P. falciparum

Challenges and Ecstasy in Genomics with patient isolates:
A case study with complicated malaria.
Ashis Kumar Das. PhD
Department of Biological Sciences
BITS Pilani (Pilani) Rajasthan
Plasmodium Lifecycle
2 Section 3 of UGC Act, 1956
BITS Pilani, Deemed to be University under
Malaria
Causative Agents in humans :
Plasmodium falciparum, P. vivax, P. malariae,
P. ovale , P. knowlesi & P. cynomolgi
Transmission
Female Anopheles Mosquito
Symptoms
Classic Symptoms : Fever, Chills, Sweating, Headaches, Muscle pains
Severe manifestations :Cerebral malaria, Anemia, Kidney failure
Severity depends on various factors
BITS Pilani, Deemed to be University under Section 3 of UGC Act, 1956
Clinical spectrum of malaria: infection & progression
Alonso et al., 2011
BITS Pilani, Deemed to be University under Section 3 of UGC Act, 1956
Patients showing severe malaria complications
1. Patient with haemoglobinurea due to
P. falciparum Infection
2. Patient with renal failure due to
P. falciparum Infection
BITS Pilani, Deemed to be University under Section 3 of UGC Act, 1956
Challenges!!
A- Collection
•
Timing - from the time the patient reports to the OPD, is clinically diagnosed
and before drug administration, all ethical consent and collection has to be
completed _ NOT POSSIBLE TO PREDICT WHEN A PATIENT WILL REPORT
•
From the time of Blood collection to initial processing & freezing at -80˚ C max time 20
mins. ( For studies with RNA) DNA provides slightly more flexibility.
B- Cold Chain Transportation
Depending on nature of material cold chain at different temperatures have to be
maintained Extraction : Different for different material and QA/QC.
C. Experimentation : For Genomics if outsourcing has to be done or for that
matter in own lab be confident of the persons involved ( more a generic
statement but CRITICAL)
D. Analysis, perception, insights, comprehensive resource management.
E. MURPHYS LAW : IF ANYTHING CAN GO WRONG IT WILL – BE
PREPARED FOR ANY EVENTUALITY .
BITS Pilani, Deemed to be University under Section 3 of UGC Act, 1956
IF IT IS SO CHALLENGING - WHY BOTHER ???
• Information about variations in genomes of pathogens are often not available.
• Transcriptome information is significantly different when in-vivo material is used
compared to in -vitro experimentation ( viz culture based – NOT down playing the
importance for the same.)
• Using data from such experimentation in silico studies ( Systems networks)
provides a more realistic view of actual happenings in- vivo.
AND much more !!!
A better chance of reaching part of the data generated into different development
processes – finally, we hope :
Bedside ---- Bench ----- > Bedside
BITS Pilani, Deemed to be University under Section 3 of UGC Act, 1956
Work Flow
Blood sample Collection
Infected RBC
Separation
PBMCs
RNA, DNA
isolation
QC check of RNA
Microarray hybridization
Microarray data
Parasite microarray data analysis
Systems Biology network
generation and analysis
Host microarray data analysis
Systems Biology network
generation and analysis
BITS Pilani, Deemed to be University under Section 3 of UGC Act, 1956
Sample collection
• Blood samples collected from patients infected with malarial parasites on informed consent
• Routine diagnosis of all malaria patients was done by
• Peripheral Blood Film
• Rapid diagnostic tests
•OptiMal test (Diamed AG, Cressier sur Morat, Switzerland)
•FalciVax test (Zephyr Biomedical Systems, Goa, India)
• Polymerase Chain Reaction based on 18S/28S rRNA gene of Plasmodium. (Das et.al.,1995;
Kochar et. al., 2005, , Pakalapati et.al.2013.a,b)
• Laboratory investigations including
• Complete blood count, platelet count, bleeding and clotting time
• Blood glucose, blood urea, serum electrolytes, serum creatinine, serum bilirubin,
• Aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase
• Complete urine analysis
• Electrocardiogram, skiagram chest,
• Tests to rule out typhoid fever, hepatitis B & C, leptospirosis, dengue & HIV.
