Supplementary Information  1

SUPPLEMENTARY INFORMATION

Supplementary Information  2

Supplementary Figure 1

Supplementary Figure 1 (a) Sequence electropherograms show the EBF3 c.625C>T mutation in DNA isolated from leukocytes and fibroblasts of the two affected siblings (subjects 1 and 2) in the heterozygous state (top rows). Sanger sequencing demonstrated mosaicism of the c.625C>T mutation in leukocyte-derived DNA of the mother. The mutation was not visible in the sequence derived from her buccal cell-derived DNA (third row). The healthy family members showed wild-type sequence in leukocyte- derived DNA. Sanger traces show the c.1101+1G>T, c.530C>T and c.469_477dup mutations in leukocyte-derived DNA of subjects 5, 6, and 10 (left bottom row), respectively, and wild-type sequence in their parents (right bottom rows). Arrows point to the position of the mutations. (b) Cloning of exon 7-containing amplicons, followed by colony PCR and sequencing revealed that ~18% of leukocytes and ~4% of buccal cells of the mother contain the heterozygous EBF3 mutation (9% and 2% of clones with the mutated allele). (c) Sequence electropherograms show heterozygosity of the c.625C>T mutation in fibroblast-derived cDNA of subjects 1 and 2. Supplementary Information  3

Supplementary Figure 2

66 EBF3_human 50 ARAHFEKQPPSNLRKSNFFHFVLALYDRQGQPVEIERTAFVDFVEKEKEPNNEKTNNGIHYKLQLLYSN EBF3_chimpanzee 50 ARAHFEKQPPSNLRKSNFFHFVLALYDRQGQPVEIERTAFVDFVEKEKEPNNEKTNNGIHYKLQLLYSN EBF3_macaque 50 ARAHFEKQPPSNLRKSNFFHFVLALYDRQGQPVEIERTAFVDFVEKEKEPNNEKTNNGIHYKLQLLYSN EBF3_mouse 50 ARAHFEKQPPSNLRKSNFFHFVLALYDRQGQPVEIERTAFVDFVEKEKEPNNEKTNNGIHYKLQLLYSN EBF3_rat 50 ARAHFEKQPPSNLRKSNFFHFVLALYDRQGQPVEIERTAFVDFVEKEKEPNNEKTNNGIHYKLQLLYSN EBF3_zebrafish 50 ARAHYEKQPPSNLRKSNFFHFVLALYDRQGQPVEIERTAFVDFVEKEKEPNSEKTNNGIHYKLQLLYSN EBF3_fruit fly 66 GRAHFEKQPPSNLRKSNFFHFVIALYDRAGQPIEIERTAFIGFIEKDSESDATKTNNGIQYRLQLLYAN EBF3_mosquito 6 GRAHFEKQPPSNLRKSNFFHFVVALYDRAGQPIEIERTAFIGFIEKDQEPDGQKTNNGIQYRLQLLYAN EBF3_clawed frog 50 ARAHFEKQPPSNLRKSNFFHFVLALYDRQGQPVEIERTAFVDFVEKDKEPNSEKTNNGIHYKLQLLYSN EBF1_human 50 ARAHFEKQPPSNLRKSNFFHFVLALYDRQGQPVEIERTAFVGFVEKEKEANSEKTNNGIHYRLQLLYSN EBF2_human 49 SRAHFEKQPPSNLRKSNFFHFVLALYDRQGQPVEIERTAFVDFVENDKEQGNEKTNNGTHYKLQLLYSN

