITIH5 NM_030569
10p14
3.168574
1.35E-07
0.00025
ITIH5 NM_032817
10p14
3.172126
1.56E-07
0.000269
FTCD NM_006657
21q22.3
7.378567
1.81E-07
0.000291
FTCD NM_206965
21q22.3
7.300812
2.96E-07
0.000427
ITIH5 NM_001001851
10p14
3.12753
3.01E-07
0.000427
GFPT2 NM_005110
5q35.3
-2.20405
4.71E-07
0.000629
SLC6A7a NM_014228
5q32
10.95675
4.95E-07
0.000629
PLEKHA6 NM_014935
1q32.1
-5.4659
6.12E-07
0.000739
CHERP NM_006387
19p13.11
2.107395
9.72E-07
0.001118
LYNX1a NM_177477
8q24.3
epidermal growth factor receptor
pathway substrate 15-like 1
inter-alpha-trypsin inhibitor heavy
chain family, member 5
inter-alpha-trypsin inhibitor heavy
chain family, member 5
formiminotransferase
cyclodeaminase
formiminotransferase
cyclodeaminase
inter-alpha-trypsin inhibitor heavy
chain family, member 5
glutamine-fructose-6-phosphate
transaminase 2
solute carrier family 6
(neurotransmitter transporter, Lproline), member 7
pleckstrin homology domain
containing, family A member 6
calcium homeostasis endoplasmic
reticulum protein
Ly6/neurotoxin 1
1.833915
1.42E-06
0.001525
LYNX1a
NM_177476
8q24.3
Ly6/neurotoxin 1
1.833122
1.45E-06
0.001525
LYNX1a NM_177457
8q24.3
Ly6/neurotoxin 1
1.827548
1.65E-06
0.001663
CAMK2A a
NM_015981
5q32
5.493911
1.88E-06
0.001814
CAMK2A a NM_171825
5q32
5.44658
2.21E-06
0.002056
MAMSTR NM_001130915
19q13.33
4.163036
2.61E-06
0.002335
GPR133 NM_198827
12q24.33
calcium/calmodulin-dependent
protein kinase II alpha
calcium/calmodulin-dependent
protein kinase II alpha
MEF2 activating motif and SAP
domain containing transcriptional
regulator
G protein-coupled receptor 133
-3.04486
2.96E-06
0.002488
ITGA8a NM_003638
10p13
integrin, alpha 8
3.139641
2.99E-06
0.002488
NR4A3a
NM_173200
9q22.33
-2.59196
5.37E-06
0.004325
NR4A3a NM_006981
9q22.33
nuclear receptor subfamily 4,
group A, member 3
nuclear receptor subfamily 4,
group A, member 3
-2.60029
6.01E-06
0.004685
90
PTGS1 NM_000962
9q33.2
2.668366
6.46E-06
0.004751
2.66779
6.49E-06
0.004751
3.828101
7.26E-06
0.005156
1q32.2
prostaglandin-endoperoxide
synthase 1 (prostaglandin G/H
synthase and cyclooxygenase)
prostaglandin-endoperoxide
synthase 1 (prostaglandin G/H
synthase and cyclooxygenase)
MEF2 activating motif and SAP
domain containing transcriptional
regulator
synaptotagmin XIV
PTGS1 NM_080591
9q33.2
MAMSTR NM_182574
19q13.33
SYT14a NM_001256006
-4.6129
8.40E-06
0.005796
RSPO4 NM_001029871
20p13
R-spondin 4
5.80926
9.46E-06
0.006098
OLFM2a NM_058164
19p13.2
olfactomedin 2
2.736299
9.52E-06
0.006098
SYT14a NM_001146264
1q32.2
synaptotagmin XIV
-4.57899
9.59E-06
0.006098
RSPO4 NM_001040007
20p13
R-spondin 4
5.786784
1.03E-05
0.00614
SYT14a NM_153262
1q32.2
synaptotagmin XIV
-4.5614
1.03E-05
0.00614
IGFBP2 NM_000597
2q35
3.950754
1.04E-05
0.00614
SYT14a NM_001146261
1q32.2
insulin-like growth factor binding
protein 2, 36kDa
synaptotagmin XIV
-4.52322
1.13E-05
0.00649
CD4a NM_001195014
12p13.31
CD4 molecule
5.139769
1.19E-05
0.00656
SYT14a NM_001146262
1q32.2
synaptotagmin XIV
-4.50598
1.22E-05
0.00656
CD4a
NM_001195016
12p13.31
CD4 molecule
5.143751
1.26E-05
0.00656
CD4a NM_001195015
12p13.31
CD4 molecule
5.143824
1.26E-05
0.00656
CD4a NM_001195017
12p13.31
CD4 molecule
5.139715
1.28E-05
0.00656
MEIS2a
NM_170674
15q14
Meis homeobox 2
-1.4652
1.42E-05
0.007031
MEIS2a NM_170677
15q14
Meis homeobox 2
-1.46453
1.43E-05
0.007031
MEOX2a NM_005924
7p21.2
mesenchyme homeobox 2
5.444285
1.49E-05
0.007193
ADAMTS7 NM_014272
15q25.1
2.62909
1.56E-05
0.007388
CD4a NM_000616
12p13.31
ADAM metallopeptidase with
thrombospondin type 1 motif, 7
CD4 molecule
5.093365
1.59E-05
0.007388
MEIS2a
NM_170675
15q14
Meis homeobox 2
-1.46041
1.71E-05
0.007653
MEIS2a
NM_170676
15q14
Meis homeobox 2
-1.4611
1.71E-05
0.007653
CDH2a NM_001792
18q12.1
-2.81553
1.80E-05
0.007927
MEIS2a NM_172315
15q14
cadherin 2, type 1, N-cadherin
(neuronal)
Meis homeobox 2
-1.45909
1.88E-05
0.008046
MEIS2a
NM_172316
15q14
Meis homeobox 2
-1.45825
1.90E-05
0.008046
MEIS2a NM_002399
15q14
Meis homeobox 2
-1.45562
1.99E-05
0.008276
MEIS2a NM_001220482
15q14
Meis homeobox 2
-1.45139
2.08E-05
0.008528
ABI3BP NM_015429
3q12.2
2.316645
2.13E-05
0.00858
TSTD1 NM_001113205
1q23.3
-8.11015
2.57E-05
0.010161
SFRP1 NM_003012
8p11.21
ABI family, member 3 (NESH)
binding protein
thiosulfate sulfurtransferase
(rhodanese)-like domain
containing 1
secreted frizzled-related protein 1
-4.11451
2.74E-05
0.010681
TSTD1 NM_001113207
1q23.3
-7.96624
2.83E-05
0.010861
KRT33B NM_002279
17q21.2
thiosulfate sulfurtransferase
(rhodanese)-like domain
containing 1
keratin 33B
3.494378
3.48E-05
0.013149
ATF5a
19q13.33
activating transcription factor 5
-1.95509
3.65E-05
0.013577
COL5A3 NM_015719
19p13.2
collagen, type V, alpha 3
2.15156
3.95E-05
0.014457
TSTD1 NM_001113206
1q23.3
thiosulfate sulfurtransferase
(rhodanese)-like domain
containing 1
-7.70873
4.39E-05
0.015817
NM_001193646
91
RPL9 NM_000661
4p14
ribosomal protein L9
-2.19224