9
BITS Pilani, Deemed to be University under Section
3 of UGC Act, 1956
Diagnosis of P. vivax / P. falciparum
P. vivax
~1400bp
~500bp
1, 2, 3, 4 = P. vivax , Amplicon Size ~500bp
5 & 6 = P. falciparum , Amplicon Size ~1.4Kb
M = 1 Kb DNA Ladder Mix
(Das et.al. 1995, Kochar et.al.2005, Pakalapati et.al.2013.a,b)
7 & 8 = Pf + Pv
BITS Pilani, Deemed to be University under Section 3 of UGC Act, 1956
Natural AntisenseTranscripts
(NATs) from Clinical isolates of P.
falciparum
BITS Pilani, Deemed to be University under Section 3 of UGC Act, 1956
Prevalence of natural antisense transcripts (NATs )
in P. falciparum patient isolates
Transcripts detected by at least 3 probes were considered
PFC
PFU
Only antisense transcripts
34
35
Both sense and antisense transcripts
736
267
Total antisense transcripts
770
302
Total number of antisense transcripts detected- 797
Protien coding788
rRNA coding 7
Non protein coding- 2
Expression range- 1.5- 1465.01 (Sense transcripts)
1.5- 240.58 (Antisense transcripts)
BITS Pilani, Deemed to be University under Section 3 of UGC Act, 1956
Validation of antisense and sense transcript detection for selected genes using
strand specific RT-PCR
Uncomplicated pool
Complicated pool
BITS Pilani, Deemed to be University under Section 3 of UGC Act, 1956
Strand specific quantitative real-time PCR analysis: Antisense and sense transcript
levels of selected genes in uncomplicated and complicated cases
BITS Pilani, Deemed to be University under Section 3 of UGC Act, 1956
Expression status of sense and antisense transcripts for six genes
in array and quantitative real-time PCR data.
Gene
Expression status in microarray
data
Expression status in real time q
PCR data
Uncomplicated
Complicated
Uncomplicated Complicated
PF07_0009
HAS-LS
NC-AS/S
HAS-LS
NC-AS/S
MAL13P1.413
HS-LAS
HS-LAS
NC-AS/S
HS-LAS
PF11_0175
NC-AS/S
NC-AS/S
NC-AS/S
HS-LAS
PFA0620c
NC-AS/S
HS-LAS
NC-AS/S
NC-AS/S
PF10_0155
NC-AS/S
HS-LAS
HS-LAS
HS-LAS
PF14_0633
NC-AS/S
HAS-LS
HS-LAS
NC-AS/S
BITS Pilani, Deemed to be University under Section 3 of UGC Act, 1956
Expression pattern of S and AS transcript pairs in two clinical conditions
Uncomplicated
Complicated
Genes
NC-AS/S
NC-AS/S
58
HS-LAS
HS-LAS
102
HAS-LS
HAS-LS
2
NC-AS/S
HS-LAS
91
NC-AS/S
HAS-LS
1
HAS-LS
NC-AS/S
3
HS-LAS
HAS-LS
1
BITS Pilani, Deemed to be University under Section 3 of UGC Act, 1956
NATs from P. vivax patient isolates
BITS Pilani, Deemed to be University under Section 3 of UGC Act, 1956
Sense and antisense transcript pairs
Median Expression ratio of AS transcripts/Median Expression ratio of S
transcripts
Log2 transformed (Fold change expression difference between AS and S
transcripts from the same gene)
Log2 ≥ 1
HAS-LS transcript
Log2 ≤ 1
HS-LAS transcript
Log2 between +1 to -1
NC:S-AS transcript
BITS Pilani, Deemed to be University under Section 3 of UGC Act, 1956
Sense and antisense transcript pairs
BITS Pilani, Deemed to be University under Section 3 of UGC Act, 1956
Experimental validation of genes
with antisense transcripts using strand specific RT-PCR
Uncomplicated pool
Complicated pool
BITS Pilani, Deemed to be University under Section 3 of UGC Act, 1956
Comparison of genes with AS transcripts between PVU & PVC
PVU
PVC
Genes
Only AS
Only AS
56
HS-LAS
HS-LAS
220
HAS-LS
HAS-LS
30