141 157 171 177 EBF3_human 119 GVRTEQDLYVRLIDSMTKQAIVYEGQDKNPEMCRVLLTHEIMCSRCCDKKSCGNRNETPSDPVIIDRFF EBF3_chimpanzee 119 GVRTEQDLYVRLIDSMTKQAIVYEGQDKNPEMCRVLLTHEIMCSRCCDKKSCGNRNETPSDPVIIDRFF EBF3_macaque 119 GVRTEQDLYVRLIDSMTKQAIVYEGQDKNPEMCRVLLTHEIMCSRCCDKKSCGNRNETPSDPVIIDRFF EBF3_mouse 119 GVRTEQDLYVRLIDSMTKQAIVYEGQDKNPEMCRVLLTHEIMCSRCCDKKSCGNRNETPSDPVIIDRFF EBF3_rat 119 GVRTEQDLYVRLIDSMTKQAIVYEGQDKNPEMCRVLLTHEIMCSRCCDKKSCGNRNETPSDPVIIDRFF EBF3_zebrafish 119 GVRTEQDLYVRLIDSMTKQAIIYEGQDKNPEMCRVLLTHEIMCSRCCDKKSCGNRNETPSDPVIIDRFF EBF3_fruit fly 135 GARQEQDIFVRLIDSVTKQAIIYEGQDKNPEMCRVLLTHEVMCSRCCDKKSCGNRNETPSDPVIIDRFF EBF3_mosquito 75 GARQEQDIFVRLIDSVTKQAIVYEGQDKNPEMCRVLLTHEVMCSRCCDKKSCGNRNETPSDPVIIDRFF EBF3_clawed frog 119 GVRTEQDLYVRLIDSMTKQAIIYEGQDKNPEMCRVLLTHEIMCSRCCDKKSCGNRNETPSDPVIIDRFF EBF1_human 119 GIRTEQDFYVRLIDSMTKQAIVYEGQDKNPEMCRVLLTHEIMCSRCCDKKSCGNRNETPSDPVIIDRFF EBF2_human 118 GVRTEQDLYVRLIDSVTKQPIAYEGQNKNPEMCRVLLTHEVMCSRCCEKKSCGNRNETPSDPVIIDRFF

COE/ZNF

209 243 EBF3_human 188 LKFFLKCNQNCLKNAGNPRDMRRFQVVVSTTVNVDGHVLAVSDNMFVHNNSKHGRRARRLDPSE EBF3_chimpanzee 188 LKFFLKCNQNCLKNAGNPRDMRRFQVVVSTTVNVDGHVLAVSDNMFVHNNSKHGRRARRLDPSE EBF3_macaque 188 LKFFLKCNQNCLKNAGNPRDMRRFQVVVSTTVNVDGHVLAVSDNMFVHNNSKHGRRARRLDPSE EBF3_mouse 188 LKFFLKCNQNCLKNAGNPRDMRRFQVVVSTTVNVDGHVLAVSDNMFVHNNSKHGRRARRLDPSE EBF3_rat 188 LKFFLKCNQNCLKNAGNPRDMRRFQVVVSTTVNVDGHVLAVSDNMFVHNNSKHGRRARRLDPSE EBF3_zebrafish 188 LKFFLKCNQNCLKNAGNPRDMRRFQVVVSTTVNVDGHVLAVSDNMFVHNNSKHGRRARRLDPSE EBF3_fruit fly 204 LKFFLKCNQNCLKNAGNPRDMRRFQVVISTQVAVDGPLLAISDNMFVHNNSKHGRRAKRLDTTE EBF3_mosquito 144 LKFFLKCNQNCLKNAGNPRDMRRFQVVIATQVAVDGPLLAISDNMFVHNNSKHGRRAKRLD-PE EBF3_clawed frog 188 LKFFLKCNQNCLKNAGNPRDMRRFQVVVSTTVNVDGHVLAVSDNMFVHNNSKHGRRARRLDPSE EBF1_human 188 LKFFLKCNQNCLKNAGNPRDMRRFQVVVSTTVNVDGHVLAVSDNMFVHNNSKHGRRARRLDPSE EBF2_human 187 LKFFLKCNQNCLKTAGNPRDMRRFQVVLSTTVNVDGHVLAVSDNMFVHNNSKHGRRARRLDPSE

Supplementary Figure 2 Amino acid sequence alignment of the human EBF3 DNA-binding domain (aa 50-251; NP_001005463) with orthologs and two paralogs [human EBF1 (NP_001277289) and human EBF2 (NP_073150)], showing conservation of amino acids N66, Y141, H157, E158, I159, G171, P177, R209 and R243 between species and within the human EBF family (EBF1, EBF2 and EBF3). Multiple alignments were gathered from http://www.ncbi.nlm.nih.gov/homologene/; alignment with EBF1 and EBF2 was performed with Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clustalo/). Conserved residues mutated in the patients are highlighted in red; arginine 243 altered in tumors is highlighted in grey. The amino acid stretch constituting the atypical zinc finger (COE/ZNF) is indicated by a black line (aa 157-170).

