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Supplementary Information
Mutations affecting components of the SWI/SNF complex cause Coffin-Siris syndrome
Yoshinori Tsurusaki, Nobuhiko Okamoto, Hirofumi Ohashi, Tomoki Kosho, Yoko Imai, Yumiko Hibi-Ko, Tadashi Kaname, Kenji Naritomi, Hiroshi Kawame, Keiko Wakui, Yoshimitsu Fukushima, Tomomi Homma, Mitsuhiro Kato, Yoko Hiraki, Takanori Yamagata, Shoji Yano, Seiji Mizuno, Satoru Sakazume, Takuma Ishii, Toshiro Nagai, Masaaki Shiina, Kazuhiro Ogata, Tohru Ohta, Norio Niikawa, Satoko Miyatake, Ippei Okada, Takeshi Mizuguchi, Hiroshi Doi, Hirotomo Saitsu, Noriko Miyake, Naomichi Matsumoto
Correspondence should be addressed to N. Matsumoto ([email protected]) or N. Miyake ([email protected]) Supplementary Methods Subjects and clinical data All the patients were diagnosed by their attending clinical geneticist/dysmorphologist. DNA samples were isolated from peripheral blood leukocytes or lymphoblastoid cell lines using QuickGene-610 (Fuji Film, Tokyo, Japan). DNA samples derived from saliva were isolated using Oragene (DNA Genotek, Kanata, Canada). Informed consent, including for showing their facial appearance, was obtained from all the participants. This study was approved by the Institutional Review Board of Yokohama City University School of Medicine. Exome sequencing Exome sequencing was performed as previously reported1-3. Five typical CSS cases (subjects 1, 4, 5, 9 and 11) were analyzed by whole exome sequencing. Briefly, 3 µg of genomic DNA was processed using a SureSelect Human All Exon Kit v1 (Agilent Technologies, Santa Clara, CA) according to the manufacturer’s instructions. Captured samples were sequenced by an Illumina GAIIx (version 4) (Illumina, San Diego, CA) with 76 pair-end reads. Each sample was run in two lanes to obtain 6.8–10.9 Gb per sample.
1Nature Genetics: doi:10.1038/ng.2219
Image analysis and base calling were performed by sequence control software 2.6/real time analysis 1.6 (Illumina) and/or offline basecaller software v1.6.0 (Illumina). Alignment was performed by CASAVA software v1.6.0. The quality-controlled (path-filtered) reads were mapped to human genome reference hg19 with Mapping and Assembly with Qualities (MAQ, http://maq.sourceforge.net/) and NextGENe software v2.00 (SoftGenetics, State College, PA). From 71.2% to 82.4% (average 79.2%) of the coding sequence was covered by ten reads or more. The variants called by MAQ were annotated using SeattleSeq SNP annotation (http://snp.gs.washington.edu/SeattleSeqAnnotation131/). Priority scheme Variants were filtered by the following conditions: 1) variants only annotated on human autosomes and chromosome X; 2) variants not in dbSNP131 or the “1000 Genomes” database (http://www.1000genomes.org/); 3) variants called in common by NextGENe and MAQ; 4) variants that were non-synonymous changes or splice site mutations (±2 bp from the exon/intron boundary) and insertions/deletions with a NextGENe score ≥10; 5) variants not in our in-house database. The variant numbers in each category are shown in Table S2. Sanger sequencing The variant calls detected by MAQ and NextGENe software were confirmed by Sanger sequencing using an ABI3500xl or ABI3100 autosequencer (Life Technologies, Carlsbad, CA) following the manufacturer’s protocol. Sequencing data were analyzed by Sequencher