4.63E-05
0.016455
ATF5a NM_012068
19q13.33
activating transcription factor 5
-1.89412
4.72E-05
0.016518
SERPINB2 NM_002575
18q21.33
3.058806
4.80E-05
0.016581
SERPINB2 NM_001143818
18q21.33
3.0513
4.96E-05
0.016866
SGK1a NM_001143677
6q23.2
-1.66558
5.60E-05
0.018674
SGK1a NM_001143676
6q23.2
-1.66351
5.67E-05
0.018674
SGK1a NM_001143678
6q23.2
-1.66198
5.72E-05
0.018674
CHST15 NM_015892
10q26.13
3.312943
5.99E-05
0.019299
SGK1a NM_005627
6q23.2
-1.66094
6.19E-05
0.019668
ADA NM_000022
20q13.12
serpin peptidase inhibitor, clade B
(ovalbumin), member 2
serpin peptidase inhibitor, clade B
(ovalbumin), member 2
serum/glucocorticoid regulated
kinase 1
serum/glucocorticoid regulated
kinase 1
serum/glucocorticoid regulated
kinase 1
carbohydrate (Nacetylgalactosamine 4-sulfate 6-O)
sulfotransferase 15
serum/glucocorticoid regulated
kinase 1
adenosine deaminase
2.19904
6.32E-05
0.019818
CCDC85A NM_001080433
2p16.1
coiled-coil domain containing 85A
-2.91549
6.46E-05
0.020009
NFATC2 NM_001136021
20q13.2
3.869965
6.58E-05
0.020107
HSD17B14 NM_016246
19q13.33
2.570196
6.82E-05
0.02051
NFATC2 NM_173091
20q13.2
3.899034
6.88E-05
0.02051
NFATC2 NM_012340
20q13.2
3.864943
7.05E-05
0.020784
BDKRB2 NM_000623
14q32.2
nuclear factor of activated T-cells,
cytoplasmic, calcineurindependent 2
hydroxysteroid (17-beta)
dehydrogenase 14
nuclear factor of activated T-cells,
cytoplasmic, calcineurindependent 2
nuclear factor of activated T-cells,
cytoplasmic, calcineurindependent 2
bradykinin receptor B2
-2.60885
7.89E-05
0.02291
RPL9 NM_001024921
4p14
ribosomal protein L9
-2.05859
7.97E-05
0.02291
CDH10a NM_006727
5p14.1
cadherin 10, type 2 (T2-cadherin)
-5.16723
8.64E-05
0.024561
FXYD1 NM_005031
19q13.12
2.638583
8.89E-05
0.024984
HLA-DPA1 NM_001242524_1
6p21.32
2.813533
9.25E-05
0.025679
FXYD1 NM_021902
19q13.12
2.638846
0.000101
0.027683
HLA-DPA1 NM_001242525_3
6p21.32
2.802061
0.000107
0.028925
HLA-DPA1 NM_033554_7
6p21.32
3.002312
0.000113
0.030465
HAND2 NM_021973
4q34.1
-1.96448
0.000118
0.031371
KRT33A NM_004138
17q21.2
FXYD domain containing ion
transport regulator 1
major histocompatibility complex,
class II, DP alpha 1
FXYD domain containing ion
transport regulator 1
major histocompatibility complex,
class II, DP alpha 1
major histocompatibility complex,
class II, DP alpha 1
heart and neural crest derivatives
expressed 2
keratin 33A
3.524094
0.000124
0.032116
PCDH7a NM_032457
4p15.1
protocadherin 7
-3.82714
0.000125
0.032116
PCDH7a NM_001173523
4p15.1
protocadherin 7
-3.82798
0.000125
0.032116
TSPAN10 NM_031945
17q25.3
tetraspanin 10
3.326484
0.000157
0.040007
KRT8 NM_001256293
12q13.13
keratin 8
-3.99615
0.000162
0.040824
KRT34 NM_021013
17q21.2
keratin 34
2.951347
0.000166
0.041394
KRT8 NR_045962
12q13.13
keratin 8
-3.93027
0.00017
0.041882
PCDH7a NM_002589
4p15.1
protocadherin 7
-3.86163
0.000172
0.042002
SOX9 NM_000346
17q24.3
-2.99962
0.000185
0.044552
F2R NM_001992
5q13.3
SRY (sex determining region Y)-box
9
coagulation factor II (thrombin)
receptor
2.493061
0.000189
0.044552
92
NPTX2a NM_002523
7q22.1
neuronal pentraxin II
4.463869
0.000189
0.044552
LOXL3 NM_032603
2p13.1
lysyl oxidase-like 3
1.489323
0.000192
0.044552
CDH13a NM_001220490
16q23.3
cadherin 13, H-cadherin (heart)
1.512014
0.000195
0.044552
CDH18a
5p14.3
cadherin 18, type 2
-4.16604
0.000198
0.044552
CDH13a NM_001220489
NM_004934
16q23.3
cadherin 13, H-cadherin (heart)
1.50877
0.000198
0.044552
COL4A5 NM_000495
Xq22.3
collagen, type IV, alpha 5
3.351482
0.000199
0.044552
COL4A5 NM_033380
Xq22.3
collagen, type IV, alpha 5
3.351482
0.000199
0.044552
OLFML2B NM_015441
1q23.3
olfactomedin-like 2B
1.573713
0.000201
0.04459
CHI3L1 NM_001276
1q32.1
-3.78675
0.000205
0.044994
CDH18a NM_001167667
5p14.3
chitinase 3-like 1 (cartilage
glycoprotein-39)
cadherin 18, type 2
-4.15824
0.000209
0.045506
CDH13a NM_001257
16q23.3
cadherin 13, H-cadherin (heart)
1.500327
0.000213
0.045926
MGP NM_001190839
12p12.3
matrix Gla protein
-3.79014
0.000218
0.046606
CDH13a
16q23.3
cadherin 13, H-cadherin (heart)
1.497982
0.000225
0.047474
GALNT5 NM_014568
2q24.1
-1.24089
0.000226
0.047474
LPHN2 NM_012302
1p31.1
UDP-N-acetyl-alpha-Dgalactosamine:polypeptide Nacetylgalactosaminyltransferase 5
(GalNAc-T5)
latrophilin 2
1.343875
0.000232
0.048233
GJC1 NM_001080383
17q21.31
1.435947
0.000236
0.048728
GJC1 NM_005497
17q21.31
1.434209
0.000238
0.048728
MGP NM_000900
12p12.3
gap junction protein, gamma 1,
45kDa
gap junction protein, gamma 1,
45kDa
matrix Gla protein
-3.80473
0.000242
0.048969
PCDH7a
4p15.1
protocadherin 7
-3.75958
0.000243
0.048969
aGene
NM_001220488
NM_032456
is associated with brain and/or neurons according to Entrez Gene or UniProtKB/Swiss-Prot.
Supplementary Table S6. Differentially expressed transcripts with FDR < 0.05 in edgeR analysis for Phe408del vs. Pro544Leu contrast. A total
of 60 transcripts derived from 42 genes were significantly differential.
