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Supplementary Information

A de novo paradigm for mental retardation

Lisenka ELM Vissers1*, Joep de Ligt1*, Christian Gilissen1, Irene Janssen1, Marloes

Steehouwer1, Petra de Vries1, Bart van Lier1, Peer Arts1, Nienke Wieskamp1, Marisol del

Rosario1, Bregje WM van Bon1, Alexander Hoischen1, Bert BA de Vries1,

Han G Brunner1#‡, Joris A Veltman1#‡

1Department of Human Genetics - 855, Nijmegen Centre for Molecular Life Sciences and Institute for Genetic and Metabolic Disorders, Radboud University Nijmegen Medical Centre, PO Box 9101, 6500 HB Nijmegen, The Netherlands.

*These authors contributed equally to this work # These authors jointly directed this work ‡To whom correspondence should be addressed

Correspondence to:

Prof. dr. Han G. Brunner or Dr. ir. Joris A. Veltman Department of Human Genetics – 855 Radboud University Nijmegen Medical Centre PO Box 9101, 6500 HB Nijmegen, The Netherlands Email: h.brunner@antrg.umcn.nl Email: j.veltman@antrg.umcn.nl Telephone: +31 24 36 14017 Fax:+31 24 36 68752

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Supplementary note regarding clinical patient description

MR Trio 1. This boy was born a term after an uneventful pregnancy with a weight of 3850g (60th centile) to a healthy 35-year-old mother and 38-year-old father. He was the first child of non-consanguineous parent and had a healthy sister. At 6 months hypotonia was noted and physical therapy started. Development was delayed. He could sit at 18 months and walk at 3 years and spoke 10 words only at the age of 5. At the age of 4 years his developmental level was tested which showed a 2 years delay. He has moderately severe mental retardation.

On physical examination at the age of 2 years he had a normal height of 85.2 cm (15th centile) and head circumference of 47.5 cm (15th centile). He had mild (facial) dysmorphism consisting of prominent forehead, plagiocephalic skull, a hypotonic face with down slanting palpebral fissures and short broad hands and feet. Additional studies included a chromosome analysis by 250 K SNP array, FMR1 repeat expansion (DNA level), metabolic studies, and MRI of the brain. These revealed no abnormalities.

MR Trio 2. This boy was born at 38 weeks as the first child of a non-consanguineous couple (maternal age 32 and paternal age 30 years) after an uncomplicated pregnancy with a weight of 3460g (50th centile). He had a younger healthy sister and a nephew with a congenital heart defect. Initially he had feeding problems due to unspecified swallowing difficulties. He walked at 18 months. At the age of 5 years he used a total of 5 words only. He attended special schooling for children with severe learning difficulties.

At the age of 5 years he had a height of 110 cm (15th centile) and head circumference of 54 cm (90th centile). He had a left epicanthic fold and mild downslanting palpebral fissures without any additional dysmorphic features. Further studies including chromosome analysis (250 K SNP array), FMR1 gene repeat expansion (DNA level), metabolic studies and MRI of the brain revealed no abnormalities.

MR Trio 3. At 36 weeks pregnancy, growth retardation and unilateral dilatation of the renal

pelvis were noted. After a caesarian section because of breech position this boy was born

with a low birth weight of 2010g (- 2.5 SD). His non-consanguineous parents had 3 older

healthy children (two girls and a boy). Parental ages at time of birth of the proband were 33

(mother) and 36 years (father).

There were no neonatal problems, except for mild hypoglycemia and hyperbilirubinemia, treated with UV therapy. As a neonate feeding remained slow and difficult and the pyelectasy was confirmed with ultrasound. His development was delayed. He could sit after 1 year and walk after 2 years 9 months. He spoke his first words at 2 years and his level was 21 months at the age of 4 years 5 months, consistent with moderate mental retardation.