software 4.10.1 (Gene Codes Corporation, Ann Arbor, MI). The PCR products were purified with ExoSap IT (GE Healthcare UK, Ltd., Little Chalfont, UK) and sequenced using a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Bedford, MA). Mutation screening All the coding sequence and exon/intron boundaries (± at least 10 bp) of the 16 SWI/SNF complex subunit genes were screened by high-resolution melting (HRM) analysis and Sanger sequencing. HRM was performed using a LightCycler 480 (Roche Diagnostics, Otsu, Japan) with the following program: preincubation [95°C for 10 min]; amplification by touchdown PCR [denaturation at 95°C for 10 s, annealing at 63–59°C (step-down, 0.5°C/cycle) for 25 s and extension at 72°C for 25 s, for 55 cycles]; HRM [denaturation at
2Nature Genetics: doi:10.1038/ng.2219
95°C for 1 min, cool-down at 4°C for 1 min, heating from 65–95°C (ramp rate, 0.01°C/sec) and final cool-down at 4°C]. The reaction was performed in 10 μl containing 10 ng of genomic DNA, 0.2 mM dNTPs, 0.125 U of ExTaq (Takara Bio, Inc., Otsu, Japan), 1× buffer and 1.5 μM SYTO9 (Invitrogen, Carlsbad, CA). A control screening was similarly performed by HRM. The primer sequences and PCR and HRM conditions are available on request. The detected variants were checked not to be observed in db132, the “1000 Genomes” database or the NHLBI GO exome sequencing project database. Copy number variation detection A GeneChip Human Mapping 250K Nsp Array (Affymetrix, Santa Clara, CA) was used to detect copy number changes for subjects 1 to 12 following the manufacturer’s instructions. The data were analyzed using Copy Number Analysis for GeneChip (Affymetrix) 4. A CytoScan HD array, a new platform by Affymetrix, was used to check the copy number changes for subjects 14, 18 and 19, in whom no point mutations in any of the 16 subunits of the SWI/SNF complex were found. The data were analyzed using Chromosome Analysis Suite Software (Affymetrix). Detection of nonsense-mediated decay Lymphoblastoid cell lines derived from the patients and their parents were subcultured at 37°C in a 5% CO2 incubator. After incubation with 1.5 μl of dimethyl sulfoxide or 1.5 μl of the protein synthesis inhibitor cycloheximide (100 mg/ml in dimethyl sulfoxide) (Sigma-Aldrich, St. Louis, MO) for 4 h, total RNA was extracted using an RNeasy Plus Mini Kit (QIAGEN, Hilden, Germany). One microgram of total RNA was used for reverse transcription using the Super Script III First-Strand Synthesis System for RT-PCR (Life Technologies). The PCR reaction contained 1ul of cDNA, 10× EX Taq Buffer, 2.5 mM each dNTP, 1.0 μM each primer and 5 units/μl EX Taq polymerase (Takara Bio). PCR products were electrophoresed on 1.0% agarose gels and stained with ethidium bromide. Sanger sequencing was performed on an ABI3500xl or ABI3130xl autosequencer (Life Technologies). Sequencing data were analyzed using Sequencher software v4.10.1 (Gene Codes Corporation). Quantitative SNP analysis was performed using an ABI Prism SNaPshot Multiplex Kit (Life Technologies) according to the manufacturer’s instructions.
3Nature Genetics: doi:10.1038/ng.2219
References 1. Doi, H. et al. Am J Hum Genet 89, 320-7 (2011). 2. Tsurusaki, Y. et al. Clin Genet 80, 161-6 (2011). 3. Tsurusaki, Y. et al. J Med Genet 48, 606-9 (2011). 4. Nannya, Y. et al. Cancer Res 65, 6071-9 (2005).