Gene / transcript name
Location
Description
logFC
PValue
FDR
CALR3 NM_145046
19p13.11
calreticulin 3
12.43883
2.65E-16
6.39E-12
HSH2D NM_032855
19p13.12
7.610412
5.88E-14
7.10E-10
CIB3 NM_054113
19p13.12
12.30106
1.93E-11
1.56E-07
SLC26A3 NM_000111
7q31.1
hematopoietic SH2 domain
containing
calcium and integrin binding family
member 3
solute carrier family 26, member 3
9.361527
1.10E-10
6.63E-07
FSCN2 NM_012418
17q25.3
6.415986
7.46E-10
3.06E-06
FSCN2 NM_001077182
17q25.3
6.39625
7.59E-10
3.06E-06
C11orf87 NM_207645
11q22.3
fascin homolog 2, actin-bundling
protein, retinal
(Strongylocentrotus purpuratus)
fascin homolog 2, actin-bundling
protein, retinal
(Strongylocentrotus purpuratus)
chromosome 11 open reading
frame 87
keratin 33B
-3.39455
1.36E-08
4.68E-05
KRT33B NM_002279
17q21.2
C19orf44 NM_032207
19p13.11
KRT33A NM_004138
17q21.2
EPS15L1 NM_021235
19p13.11
CD4a NM_001195014
12p13.31
5.132913
1.88E-08
5.67E-05
chromosome 19 open reading
frame 44
keratin 33A
3.041555
3.14E-08
8.42E-05
5.086512
2.74E-07
0.000662
epidermal growth factor receptor
pathway substrate 15-like 1
CD4 molecule
2.302651
4.26E-07
0.000884
6.174942
5.45E-07
0.000884
93
CD4a NM_001195016
12p13.31
CD4 molecule
6.205923
5.48E-07
0.000884
CD4a NM_001195015
12p13.31
CD4 molecule
6.205995
5.48E-07
0.000884
CD4a NM_001195017
12p13.31
CD4 molecule
6.199787
5.56E-07
0.000884
CD4a
12p13.31
CD4 molecule
6.22002
5.85E-07
0.000884
KRT34 NM_021013
NM_000616
17q21.2
keratin 34
4.104666
6.75E-07
0.000959
F8 NM_000132
Xq28
2.848212
8.35E-07
0.001121
TBX2 NM_005994
17q23.2
coagulation factor VIII,
procoagulant component
T-box 2
4.024194
1.24E-06
0.001574
VAT1La NM_020927
16q23.1
-5.01068
1.47E-06
0.001771
GDF6 NM_001001557
8q22.1
vesicle amine transport protein 1
homolog (T. californica)-like
growth differentiation factor 6
-3.50212
2.20E-06
0.002454
EMCN NM_016242
4q24
endomucin
-4.14756
2.24E-06
0.002454
SAMD14 NM_174920
17q21.33
-2.9259
2.69E-06
0.002829
ADAMTS7 NM_014272
15q25.1
2.853724
3.40E-06
0.003418
EMCN NM_001159694
4q24
sterile alpha motif domain
containing 14
ADAM metallopeptidase with
thrombospondin type 1 motif, 7
endomucin
-4.08362
3.57E-06
0.00345
CHERP NM_006387
19p13.11
1.976249
3.96E-06
0.003681
HAPLN1 NM_001884
5q14.3
-4.68349
9.16E-06
0.008196
MAMSTR NM_001130915
19q13.33
3.863409
9.71E-06
0.008381
LOC100507404 NR_039991
Xq28
calcium homeostasis endoplasmic
reticulum protein
hyaluronan and proteoglycan link
protein 1
MEF2 activating motif and SAP
domain containing transcriptional
regulator
TMLHE antisense RNA 1
3.461318
1.59E-05
0.013207
MAMSTR NM_182574
19q13.33
3.629046
1.77E-05
0.014233
AK8 NM_152572
9q34.13
MEF2 activating motif and SAP
domain containing transcriptional
regulator
adenylate kinase 8
-3.70045
1.87E-05
0.014554
GFPT2 NM_005110
5q35.3
-1.83738
2.11E-05
0.015966
MYH4 NM_017533
17p13.1
-5.76038
2.36E-05
0.017295
RPL9 NM_000661
4p14
glutamine-fructose-6-phosphate
transaminase 2
myosin, heavy chain 4, skeletal
muscle
ribosomal protein L9
-2.27753
2.46E-05
0.017356
CDH2a
NM_001792
18q12.1
-2.76042
2.51E-05
0.017356
KRT31 NM_002277
17q21.2
cadherin 2, type 1, N-cadherin
(neuronal)
keratin 31
4.300765
3.27E-05
0.021915
MYH1 NM_005963
17p13.1
-5.46032
3.98E-05
0.024982
COL4A5 NM_000495
Xq22.3
myosin, heavy chain 1, skeletal
muscle, adult
collagen, type IV, alpha 5
3.772011
4.03E-05
0.024982
COL4A5 NM_033380
Xq22.3
collagen, type IV, alpha 5
3.772011
4.03E-05
0.024982
RPL9 NM_001024921
4p14
ribosomal protein L9
-2.13395
4.52E-05
0.027308
ITGA4 NM_000885
2q31.3
-1.27713
5.11E-05
0.029384
MYH2 NM_017534
17p13.1
-4.6088
5.24E-05
0.029384
MYH2 NM_001100112
17p13.1
-4.60333
5.30E-05
0.029384
LDHC NM_002301
11p15.1
integrin, alpha 4 (antigen CD49D,
alpha 4 subunit of VLA-4 receptor)
myosin, heavy chain 2, skeletal
muscle, adult
myosin, heavy chain 2, skeletal
muscle, adult
lactate dehydrogenase C
1.577383
5.55E-05
0.029384
LDHC NM_017448
11p15.1
lactate dehydrogenase C
1.577383
5.58E-05
0.029384
NMNAT2a NM_170706
1q25.3
3.46054
5.78E-05
0.029384
NMNAT2a NM_015039
1q25.3
3.46054
5.81E-05
0.029384
TRIM55 NM_033058
8q13.1
nicotinamide nucleotide
adenylyltransferase 2
nicotinamide nucleotide
adenylyltransferase 2
tripartite motif containing 55
-4.80192
5.84E-05
0.029384
94
TRIM55 NM_184085
8q13.1
tripartite motif containing 55
-4.77795
6.11E-05
0.030132
TRIM55 NM_184086
8q13.1
tripartite motif containing 55
-4.75011
6.70E-05
0.032387
TSPAN10 NM_031945
17q25.3
tetraspanin 10
3.495612
8.12E-05
0.038001
BRCC3 NM_024332
Xq28
1.786308
8.18E-05
0.038001
ADA NM_000022
20q13.12
BRCA1/BRCA2-containing complex,
subunit 3
adenosine deaminase
2.159596
8.34E-05
0.038001
TSC1 NM_001162426
9q34.13
tuberous sclerosis 1
-2.16923
8.80E-05
0.038657
TSC1 NM_000368
9q34.13
tuberous sclerosis 1
-2.16904
8.80E-05
0.038657
TSC1 NM_001162427
9q34.13
tuberous sclerosis 1
-2.14949
9.47E-05
0.040467
SRD5A2 NM_000348
2p23.1
-6.29054
9.55E-05
0.040467
ITGA8a NM_003638
10p13
steroid-5-alpha-reductase, alpha
polypeptide 2 (3-oxo-5 alphasteroid delta 4-dehydrogenase
alpha 2)
integrin, alpha 8
2.539064
0.000107
0.044441
BRCC3 NM_001018055
Xq28
1.742028
0.00012
0.048532
1.742028
0.000121
0.048532
BRCA1/BRCA2-containing complex,
subunit 3
BRCC3 NM_001242640
Xq28
BRCA1/BRCA2-containing complex,
subunit 3
aGene is associated with brain and/or neurons according to Entrez Gene or UniProtKB/Swiss-Prot.
Supplementary Table S7. Differentially expressed transcripts with FDR < 0.05 in edgeR analysis for nonsense mutation vs. WT contrast. A
total of 44 transcripts derived from 27 genes were significantly differential.