At the age of 33 months his height was 84 cm (-3 SD) and head circumference 48.5 cm (15th centile). He had telecanthus, down slanting palpebral fissures, high nasal bridge, and a broad mouth. The dysmorphic features were not suggestive of any specific syndrome diagnosis according to an experienced clinical geneticist (B.B.A.d.V.). Additional studies including chromosome analysis (250 K SNP array), FMR1 analysis (DNA level), metabolic studies and ophthalmological investigations revealed no abnormalities.

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MR Trio 4. This girl was born after 36 weeks following an uncomplicated pregnancy with a weight of 3660g (98th centile) as the first child of non-consanguineous parents (maternal age 32 and paternal age 31 year). Her motor development was delayed due to hypotonia. With support of physical therapy, she sat at 14 months and walked at 2 years. She spoke her first words after 2 years and at the age of 3 years 8 months she used 30 words. At that age she had a height of 100 cm (30th centile) and a head circumference of 47.5 cm (10th centile). She had thin blond hair, periorbital fullness and a broad nose with full tip. No syndrome diagnosis was made. Further studies including chromosome analysis (250 K SNP array), FMR1 repeat expansion (DNA level), metabolic studies and MRI investigation of the brain which revealed no abnormalities.

MR Trio 5. This boy was born at 41w 2d after an uncomplicated pregnancy with a low birth weight of 2600g (3rd centile) as the only child of a non-consanguineous couple (maternal age 32 and paternal age 35 years). His development was clearly delayed. He walked at 17 months but at the age of 6 years he still could not speak. His behavior was happy and he seldom cried.

On physical examination at the age of 2y 10m he had a low normal height of 91 cm (5th centile) and his head circumference was 49.2 cm (25th centile). He had thin fair hair, upward slanting palpebral fissures, epicanthic folds, almond shaped eyes, proximal implant of the thumbs and a scrotal raphe. An initial clinical suspicion of Angelman syndrome led to methylation study of the 15q11-q13 region and mutation analysis of UBE3A. These revealed no aberrations. Further studies including chromosome analysis (250 K SNP array), FMR1 repeat expansion (DNA level) and metabolic studies and ophthalmological investigations revealed no abnormalities.

MR Trio 6. This boy was born at 38w 3d after an uncomplicated pregnancy by cesarean section with a weight of 3000g (40th centile) as the second child of a non-consanguineous couple (maternal age 29 and paternal age 27 years). His development was delayed. He sat at 15 months, walked at 22 months and spoke his first words after 21 months and at the age of 3 years used 20 words only.

At the age of 21 months his height was 80.5 cm (5th centile) and head circumference 47.3 cm (15th centile). He had no evident facial dysmorphisms. Further studies including chromosome analysis (250 K SNP array), FMR1 repeat expansion (DNA level), metabolic studies and MRI investigation of the brain revealed no abnormalities.

MR Trio 7. This 19-year-old male was the second child of non-consanguineous parents and born at 40 weeks after an uncomplicated pregnancy with a weight of 3500g (50th centile). Parental ages at time of birth were 28 and 29 years for mother and father, respectively. Early motor development was slow but within normal range. Speech development was severely delayed, he spoke his first words at the age of 4 years. He attended special school and his developmental level was 3-3,5 years at the age of 19 years. He is severely retarded. He showed variable aggressive behavior.

On physical examination at the age of 19 years, a low-normal height of 172 cm (5th centile) and normal head circumference of 56 cm (15th centile) was noted. He had minor facial dysmorphisms; upward slanting palpebral fissures, slightly everted ears, thin upper lip with full everted lower lip without additional physical features. Further studies including

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chromosome analysis (250 K microarray), FMR1 expansion (chromosome level), EHMT1 gene and metabolic studies revealed no abnormalities.

MR Trio 8. This girl was born at 36w 5d after an uncomplicated pregnancy by cesarean section due to breech position with a weight of 2800g (50th centile) as the only child of a non-consanguineous couple (maternal age 32 and paternal age 29 years).

Her motor development was delayed due to hypotonia, she sat at 19 months. MRI of the brain revealed mild delay of myelinization at 10 months. A muscle biopsy at the age of 20 months did not show any abnormalities. At the age of 4 ½ months she did not speak any words. At age 4 years epilepsy was diagnosed.