Supplementary Information  4

Supplementary Figure 3

Supplementary Figure 3 (a) Model of the DNA-binding domain of an EBF3 monomer (cyan ribbon, affected residues shown as sticks) bound to DNA (sticks). Hydrogen bonds are represented by yellow lines. Major interactions of affected residues are shown in (b-f). (b) Asn66 forms a hydrogen bond with a DNA phosphate group (left panel), which is disrupted by the mutation (right panel). In addition, the negative charge of aspartate at position 66 likely leads to electrostatic repulsion of the phosphate group. (c) Gly171 is part of the protein-DNA interface (left panel). Mutation of Gly171 to the negatively charged asparagine could lead to an electrostatic repulsion of the DNA backbone (right panel). (d) Pro177 is localized in close proximity to Asn174, which forms a hydrogen bond with the DNA (left panel), and to the zinc finger (right panel). Replacement of Pro177 by leucine may lead to a conformational change altering the position of Asn174 and possibly of the zinc finger, reducing the DNA-binding capacity of EBF3. His157 together with Cys161, Cys164 and Cys170 are invariant residues coordinating Zn2+ (right panel). In-frame duplication of the amino acids His157, Glu158 and Ile159 may cause a conformational change of the zinc finger reducing DNA-binding capability of EBF3. (e) Arg209 forms hydrogen bonds with the backbone of Cys198 and Asn197, with the latter forming a hydrogen bond with the DNA (left panel). Mutation of Arg209 leads to disruption of these hydrogen bonds, probably affecting the positioning of Asn197 (right panel). (f) Tyr141 is localized within a loop which is not directly involved in DNA-binding but in EBF3 dimer formation (pink ribbon). Mutation of Tyr141 may lead to a conformational change at the dimer interface, resulting in reduced stability of the EBF3 dimer and interfering with its ability to bind to DNA. Supplementary Information  5

Supplementary Figure 4

Supplementary Figure 4 Co-expression of wild-type EBF3 and EBF3 mutants in transactivation assays. (a) HEK 293T cells were transfected with EBF3 mutant-expressing vectors together with a plasmid expressing wild-type EBF3 (1:1) or with empty vector alone (control). Expression of only wild-type EBF3 (green bar) led to a 4- to 5-fold elevated promoter activity compared with cells transiently transfected with empty vector (white bar). Co-expression of EBFWT H157A and the DNA binding-deficient EBF3 mutant (yellow bar) or either of the disease-associated EBF3 mutants N66D, Y141C, Q305*, H157_I159dup and R303* (blue bars) resulted in ~50% reduction of luciferase reporter activity. The three mutants P177L, R209W and G171D did not cause any significant reduction in reporter activity. The normalized luciferase activity (mean ± s.d.) of three independent experiments is depicted as the fold induction relative to cells transfected with a control vector. All comparisons are in reference to wild-type EBF3, and P values were calculated N66D Q305* using the two-sided Student’s t test. (b) Increasing amounts of EBF3 (left panel; blue bars) and EBF3 (right panel; blue bars) reduce promoter activity indicating a dominant-negative effect on wild-type EBF3. HEK 293T cells were transiently transfected with a constant amount of wild-type EBF3 (4 µg; +), together with the reported amount of mutant EBF3, with 8 µg wild-type EBF3 (++; green bar) or with empty vector alone (-; white bar). Dual luciferase assays were done with the extracts of transfected cells 48 hours after transfection. The normalized luciferase activity (mean ± s.d.) of three independent experiments is depicted as the fold induction relative to cells transfected with a control vector. For all comparisons, P values were calculated using the two-sided Student’s t test. *P <0.05, **P < 0.005, ***P < 0.0005; n.s.: not significant. Supplementary Information  6

Supplementary Figure 5

Supplemental Figure 5 Boxplots of variance stabilized counts for significantly differentially expressed genes (FDR <0.05) between untransfected SK-N-SH cells (blue), EBF3WT (green), and EBF3P177L (red) expressing cells.