Gene / transcript name
Location
Description
logFC
PValue
FDR
PPP1R12B NM_032104
1q32.1
4.72081
1.92E-20
4.64E-16
PPP1R12B NM_032103
1q32.1
4.614609
1.52E-19
1.83E-15
PPP1R12B NM_002481
1q32.1
4.436191
2.62E-19
2.11E-15
HSH2D NM_032855
19p13.12
8.604616
1.14E-14
6.91E-11
CALR3 NM_145046
19p13.11
protein phosphatase 1, regulatory
subunit 12B
protein phosphatase 1, regulatory
subunit 12B
protein phosphatase 1, regulatory
subunit 12B
hematopoietic SH2 domain
containing
calreticulin 3
10.77506
2.56E-13
1.24E-09
PPP1R12B NM_001197131
1q32.1
4.037987
3.19E-13
1.29E-09
CIB3 NM_054113
19p13.12
11.79294
1.87E-10
6.47E-07
KDM5B NM_006618
1q32.1
protein phosphatase 1, regulatory
subunit 12B
calcium and integrin binding
family member 3
lysine (K)-specific demethylase 5B
2.353923
2.71E-09
8.18E-06
C19orf44 NM_032207
19p13.11
3.632156
2.80E-08
7.51E-05
SLC6A8a NM_005629
Xq28
-3.2085
8.91E-08
0.000215
SLC6A8a NM_001142806
Xq28
-3.20142
1.27E-07
0.000259
SLC6A8a NM_001142805
Xq28
-3.20027
1.29E-07
0.000259
PTGIS NM_000961
20q13.13
-6.20105
2.39E-07
0.000445
EPS15L1 NM_021235
19p13.11
2.866496
2.76E-07
0.000476
EPB41L3 NM_012307
18p11.31
-2.74367
5.20E-07
0.000837
CHERP NM_006387
19p13.11
chromosome 19 open reading
frame 44
solute carrier family 6
(neurotransmitter transporter,
creatine), member 8
solute carrier family 6
(neurotransmitter transporter,
creatine), member 8
solute carrier family 6
(neurotransmitter transporter,
creatine), member 8
prostaglandin I2 (prostacyclin)
synthase
epidermal growth factor receptor
pathway substrate 15-like 1
erythrocyte membrane protein
band 4.1-like 3
calcium homeostasis endoplasmic
reticulum protein
2.543635
1.77E-06
0.002671
95
AGTR1 NM_031850
3q24
angiotensin II receptor, type 1
3.782574
2.16E-06
0.003074
HAND2 NM_021973
4q34.1
-4.34468
2.73E-06
0.00332
AGTR1 NM_004835
3q24
heart and neural crest derivatives
expressed 2
angiotensin II receptor, type 1
3.773031
2.74E-06
0.00332
AGTR1 NM_000685
3q24
angiotensin II receptor, type 1
3.772552
2.78E-06
0.00332
AGTR1 NM_032049
3q24
angiotensin II receptor, type 1
3.778037
2.89E-06
0.00332
AGTR1 NM_009585
3q24
angiotensin II receptor, type 1
3.762507
3.78E-06
0.004153
TSPAN18 NM_130783
11p11.2
tetraspanin 18
2.849367
4.31E-06
0.00453
NDNFa NM_024574
4q27
3.245657
6.85E-06
0.006893
HMCN1a NM_031935
1q25.3
neuron-derived neurotrophic
factor
hemicentin 1
2.560517
1.01E-05
0.009771
FTCD NM_006657
21q22.3
6.335793
1.44E-05
0.013072
IGFBP7 NM_001553
4q12
2.872799
1.51E-05
0.013072
IGFBP2 NM_000597
2q35
4.476771
1.52E-05
0.013072
IGFBP7 NM_001253835
4q12
2.848819
1.57E-05
0.013105
FTCD NM_206965
21q22.3
6.249194
2.20E-05
0.01773
CXCL12 NM_001033886
10q11.21
formiminotransferase
cyclodeaminase
insulin-like growth factor binding
protein 7
insulin-like growth factor binding
protein 2, 36kDa
insulin-like growth factor binding
protein 7
formiminotransferase
cyclodeaminase
chemokine (C-X-C motif) ligand 12
-2.98868
2.42E-05
0.018869
LAMA4 NM_001105206
6q21
laminin, alpha 4
-2.55132
2.89E-05
0.02061
LAMA4 NM_002290
6q21
laminin, alpha 4
-2.55117
2.89E-05
0.02061
LAMA4 NM_001105207
6q21
laminin, alpha 4
-2.55122
2.90E-05
0.02061
CXCL12 NM_000609
10q11.21
chemokine (C-X-C motif) ligand 12
-2.78343
3.93E-05
0.027125
ADH1C NM_000669
4q23
-6.80196
4.08E-05
0.027386
MOXD1 NM_015529
6q23.2
alcohol dehydrogenase 1C (class I),
gamma polypeptide
monooxygenase, DBH-like 1
-3.09077
4.82E-05
0.031498
CXCL12 NM_001178134
10q11.21
chemokine (C-X-C motif) ligand 12
-2.93299
5.06E-05
0.032089
NGEFa
2q37.1
-13.8292
5.27E-05
0.032089
-13.8018
5.31E-05
0.032089
2.809683
7.86E-05
0.046183
NM_001114090
NGEFa NM_019850
2q37.1
LOC255130 NR_034081
4q12
neuronal guanine nucleotide
exchange factor
neuronal guanine nucleotide
exchange factor
uncharacterized LOC255130
C10orf116 NM_006829
10q23.2
adipogenesis regulatory factor
-3.27284
8.03E-05
0.046183
CXCL12 NM_199168
10q11.21
chemokine (C-X-C motif) ligand 12
-2.90411
8.33E-05
0.046794
ADH1B NM_000668
4q23
-6.77443
8.59E-05
0.047164
alcohol dehydrogenase 1B (class I),
beta polypeptide
aGene is associated with brain and/or neurons according to Entrez Gene or UniProtKB/Swiss-Prot.
Supplementary Table S8. Differentially expressed transcripts with FDR < 0.05 in edgeR analysis for SLC6A8 del1–13 vs. WT contrast. A total of
34 transcripts derived from 20 genes were significantly differential.
Gene / transcript name
Location
Description
logFC
PValue
FDR
UBE2Q2P2 NR_004847
15q25.2
5.674606
1.80E-42
4.36E-38
UBE2Q2P3 NR_024474
15q25.2
5.688236
2.43E-41
2.94E-37
GOLGA6L9 NM_198181
15q25.2
ubiquitin-conjugating enzyme E2Q
family member 2 pseudogene 2
ubiquitin-conjugating enzyme E2Q
family member 2 pseudogene 3
golgin A6 family-like 9
5.610332
2.09E-37
1.68E-33
LOC727849 NR_033936
15q25.2
golgin A2 pseudogene
5.2362
2.92E-35
1.76E-31
LOC440297 NR_033579
15q25.2
chondroitin sulfate proteoglycan 4
pseudogene
5.472136
1.18E-34
5.70E-31
96
AGSK1 NR_026811
15q25.2
golgin subfamily A member 2-like
5.266418
1.76E-34
7.07E-31
BCAP31 NR_024450
Xq28
-6.17514
2.45E-23
7.44E-20
BCAP31 NM_001256447
Xq28
-6.17514
2.46E-23
7.44E-20
BCAP31 NM_001139441
Xq28
-6.17703
3.18E-23
8.12E-20
BCAP31 NM_005745
Xq28
-6.17079
3.40E-23
8.12E-20
BCAP31 NM_001139457
Xq28
-6.17664
3.70E-23
8.12E-20
SLC6A8a NM_005629
Xq28
-5.38797
5.38E-15
1.08E-11
SLC6A8a NM_001142805
Xq28
-5.37008
1.18E-14
2.15E-11
SLC6A8a NM_001142806
Xq28
-5.3615
1.25E-14
2.15E-11
CCDC85A NM_001080433
2p16.1
B-cell receptor-associated protein
31
B-cell receptor-associated protein
31
B-cell receptor-associated protein
31
B-cell receptor-associated protein
31
B-cell receptor-associated protein
31
solute carrier family 6
(neurotransmitter transporter,
creatine), member 8
solute carrier family 6
(neurotransmitter transporter,
creatine), member 8
solute carrier family 6
(neurotransmitter transporter,
creatine), member 8
coiled-coil domain containing 85A
-8.86819
6.30E-08
0.000101
THBS4 NM_003248
5q14.1
thrombospondin 4
4.712385
1.20E-07
0.000181
COL15A1 NM_001855
9q22.33
collagen, type XV, alpha 1
3.016657
3.47E-07
0.000493
ITIH5 NM_030569
10p14
3.255986
4.51E-06
0.006058
ITIH5 NM_032817
10p14
3.255208
5.17E-06
0.006579
ITIH5 NM_001001851
10p14
3.233844
7.60E-06
0.009186
FTCD NM_006657
21q22.3
6.493575
9.67E-06
0.011129
AQP1 NM_198098
7p14.3
inter-alpha-trypsin inhibitor heavy
chain family, member 5
inter-alpha-trypsin inhibitor heavy
chain family, member 5
inter-alpha-trypsin inhibitor heavy
chain family, member 5
formiminotransferase
cyclodeaminase
aquaporin 1 (Colton blood group)
3.765411
1.18E-05
0.012642
AQP1 NM_001185062
7p14.3
aquaporin 1 (Colton blood group)
3.729217
1.24E-05
0.012642
AQP1 NM_001185060
7p14.3
aquaporin 1 (Colton blood group)
3.73262
1.26E-05
0.012642
AQP1 NM_001185061
7p14.3
aquaporin 1 (Colton blood group)
3.719051
1.31E-05
0.012642
GBP3 NM_018284
1p22.2
guanylate binding protein 3
-2.45365
1.43E-05
0.012975
FTCD NM_206965
21q22.3
6.417781
1.45E-05
0.012975
ID3 NM_002167
1p36.12
-2.57998
3.65E-05
0.031495
C13orf15 NM_014059
13q14.11
formiminotransferase
cyclodeaminase
inhibitor of DNA binding 3,
dominant negative helix-loop-helix
protein
regulator of cell cycle
3.471337
4.22E-05
0.035179
TRIM55 NM_184086
8q13.1
tripartite motif containing 55
5.459388
5.51E-05
0.043195
TRIM55 NM_184085
8q13.1
tripartite motif containing 55
5.450672
5.54E-05
0.043195
TRIM55 NM_033058
8q13.1
tripartite motif containing 55
5.443594
5.76E-05
0.043506
RHD NM_016124
1p36.11
Rh blood group, D antigen
-7.13963
6.60E-05
0.048217
CMKLR1 NM_001142343
12q23.3
chemokine-like receptor 1
4.411888
6.79E-05
0.048217
aGene
is associated with brain and/or neurons according to Entrez Gene or UniProtKB/Swiss-Prot.