On physical examination at the age of 11 months she had a normal height of 70 cm (10th centile) and head circumference of 46.3 cm (60th centile). She had a high forehead, upward slanting palpebral fissures, mild hypertelorism, epicantical folds, low nasal bridge and full lips with everted lower lips. A general, predominantly axial, hypotonia was present. Further studies, including chromosome analysis (250 K SNP array), DNA-analysis of FMR1, myotonic dystrophy, Prader Willi syndrome, as well as metabolic studies and ophthalmological investigation, all revealed no abnormalities.

MR Trio 9. This boy was born at 39w 1d after an uncomplicated pregnancy with a weight of 3520g (50th centile) as the second child of a non-consanguineous couple (maternal age 31 and paternal age 37 years) . There were neonatal feeding difficulties. His development was delayed. He sat at 12 months, walked at 18 months and spoke his first words at 2 years. At the age of 3 2/12 years he used 5-10 words only. He was moderately mentally retarded. On physical examination at that age he had a normal height of 103 cm (75th centile) and head circumference of 50.5 cm (50th centile). He had mild (facial) dysmorphism consisting of upward slanting palpebral fissures, short palpebral fissures, flattened upper helices and broad nose with full tip. Further studies including chromosome analysis (250 K SNP array), FMR1 repeat expansion (DNA level) and metabolic studies revealed no abnormalities.

MR Trio 10. This boy was born after a normal gestation at 39w 6d with a weight of 3270g (25th centile) as the second child of non-consanguineous parents (maternal age 29 and paternal age 31 years). At 6 months, delayed development was noted. , He sat at 10-11 months, walked at 13-14 months and at the age of 4 years he only used 4 words and his developmental level at that age was 15-18 months. On physical examination at the age of 2 4/12 years he had a height of 89.5 cm (25th centile) and head circumference of 49 cm (30th centile). He had a metopic ridge, hypotelorism (ICD 2cm, < 3rd centile), mild ptosis, short palpebral fissures and a flat palate. He had a broad, uncertain gait. Further studies, including G-banded chromosomes and FMR1 repeat expansion analysis (DNA level) were normal. Additional metabolic and chromosome analysis (250K SNP array) revealed no abnormalities.

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Supplementary Tables Supplementary Table 1. Overview of exome-sequencing performance