4Nature Genetics: doi:10.1038/ng.2219
SMARCB1
…DAEMEKKIRDQ……TRRMRRLANTA……DAEMEKKIRDQ……TRRMRRLANTA……DAEMEKKIRDQ……TRRMRRLANTA……DAEMEKKIRDQ……TRRMRRLANTA……DAEMEKKIRDQ……TRRMRRLANTA…
R.norvegicusG.gallusD.rerio
…DAEMEKKIRDQ……TRRMRRLANTA…
Subject 11
Exon 9c.1130G>A
p.Arg377His
Arg Arg LeuA G G C G T C T TNormal allele
Mutant allele A G G C A T C T TArg His Leu
…DAEMEKKIRDQ……TRRMRRLANTA…
…DAEMEKKIRDQ……TRRMRRLANTA…
1 9 8
Subject 4
Exon 8c.1091_1093delAGA
p.Lys364del
Lys Lys IleA A G A A G A T CNormal allele
Mutant allele A A G A T C C G CLys Ile Arg
Subject 21
Subject 22
Father
Mother
Father
Mother
N’ C’
1 385
SNF5178 373
p.Lys364del p.Arg377His
H.sapiensP.troglodytesC.lupusB.taurusM.musculus
p.K364 p.R377
D.melanogaster …DAEMEKKIRDQ……TRRMRRLANTT… A.gambiae …DAEMEKKIRDQ……TRRMRRLANTF… C.elegans …DAEIEKKMRDQ……TRRMRRLVGGG…
255
250
SNF5
Supplementary Figure 1 SMARCB1 mutations
One 3-bp deletion and one missense mutation in SMARCB1 were identified in four subjects. The upper panels
show the gene structure with nine exons and the evolutionary conservation of the mutated (or deleted) amino
acid through 11 different species (from Caenorhabditis elegans to Homo sapiens). The mutations were
confirmed as de novo in subjects 4 and 11. The middle and lower panels show electropherograms around the
mutations and the localization of the mutations. The changed nucleotides are highlighted in yellow boxes.
SMARCB1 contains two sucrose nonfermenting 5 (SNF5) domains. The domains (for this and the following
figures) were annotated by SMART (http://smart.embl-heidelberg.de/).5
Nature Genetics: doi:10.1038/ng.2219
Subject 5
His Arg MetC A C C G C A T GNormal allele
Mutant allele C A C T G C A T GHis Cys Met
Subject 7
Exon 18 c.2576C>T
p.Thr859Met
Normal allele
Mutant allele
TyrThr
A C G A T G T A C
ThrA C G A C G T A C
Thr Met Tyr
Subject 16
Exon 19c.2761C>T
p.Leu921Phe
Glu Leu TrpG A G C T C T G G
Normal allele
Mutant allele G A G T T C T G GGlu Phe Trp
Subject 17
Exon 25 c.3469C>G
p.Arg1157Gly
Thr Arg AlaA C C C G G G C T
Normal allele
Mutant allele A C C G G G G C TThr Gly Ala
Subject 25
His Met GlnC A C A T G C A GNormal allele
Mutant allele C A C A C G C A GHis Thr Gln
…LIDQKKDKRLA…NVLLTTYEYII…VDEGHRMKNHH…NKLPELWALLN…VLYRHMQAKGV…FLLSTRAGGLG… …LIDQKKDKRLA…NVLLTTYEYII…VDEGHRMKNHH…NKLPELWALLN…VLYRHMQAKGV…FLLSTRAGGLG…
H.sapiens
…LIDQKKDKRLA…NVLLTTYEYII…VDEGHRMKNHH…NKLPELWALLN…VLYRHMQAKGV…FLLSTRAGGLG… P.troglodytesC.lupusB.taurus …LIDQKKDKRLA…NVLLTTYEYII…VDEGHRMKNHH…NKLPELWALLN…VLYRHMQAKGV…FLLSTRAGGLG…
…LIDQKKDKRLA…NVLLTTYEYII…VDEGHRMKNHH…NKLPELWALLN…VLYRHMQAKGV…FLLSTRAGGLG… M.musculusR.norvegicusD.rerio
…LIDQKKDKRLA…NVLLTTYEYII…VDEGHRMKNHH…NKLPELWALLN…VLYRHMQAKGV…FLLSTRAGGLG… …LIDQKKDKRLA…NVLVTTYEYII…VDEGHRMKNHH…NKLPELWALLN…VLYRHMQAKGV…FLLSTRAGGLG…
