Supplementary Full Methods
De novo mutations in sporadic cases of Childhood Onset Schizophrenia
This file includes:
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Clinical diagnosis
Exome sequencing
Likelihood analysis
Copy Number Variations (CNV)
Supplementary Figures (1 to 4)
Supplementary References
Supplementary Table 1
Supplementary Table 2
Clinical diagnosis:
Patients meeting DSM -IIIR/DSM-IV criteria for schizophrenia with onset of psychosis before age 13 were
recruited nationally. To address the concern of false positives resulting from inclusion of language
disorders, we included only patients with clear positive symptoms (delusions or hallucinations) in this
study. Medical or neurological disorders, or IQ under 70 were exclusionary criteria. Patients and their
available first-degree relatives were interviewed for lifetime and current psychiatric disorders using
structured psychiatric interviews and Autism Symptom Questionnaire 1,2. Diagnosis was confirmed with
inpatient medication-free observation. A total of 361 patients were screened and among that, only 20
patients were selected for the current study whose parents are not affected. This study was approved
by the Institutional Review Board of The National Institute of Mental Health. All participants provided
written assent/consent with written informed consent from a parent or legal guardian for minors.
Exome sequencing:
The genomic DNA of 60 samples (20 COS trios) was prepared using a classical mammalian DNA
purification method. Each trio is formed of a COS proband and his/her two parents in whom no
psychiatric disorders has been observed. The DNA of probands was extracted from blood and DNA of
parents was prepared from lymphoblastoid cell lines. The probands were not from a single ethnic group
and belonged to various groups (Hispanic, African American, Indian, and Arabic). We prepared samples
for exome sequencing in three different batches. The exome capture of every individual was performed
using Sure Select XT human all exome V4 kit (Agilent Technologies Inc.). The first batch of 42 samples
was captured and sequenced using an Illumina Hiseq2000 platform at the McGill Genome Centre,
Montreal, Canada. The last batch of 18 samples was captured using Sure Select XT2 and were sequenced
using the Illumina Hiseq2000 platform at the pharmacogenomics Centre of Montreal heart institute at
the Montreal Heart Institute (Montreal, Canada).
The processing and storage of data needs high computing facility to perform the analysis and the access
to such a requisite is possible through RQCHP-Calcul Quebec —a local supercomputing facility. The
sequenced reads from Illumina Hiseq2000 were mapped to reference genome using Burrow Wheeler’s
Algorithm3. The aligned reads were converted to binary format for the convenience of further analysis
using SAM tools4. The variant calling was performed using Genome Analysis Tool Kit5. This process
identified single nucleotide variants and small insertions or deletions at different levels of stringency
according to their quality scores. The variants identified were annotated with ANNOVAR tool to state
the position of genes and their chromosomes.
Likelihood analysis
A likelihood analysis involving PolyPhen-2 scores of all the variants from EVS and RVIS percentiles of all
genes was performed using a simulation. We wrote a small program to randomly select 20 variants from
EVS and repeated the selection for 1000 times. The mean PolyPhen-2 score of every selection of 20
variants was calculated. Next, we distributed all the mean scores from the simulation (Supplementary
Figure 2.a) and acquired a normal distribution with bins ranging from 0.1 to 0.9 (PolyPhen-2 scores). We
then inferred the probability of each bin of PolyPhen-2 score based on the frequency. In case of RVIS we
randomly selected 20 genes from the list of all genes reported in the RVIS percentile in the RVIS
percentile study6, applying the simulation program mentioned earlier. We followed the same method,
using the simulated mean RVIS percentiles, to infer their probability of occurrence (Supplementary
Figure 2. b). We calculated the P-value using the permutation test.
Copy Number Variations (CNV):
Our collaborator, Dr Judith Rapoport’s group has previously examined CNVs in 126 probands affected by
COS, which also includes the probands of this study7. Ahn et al showed the disease related CNVs that
were significantly different in COS compared to their controls. CNVs identified in the 17 probands of the
current study are not disease related except for one in proband COS885 (Supplementary Table 1). The
proband COS885 has two disease related CNVs, and one of these two (10q22.3) is significantly enriched
in cases by comparison to controls7. The other one, 1q21.3 was not significantly associated with COS,
although we observed a de novo missense variant (GPR153:NM_207370.1:c.217C>T) in the same
proband. The de novo mutation rate for 16 probands excluding this COS885 with CNV is 1.83x10-8 per
base pair.
Supplementary Figure 1. Capture efficiency of target regions from exome capture kits a) SureSelect XT
b) SureSelect XT2 Target efficiency is calculated as the percentage of reads mapped onto the target exon
regions out of the number of reads that are uniquely mapped to the whole genome reference.
Supplementary Figure 2. Mean PolyPhen-2 score for a random set of 15 mutations from EVS is selected.
This random selection was simulated for about 1000 times along with the mean score of de novo
missense mutation. Similarly mean RVIS percentile score of 20 mutations were selected in random from
the list of genes provided from a published study6. The simulation plots are shown in Supplementary
Figure 2. Random simulation of mutations from EVS and their distribution of prediction scores a)
PolyPhen-2 b) RVIS.