reads sequenced

reads mapped

bps mapped

% mapping to targeted

sequence*

MR trio 1 Mother 75,539,970 62,421,598 2,994,163,125 84.3

Father 60,482,344 49,757,641 2,359,196,222 79.7

Patient 83,750,821 63,968,695 2,993,659,326 82.4

MR trio 2

Mother 61,213,042 45,635,581 2,129,184,998 79.5

Father 81,822,660 64,920,388 3,101,222,088 83.8

Patient 81,274,402 63,413,997 2,998,226,897 80.1

MR trio 3

Mother 91,218,082 65,444,907 3,111,727,311 82.4

Father 90,482,575 66,637,781 3,174,885,071 83.4

Patient 89,089,332 63,253,491 2,987,977,994 82.5

MR trio 4

Mother 89,715,373 63,797,713 3,045,658,184 83.3

Father 80,613,693 64,548,447 3,100,745,735 82.7

Patient 84,917,415 65,900,711 3,147,306,800 83.7

MR trio 5

Mother 95,997,554 75,047,477 3,635,298,749 77.3

Father 85,743,467 61,089,261 2,939,733,902 74.3

Patient 72,400,831 55,557,595 2,658,661,406 71.5

MR trio 6

Mother 77,312,957 66,715,554 3,208,045,277 70.1

Father 82,262,575 63,187,587 3,006,761,015 71.8

Patient 74,889,401 57,633,832 2,769,859,304 71.7

MR trio 7

Mother 88,884,506 67,665,146 3,207,116,037 76.3

Father 93,174,731 72,970,692 3,483,792,759 81.0

Patient 76,030,490 58,908,643 2,789,555,317 81.2

MR trio 8

Mother 82,506,855 66,109,620 3,185,522,297 70.4

Father 55,116,325 45,653,877 2,163,335,572 76.5

Patient 81,579,330 63,654,731 3,047,698,346 69.9

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MR trio 9

Mother 88,622,619 69,950,138 3,363,461,864 76.3

Father 95,568,766 82,036,334 3,985,154,541 77.9

Patient 83,296,156 62,687,172 3,032,856,030 77.4

MR trio 10

Mother 90,342,631 73,134,022 3,537,828,588 77.8

Father 95,285,830 79,698,200 3,875,693,431 81.6

Patient 87,139,147 67,783,275 3,255,909,349 80.6

Over all 30 individuals

Median 83,523,489 64,258,571 3,074,222,041 79.6

Min 55,116,325 45,635,581 2,129,184,998 69.9

Max 95,997,554 82,036,334 3,985,154,541 84.3

*Defined as the sum of bases mapping to the SureSelect bait and one read length (50 bp) away from the Sure Select bait.

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Supplementary table 2: All potential de novo variants per trio followed up by Sanger

sequencing

The columns represent and should be interpreted as follows: Chromosome: chromosome Start: genomic start position of variant base(s), mapped on hg18 End: genomic end position of variant base(s), mapped on hg18 Reference: wildtype sequence for the genomic position Variant: variant sequence for the genomic position Reads: number of reads for the genomic position (e.g. total coverage) Variant reads: number of reads showing the variant base(s) Unique starts: number of variant reads with an unique start position % variation: the percentage of reads that show the variant (based on variant

reads/reads). Low covered variants (<15%) are hereby distinguished from heterozygous (15-80%) and homozygous/hemizygous mutations (>80%).

Gene name: gene in which the variant occurs Gene ID: NM used to annotate the variants Total exons: total number of exons known to be included in the gene ID used Variant in exon: exon in which the variant occurred Reference aa: wildtype amino acid for this genomic position Variant aa: variant amino acid (‘*’ denotes premature stop) PhyloP score: conservation score for the genomic nucleotide the variant starts Grantham score: conservation score indicating the physiochemical changes due to amino acid change Cells with red shading and text in bold represent validated and de novo mutations, in gray shading and gray text represent validated but inherited variants, and cells without shading and gray text represent variants that could not be validated in the patients, hence parents were not tested. Notes:

For indels, unique start sites cannot be determined and are therefore not presented

For frameshift variants, the first two amino acid changes are provided

Grantham scores for nonsense and frameshift variations are not provided. Nonsense variants do not have a Grantham score. Grantham scores for frameshift variants are not representative of the change, as in many cases a premature stop occurs downstream of the mutation.

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Supplementary Table 2 – continued

Ch

rom

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me

Star

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Var

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ce a

a

Var

ian

t a

a

ph

ylo

P S

core

Gra

nth

am S

core

MR trio 1

chr14 101575506 101575506 A C 35 19 15 54 DYNC1H1 NM_001376 78 61 H P 5.50 77

chr13 36437769 36437769 T G 21 4 4 19 ALG5 NM_013338 10 8 K T 5.12 78

chr6 33854077 33854077 G A 47 8 5 17 LEMD2 NM_181336 9 6 A V 1.73 64

chr19 39943014 39943014 G A 60 29 22 48 ZNF599 NM_001007248 4 4 L F -1.45 22

MR trio 2

chrX 154143367 154143367 C T 44 42 24 95 RAB39B NM_171998 2 2 W * 4.85 -

chr4 170870853 170870853 G A 45 7 5 16 CLCN3 NM_173872 14 12 C Y 3.92 194

chr3 65347895 65347899 CTCTCTCTCAC CTCTCTCAC 5 5 100 MAGI1 NM_004742 23 15 E D 3.74 45

chr4 55297463 55297463 A G 50 8 5 16 KIT NM_001093772 21 18 N D 2.01 23

chr7# 150274912 150274912 C T 8 4 4 50 KCNH2 NM_000238 15 14 D N 0.39 23

chr19 12499218 12499218 A C 53 8 7 15 ZNF564 NM_144976 4 4 I S -1.16 97

chr1 150351392 150351392 T C 35 6 5 17 TCHH NM_007113 2 2 K E -2.07 56

# the mother of the patient did not have coverage for this variant, as such it was validated – it appeared to be maternally inherited after Sanger sequencing

