Supplement 1 - Biological Psychiatry

Franke et al.
Supplemental Information
Neuroimaging procedures
All scans covered the entire brain and had a voxel-size of 1 x 1 x 1 mm3. Automated
volumetry as implemented in the FIRST module (version 1.1) of FSL (version 4.1)
(http://www.fmrib.ox.ac.uk/fsl/first/) (1) was applied to segment brainstem (defined as
medulla, pons and midbrain, bordering the ventral diencephalon, the fourth ventricle and the
cerebellum) as well as bilateral hippocampus, amygdala, nucleus accumbens, caudate nucleus,
thalamus and globus pallidus. Volumes of bilateral structures were added-up for genetic
testing. Segmentation into gray matter and white matter was performed using the VBM5.1
toolbox in SPM5 using priors (default settings). This method uses an optimized voxel-based
morphometry (VBM) protocol (2) as well as a model based on Hidden Markov Random
Fields (HMRF) developed to increase signal-to-noise ratio. Total volumes of gray matter and
white matter were calculated by adding the resulting tissue probabilities. Traits were checked
for normal distribution of values before further analysis; no transformation was required.
Whole-brain VBM was performed in SPM5, separately for data acquired at 1.5T and
3T. Data were analyzed using the gray and white matter images in two separate analyses.
Diffeomorphic image registration was performed using DARTEL (3) followed by calculation
of Jacobian-scaled images and transformation to Montreal Neurological Institute (MNI) space
using affine transformation. Finally, all data were smoothed with an 8 mm FWHM Gaussian
smoothing kernel. A full-factorial ANCOVA was applied using genotype as factor and
participants' age, gender, total brain volume, and scan protocol were added to the model as
covariates. Statistics were corrected for non-stationarity and were applied at p(whole brain
uncorrected) <
1.0E-03 and subsequent cluster statistics at p(FWE) < 5.0E-02.
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Franke et al.
Table S1. Nominal association p-values at the CACNA1C locus for brain structures in 585 healthy individuals (18 to 36 years of age). SNPs were
included in the table if they showed association with at least one of the structures at p < 5.00E-02. Positions on the chromosome are according to
Genome Build 36.3. In light gray shading is the SNP associated with bipolar disorder (rs1006737), in bold SNPs showing nominal association
with a trait.
MAF
Position
on
chrom.
12 in bp
Position
in gene
Gray
matter
White
matter
Nucleus
accumbens
Amygdala
Caudate
nucleus
Globus
pallidus
Hippocampus
Thalamus
Brainstem
rs4765880
0.052
2010886
upstream
5.49E-01
8.69E-01
2.83E-01
2.87E-01
2.93E-02
2.63E-01
2.60E-02
4.35E-03
1.15E-01
SNP
rs-number
rs3794299
0.128
2156245
intron 3
4.87E-02
2.93E-01
1.90E-01
7.05E-01
8.99E-01
7.00E-03
8.89E-01
2.36E-01
4.69E-01
rs1006737
0.283
2215556
intron 3
3.56E-01
5.09E-02
5.71E-01
7.11E-01
7.82E-01
9.64E-01
2.33E-01
6.27E-01
3.10E-01
rs2283295
0.100
2294172
intron 3
5.43E-02
1.99E-02
9.53E-01
9.25E-02
3.42E-01
8.29E-02
6.45E-02
8.11E-02
1.24E-04
rs2238061
0.100
2296049