Supplementary Figure 3. Violin plot showing the distribution of GERP score for de novo and private
inherited variants in our study for prediction of conservation. There is no significant difference between
the groups.
Supplementary Figure 4. COS trio having the RYR2 variant with Sanger sequencing result showing two
variants in the same codon.
Supplementary References
1.
Kumra S, Frazier JA, Jacobsen LK et al: Childhood-onset schizophrenia. A double-blind clozapinehaloperidol comparison. Archives of general psychiatry 1996; 53: 1090-1097.
2.
Shaw P, Sporn A, Gogtay N et al: Childhood-onset schizophrenia: A double-blind, randomized
clozapine-olanzapine comparison. Archives of general psychiatry 2006; 63: 721-730.
3.
Li H, Durbin R: Fast and accurate long-read alignment with Burrows-Wheeler transform.
Bioinformatics 2010; 26: 589-595.
4.
Li H, Handsaker B, Wysoker A et al: The Sequence Alignment/Map format and SAMtools.
Bioinformatics 2009; 25: 2078-2079.
5.
McKenna A, Hanna M, Banks E et al: The Genome Analysis Toolkit: a MapReduce framework for
analyzing next-generation DNA sequencing data. Genome Res 2010; 20: 1297-1303.
6.
Petrovski S, Wang Q, Heinzen EL, Allen AS, Goldstein DB: Genic intolerance to functional
variation and the interpretation of personal genomes. PLoS genetics 2013; 9: e1003709.
7.
Ahn K, Gotay N, Andersen TM et al: High rate of disease-related copy number variations in
childhood onset schizophrenia. Molecular psychiatry 2014; 19: 568-572.
8.
Gilissen C, Hehir-Kwa JY, Thung DT et al: Genome sequencing identifies major causes of severe
intellectual disability. Nature 2014; 511: 344-347.
9.
Fromer M, Pocklington AJ, Kavanagh DH et al: De novo mutations in schizophrenia implicate
synaptic networks. Nature 2014; 506: 179-184.
10.
Gulsuner S, Walsh T, Watts AC et al: Spatial and temporal mapping of de novo mutations in
schizophrenia to a fetal prefrontal cortical network. Cell 2013; 154: 518-529.
11.
Neale BM, Kou Y, Liu L et al: Patterns and rates of exonic de novo mutations in autism spectrum
disorders. Nature 2012; 485: 242-245.
12.
Sanders SJ, Murtha MT, Gupta AR et al: De novo mutations revealed by whole-exome
sequencing are strongly associated with autism. Nature 2012; 485: 237-241.
Supplementary Table.1 Clinical information of the COS probands and their comorbidities as diagnosed by the NIMH
Age
of
onse
t
Gender
Race
Rare
CNV
COS451
COS483
COS630
COS691
COS755
COS885*
8
11
11
9.5
11
12
Male
Male
Female
Female
Male
Male
white
white
hispanic
hispanic
white
others
no
yes
no
yes
yes
yes
COS1012
8
Male
white
yes
COS1141
10
Male
white
no
COS1251
COS1553
COS1677
COS1785
10
12
10
10
Male
Female
Female
Female
white
white
white
African
American
yes
no
yes
no
COS1801
8
Male
others
no
COS1814
COS1855
COS1870
7
12
9
Male
Female
Male
white
others
white
no
no
yes
COS2720
13
Male
others
no
Proband ID
Chr
CNV
2q31.2-31.3
deletion
16q23.3
8p22, 10p11.23
1q21.3
10q22.3
Yq11.221
duplication
duplication
duplication
deletion
duplication
18q22.1
duplication
5p12.3
duplication
6q22.31
* Proband with disease related CNV Ahn et al 7.
deletion
Disease
related
CNV
Comorbidity1
Comorbidity2
Comorbidity3
yes
Generalized
Anxiety
Disorder
Generalized
Anxiety
Disorder
Asperger's Disorder
AttentionDeficit/Hyperactivity
Disorder
Pervasive Developmental
Disorder NOS
Asperger's Disorder
Asperger's Disorder
Obsessive-Compulsive
Disorder
Mathematics
Disorder
Separation Anxiety
Disorder
Expressive
Language Disorder
Asperger's
Disorder
Supplementary Table 2.
De novo mutation rates per exome in recent studies
Studies
Subjects
No of
cases
No of
mutations
Rate per
exome
% Target
covered
Depth of
coverage
Current study
COS
17
20
1.17
92
10X
Gillisen8 et al.
Intellectual
disability
79
84
1.68
94
40X
Fromer9 et al.
Schizophrenia
617
637
1.03
93
10X
Gulsuner10 et al.
Schizophrenia
105
103
0.98
93
10X
Neale11 et al.
Autism
175
167
0.95
90
10X
Sanders12 et al.
Autism
238
167
0.70
83
8X