MR trio 3

chr14 99813583 99813583 G T 70 19 16 27 YY1 NM_003403 5 5 D Y 6.94 160

chr10 104669687 104669687 A G 22 9 7 41 CNNM2 NM_199076 7 1 E G 5.03 98

chr20 31090416 31090416 G A 24 7 5 29 BPIL3 NM_174897 15 9 R H 0.58 89

MR trio 4

chr17 1511106 1511106 CG CACG 5 4 80 PRPF8 NM_006445 43 28 G A 5.91 60

chr1 84894680 84894680 T C 21 5 4 24 SSX2IP NM_001166293 14 10 K R 1.62 26

chr1 152057903 152057903 TT TTT 26 22 85 GATAD2B NM_020699 11 4 N K 0.93 94

chrX 75565439 75565439 C G 26 5 4 19 MAGEE1 NM_020932 1 1 P A 0.69 27

chr11 60775220 60775220 T C 268 102 44 38 PGA5 NM_014224 9 9 V A 0.65 64

chr11 62060131 62060131 CTCCTCCT CTCCTCCTCCT 6 6 100 AHNAK NM_001620 5 3 T RX 0.36 -

chr1 7996012 7996012 TC 4 4 100 ERRFI1 NM_018948 4 4 R E -0.66 80

MR trio 5

chr16 22834028 22834028 A G 44 8 4 18 HS3ST2 NM_006043 2 2 I V 5.24 29

chr11 676979 676979 A C 35 17 7 49 DEAF1 NM_021008 12 5 I S 4.91 142

chr7 86912218 86912218 TGAAAAAAAAA TGAAAAAAAA 4 4 100 ABCB4 NM_018849 28 10 S Q 2.83 43

chr8 27450247 27450247 CTGAGTC CTGAGTCGTC 4 4 100 EPHX2 NM_001979 19 13 N NX 1.60 -

chrY 5028095 5028095 G A 31 6 4 19 PCDH11Y NM_032973 5 2 V M 0.97 21

chr12 74179860 74179860 ACTTTTTTTTCT ACTTTTTTTCT 4 4 100 KRR1 NM_007043 10 10 K S 0.92 26

chr2 73529542 73529542 G T 30 12 5 40 ALMS1 NM_015120 23 8 A S -1.91 112

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MR trio 6

chr19 47486234 47486234 C T 36 18 15 50 CIC NM_015125 20 10 R W 2.57 177

chr12 52619000 52619000 TTAT TTATTAT 5 5 100 HOXC13 NM_017410 2 1 L LX 1.70 -

MR trio 7

chr3 49257117 49257117 CAGT CAGTGAC 5 4 80 CCDC36 NM_001135197 8 6 L PX 2.76 -

chr18 32054137 32054137 T C 31 8 5 26 MOCOS NM_017947 15 9 F S -1.87 155

MR trio 8

chr11 71619753 71619753 G A 36 6 4 17 INPPL1 NM_001567 28 12 E K 6.31 56

chr6 33513701 33513702 ACTGTGCC ACTGCC 25 8 32 SYNGAP1 NM_006772 19 8 TV TA 3.26 64

chr22 364502674 36450276 GCCTCCTCTC GCCTCTC 7 5 71 TRIOBP NM_001039141 24 7 AS ASX 1.71 -

chr11 51268086 51268086 C T 16 5 4 31 OR4A5 NM_001005272 1 1 E K 1.50 56

chr19 14604762 14604762 CTATTGA CTTTGA 9 8 89 EMR3 NM_032571 16 13 N K 0.62 94

chr19 42794493 42794493 T C 23 4 4 17 ZNF540 NM_152606 5 5 F L 0.25 22

MR trio 9

chr17 21260004 21260004 T TT 4 4 100 KCNJ12 NM_021012 3 3 V F 6.48 50

chr12 50009865 50009865 GGGGGC GGGGGGC 5 4 80 CELA1 NM_001971 8 7 L R 4.99 102

chr3 53885149 53885149 GGGGGGGA GGGGGGGGA 5 5 100 ACTR8 NM_022899 13 7 4.82 -

chr17 8337932 8337933 TGTGTTTTTTTT TGTGTTTTTTT 4 4 100 MYH10 NM_005964 41 29 4.60 -

chr10 78399792 78399792 GCTTTTTTTTTG GCTTTTTTTTG 6 6 100 KCNMA1 NM_002247 27 19 K S 4.57 121

chr8 101145241 101145241 T C 18 6 4 33 RGS22 NM_015668 28 8 T A -0.54 58

MR trio 10

chr4 151739681 151739681 G A 30 5 4 17 LRBA NM_006726 58 38 R W 6.95 101