A A G G A C A A G
Lys Lys AspA A G A A G G A CNormal allele
Mutant allele Lys Asp Lys
Subject 9
Father
Mother
Father
Mother
Father
Mother
Father
Mother
Father
Mother
SMARCA4 10 36 19 18 21 1 25
N’ C’
QLQ HSA BRK DEXDc HELICc BROMO170 206
1 1679
460 532 612 656 750 942 1110 1194 1487 1598
p.Lys546del p.Thr859Met p.Arg885Cys p.Leu921Phe p.Met1011Thr p.Arg1157Gly
Father
Mother
p.K546 p.T859 p.R885 p.L921 p.M1011 p.R1157
D.melanogasterC.elegans
…LIDQKKDKRLA…NVLLTTYEYVI…IDEGHRMKNHH…NKLPELWALLN…VLYKHMQSKGV…FLLSTRAGGLG… …LLDEKKDQRLV…NVLMTTYEYVI…IDEGHRLKNHN…NKLPELWALLN…VIYRHMQ-KGL…FMLSTRAGGLG…
Supplementary Figure 2 SMARCA4 mutations
One 3-bp deletion and five missense mutations in SMARCA4 were found in six subjects. All the mutated (or deleted)
amino acids are evolutionarily conserved. All the mutations occurred de novo. SMARCA4 contains a conserved Gln,
Leu, Gln (QLQ) motif, a helicase/SANT-associated (HSA) domain, a Brahma and Kismet (BRK) domain, DEAD-like
helicases superfamily (DEXDc) and helicase superfamily c-terminal (HELICc) domains and a bromodomain (BROMO).
Exon 19 c.2653C>T
p.Arg885Cys
Exon 21 c.3032T>C
p.Met1011Thr
Exon 101636_1638delAAG
p.Lys546del
6Nature Genetics: doi:10.1038/ng.2219
SMARCE1 1
H.sapiensP.troglodytesC.lupusB.taurusM.musculusR.norvegicusG.gallusD.rerio
…KPLMPYMRYSR……KPLMPYMRYSR……KPLMPYMRYSR……KPLMPYMRYSR……KPLMPYMRYSR……KPLMPYMRYSR……KPLMPYMRYSR……KPLMPYMRYSR…
10 5
N’ C’
HMG
411
65 135
1
p.Tyr73Cys
C C C T G C A T G
Pro TyrC C C T A C A T G
MetNormal allele
Mutant allelePro Cys Met
Subject 24
Father
Mother
Exon 5
p.Tyr73Cysc.218A>G
p.Y73
D.melanogasterA.gambiaeC.elegans
…KPILPYMRYSK……KPLMPYMRYSR……RPLQPYMRYSR…
Supplementary Figure 3 SMARCE1 mutation
A de novo missense mutation was identified in subject 24. The amino acid residue p.Y73 is evolutionarily
conserved through 11 different species and is located within a high-mobility group (HMG) domain.
7Nature Genetics: doi:10.1038/ng.2219
ARID1A
Subject 3
Ala Ser SerG C C A G C A G C C T G G G C A A C C C G C C G C C G C C G C C GNormal allele
Mutant allele G C C G C C G C C C T C G G A G C T G A A G A A A G C C G A G C AAla Ala Ala
Leu Gly Asn Pro Pro Pro Pro Pro
Subject 6
Exon 9 c.2758C>T
Asn Gln GlyA A T C A A G G GNormal allele
Mutant allele A A T T A A G G GAsn X
Subject 8
Gln Arg HisC A A C G A C A TNormal allele
Mutant alleleC A A T G A C A T
Gln X
Leu Gly Ala Glu Glu ArgSer Ala
Father
Mother
1 20 9 16
N’ C’ARID
22851014 1104
p.Ser11AlafsX91
1
p.Gln920X p.Arg1335X
ARID1014 1104
exon / intron border
p.Gln920X
Exon 1 c.31_56delAGCAGCCTGGGCAACCCGCCGCCGCC
p.Ser11AlafsX91
Exon 16 c.4003C>T
p.Arg1335X
Exon 16 Exon 17
Exon 16 Intron 16
c.DNA
g.DNAC A A C G G T G A Normal allele
Mutant allele C A A T G G T G A
Supplementary Figure 4 ARID1A mutations
