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MATERIALS AND METHODS.
Discovery sample additional clinical characteristics.
The mean Positive and Negative Symptoms Scale (PANSS (1),) total score was 72 (ranging from
48 to 104, with a standard deviation of 14). The negative symptom subscale scores ranged from 9
to 26, with a mean and standard deviation of 19 and 4.3, respectively; the positive symptom scale
scores ranged from 9 to 28, with a mean and standard deviation of 16 and 4.5. All subjects had
stable clinical states and were on stable doses of atypical antipsychotic medications, except for
two who were on conventional antipsychotic medications. Six subjects were also on mood
stabilizers, four on antidepressants, and two on antiparkinson agents. All subjects were Caucasian
(three Hispanic) except for two African-Americans. While this is a small sample, the profile of
scores is typical of chronic schizophrenic patients in treatment with stable symptoms.
Corroborative sample clinical characteristics.
The mean global summary score for the Schedule for Assessment of Positive Symptoms
(SAPS(2)) score was 8.9 (with a standard deviation of 3.3) and the mean global summary score
for the Schedule for Assessment of Negative Symptoms (SANS(3)) score was 8.9 (with a
standard deviation of 4.42). The Global Assessment of Function Scale (GAF(4)) score was 53.9
(with a standard deviation of 13).
fMRI parameter additional details
The fMRI parameters were a 24 cm Field of View, 28 slices, 5 mm thick with no gap, interleaved,
axially oriented; TR = 3s, TE = 40 ms, 90 deg flip angle; 80 volumes collected in each scan.
Coordinates of the analyzed images were converted from MNI space to the space defined by
Talairach and Tournoux using a non-linear combination of two linear transformations
(http://imaging.mrccbu.cam.ac.uk/imaging/MniTalairach).
Genotyping methods: Discovery sample whole genome scan and quality control
The Discovery sample whole-genome scan was performed using the Illumina Infinium Human1
(with 109,365 gene-centered SNPs) and the HumanCNV370-Duo DNA Analysis BeadChip
(with the standard content featured on HumanHap300-Duo with an additional 52,167 markers
designed to target nearly 14,000 copy number variant regions of the genome, for a total of over
370,000 markers). The Discovery sample used the HumanCNV370 as a cost effective method to
genotype additional RSRC1 and ARHGAP18 SNPs. Approximately 750ng of genomic DNA was
used to genotype each subject of the discovery sample according to the Illumina Infinium 2 assay
manual. Each sample was whole-genome amplified, fragmented, precipitated and hybridized
overnight for a minimum of 16 hours at 48°C to allele-specific (Human1) or locus-specific
(Hap300) probes on the BeadArray. Non-specifically hybridized fragments were removed by
washing while remaining specifically hybridized DNA were processed for the single base
extension reaction, stained and imaged on an Illumina Bead Array Reader. Normalized bead
intensity data obtained for each sample were loaded into the Illumina Beadstudio 2.0 software
which generated SNP genotypes from fluorescent intensities using the manufacturer's default
cluster settings (Suppl Figure 1).
Statistical analysis of the Discovery sample were performed on Human I and supplemented by
ARHGAP18 and RSRC1 SNPs obtained from the HumanCNV370-Duo, for 105,950 autosomic
SNPs.
Samples successfully genotyped in less than 90% of markers on either array were
excluded from analysis. We removed 5,297 SNPs with missing genotypes > 10% across subjects
and 2,117 SNPs with MAF < 1% that partially overlap with the previous category. After removal
of SNPs that did not pass the quality control measures we had 98,648 SNPs to analyze, with a
mean call rate of 98.2%.
Corroborative sample whole genome scan and quality control
To verify the results of the Discovery sample, we focused only on the SNPs mapping the
RSRC1and ARHGAP18 genes, with 29 and 61 SNPs respectively. Corroborative samples were
assayed with the Illumina Infinium HumanHap300 BeadArrays and Taqman® SNP Genotyping
Assay (Applied Biosystems, Foster City, CA, U.S.A.) for those markers selected for confirmation
that were not present on the HumanHap300. The HumanHap300 BeadArray assayed 317,503
SNPs, derived from the Phase I HapMap and selected to tag haplotype blocks, with a mean call
rate of 99.7%. Quality controls criteria were the same applied to Discovery sample whole genome
scan.
Six RSRC1 SNPs and 1 ARHGAP18SNP were genotyped by Custom Taqman® SNP
Genotyping Assay (Applied Biosystems, Foster City, CA, U.S.A.). Amplification was performed
in 10 µl final volume with 20 ng of genomic DNA. SNP variation was assessed by means of the
allelic discrimination assay employing the Applied Biosystems Software Package SDS 2.1.