97
Supplementary Fig. 1. Venn diagrams of differentially expressed transcripts in SLC6A8 deficiency. Each of the SLC6A8 mutations
versus WT contrasts is compared with one another. Shown are the number of significantly differentially expressed transcripts
(FDR < 0.05) identified with edgeR analysis. The name of genes and transcripts that overlap between two contrasts are shown
below.
98
99
Chapter 7
Summary, concluding remarks and perspectives.
"No research will answer all queries that the future may raise. It is wiser to praise
the work for what it has accomplished and then to formulate the problems still to be
solved"
Theobald Smith
100
101
Summary and concluding remarks
Continuous loss of creatine/phosphocreatine in the form of creatine means there is a need for creatine
replacement either through biosynthesis or from the diet. The physiological relevance of creatine is
evident in individuals with a genetic defect of creatine synthesis – AGAT (OMIM: 602360) or GAMT
(OMIM: 601240) or a genetic defect of creatine transport – SLC6A8 (OMIM: 300036) characterized by
symptoms such as; intellectual disability, autistic behavior, speech/language and hypotonia delay. Dietary
creatine supplementation has proved useful in treating these patients, especially in the case of patients
with defects in AGAT and GAMT, and also heterozygous SLC6A8 female patients [1]. Being an
endogenously synthesized compound, it is not surprising that the few issues raised on the side effects of
creatine can be attributed to the purity of the supplemented creatine itself. Although patients will require
lifelong creatine supplementation in order to avoid a relapse of symptoms, consolation can be drawn from
the fact that near-complete remittance from (or prevention of onset of) symptoms seems to be possible.
It is evident that phenotypic outcome in response to treatment of AGAT and GAMT deficiency depends
on; timely diagnosis, early treatment onset and the duration of treatment (in this order), reason why we
cannot sufficiently emphasize the importance of early diagnosis. In this regard we have developed an
online database for both AGAT (www.lovd.nl/GATM) (Chapter 2) and GAMT (www.lovd.nl/GAMT)
(Chapter 3) deficiency, which provides information on diagnosis and functional analysis of nontruncating
variants. This initiative targets the provision of information to clinicians seeking confirmatory diagnosis
and therapeutic approaches in patients suspected of creatine deficiency. As such we recommend that the first step towards diagnosis of a suspected creatine deficiency patient should be metabolite testing and/or DNA sequencing of either the GATM, GAMT or SLC6A8 genes, followed by
online verification of any identified variants to check if they have been identified in previous patients and
whether or not they are pathogenic. In chapter 3 we described the functional analysis of missense variants
in GAMT, which is of pivotal importance for proper interpretation.
Evidence in the form of creatine synthesis deficient patients suggests that endogenous creatine synthesis
is quantitatively insufficient to fully make up for the CNS creatine requirement - be it as ATP provider or
neurotransmitter. The relevance of studies aimed at unravelling how the creatine transporter can be
stimulated and/or inhibited is highlighted by the fact that biosynthesis patients rely on creatine transport
to benefit from therapy, while stimulation of creatine synthesis (via arginine and glycine supplementation)
has shown some therapeutic potential in SLC6A8 deficient individuals. In fact there are studies that have
shown a repression of AGAT expression in response to elevated creatine levels [2–5]. We have provided
insight on two key areas (Chapter 4/5) that affect transcript and protein levels of the creatine transporter.
In Chapter 4, we show that overexpression of CTR1 splice variants is associated with an increase in
expression levels of the of the full-length CTR1 transporter. This highlights a potent avenue through which
the expression levels of the creatine transporter can be manipulated.
Differential transcriptional potential of the SLC6A8 promoter seen across cell types, suggests that
alternative transcription factor expression is one of the ways that SLC6A8 transcript levels between
different cell types are regulated (Chapter 5). It remains to be determined if genetic variants in this region
can lead to SLC6A8 deficiency as a result of downregulated mRNA expression. By identifying the previously
unknown promoter region of SLC6A8, we uncover a functional area that can also be screened for the
presence of variants that could potentially alter the expression/function of the SLC6A8 creatine
transporter. Surprisingly we found out that the SLC6A10P pseudogenes have a very high transcriptional
102
potential – another indication of their potentially significant, but yet unknown endogenous function(s)
(Chapter 5).
The most common symptoms of creatine synthesis or transport (ID, language delay and behavioral
disorders) indicate a disturbance of higher cortical function [6]. Some of these features are replicated in
the animal models of inborn errors of creatine metabolism [7–10] and they will be crucial in understanding
the pathophysiology of these diseases. Nonetheless, unravelling the molecular events associated with
creatine deficiency could provide important clarifications on its phenotypic outcome as well as potential
therapeutic targets. Global transcription analysis revealed that the major outcome of the energy
dysfunction due to creatine deficiency at the cellular level could be a disruption of the extracellular matrix
with the potential consequence of compromised cellular integrity (Chapter 6). This provides an exciting
avenue to explore the pathogenicity of creatine deficiency, whilst also providing a previously
unappreciated role of creatine.
Perspectives
Since all creatine deficiencies share a cluster of symptoms, it is safe to assume that the primary
determinant of their pathological consequence is creatine. And that the organs most affected will be those
without secondary mechanisms to maintain/replenish their phosphocreatine stores. A case in point is
that of SLC6A8 deficient patients, where it has been shown in vitro that in recombinant cells whereof the
mutated transporter was still capable of partial creatine transport there was a corresponding mild
phenotype in the individuals harboring these mutations [11]. Furthermore, muscle creatine levels are not
as severely depleted as brain creatine levels in patients with either GAMT or SLC6A8 deficiency [12,13].
This is all very encouraging as it highlights the therapeutic potential of replenishing depleted creatine
stores in these patients. Understanding how creatine transport is regulated could prove very useful for
biosynthesis deficient patients as they rely on a properly functioning creatine transporter for treatment
to be effective. In particular the function of the SLC6A10P pseudogenes should be explored, especially
with their potential implication in the autistic phenotype of a patient. In view of the open questions on
alternative creatine transport mechanisms in tissues less susceptible to creatine deficiency, as well as a
reported patient with a deletion in the promoter region of SLC6A10pA, we recommend functional studies
on the physiological relevance of these pseudogenes. Understanding the mechanisms through which
creatine transport can be regulated has the potential to impact many different areas of neuroscience
research and drug use in general, but particularly those patients that have been diagnosed with inborn
errors of creatine metabolism and also those patients suffering from neurodegenerative diseases who
currently benefit from creatine supplementation.