One frameshift mutation and two nonsense mutations in ARID1A were identified in subjects 3, 6 and 8. For
subject 8, the first position of p.Arg1335 was altered from C to T. The exon 16/intron 16 boundary is shown in
the electropherograms. The mutation reads as a stop codon in the cDNA. ARID1A contains an ARID/BRIGHT
DNA-binding (ARID) domain.8
Nature Genetics: doi:10.1038/ng.2219
Subject 15
Ser Gln GlnT C C C A G C A G
Normal allele
Mutant allele T C C T A G C A GSer X
Phe Arg LeuT T C C G A C T C
Normal allele
Mutant allele T T C T G A C T CPhe X
Father
Mother
Subject 23
Father
Mother
Pro Gly Ser Asp Ser Arg
Pro Ile Gly Ile Gln Gly
ARID1B
H.sapiensP.troglodytesC.lupusM.musculusR.norvegicusG.gallus
Subject 1 Subject 10
Asn Pro AlaA A C C C G G C G
Normal allele
Mutant allele A A C C T G G C GAsn Leu Ala
Subject 10
Ile Asp AspA T C G A T G A CNormal allele
Mutant allele A T C A T G A C GIle Met Thr
Exon 4 c.1903C>T p.Gln635X
Exon 12 c.3304C>T p.Arg1102X
1 20
Father
Mother
2
C C A A T T G G C A T C C A G G G TNormal allele
Mutant allele
4 6 12
N’ C’
ARID
2285
1063 1153
1
p.Ile560GlyfsX89 p.Gln635X p.Pro715Leu p.Arg1102X p.Asp1878MetfsX96
C C A G G G T C G G A C T C C C G G
p.P715
D.rerioA.gambiaeC.elegans
…GDQSNPAQSPF……GDQSNPAQSPF……GDQSNPAQSPF……GDQSNPAQSPF……GDQSNPAQSPF……GEQSNPAQSPF……GEQSNPAQSPF……AAQGYPAQPPH……--QHHPQHPGM…
Exon 2 c.1678_1688delATTGGCATCCA
p.Ile560GlyfsX89
Exon 6 c.2144C>T
p.Pro715Leu
Exon 20 c.5632delG
p.Asp1878MetfsX96
Supplementary Figure 5 ARID1B mutations
Two frameshift, two nonsense and one missense mutation in ARID1B were identified in four subjects. In
subject 10, both p.Pro715Leu and p.Asp1878MetfsX96 mutations were identified, but they resided on different
alleles. ARID1B contains an ARID/BRIGHT DNA-binding (ARID) domain.
9Nature Genetics: doi:10.1038/ng.2219
Chromosome 6
qp
158 159 160 161 162 163 164 165 1666q25.3 6q26 6q27
AGPA
T4
PARK
2
PACR
G
cen telMb157
ARID
1B
SNX9
C6or
f35
ZDHH
C14
SYNJ
2TU
LP4
SYTL
3
DYNL
T1
SERA
C1G
TF2H
5
TMEM
181
FNDC
1
TAG
AP
EZR
RSPH
3
OST
CLC6
orf9
9
WTA
P
SOD2
ACAT
2
LOC1
0012
9518
TCP1
MRP
L18
PNLD
C1M
AS1
IGF2
RLP
AL2
SLC2
2A3
SLC2
2A1
SLC2
2A2
LPA
PLG
MAP
3K4
QKI C6or
f118
PDE1
0A
T
3.7 Mb deletion5.5 Mb deletion
Supplementary Figure 6 Microdeletion involving ARID1B in subject 12
Microdeletion involving ARID1B in subject 12. A human mapping 250K SNP array detected two microdeletions
at 6q25.3–q27 (bidirectional red arrows, top). The red bars indicate the proximal 3.7-Mb and distal 5.5-Mb
deleted regions (bottom). An ideogram of chromosome 6 is shown in the middle. The proximal 3.7-Mb deletion
(chromosome 6 coordinates: 156,706,749–160,432,331 bp based on UCSC 2009 Feb) involves the entire
ARID1B gene. Inheritance of the deletions was not confirmed because parental samples were unavailable.