Supplementary Information  7

Supplementary Figure 6

Supplementary Figure 6 Gene Ontology (GO) term analysis of genes significantly differentially expressed between EBF3WT and EBF3P177L versus SK-N-SH control. (a) EBF3WT top 10 most significantly enriched GO terms by p-value for all GO term categories. (b) EBF3P177L top 10 most significantly enriched GO terms by p-value for all GO term categories (BP: Biological Processes, CC: Cellular Compartment).

Supplementary Information  8

Supplementary Figure 7

Supplementary Figure 7 Schematic of EBF3WT and EBF3P177L expression vectors. EBF3WT and EBF3P177L cDNA were inserted into an expression vector with a C-terminal 3X-FLAG epitope and 2A self-cleaving peptide linked neomycin resistance.

Supplementary Information  9

Supplementary Figure 8

Supplementary Figure 8 Wild-type EBF3 dose-dependent activation of the p21 (CDKN1A) promoter. Transactivation assays were performed using transiently transfected HEK 293T cells expressing wild-type EBF3 in the presence of the promoter region of the human CDKN1A gene. Cells were transfected with 2 µg of pGL2-p21 promoter-Luc and 2 µg pREN constructs, together with the reported amount of wild-type EBF3 or with empty vector alone (-). Maximal activation was attained with 6 µg wild-type EBF3 DNA. The normalized luciferase activity (mean ± s.d.) of three independent experiments is depicted as the fold induction relative to cells transfected with a control vector. All comparisons are in reference to the control vector and P values were calculated using the two-sided Student’s t test. n.s.: not significant; **P < 0.005; ***P < 0.0005.

Supplementary Information  10

Supplementary Table 1

ExAC Genic Genomic mRNA reference Nucleotide Amino acid ExAC Chr Gene EVS intolerance position number change change metrics dbSNP (%ExAC_RVIS) Z=5.09 19 47207871 PRKD2 NM_016457.4 c.547A>G p.(S183G) 0 9.45 pLI=1.00 Z=4.89 10 131676043 EBF3 NM_001005463.2 c.625C>T p.(R209W) 0 6.78 pLI=1.00 Z=1.72 1 204437988 PIK3C2B NM_002646.3 c.933+10T>A  0 12.58 pLI=0.98 Z=2.24 1 203667366 ATP2B4 NM_001001396.2 c.275C>A p.(T92N) 0 19.32 pLI=0.52 Z=0.59 22 18301687 MICAL3 NM_015241.2 c.3740C>G p.(P1247R) 0 78.31 pLI=1.00 Z=-1.38 1 32671286 IQCC NM_001160042.1 c.4G>C p.(E2Q) 0 80.71 pLI=0.00 Z=-2.42 3 37368154 GOLGA4 NM_001172713.1 c.4843C>G p.(Q1615E) 0 49.22 pLI=0.00 3 156638378 LEKR1 NM_001004316.2 c.278G>T p.(G93V) 0 NA NA Z=-1.04 7 1589968 TMEM184A NM_001097620.1 c.449T>G p.(M150R) 0 98.22 pLI=0.00 Z=0.73 7 8126010 GLCCI1 NM_138426.3 c.1486C>G p.(Q496E) 0 15.48 pLI=0.25 Z=-0.24 12 109540699 UNG NM_003362.3 c.562G>A p.(G188S) 0 59.37 pLI=0.05 Z=-3.14 16 77359930 ADAMTS18 NM_199355.3 c.1865A>T p.(Q622L) 0 93.74 pLI=0.00 Z=0.62 17 28601119 BLMH NM_000386.3 c.742A>G p.(I248V) 0 20.28 pLI=0.01 17 79402471 BAHCC1 NM_001291324.1 c.179-5G>A  0 NA NA Z=-1.13 19 1064227 ABCA7 NM_019112.3 c.6019G>C p.(G2007R) 0 99.1 pLI=0.00 18121005 Z=1.79 19 ARRDC2 NM_015683.1 c.850_890del p.(V284Afs*40) 0 57.67 _18121045 pLI=0.00