In summary it seems obvious that understanding the full scope of the physiological relevance of creatine,
creatine synthesis and creatine transport will shed more light on the pathophysiology of creatine
deficiency.
103
References
[1]
S. Mercimek-Mahmutoglu, S. Stöckler-Ipsiroglu, G.S. Salomons GS. Creatine Deficiency
Syndromes. 2009 Jan 15 [Updated 2011 Aug 18]. In: Pagon RA, Adam MP, Ardinger HH, et al.,
editors. GeneReviews® [Internet]. Seattle (WA): University of Washington, Seattle; 1993-2014.
Available from: http://www.ncbi.nlm.nih.gov/books/NBK3794/.
[2]
D.M. McGuire, M.D. Gross, J.F. Van Pilsum, H.C. Towle, Repression of rat kidney L-arginine:glycine
amidinotransferase synthesis by creatine at a pretranslational level., J. Biol. Chem. 259 (1984)
12034–8.
[3]
J.F. van Pilsum, D. M. McGuire, and C. A. Miller. "The antagonistic action of creatine and growth
hormone on the expression of the gene for rat kidney L-arginine: glycine
amidinotransferase." Guanidino compounds in biology and Medicine (1992): 147-151.
[4]
R. da Silva, I. Nissim, Creatine synthesis: hepatic metabolism of guanidinoacetate and creatine in
the rat in vitro and in vivo, … Metab. 9 (2009) 256–261.
[5]
P. Guthmiller, J. Van Pilsum, Cloning and sequencing of rat kidney L-arginine: glycine
amidinotransferase. Studies on the mechanism of regulation by growth hormone and creatine., J.
Biol. …. 269 (1994) 17556–17560.
[6]
V. Leuzzi, M. Mastrangelo, R. Battini, G. Cioni, Inborn errors of creatine metabolism and epilepsy.,
Epilepsia. 54 (2013) 217–27.
[7]
A. Schmidt, B. Marescau, E. a Boehm, W.K.J. Renema, R. Peco, A. Das, et al., Severely altered
guanidino compound levels, disturbed body weight homeostasis and impaired fertility in a mouse
model of guanidinoacetate N-methyltransferase (GAMT) deficiency., Hum. Mol. Genet. 13 (2004)
905–21.
[8]
M.R. Skelton, T.L. Schaefer, D.L. Graham, T.J. Degrauw, J.F. Clark, M.T. Williams, et al., Creatine
transporter (CrT; Slc6a8) knockout mice as a model of human CrT deficiency., PLoS One. 6 (2011)
e16187.
[9]
C.I. Nabuurs, C.U. Choe, a Veltien, H.E. Kan, L.J.C. van Loon, R.J.T. Rodenburg, et al., Disturbed
energy metabolism and muscular dystrophy caused by pure creatine deficiency are reversible by
creatine intake., J. Physiol. 591 (2013) 571–92.
[10]
C. Choe, C. Nabuurs, M.C. Stockebrand, A. Neu, P. Nunes, F. Morellini, et al., L-arginine:glycine
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O.T. Betsalel, A. Pop, E.H. Rosenberg, M. Fernandez-Ojeda, C. Jakobs, G.S. Salomons, Detection of
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[12]
R. Ensenauer, T. Thiel, K.O. Schwab, U. Tacke, S. Stöckler-Ipsiroglu, A. Schulze, et al.,
Guanidinoacetate methyltransferase deficiency: differences of creatine uptake in human brain
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G.J. Pyne-Geithman, T.J. deGrauw, K.M. Cecil, G. Chuck, M. a Lyons, Y. Ishida, et al., Presence of
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105
Appendices
Nederlandse samenvatting
Acknowledgements
Curriculum vitae
List of Publications
"Let me tell you the secret that has led me to my goal. My only strength lies in my tenacity"
Louis Pasteur
106
107
NEDERLANDSE SAMENVATTING
Creatine Deficiëntie Syndromen: Klinische,
Moleculaire en Functionele Aanpak.
108
NEDERLANDSE SAMENVATTING
Creatine deficiëntie syndromen (CDS) zijn aangeboren stofwisselingsziekten met als biochemisch
kenmerk, de afwezigheid van creatine/fosfocreatine in de hersenen. Klinische manifestaties worden
gekenmerkt door verstandelijke beperking, vertraagde spraak- en taalontwikkeling, autistisch gedrag en
epilepsie. Er wordt geschat dat de endogene creatine synthese (door arginine:glycine amidinotransferase
– AGAT en guanidinoacetaat methyltransferase – GAMT) voorziet in ongeveer de helft van onze dagelijkse
behoefte aan creatine. De andere helft wordt verkregen uit de voeding. De creatine transporter (CT1
wordt door het SLC6A8 gen gecodeerd) is verantwoordelijk voor de opname van creatine in de cellen.
Genetische afwijkingen in ofwel AGAT, GAMT of SLC6A8 veroorzaken CDS. Creatine suppletie heeft
opmerkelijke successen opgeleverd in de behandeling van individuen met een AGAT - of GAMT deficiëntie.
Creatine suppletie is tot nu toe niet effectief bewezen in de behandeling van patiënten met een SLC6A8
deficiëntie.
De afwezigheid van klinische verschijnselen bij pre-symptomatische individuen met een AGAT - of GAMT
deficiënte waarbij de behandeling vroeg in het leven werd gestart, illustreert het belang van vroegtijdige
diagnose en behandeling. Het is dan ook belangrijk om bekendheid met deze aandoening te vergroten.
Hiertoe hebben wij het klinische fenotype en de lange termijn behandelingsresultaten beschreven van
een patiënt aangedaan met AGAT deficiëntie (hoofdstuk 2), als ook een overzicht gemaakt van de tot dan
toe in de literatuur beschreven AGAT deficiënte patiënten. GAMT deficiëntie was al wel grondig
beschreven, hoewel studies naar missense mutaties in het GAMT gen nog ontbraken. Aangezien missense
mutaties meestal moeilijk te interpreteren zijn, hebben wij een functionele studie opgezet voor het
karakteriseren van dit type mutaties. We onderzochten de tot nu toe bekende missense mutaties,
inclusief nieuwe missense mutaties gevonden in 13 GAMT patiënten (hoofdstuk 3). Wij hebben ook een
online database ontwikkeld voor zowel AGAT (www.lovd.nl/GATM) (hoofdstuk 2) als GAMT
(www.lovd.nl/GAMT) (hoofdstuk 3) deficiëntie. Deze databases bevatten informatie over de
pathogeniciteit van de varianten en de functionele analyses van diverse varianten. Dit initiatief richt zich
vooral op de verstrekking van informatie aan diagnostici en clinici, die op zoek zijn naar de
bevestiging/uitsluiting van een diagnose.
De behandeling van AGAT en GAMT deficiëntie is afhankelijk van functioneel creatine transport. Door het
onderzoek naar de SLC6A8 genregulatie, kunnen deze behandelingsmethoden mogelijk worden
verbeterd. Daarom onderzochten wij de mogelijke aanwezigheid van alternatieve splice varianten van de
creatine transporter. Wij hebben twee splice varianten geïdentificeerd met een vergelijkbaar expressie
profiel als dat van het creatine transporter gen. Door middel van overexpressie experimenten is
onderzocht of deze splice varianten in staat zijn creatine transport te reguleren. Inderdaad bleek
overexpressie van deze varianten CT1 expressie positief te reguleren (hoofdstuk 4).