10Nature Genetics: doi:10.1038/ng.2219
TAAGAGCAGCAGTTCCATA
cDNA
TAAGAGCAGCCATCAGGTC
CATCTCTGACAGTTCCATA
Genomic DNA
2,094,285 bp
2,147,059 bp
Supplementary Figure 7 Partial deletion of SMARCA2 in subject 19
A CytoScan HD array revealed a 55-kb interstitial deletion of SMARCA2 (chromosomal 9 coordinates:
2,093,551–2,144,765 bp based on UCSC 2009 Feb). The Log 2 ratio of probe signal intensity indicated
a heterozygous deletion, which was also supported by allele peaks showing hemizygous SNPs. Deletion
breakpoints were determined by PCR and sequencing. RT-PCR analysis revealed the skipping of exons
20–27.
Ex 19 Ex 28
T C C C G A A A A A G C C A T C G A A G・・・・・・・・・・
・・・・・・・・・・
proximal
distaldeletion
11Nature Genetics: doi:10.1038/ng.2219
ARID1A c.2758C>T p.Gln920X
Subject 6
TC
Genomic DNA
cDNACHX (-)
ARID1B c.3304C>T p.Arg1102X
Subject 23
TC
TC
Genomic DNA
cDNACHX (+)
cDNACHX (-)
cDNACHX (+)
T T C T G AA A T T A A
AsnA A T C A A
GlnNormal allele
Mutant allele Asn X
Phe ArgT T C C G A Normal allele
Mutant allele Phe X
A T C A T G
ARID1B p.Ile560GlyfsX89
Subject 1
cDNACHX (-)
cDNACHX (+)
Genome DNA
ARID1B p.Asp1878MetfsX96
Subject 10
Genome DNA
Pro IleC C A A T T
Normal allele
Mutant allelePro Gly
C C A G G G
Ile AspA T C G A TNormal allele
Mutant alleleIle Met
cDNACHX (-)
cDNACHX (+)
TC
TC
Supplementary Figure 8 Nonsense-mediated decay in subjects 1, 6, 10 and 23
Electropherograms of amplicons from genomic DNA and cDNA with/without cycloheximide (CHX) treatment
are shown. In subjects 6 and 23, the wild-type allele (black peak) and mutant allele (red peak) in genomic DNA
and cDNA with/without CHX treatment are shown by SNaPshot analysis. The mutant alleles were rescued by
CHX treatment. Peak areas are shown in the bottom table. rfu: relative fluorescent units. In subject 10, the
mutation associated with the premature stop codon in the last exon was not subject to nonsense-mediated
decay as expected.
TC
12Nature Genetics: doi:10.1038/ng.2219
Table S1 Clinical features in CSS
Clinical features
Neurodevelopment
developmental delay 5 / 5 3 / 3 4 / 4 6 / 6 1 / 1 1 / 1 20 / 20 3 / 3
hypototnia 4 / 5 2 / 3 4 / 4 4 / 6 1 / 1 1 / 1 16 / 20 3 / 3
microcephaly 1 / 5 1 / 3 2 / 3 4 / 5 1 / 1 1 / 1 10 / 18 2 / 3
small cerebellum 0 / 5 1 / 2 2 / 3 0 / 3 0 / 1 3 / 14 0 / 1
seizures 2 / 5 0 / 2 2 / 4 2 / 6 1 / 1 7 / 18 1 / 3
Dandy-Walker 0 / 5 1 / 3 0 / 2 1 / 5 0 / 1 2 / 16 0 / 2
abnormal corpus callosum 1 / 2 3 / 3 2 / 2 1 / 1 0 / 1 7 / 9 0 / 1