Supplementary Table 1 Variants in subjects 1 and 2 identified by filtering the whole-exome sequencing data for heterozygous variants absent in the exome dataset of the healthy sibling and population databases (ExAC Browser, Exome Variant Server, 1000 Genomes Browser, dbSNP138). Variants were ranked according to the Z and pLI scores derived from ExAC (scores determining the tolerance of a gene against missense or loss-of-function mutations). The top five candidates are listed according to their ranked Z and pLI scores, the remaining variants according to their genomic position starting with chromosome 1. The genomic position is given according to the human reference genome hg19. Nucleotide and amino acid changes are given according to the longest transcript variant. Chr: chromosome; NA: not available.

Supplementary Information  11

Supplementary Table 2

Gene Patient 1 Patient 2 Sibling 1 Sibling 2 Sibling 3 Father Mother

PRKD2 c.547A>G c.547A>G WT WT WT c.547A>G WT

EBF3 c.625C>T c.625C>T WT WT WT WT c.[=/625C>T] PIK3C2B c.933+10T>A c.933+10T>A WT WT WT WT c.933+10T>A ATP2B4 c.275C>A c.275C>A WT WT WT WT c.275C>A MICAL3 c.3740C>G c.3740C>G WT c.3740C>G c.3740C>G WT c.3740C>G

Supplementary Table 2 Segregation analysis of the five most promising variants in family 1 by Sanger-sequencing of leukocyte-derived DNA samples. For the variants in ATP2B4, PIK3C2B, PRKD2 and MICAL3 one of the parents was found to be a heterozygous carrier, excluding these variants to be pathogenic in the two affected siblings. In the case of MICAL3, also two healthy siblings showed the variant. Analysis of the EBF3 variant c.625C>T revealed mosaicism in leukocyte-derived DNA from the mother. All healthy siblings showed wild-type sequence for the EBF3 variant. WT: wild-type.

Supplementary Information  12

Supplementary Table 3

Nucleotide Amino acid CADD GERP++ Patient change change Score Score

1 + 2 c.625C>T p.(R209W) 28.8 5.1 3 c.913C>T p.(Q305*) 38.0 4.88

4 c.196A>G p.(N66D) 22.9 3.27

5 c.1101+1G>T ̶ 22.3 5.77 6 c.530C>T p.(P177L) 19.74 5.95 7 c.422A>G p.(Y141C) 23.6 4.32

8 c.512G>A p.(G171D) 19.58 5.95

9 c.907C>T p.(R303*) 37 3.59 10 c.469_477dup p.(H157_I159dup) 21.7 4.41

Supplementary Table 3 In silico pathogenicity prediction of mutations in EBF3. The functional impact of the identified variants were predicted by the Combined Annotation Dependent Depletion (CADD) and GERP++ scoring systems, using ANNOVAR (2016 Feb 01), an integrated database of functional annotations from multiple sources for the comprehensive collection of human non-synonymous SNPs. CADD is a framework that integrates multiple annotations into one metric by contrasting variants that survived natural selection with simulated mutations. Reported CADD score is a phred-like rank score based upon the rank of that variant’s score among all possible single nucleotide variants of hg19, with 10 corresponding to the top 10%, 20 at the top 1%, and 30 at the top 0.1%. The larger the score the more likely the variant has deleterious effects; the score range observed here is strongly supportive of pathogenicity, with all observed variants ranking above ~99% of all variants in a typical genome and scoring similarly to variants reported in ClinVar as pathogenic (~85% of which score >15). GERP++ scores1,2 measure evolutionary constraint across mammalian species at the single-nucleotide level and have previously been shown to correlate with mutational deleteriousness3 and to be useful in the identification of highly penetrant mutations4.