Bij patiënten met een (X-gebonden) verstandelijke beperking is de kans op een defect in de creatine
transporter aanwezig. Bij deze patiënten kan DNA analyse van het SLC6A8 gen worden ingezet. Echter, bij
deze screening is geen rekening gehouden met de nog onbekende promotor regio van het SLC6A8 gen.
Uit diverse whole genome sequencing projecten is gebleken dat ziekte veroorzakende varianten ook
gevonden kunnen worden binnen niet-coderende gebieden, waardoor wij de promotor van het SLC6A8
gen als ook dat van het SLC6A8 pseudogen op chromosoom 16 (SLC6A10P) graag wilde
identificeren(hoofdstuk 5). In tegenstelling tot SLC6A8, is de expressie van het SLC6A10P pseudogen alleen
gerapporteerd in de hersenen en de testis. Vervolgens hebben wij zowel de promotor regio van het
pseudogen als van SLC6A8 gekloneerd en onderzocht op het transcriptie potentiaal. Verassend genoeg
bleek ten opzichte van de SLC6A8 promotor het transcriptie potentiaal van de SLC6A8P promotor in
verschillende celtypen hoger te liggen dan dat van SLC6A8 (hoofdstuk 5).
109
NEDERLANDSE SAMENVATTING
Tot nu toe zijn pogingen voor de behandeling van patiënten met een SLC6A8 deficiëntie nog niet succesvol
gebleken. Door een beter inzicht te verkrijgen in de moleculaire pathofysiologie van creatine deficiëntie,
kunnen alternatieve behandelingsstrategieën worden ontwikkeld. Voor verdere verbetering van onze
kennis over de achtergrond van creatine deficiëntie pathways is er een differentieel genome–wide RNAexpressie experiment uitgevoerd in SLC6A8 deficiënte fibroblasten. In vergelijking met controle
fibroblasten, bleken in fibroblasten van verschillende patiënten diverse genen extracellulaire matrix
betroken. Ook genen die coderen voor sommige synaptische eiwitten bleken differentieel tot expressie
te komen in fibroblasten van patiënten (hoofdstuk 6).
110
ACKNOWLEDGEMENTS
Dear Supervisors, Colleagues, Friends and Family,
Several years of work have eventually led to some good publications and finally my thesis is ready. To
quote one of the members on my thesis review committee “…. I think that it is of very high quality. I
suggest that you proceed with the public defense of this thesis. Ndika’s work will contribute significantly
to our knowledge about the syndromes of Creatine Deficiency.”
It is tempting to be enveloped with a sense of smug satisfaction with myself, but I do not indulge, for I
realize that none of this would have been possible without your tremendous help and support all these
years. It is of course well documented how the right attitude, hard work, some luck and being at the right
place at the right time are the key ingredients to any kind of success, but I will want to add that for me it
has also been about being surrounded by the right people. In one way or the other, directly or indirectly,
you have all been extremely helpful in putting together the pieces which make up the final picture. I could
go on and on about how each of you have been an intricate part of the whole stuff, from its conception
to where it is today, but first I want to say that I am overwhelmingly grateful to those who provided the
pieces to the puzzle. These are my promoters; Gajja Salomons and Cornelis Jakobs, who first of all gave
me the opportunity to do my PhD in this lab. Karel you have the ability to be extremely critical and very
encouraging at the same time – the perfect combination for molding anyone into a top scientist. It was
easy for me to settle in when I just started because every now and then Karel will stop by to ask how
things (especially besides work) were going. You once told me work can take care of itself but family is
very important! It is an honor for me to have been your student.
At face view my relationship with Gajja seems to be about a decent amount of cancelled appointments (I
actually still have our monthly lunch meeting in my calendar), but what you don’t see is the numerous
hours behind separate computers and way into the night getting these manuscripts ready for submission
and resubmission. As a young researcher our minds are typically running wild with all these amazing
experiments we will like to do, and I can still hear Gajja’s voice saying something along the lines of “yes
you will conquer the world, but one day at a time – make a deadline and let’s send out the paper firstJ”.
Gajja you have always encouraged us to drop by your office anytime – even if it wasn’t about work
(jeeeej!). It has been all fun working in your group, and you are such an easy-going person. Thanks for the
all-round support, especially with de laatste loodjes. Those who have done it will agree with me that when
you leave most of your family behind and go abroad to work, the hospitality of your bosses and colleagues
become extremely crucial to your performance at work. It has been a bumpy (nonetheless merry) ride all
the way and they (Gajja and Karel) have been the catalyst of such a pleasant working atmosphere.
Cristina Martinez – my copromoter and weekly supervisor during my first year, was the hand in charge of
steadying the ship. In her research life Cris has been in the thick of it and still came out on top, and this is
the tenacity that she passed on to me. A moment I will like to recall was some 6 months into my PhD
training, when you told me that in the ideal world it will be awesome as a PhD student to have a senior
researcher in your same line of research to help you out along the way when you get stuck. But you were
going to only be around for another half year – although not ideal, the advantage will be that I will have
the opportunity to learn how to become an independent research scientist. I looked on the bright side
like you said, kept at it, and in my current position as research scientist I am glad to say I am reaping the
benefits you talked about. Words cannot sufficiently express my gratitude for your immense support and
guidance, but I must say that it was great working with you and to date I haven’t learnt more in a single
year than I did while working with you.
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ACKNOWLEDGEMENTS
It dawned on me just how much this lab meant to me when I had to leave. Being such an adventurous
person who has always been on the lookout for the next new experience, this came as a surprise to me.
I still remember my first day at the lab, when after my interview Cris gave me a tour of the lab. There was
an immediate connection, but at first I couldn’t put my finger on it. It must have been the enthusiasm and
openness of Cris when she talked about research at the Metabool lab, or that we were making a joke
about Silvy’s birthday in Gajja’s office or even that Ofir (who has always been my go-to guy from day 1)
had already been asking me if I liked my potential sitting spot, and what kind of configurations I will like
for my PC. I mean, all these with the “potential” PhD student. So much for the “foreigners-should-avoiddoing-their-first-internships-at-the-VUMC” speech we got from our tutors during the Masters at the
University. Obviously there may have been a couple of issues involving international students who had
been doing their internships at the VUMC, and the general feeling was that the VUMC had some issues
with integrating different cultures. But this was definitely not the case of the Metabool lab or the Clinical
Chemistry department for that matter. I had just seen some Romanians, Surinamese, Moroccans,
Peruvians, Vietnamese…and finally even Dutch people! J
Those of you who worked very closely with me may think my favorite moment in the lab, will have to be
either when we finally cloned the creatine transporter (took us about a year) and if not maybe when we
were first able to set up the optimized creatine uptake assay using the depletion method, or maybe when
we were finally able to clone and sequence the GC-rich CT1 promoter…or maybe it is when each of the
articles presented in this thesis got accepted….well, NO! My ultimate best memories from the lab, are all
the parties we had! At Karel’s boat, at the coffee corner (Christmas, New Year, Rien’s birthday…), at “The
Basket”, at Gajja’s place, at Silvy’s place, at my place, at “Coco’s outback” …. Etc. And I’ll tell you why –
with such familiarity at work, work became a way of life. Gone is the stress when things are not working,
gone is the “nine-to-five” mentality and you get tremendous support from colleagues when you need help
with experiments. This is the memory I will always have of this lab. When I talk about how I miss
Amsterdam my colleagues in Helsinki sometimes ask me if I have family back there….well yes – my brother
and the Metabool lab! What I missed doing my research, in terms of state of the art equipment was more
than sufficiently compensated for by amazing colleagues. We didn’t for example have a (?00000 €) cell
culture robot like the one they have at the CNCR, but I always had the girls – Ana, Matilde Fernandez,
Silvy and Warsha to look after my precious cells in the ML2 when I couldn’t … priceless! I am extremely
grateful for the tremendous help I got from Warsha Kanhai and Silvy van Dooren. We had already worked
together on a couple of projects, BUT, even when I left the lab and the reviewers asked for additional
experiments they stepped in to do them for me. Without you guys this will have been by no means be
over when it did.