chr1 44195800 44195800 T G 57 11 8 19 IPO13 NM_014652 20 7 F C 4.99 205

chr2 196448664 196448664 A G 20 4 4 20 DNAH7 NM_018897 65 38 M T 4.85 81

chr19 60516183 60516183 C A 19 5 5 26 TMEM150B NM_01085488 8 8 W C 1.76 215

chr7 72892397 72892398 TGTGTGTGGG TGTGTGGG 4 4

100 WBSCR27 NM_152559 6 4 1.50 -

chr12 11397965 11397967 AGGAGGTGGG AGGTGGG 4 4 100 PRB1 NM_005039 4 3 PP PGX 1.42 -

chr6 26125843 26125843 C T 21 7 4 33 HIST1H1A NM_005325 1 1 A T 1.42 58

Validated, de novo

Validated, not de novo

Not validated

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Supplementary Figures

Supplementary Figure 1: Coverage plots of all 30 individuals

Figure legend

Coverage for all exons targeted by enrichment was evaluated. The median coverage for all 30 individuals was 42-fold, with on average 90% of all targets covered at least 10-fold. The numbers in the figure legends refer to the corresponding MR trios; M: Mother; F: Father; C: Child

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Supplementary Figure 2: Variant calling per trio

Figure legend

Schematic representation of number of variants identified per individual, per trio. On average, 22,272 variants are identified per individual, of which 19,212 are located in genes (intronic and exonic together), and 12,341 are located within the exons and/or canonical splice-sites. The number on the x-axis refer to the corresponding MR trios, whereas M to Mother, F to Father and C to child.

0

5,000

10,000

15,000

20,000

25,000

30,000

Nu

mb

er

of

vari

ants

non genicintronicexonic

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Supplementary Figure 3: IGV browser overview for validated de novo mutations

a

c

b

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d

e

f

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g

h

i

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Figure Legend

IGV browser plots for the nine de novo and one X-linked inherited mutations identified in the

patients with mental retardation. (a) DYNC1H1 - trio 1, (b) ZNF599 –trio 1, (c) RAB39B –trio

2; (d) YY1 –trio 3; (e) BPIL3 –trio 3; (f) PGA5 –trio 4; (g) DEAF1 –trio 5; (h) CIC –trio 6; (i)

SYNGAP1 –trio 8; (j) JARID1C –trio 10. For each overview, top panel show chromosome with

mutation location indicated by a red box. The second panel from the top shows the relative

coverage per base pair for patient, mother and father. Colored coverage columns indicate

the location of the mutation with the relative height of the color representing the

percentage of variant reads observed. Middle panels show the individual reads (zoom-in to

~10-15 reads of all reads) with bases deviant from the wildtype sequence highlighted in

colors. Lower panel shows wildtype sequence and translated amino acids.