Supplementary Information  13

Supplementary Table 4 Clinical features of patients with mutations in EBF3

Family 1 Family 2 Family 3 Family 4 Family 5 Family 6 Family 7 Family 8 Family 9 Individual Patient 1 Patient 2 Mother Patient 3 Patient 4 Patient 5 Patient 6 Patient 7 Patient 8 Patient 9 Patient 10 Mutation in EBF3a c.625C>T c.625C>T c.[=/625C>T] c.913C>T c.196A>G c.1101+1G>T c.530C>T c.422A>G c.512G>A c.907C>T c.469_477dup p.(R209W) p.(R209W) p.(Q305*) p.(N66D) p.(P177L) p.(Y141C) p.(G171D) p.(R303*) p.(H157_I159dup) Origin Inherited Inherited de novo de novo de novo de novo de novo de novo de novo de novo de novo (Mosaicism) Sex Female Male Female Male Male Male Female Male Female Female Male Age at assessment 9 years 3 months 3 years 4 months 33 years 5 years 9 months 16 years 6 4 years 6 months 2 years 7 months 1 year 11 months 13 years 25 years 3 year 5 months months Birth weight 2500/-2 1780/-3.5 NA 2495/-2 3500/-0.2 4054/+0.7 3033/-0.7 2891/-1 3068/-0.6 2900/-1 3190/-0.7 (grams/SD) Birth length (cm/SD) NA NA NA 45.7/-2 50.8/+0.3 NA NA 48.2/-0.7 49.5/0 NA 49/-0.25 OFC at birth (cm/SD) NA NA NA 33.5/-1.3 NA NA NA 35.5/-0.25 NA NA 36/+1 Weight (kg/SD) 22/-2 11/-2 NA 21/+0.5 45.8/-2.2 25.67/+3.4 13.3/0 10.6/-1.5 42.2/-1 55/-0.5 12.4/-1 Length (cm/SD) 119/-3 91/-2 NA 108/-1.2 168.6/-0.8 110.49/+1 90.2/-0.2 NA NA (severe hip 167/+0.8 95/-1 and knee contractures) OFC (cm/SD) 50/-3 48.3/-2.5 NA 51.5/-1 57/+0.7 NA 49.75/+1 49.5/+0.7 51.9/-1.7 NA 51/+0.5 Neurological abnormalities Intellectual disability + + ̶ + + + + + + + + Motor + + ̶ + + + + + + + + developmental delay Speech delay + + ̶ + + + + + + + + Ataxia + + ̶ + + NA + ̶ Wide based bent ̶ NA knee dystonic gait Seizures + + ̶ ̶ ̶ ̶ ̶ ̶ NA ̶ ̶ Tone Normal Normal Normal Mild hypotonia in Normal Truncal hypotonia Truncal hypotonia Hypotonia Hypotonia as Normal Truncal hypotonia early childhood, infant; dystonia normal now now Deep tendon reflexes Normal Normal Normal Normal Normal NA Brisk Normal Brisk Normal Normal Magnetic resonance Normal ND ND Normal Cerebellar Normal Normal Normal Normal ND Cerebellar imaging of the brain vermian vermian hypoplasia hypoplasia Supplementary Information  14