vision problem 1 / 4 1 / 2 2 / 3 5 / 6 1 / 1 10 / 16 1 / 2
hearing loss 1 / 5 1 / 2 3 / 4 3 / 6 1 / 1 1 / 1 10 / 19 0 / 2
Ectodermal
absent/hypoplastic fifth finger/toenails 5 / 5 3 / 3 4 / 4 6 / 6 1 / 1 0 / 1 19 / 20 3 / 3
hirsutism 5 / 5 3 / 3 3 / 4 6 / 6 1 / 1 1 / 1 19 / 20 2 / 2
sparse scalp hair 3 / 5 3 / 3 4 / 4 3 / 6 1 / 1 0 / 1 14 / 20 0 / 2
thick eyebrow 5 / 5 2 / 3 4 / 4 6 / 6 1 / 1 1 / 1 19 / 20 3 / 3
long eyelashes 4 / 5 3 / 3 4 / 4 6 / 6 1 / 1 1 / 1 19 / 20 2 / 3
abnormal/delayed dentition 5 / 5 2 / 2 3 / 3 3 / 5 1 / 1 1 / 1 15 / 17 0 / 2
non-functioning/absent tear duct 0 / 1 0 / 2 2 / 3 1 / 4 0 / 1 0 / 1 3 / 12 0 / 2
Facial
coarse appearance 5 / 5 3 / 3 4 / 4 6 / 6 1 / 1 1 / 1 20 / 20 3 / 3
flat nasal bridge 5 / 5 2 / 3 3 / 4 4 / 6 1 / 1 1 / 1 16 / 20 2 / 3
broad nose 5 / 5 2 / 3 4 / 4 2 / 6 1 / 1 1 / 1 15 / 20 2 / 3
wide mouth 3 / 5 3 / 3 4 / 4 3 / 6 1 / 1 1 / 1 15 / 20 2 / 3
thick lips 5 / 5 3 / 3 4 / 4 5 / 6 1 / 1 1 / 1 19 / 20 3 / 3
abnormal ears 4 / 5 3 / 3 4 / 4 5 / 6 1 / 1 1 / 1 18 / 20 0 / 2
high palate 5 / 5 2 / 3 4 / 4 5 / 5 1 / 1 0 / 1 17 / 19 2 / 3
cleft palate 0 / 5 2 / 3 2 / 4 3 / 6 1 / 1 0 / 1 8 / 20 0 / 3
ptosis 0 / 5 0 / 3 3 / 4 5 / 6 1 / 1 1 / 1 10 / 20 2 / 3
macroglossia 0 / 5 0 / 3 3 / 4 2 / 6 1 / 1 0 / 1 6 / 20 0 / 2
short philtrum 0 / 5 1 / 3 0 / 4 3 / 6 1 / 1 0 / 1 5 / 20 1 / 3
long philtrum 1 / 5 1 / 3 2 / 4 0 / 6 0 / 1 0 / 1 4 / 20 1 / 3
Skeletal
absent/hypoplastic fifth phalanx (hand) 5 / 5 2 / 2 1 / 1 4 / 5 1 / 1 0 / 1 13 / 15 1 / 3
absent/hypoplastic fifth phalanx (foot) 4 / 5 2 / 2 1 / 1 3 / 3 1 / 1 0 / 1 11 / 13 1 / 2
short stature 2 / 5 2 / 3 4 / 4 4 / 5 1 / 1 1 / 1 14 / 19 3 / 3
spinal anomalies 3 / 4 1 / 2 3 / 4 1 / 4 1 / 1 1 / 1 10 / 16 0 / 2
delayed bone age 0 / 1 1 / 2 1 / 1 2 / 4 3 / 3
Gastrointestinal
feeding problems 4 / 5 3 / 3 4 / 4 5 / 6 1 / 1 1 / 1 18 / 20 2 / 2
sucking problems 4 / 5 3 / 3 4 / 4 5 / 6 1 / 1 17 / 19 3 / 3
intestinal anomalies 1 / 5 2 / 2 1 / 4 2 / 5 1 / 1 7 / 17 1 / 1
Others
frequent infections 5 / 5 3 / 3 3 / 4 4 / 6 1 / 1 0 / 1 16 / 20 1 / 2
Intrauterine growth retardation 1 / 5 1 / 3 2 / 4 2 / 6 1 / 1 1 / 1 8 / 20 2 / 3
joint laxity 2 / 4 2 / 3 2 / 3 2 / 6 1 / 1 0 / 1 9 / 18 2 / 3
cardiac findings 1 / 5 3 / 3 2 / 4 2 / 6 1 / 1 0 / 1 9 / 20 2 / 3
genital findings 1 / 4 1 / 2 1 / 2 1 / 6 0 / 1 1 / 1 5 / 16 0 / 2
inguinal hernia 0 / 5 1 / 3 2 / 4 2 / 6 0 / 1 1 / 1 6 / 20 1 / 3
umbilical hernia 0 / 4 0 / 3 0 / 4 1 / 6 0 / 1 1 / 1 2 / 19 1 / 3