j

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Patient

Father

Mother

Patient

Father

Mother

Patient

Father

Mother

Patient

Father

Mother

Supplementary Figure 4: Sanger validation of de novo mutations

G

a

b

c

d

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Patient

Father

Mother

Patient

Father

Mother

Patient

Father

Mother

Figure legend

Partial electropherograms showing the de novo occurrence for the nine mutations identified. Mutations are highlighted in yellow. (a) trio 1, left panel DYNC1H1 with mutation c.11465A>C leading to p.His3822Pro and right panel ZNF599 with mutation c.532C>T leading to p.Leu187Phe. (b) trio 2, RAB39B with mutation c.557G>A leading to a premature stop p.Trp186X. (c) trio 3, left panel YY1 with mutation c.1138G>T leading to p.Asp380Tyr and right panel BPIL3 with mutation c.887G>A leading to p.Arg269His. (d) trio 4 with mutation c. 1058T>C in PGA5, leading to Val353Ala. (e) trio 5, with mutation c.683T>G in DEAF1 leading to p.Ile228Ser. (f) trio 6 with mutation c.1474C>T in CIC leading to p.Arg492Trp and (g) trio 8, with mutation c.998_999del in SYNGAP1 leading to p.Val333Alsfs70X.

f

e

g

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Supplementary Figure 5: Distribution of PhyloP and Grantham scores for dbSNP, HGMD

and the de novo mutations identified in this study

0

50

100

150

200

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PhyloP score

'benign'

'pathogenic'0.0

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dbSNP

HGMD

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Figure legends

(a) Relative distribution of PhyloP scores for non-synonymous in dbSNP (green surface) and HGMD (red surface) to represent evolutionary scores observed for benign and pathogenic variants. (b) Evolutionary conservations for the 3 functionally benign (green diamonds) and 4 functionally pathogenic de novo mutations, as well as the X-linked inherited mutation (red diamonds). This analysis was further extended to analyse these combined scores for all non-synonymous variants in dbSNP and HGMD to visualize the probability for the de novo variants to be pathogenic or benign. Heat maps for distribution and frequency for the combined PhyloP and Grantham scores in dbSNP (c) and HGMD (d). Functionally benign and pathogenic de novo mutations, as well as the inherited JARID1C mutation, are represented by squares and triangles respectively. Benign de novo variants show a better overlap with dbSNP as indicated by the their location mostly restricted to dark green areas in c and not in d, whereas the pathogenic de novo variants show a better overlap with HGMD, as indicated by their location mostly being in dark green areas in d and not in c. Statistical probability scores for these analyses are provided in Table 2.

Note: Two de novo mutations, a nonsense and frameshift mutation, identified in our patient cohort are not depicted because such mutations do not have representative Grantham scores. Their functional impact on the protein level will however be most severe and both are located in known mental retardation genes.

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References to Supplementary Information

1. Marth,G.T. et al. A general approach to single-nucleotide polymorphism discovery. Nat

Genet. 23, 452-456 (1999). 2. Ng,S.B. et al. Targeted capture and massively parallel sequencing of 12 human exomes.

Nature 461, 272-276 (2009). 3. Pushkarev,D., Neff,N.F., & Quake,S.R. Single-molecule sequencing of an individual human

genome. Nat Biotechnol. 27, 847-852 (2009). 4. Wang,J. et al. The diploid genome sequence of an Asian individual. Nature 456, 60-65

(2008). 5. Venables, W.N. and Ripley, B D. Modern Applied Statistics with S. Springer, Fourth

edition (2002). 6. http://www.r-project.org/ 7. Lilliefors, H. On the Kolmogorov–Smirnov test for normality with mean and variance

unknown", Journal of the American Statistical Association 62, 399–402 (1967).

Web resources

For variant visualization we used the IGV browser developed at: http://www.broadinstitute.org/igv

For in-house variant database: http://www.1000genomes.org

For genome browser annotations: http://genome.ucsc.edu

For variant effect of identified mutations: http://sift.jcvi.org/ http://genetics.bwh.harvard.edu/pph/

Nature Genetics: doi: 10.1038/ng.712