intron 3
5.15E-02
1.88E-02
9.59E-01
9.07E-02
3.29E-01
8.29E-02
6.87E-02
8.19E-02
1.22E-04
rs2239044
0.082
2300439
intron 3
2.44E-02
3.22E-02
6.98E-01
3.01E-01
6.62E-01
1.08E-01
2.71E-01
2.66E-01
4.05E-04
rs2283298
0.100
2305039
intron 3
5.43E-02
1.99E-02
9.53E-01
9.25E-02
3.42E-01
8.28E-02
6.45E-02
8.11E-02
1.24E-04
rs3819529
0.082
2306920
intron 3
2.40E-02
3.70E-02
9.32E-01
4.61E-01
9.33E-01
2.89E-01
2.97E-01
4.44E-01
5.76E-04
rs11062192
0.108
2310961
intron 3
2.68E-02
1.13E-02
7.50E-01
1.21E-01
4.97E-01
5.55E-02
1.75E-01
7.74E-02
6.79E-05
rs11062193
0.082
2310981
intron 3
3.90E-02
5.19E-02
9.27E-01
4.34E-01
9.81E-01
2.62E-01
3.38E-01
4.30E-01
7.18E-04
rs2051992
0.108
2311587
intron 3
2.40E-02
9.55E-03
8.70E-01
1.14E-01
4.70E-01
5.38E-02
1.45E-01
6.02E-02
5.71E-05
rs2238064
0.083
2312442
intron 3
3.53E-02
3.92E-02
9.66E-01
4.68E-01
9.28E-01
2.69E-01
3.97E-01
4.90E-01
6.45E-04
rs2239050
0.109
2317675
intron 3
1.75E-02
9.51E-03
7.18E-01
1.17E-01
5.33E-01
6.70E-02
1.33E-01
5.68E-02
5.91E-05
rs7959938
0.107
2320726
intron 3
1.92E-02
6.84E-03
9.96E-01
7.61E-02
3.56E-01
3.61E-02
9.76E-02
5.02E-02
3.62E-05
rs2024077
0.295
2320856
intron 3
1.39E-02
4.58E-01
9.93E-02
1.04E-02
1.13E-01
5.02E-03
4.57E-02
4.16E-03
2.09E-02
rs2239052
0.100
2323349
intron 3
5.43E-02
1.99E-02
9.53E-01
9.25E-02
3.42E-01
8.28E-02
6.45E-02
8.11E-02
1.24E-04
rs4765958
0.061
2534370
intron 10
7.12E-01
3.67E-01
7.86E-01
4.29E-03
6.19E-01
8.05E-01
5.99E-01
6.07E-01
2.19E-02
rs12369334
0.088
2649216
Intron 38
7.10E-01
7.66E-02
3.30E-01
9.89E-01
1.46E-03
9.60E-01
1.47E-01
2.97E-01
1.02E-01
SNP, single nucleotide polymorphism; MAF, minor allele frequency
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Franke et al.
rs2239050
rs1006737
rs2051992
rs7959938
Figure 1: Linkage Disequilibrium (LD) plot of the CACNA1C gene (and surrounding 20 kb) as derived from 585 data sets in the Brain Imaging
Genetics (BIG) study. The location of the three SNPs with strongest evidence of association with brainstem volume is shown (rs2051992,
rs2239050, rs7959938), as is the location of rs1006737, the SNP genome-wide significantly associated with bipolar disorder by others.
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Franke et al.
Figure S2. Sagittal (left) and coronal (right) images showing the results of the VBM analyses projected on a standard brain in MNI space.
Increased white matter voxel-based volume for rs7959938 A-carriers compared to C homozygotes was found in the 1.5T (red, p(FWE) = 0.001) and
3T data (blue, p(FWE) = 0.003). Strong overlap (pink) was found in the midbrain and pons.
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Franke et al.
1. Smith SM, Jenkinson M, Woolrich MW, Beckmann CF, Behrens TE, Johansen-Berg H, et
al. (2004): Advances in functional and structural MR image analysis and implementation
as FSL. Neuroimage 23 Suppl 1: S208-S219.
2. Ashburner J, Friston KJ (2000): Voxel-based morphometry--the methods. Neuroimage 11:
805-821.
3. Ashburner J (2007): A fast diffeomorphic image registration algorithm. Neuroimage 38:
95-113.
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