renal findings 0 / 4 0 / 2 0 / 3 0 / 4 0 / 1 1 / 1 1 / 15 0 / 2
diaphragmatic hernia 0 / 5 0 / 3 1 / 4 0 / 5 0 / 1 0 / 1 1 / 19 0 / 3
Mutated gene Positive Negative
1B 1A B1 A4 E1 A2 (mutation) (mutation)
13Nature Genetics: doi:10.1038/ng.2219
Supplementary Table 2 Flow of informatics analyses using two different variant call programs
Subject 1 Subject 4 Subject 5 Subject 9 Subject 11
Next MAQ Next MAQ Next MAQ Next MAQ Next MAQ
GENe (SeattleSeq) GENe (SeattleSeq) GENe (SeattleSeq) GENe (SeattleSeq) GENe (SeattleSeq)
Total variants called 71,209 146,394 162,417 269,309 52,471 241,286 90,582 196,493 79,774 169,131
Autosome + chr X 67,903 145,622 158,602 268,650 48,625 240,253 86,595 195,575 77,105 168,584
Unknown SNP variants 12,927 24,001 64,056 85,259 10,883 69,505 15,348 40,629 12,546 32,001
(dbSNP 131, 1000 genome)
Overlap of NextGENe and MAQ 1,508 4,701 988 1,883 1,721
NS/SS/In-Del (In-Del) 308 (68) 436 (143) 198 (44) 372 (111) 350 (85)
[NextGENe score ≥10]
Unknown variants 239 (25) 339 (85) 152 (20) 275 (41) 244 (27)
[in-house database]
Variants not found in parental 23 (12) 64 (17) 58 (13)
samples (subjects 1, 5, 11)
Variants found in two or more patients 76
After removal of variants associated 51
with segmental duplications
NS: non-synonymous; SS: splice site (±2 bp); In-del: insertions/deletions. Variants identified by MAQ were subsequently annotated with SeattleSeq (physical
position, gene name, genotype, dbSNP131 and 1000 Genomes variation, function (e.g., missense) and amino acid alteration, position and distance to nearest
splice-site).
14Nature Genetics: doi:10.1038/ng.2219
Table S3 Mutation analysis of SWI/SNF subunits in CSS
Total Truncation Non-truncation total/partial deletionSMARCB1 601607 BAF47/hSNF5 4 0 4 0
SMARCA4 603254 BRG1 6 0 6 0SMARCA2 600014 BRM 1 0 0 1
SMARCC1 601732 BAF155 0 0 0 0
SMARCC2 601734 BAF170 0 0 0 0
ARID1A 603024 BAF250A 3 3 0 0
ARID1B - BAF250B 5 4* 1* 1
BRD7 - BRD7 0 0 0 0
ARID2 609539 BAF200 0 0 0 0
PBRM1 606083 BAF180 0 0 0 0
SMARCE1 603111 BAF57 1 0 1 0
SMARCD1 601735 BAF60A 0 0 0 0
SMARCD2 601736 BAF60B 0 0 0 0
SMARCD3 601737 BAF60C 0 0 0 0
ACTL6A 604958 BAF53a 0 0 0 0
ACTL6B 612458 BAF53b 0 0 0 0
Total 20 7 11 2
Core
BAF-specific
PBAF-specific
Classification Gene MIM
The mutation-positive genes are shown in red. *One patient (subject 10) has two variants.
SubunitNumber of CSS patients
Others
15Nature Genetics: doi:10.1038/ng.2219