NC: S-AS
NC: S-AS
245
Only AS
HAS-LS
1
Only AS
NC: S-AS
5
HAS-LS
Only AS
3
NC: S-AS
Only AS
8
HS-LAS
NC: S-AS
11
HAS-LS
NC: S-AS
30
NC: S-AS
HS-LAS
111
NC: S-AS
HAS-LS
31
BITS Pilani, Deemed to be University under Section 3 of UGC Act, 1956
AS/S transcript level measurement by specific q PCR in PVU and PVC and its
comparison between the conditions
BITS Pilani, Deemed to be University under Section 3 of UGC Act, 1956
Expression status of sense and antisense transcripts for six genes in array and
quantitative real-time PCR data
Gene
Array data
Quantitative real-time PCR
PVX_091425
HS-LAS in both PVU and PVC
NC:S-AS in both PVU and PVC
PVX_096080
HS-LAS in both PVU and PVC
HS-LAS in both PVU and PVC
PVX_003775
HAS-LS in both PVU and PVC
HAS-LS in both PVU and PVC
PVX_094790
HAS-LS in both PVU and PVC
HS-LAS in PVU to NC:S-AS in PVC
PVX_096365
NC:S-AS in PVU to HS-LAS in PVC
HS-LAS in PVU to decrease in sense in
PVC compared to the antisense level
in both
PVX_003920
NC:S-AS in PVU to HS-LAS in PVC
NC:S-AS in PVU to HS-LAS in PVC
BITS Pilani, Deemed to be University under Section 3 of UGC Act, 1956
Overlapping gene clusters
Overlapping gene pairs : extent of overlap ( 20-650 bp)
13 pairs of overlapping genes
Divergent gene pairs : 3
Convergent gene pairs : 10
BITS Pilani, Deemed to be University under Section 3 of UGC Act, 1956
Antisense expression by various mechanisms
Independent antisense-specific
promoter
Tandem
Non-overlapping
head to head gene pairs
Bidirectional promoter /Antisensespecific promoter
Non-overlapping
head to head gene pairs
Tandem
Transcriptional read through
Non-overlapping Tail to tail gene pairs
BITS Pilani, Deemed to be University under Section 3 of UGC Act, 1956
Functional & Pathway enrichment of genes with AS transcripts
BITS Pilani, Deemed to be University under Section 3 of UGC Act, 1956
Mapping of genes with AS transcripts in the Central Carbon
Metabolism
BITS Pilani, Deemed to be University under Section 3 of UGC Act, 1956
More fun with P. falciparum
BITS Pilani, Deemed to be University under Section 3 of UGC Act, 1956
Expression of var genes in P. falciparum clinical
isolates
Var families B & C upregulated
in J,J/RF cases. CD 36 transcript
levels (host) high in same.
Network connectivity of differentially regulated genes and functional
analysis
BITS Pilani, Deemed to be University under Section 3 of UGC Act, 1956
Connectivity between virulence proteins and exportome
proteins
BITS Pilani, Deemed to be University under Section 3 of UGC Act, 1956
Clusters showing highly connected hub genes
BITS Pilani, Deemed to be University under Section 3 of UGC Act, 1956
Acknowledgements
Isha Pandey, Ramandeep Kaur , Zarna Pala
Aatif Mehraj Kababi , Debjani Bhattacharya
All Patients who volunteered for the study.
Department of Biotechnology (DBT), New Delhi, India for the financial support through the grant
BT/PR7520/BRB/10/481/2006
All Scientists involved in data generation/deposition or curation in the Plasmo DB, NCBI and KEGG
Databases.
BITS Pilani, Deemed to be University under Section 3 of UGC Act, 1956
Conti……..
BITS Pilani, Deemed to be University under Section 3 of UGC Act, 1956
BITS Pilani, Deemed to be University under Section 3 of UGC Act, 1956

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