After working at such a bustling lab for half a decade I now have a huge task of trying to remember all of
you who have been an integral part of my VUMC life. I especially have fond memories of;
-Former AIO’s; Martijn Kranendijk, Leonard van der Zwan, Daan van Abel, MARISKA Davids, Ofir
Betsalel, Hong Bui, Jiddeke van de Kamp, Saadet Mahmutoglu, the Portuguese crew; MONICA Rocha,
Ruben Esse, Marisa Mendes. It was fun to have worked with you guys. It seems like a lifetime ago that
we were all working together. I am happy that we are all moving forward with our careers. Special
thanks to Ofir – my software expert, movie dealer, manuscript figures editor …
-The metabolite guys; Henk Blom, Eduard Struys, Birthe Roos, Abdellatif Bakkali, Bram Grob,
Ulbe Holwerda, ERIC (dude) Wever, Willeke Klappe, Fariza El Aomari-Bel, Mirjam Wamelink, Desirée
Smith, Erwin Jansen…it was great working with you guys. Eric thanks a lot for always taking care of my PC
issues.
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ACKNOWLEDGEMENTS
On an entirely different note, we should definitely have a few more beers for the roadJ. Abdelatif we live
today to talk politics another day. Erwin please let me know when you set up the betting pool for the
upcoming FIFA world cup, the Dutch team is not looking in great shape this time around so I fancy my
chances of making some quick cash. My sincere gratitude to the GC-MS guys (Erwin and Ulbe) and the LCMS girl (Desire), you ran countless samples for me.
-The chemists; Herman ten Brink, Peter Bier, thanks for the “home-made” isotope-labelled creatine.
-My students; Vera Lusink, Claudine Beaubrun, Fay Visser, Nandaja Anand and Gaby Steba. It was a great
experience and a learning moment for both supervisor and student. You all did such great work on your
projects and I wish you guys all the best to come.
-The MBL lab guys; Marie van Dijk, Omar Michel, Daan, Hari Thulluru, we had such a great collaboration
– sharing lab space, lab equipment, enzymes and even cell lines. Thanks guys.
-The present AIO’s; Ana Pop, Hari, Arjan Malekzadeh (my other movie dealer) – we all get there! Wish
you guys all the best as you approach your final stages. Ana, you are just starting but I am very sure you
will quickly find out that it is not all different from what you have been doing all these years, well except
maybe more fun. -My other go-to ladies – Anneke Tekkelenburg, Karin Studer and Anne Zijtregtop,
thanks for all your great help with the never-ending administrative and immigration hassles.
-And finally at the end I got my postdoc – Ben Nota! “… we don’t do duplicates – they are useless for
statistics”. Oh, I forgot you did mention that there is another kind of statistics for duplicates and single
samples … It was great having you around and working with you. You came with the next generation flow,
and we learnt a great deal together and I learnt a lot from you – yes statisticsJ.
Well, all this talk about colleagues, but the real architects of the whole thing…way back when – my
parents, Henry Tanyi (in loving memory) and Beatrice Tanyi. Mum and Dad I can never thank you enough
for your ever-present and ongoing support, and above all your unwavering confidence in me.
And finally, from the bottom of my heart I want to thank the one person who kept all of this neatly bound
together; my rock, my loving wife Jane Besong-Ndika. For nine years you have always been there picking
up the pieces and offering the most support even though it was you who sacrificed the most. With you by
my side I feel I only need to want it to get it. Like Martijn said, being a father is the greatest title I will ever
get, and that is all thanks to you and our little jumpy boy Jaycie Ndika.
In a nutshell, for me this is no “happy ending”, only a cherished moment in time to be continued someday
somehow ….
I wish you all good health and lots of happiness. And for the AIO’s, 1000 publications!
Sincerely,
Joe.
113
CURRICULUM VITAE
Joseph Ndika was born in Bamenda, Cameroon on December 5 th 1983. From 2001 – 2004, he was an
undergraduate student at the University of Buea, where he obtained a BSc. degree, with honors, in
Biochemistry/Medical Laboratory Technology.
In the same year he took up a Biochemistry/Genetic Engineering postgraduate study program at the
Université de Yaoundé I. In 2006, he gave up this rather theoretical study program and moved to
Amsterdam to pursue a Masters degree in Molecular Cell Biology at Vrije University.
He obtained his MSc. in 2008, after two internships; first in the group of dr. Henk Schallig [Royal Tropical
Institute (KIT) – Parasitology] – where he was involved in the development of PCR-based rapid diagnostic
tests for Malaria, and subsequently in the group of prof. Sander Tans [FOM Institute for Atomic and
Molecular Physics (AMOLF) – Biophysics] – where he applied a directed evolution approach to elucidate
the mutational trajectories during the adaptive evolution of a novel regulatory function in a variable
environment, using the lac repressor as a model system.
In 2008 he was admitted as a graduate student of the Neuroscience Campus Amsterdam, to carry out his
PhD research at the VU, University Medical Center, in the group of prof. Gajja Salomons (Metabolic Lab –
DNA group) at the Department of Clinical Chemistry. The results of this work are described in this thesis.
Since 2013, Joe has been working as a Research Scientist at the Finnish Institute of Occupational Health,
in the group of prof. Harri Alenius (Systems Toxicology), where he employs high-throughput proteomics
and a systems biology approach to; 1 – study the health effects of engineered nanomaterials and, 2 –
characterize the genetic and/or environmental components of exposure-linked allergic reactions.
Joe is married to Jane Besong-Ndika and they live together with their son Jaycie in Helsinki.
114
LIST OF PUBLICATIONS
[1]
J.D.T. Ndika, K. Johnston, J. A Barkovich, M.D. Wirt, P. O’Neill, O.T. Betsalel, et al., Developmental
progress and creatine restoration upon long-term creatine supplementation of a patient with
arginine:glycine amidinotransferase deficiency., Mol. Genet. Metab. 106 (2012) 48–54
[2]
M. Davids, J.D.T. Ndika, G.S. Salomons, H.J. Blom, T. Teerlink, Promiscuous activity of
arginine:glycine amidinotransferase is responsible for the synthesis of the novel cardiovascular risk
factor homoarginine., FEBS Lett. 586 (2012) 3653–7
[3]
M.G.J. de Vos, F.J. Poelwijk, N. Battich, J.D.T. Ndika, S.J. Tans, Environmental dependence of
genetic constraint., PLoS Genet. 9 (2013) e1003580
[4]
J.D.T. Ndika, V. Lusink, C. Beaubrun, W. Kanhai, C. Martinez-Munoz, C. Jakobs, et al., Cloning and
characterization of the promoter regions from the parent and paralogous creatine transporter
genes., Gene. 533 (2014) 488–93.
[5]
J. Ndika*, S. Mercimek-Mahmutoglu*, W. Kanhai, T. Billette de Villemeur, D. Cheillan, E.
Christensen, et al., Thirteen new patients with guanidinoacetate methyltransferase deficiency and
functional characterization of nineteen novel missense variants in the GAMT gene, Hum. Mutat.
35 (2014) 462-469
[6]
J.D.T. Ndika, C. Martinez-Munoz, N. Anand, S.J.M. van Dooren, W. Kanhai, D.E.C. Smith, et al., Posttranscriptional regulation of the creatine transporter gene: Functional relevance of alternative
splicing., Biochim. Biophys. Acta. (2014)- General Subjects. 1840 (6) 2070-2079
[7]
Benjamin Nota, Joseph D. T. Ndika, Jiddeke M. van de Kamp, Warsha A. Kanhai, Silvy J. M. van
Dooren, Mark A. van de Wiel, Gerard Pals, and Gajja S. Salomons., RNA sequencing of creatine
transporter (SLC6A8) deficient fibroblasts reveals impairment of the extracellular matrix.
Submitted article.
*Contributed equally
116

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