Genotyping was performed with the 5' nuclease assay technology for allelic discrimination using
fluorogenic Taqman® probes on a 7500 Fast Real Time system (Applied Biosystems Foster City
CA). Amplification was performed in 10 µl final volume with 20 ng of genomic DNA and the
following conditions: 95° C for 20 s, and 40 cycles each of 95°C for 3 s and 60° C for 30 s. SNP
variation was assessed by means of the allelic discrimination assay employing the Applied
Biosystems Software Package SDS 2.1. All genotyping ambiguities were manually resolved by
checking raw fluorescence data and in any case were tested twice.
In performing the same QA for the genotyping in the Corroborative sample, we removed 6,309
SNPs with missing genotypes > 10% across subjects and 231 with MAF < 1%. We removed 6
subjects with missing genotypes > 10% across SNPs. We controlled for departure from HardyWeinberg equilibrium in controls, with a p ≤ 0.001 for each SNP. After removal of SNPs that did
not pass the quality control measures we had 300,855 (autosomic) to analyze, with a mean call
rate of 98.3% indicating a very high rate of successful genotyping.
Supplemental Figure 1. Representative genotype clusters obtained using Illumina
BeadArrays.
We used intensity data from the Human1 and Hap300 bead arrays to establish sample genotypes
based on reference clusters obtained by Illumina BeadStudio 2.0. Plots are shown for DNA
samples genotyped at loci confirmed in the corroborative study. Dark points within shaded areas
indicate signal intensities corresponding to successful homozygous (AA: red, BB: blue) and
heterozygous (purple) genotype calls.
Supplementary Table 1 Legend. QT (quantitative trait) analysis on the discovery sample (left
half) and case-control association analysis on the Corroborative sample (right half) for SNPs
related to the RSRC1 gene in the Human 1 and HumanHap300 BeadArrays supplemented by
Taqman*.
For the QT Analysis (left half): Beta = slope of the regression analysis; SE = standard error of the
Beta; R² = total variance explained by the regression; T = regression statistical test value; P =
probability of the likelihood ratio test between the models with and without the SNP; Wald test =
Likelihood ratio; Empirical p-value = permutation-determined p value. #permut = number of
permutations to obtain that p value. (Analyses performed using PLINK.)
For the case-control analysis (right half) logistic regression analysis (GLM BINREG): MAF =
minor allele frequency; OR = odds ratio with standard error; Z = square root of Chi Square;
BINREG P values and 95% CI intervals for the OR.
Yellow highlights indicate statistical significance and red text indicates SNPs positive in both QT
and case-control analyses.
Supplementary Table 2 Legend. QT (quantitative trait) analysis on the discovery sample (left
half) and case-control association analysis on the Corroborative sample (right half) for SNPs
related to the ARHGAP18 gene in the Human 1 and HumanHap300 BeadArrays supplemented
by Taqman*. Table headings same as in Table 1.
Supplementary Figure 2. The LD matrix map for RCRC1 in cases (schizophrenia) from the
Corroborative case-control sample. Figures generated with Haploview 4.0 (5). Red squares
indicate high degree of LD (D’); white squares indicate low of absence of LD. Black triangles
represent LB blocks based on the 4 gamete rule algorithm. Comparison with Figure S3 suggests
a small different LD structure for cases and controls.
Supplementary Figure 3. The LD matrix map for RCRC1 in healthy controls from the
Corroborative case-control sample. Figures generated with Haploview 4.0 (5). Red squares
indicate high degree of LD (D’); white squares indicate low of absence of LD.
Supplementary Figure 4. The LD matrix map for ARHGAP18 in cases (schizophrenia) from the
Corroborative case-control sample. Figures generated with Haploview 4.0 (5). Red squares
indicate high degree of LD (D’); white squares indicate low of absence of LD. Black triangles
represent LB blocks based on the 4 gamete rule algorithm. Comparison with Figure S5 suggests
a difference in the LD structure for cases and controls.
Supplementary Figure 5. The LD matrix map for ARHGAP18 in healthy controls from the
Corroborative case-control sample. Figures generated with Haploview 4.0 (5). Red squares
indicate high degree of LD (D’); white squares indicate low of absence of LD.
References:
1.
Kay SR, Fiszbein A, Opler LA. The positive and negative syndrome scale
(PANSS) for schizophrenia. Schizophr Bull 1987; 13(2): 261-276.
2.
Andreasen NC. Scale for the Assessment of Positive Symptoms (SAPS).
University of Iowa: Iowa City, 1984.
3.
Andreasen NC. Modified Scale for the Assessment of Negative Symptoms
(SANS). University of Iowa: Iowa City, 1984.
4.
Schwartz RC. Concurrent validity of the Global Assessment of Functioning Scale
for clients with schizophrenia. Psychol Rep 2007 Apr; 100(2): 571-574.
5.
Barrett JC, Fry B, Maller J, Daly MJ. Haploview: analysis and visualization of
LD and haplotype maps. Bioinformatics 2005 Jan 15; 21(2): 263-265.