Craniofacial abnormalities Long face + + + ̶ + NA NA ̶ + + ̶ Deep philtrum + + + ̶ + NA + ̶ + ̶ ̶ Tall forehead + + + ̶ + NA + ̶ + + ̶ High nasal bridge + + + ̶ + NA NA ̶ + + ̶ Straight eyebrows + + + ̶ + NA NA ̶ ̶ ̶ + Strabismus + + ̶ + + + + + + ̶ + Ears Low set, Low set, Low set ̶ Small ear lobes NA NA ̶ normal NA Low set posteriorly posteriorly posteriorly rotated, small ear rotated rotated lobes Short and broad chin + + + ̶ + NA NA ̶ Prominent chin Broad chin ̶ Other craniofacial Thick vermillion ̶ ̶ Small mouth, Thick vermillion Flat nasal bridge Relative macro- Hypertelorism Facial asymmetry, Upward slanted Thin lower lip abnormalities of upper and short philtrum, of upper and cephaly, mild submucous cleft palpebral fissures, lower lips micrognathia lower lips muscular hyper- palate broad nasal trophy, promi- bridge, smooth nent shoulders, philtrum, thin narrow waist, upper lip, and upward slanting chin dimple palpebral fissures, long eyelashes, straight hair Other findings ̶ ̶ ̶ 2-3 toe syndac- Dysarthria, pectus ̶ ̶ Congenital heart Complete 2-3 toe ̶ Bilateral talipes tyly, inguinal excavatum, thin disease (atrial syndactyly on equinovarus, phi- hernia (repaired) and scooped septal defect) right, partial 2-3 mosis, recurrent nails, orchiopexy toe syndactyly on lower urinary for undescended left, limited facial tract infections, testicles, very expression, neu- constipation mild hypospadias, rogenic bladder strabismus sur- (vesicostomy), gery, intoeing due feeding difficul- to femoral ante- ties, can swallow version, attention semi-solid food deficit disorder, only, mild sco- gastroesophageal liosis, dysplastic reflux, possible right kidney, Supplementary Information  15

eosinophilic eso- bilateral vesico- phagitis ureteric reflux, megacolon with intractable consti- pation Karyotype Normal Normal ND ND Normal Normal Normal Normal Normal ND ND Chromosomal Normal Normal ND Normal Chr7q34 Normal Normal Normal Normal ND Normal microarray (139,737,216- 140,035,653)x3; chr12q24.11 (107,875,652- 108,235,158)x3; both paternally inherited Other investigations Normal tandem ̶ ̶ Normal lactate, ̶ Normal testing Normal results for ̶ Normal results for ̶ Normal results for mass spectro- amino acids, for Fragile X, ele- Fragile X, MECP2 7-dehydrochol- blood lactic acid, metry, normal urine metabolic vated blood crea- and UBE3A gene esterol assay, creatine phospho- electroencepha- screen, NGS tine phospho- testing, blood myotonic dystro- kinase, urine logram, normal ataxia panel, kinase (285 IU/L), lactic acid normal, phy DNA testing, creatine and thyroid functions, nerve conduction normal plasma creatine phospho- acylcarnitines, plasma creatine, normal complete velocity, normal amino acids, nor- kinase normal, creatine phospho- normal organic blood count screening for mal organic acids normal urine kinase and aldo- acids and oligo- congenital disor- creatine and lase saccharides, ders of glycosy- plasma creatine, normal screening lation, alpha feto- normal organic for congenital protein, cerebro- acids and oligo- disorders of spinal amino saccharides glycosylation acids, tetrahydro- folate and neuro- transmitters a according to NCBI reference sequence NM_001005463.2; +: present; ̶ : absent; NA: not available; ND: not determined; NGS: next-generation sequencing; OFC: occipital frontal circumference; SD: standard deviation.

References: 1. Cooper, G.M. et al. Distribution and intensity of constraint in mammalian genomic sequence. Genome Res 15, 901-13 (2005). 2. Davydov, E.V. et al. Identifying a high fraction of the human genome to be under selective constraint using GERP++. PLoS Comput Biol 6, e1001025 (2010). 3. Goode, D.L. et al. Evolutionary constraint facilitates interpretation of genetic variation in resequenced human genomes. Genome Res 20, 301-10 (2010). 4. Cooper, G.M. et al. Single-nucleotide evolutionary constraint scores highlight disease-causing mutations. Nat Methods 7, 250-1 (2010).