Supporting Information

Collin et al. 10.1073/pnas.1220864110 SI Materials and Methods Mutation Analysis of PASK and ZNF408. The two candidate variants Human Subjects. A detailed clinical description of familial exudative that were left after linkage and exome analysis were analyzed in vitreoretinopathy (FEVR) family W05-215 has been reported family W05-215 with Sanger sequencing of exon 6 of proline- previously (1), and clinical details of the affected individuals alanine-rich ste20-related kinase (PASK) and exon 5 of zinc studied using next-generation sequencing (NGS) (III:5 and V:2) finger protein 408 (ZNF408). Primer sequences are available are summarized below. Eight affected individuals, three un- on request. The presence of these two changes in additional affected individuals, and three spouses participated in the ge- Dutch FEVR probands and 110 control individuals was ana- netic analysis (Fig. S1A). After linkage and exome sequencing lyzed via restriction fragment length polymorphism analysis, analysis were performed, three additional affected relatives using AflIII for the c.791dup change in PASK and SfaNI for the (IV:10, IV:11, and V:7) were sampled. Clinical details of the c.1363C>T change in ZNF408. All exons and intron–exon two individuals with FEVR from family W05-220 (IV:3 and boundaries of ZNF408 were amplified under standard PCR V:2) are summarized below. Furthermore, 132 individuals with conditions using primers that are available on request. Sanger FEVR (8 from The Netherlands, 64 from the United Kingdom, sequence analysis was performed with the ABI PRISM Big Dye 55 from Japan, and 5 from Switzerland) participated in this Terminator Cycle Sequencing V2.0 Ready Reaction Kit and study, along with 110 ethnically matched Dutch and 191 eth- an ABI PRISM 3730 DNA analyzer (Applied Biosystems). nically matched Japanese controls. Written informed consent The occurrence of the c.377G>A (p.Ser126Asn) variant in was obtained from all participants, and the study was approved 191 Japanese controls was assessed via Sanger sequencing by the local Ethics Committee, adhering to the tenets of the analysis. All PASK exons were analyzed using DNA of indi- Declaration of Helsinki. viduals IV:3 and V:2 of family W05-220 by Sanger sequencing, as described above. Linkage Analysis. Genomic DNA from all participating individuals of family W05-215 was extracted from peripheral blood lympho- Generation of Plasmids. An Image clone containing the full ORF cytes according to standard protocols (2), except for DNA of in- of ZNF408 (ImaGenes; accession no. NM_024741.2) was used dividuals IV-10, IV-11, and V-7 of family W05-215, which was as a template to amplify a cDNA molecule encoding the full- extracted from saliva using Oragene containers (DNA Genotek) length WT ZNF408 protein but lacking the start methionine according to the protocol supplied by the manufacturer. Eight to generate an N-terminally tagged fusion construct. PCR analysis affected individuals of family W05-215 were genotyped using was performed using the following primers: forward, 5′-GGGG- the Infinium II HumanLinkage-12 Panel (HumanOmniExpress; ACAAGTTTGTACAAAAAAGCAGGCTTCGAGGAGGCG- Illumina), which contains 733,202 SNP markers. Multipoint link- GAGGAGCTG-3′; reverse, 5′-GGGGACCACTTTGTACAA- age analysis was performed with the GeneHunter 2.1r5 program GAAAGCTGGGTTCAGGTGCCCATCTCCACC-3′ (attB1- and in the easyLinkage v5.052beta software package (3) using the attB2- sites for Gateway cloning are underlined). Using Gateway MarshfieldSNPmapandCaucasianallelefrequencies.Forthe technology (Invitrogen), the cDNA encoding human full-length microsatellite markers, two-point logarithm of the odds (LOD) ZNF408 was cloned into the pDONR201 vector as described scores were calculated using the SuperLink v1.6 program in the previously (6). To introduce the missense variants that were easyLinkage software. For each calculation, an autosomal dom- identified in this study, site-directed mutagenesis was performed inant mode of inheritance was assumed with a penetrance of by PCR with Phusion Taq (Finnzymes) and corresponding 0.75, and the disease allele frequency was estimated at 0.0001. primers (available on request). The DNA constructs after site- directed mutagenesis were validated by Sanger sequencing of Exome Sequencing Analysis and Variant Filtering. The exomes of the entire coding regions of the WT and mutant ZNF408 cDNAs. individuals III:3 and V:2 of family W05-215 (Fig. S1A) were The cDNAs were subsequently cloned into either the HA-tagged enriched using the SureSelect Human Exome Enrichment Kit or enhanced cyan fluorescent protein (eCFP)-tagged destina- V1 (Agilent) and sequenced using one-quarter of a SOLiD se- tion vectors (pcDNA3-HA/DEST and pcDNA3-eCFP/DEST; quencing slide (Life Technologies). The SureSelect Human Invitrogen), as described previously (6, 7). Exome Enrichment Kit V1 covers 1.22% of human genomic re- gions corresponding to the National Center for Biotechnology Transient Transfection of COS-1 Cells. HA-tagged or eCFP-tagged Information’s Consensus Coding DNA Sequence Database WT and mutant ZNF408 were expressed using the mammalian (CCDS), including >700 human miRNAs from the Sanger v13 expression vector pcDNA3-HA/DEST or pcDNA3-eCFP/DEST, database and >300 additional human noncoding RNAs, such both driven by a cytomegalovirus (CMV) promoter. COS-1 cells as small nucleolar RNAs (snoRNAs) and small Cajal body- were transiently transfected using Effectene (Qiagen), according specific RNAs (scaRNAs). This design covers >37 Mb of the to the manufacturer’s instructions. At 24 h after transfection, cells human genome. Color space reads were mapped to the hg19 were washed with PBS and then prepared for (immuno)fluores- reference genome with SOLiD BioScope version 1.3, which used cence analysis. an iterative mapping approach. Single-nucleotide variants were subsequently called by the diBayes algorithm with conservative Subcellular Localization of WT and Mutant ZNF408 in COS-1 Cells. To call stringency. Small insertions and deletions were detected using determine the subcellular localization of the HA- or eCFP-tagged the SOLiD Small InDel Tool. Called SNP variants and indels WT and mutant ZNF408 proteins, transiently transfected cells were combined and annotated using a custom analysis pipeline, were fixed with 4% paraformaldehyde and either directly mounted with information concerning the effect of the variants on the (in case of eCFP fusion proteins) or incubated with primary anti- amino acid translation, overlap of variants with polymorphisms HA and secondary goat anti-mouse Alexa Fluor 680 antibodies from dbSNP132, overlap with variants from our own in-house before mounting with Vectashield containing DAPI (Vector database, and evolutionary conservation scores for the affected Laboratories). Images were obtained with a Zeiss AxioImager nucleotide (4, 5). Z1 upright fluorescent microscope and processed with a Zeiss

Collin et al. www.pnas.org/cgi/content/short/1220864110 1of8 ApoTome slider module. All experiments were performed in Left eye: 20/125; refraction: sphere, −12/cylinder, −1.0 × 0°. duplicate. Anterior segments: Slight cortical cataract in both eyes. Fundi. Evolutionary Conservation of ZNF408. To assess the evolutionary Stretched course of the temporal retinal vessels; some conservation of the mutated amino acids, amino acid sequences of temporal ectopia of the macula. Local areas of atrophy of choroid orthologous ZNF408 proteins were aligned with the protein se- and retinal pigment epithelium (RPE) (posterior pole). Avascular quence of human ZNF408 using the Align program in Vector NTI peripheral retina temporally anterior to the equator. Left eye with version 11 (Invitrogen). Protein identification numbers, derived large pigmented scars of choroid and retina in the inferotemporal from GenBank and/or Uniprot, were as follows: human ZNF408: quadrant (secondary to surgical procedures). No retinal exudates, Q9H9D4; bovine ZNF408: NP_001180086; canine ZNF408: F1:00 defects, or detachment. PMD3; mouse Znf408/Zfp408: NP_001028623; zebrafish ZNF408: Patient V:2. NP_001002305. Family D, daughter of V-12 of van Nouhuys (1). History. No history of premature birth. Eye examination at age 3 y Morpholinos and Zebrafish Embryo Manipulations. Tg(fli1:eGFP) fi because of family history; visual impairment and FEVR diagnosed. zebra sh were bred and raised under standard conditions (8), Support at school age because of a considerable visual handicap. in accordance with international and institutional guidelines. Examination at age 15 y; best corrected visual acuities: Zebrafish eggs were obtained from natural spawning. Trans- lation (5′-TCCCAGTGACTGTAGACATCAGGGC-3′)and Right eye: Finger counting; poor fixation. splice-blocking (5′-GCAACCCCTGAAAAATAACAACACA-3′) Left eye: 20/100; pseudoexotropia due to a large positive morpholinos (MOs) for znf408 were designed by Gene Tools kappa angle. and diluted in deionized, sterile water supplemented with 0.5% phenol red. To determine the most effective dose of the znf408 Anterior segments: No abnormalities. MOs, 1 nL of diluted MO (containing 2, 6, and 10 ng) was injected Fundi. Right eye: Prominent falciform retinal fold running from into the yolk of one- to two-cell stage embryos using a Pneumatic the optic nerve head across the center of the posterior pole in the PicoPump pv280 (World Precision Instruments). For in vivo rescue inferotemporal direction. Marked chorioretinal atrophy and ret- experiments, human ZNF408 and mutant p.His455Tyr ZNF408 inal pigmentations along both sides of the fold. No retinal breaks mRNAs were prepared using the mMESSAGE mMACHINE or exudates. Kit (Ambion) according to the manufacturer’s instructions. The Left eye: Posterior pole showing a dragged disk and marked mRNAs (150 pg) were (co)injected together with MOs or alone deformation of the retina. Retinal vessels with an abnormally as described above. A minimum sample size of 50 was used in stretched course to the inferotemporal area, with a whitish mass each case. After injection, embryos were cultured at 28 °C in E3 of fibrous tissue noted. Several retinal exudates located central to embryo medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, this mass. and 0.33 mM MgSO4, supplemented with 0.1% methylene Deformed retinal network in both fundi detected on fluores- blue) and subsequently phenotyped at 3 days postfertilization cein angiography, with leakage of dye particularly from dilated (dpf). Injected embryos were classified into three classes of capillaries in the left posterior pole. phenotypes on the basis of the relative severity compared with Course/Treatment. Fromage19yonward,severaltreatmentsbydiode- stage-matched standard control (5′-CCTCTTACCTCAGTTA- laser coagulation in the left eye because of slowly extending retinal CAATTTATAC-3′; Gene Tools) MO-injected (10 ng) embryos exudates. Progressive posterior vitreous detachment and tractional of the same clutch. Images were obtained with an Zeiss Axioplan retinal detachment treated with an encircling procedure and sub- 2imagingfluorescence microscope equipped with a Zeiss sequently with lensectomy and several pars plana vitrectomies, in- DC350FX camera. cluding endolaser and silicone oil procedures. At age 26 y, visual To determine the efficiency of splice blocking, RNA was iso- acuity of the left eye of finger counting with an attached retina. The lated from 50 control MO and znf408 splice MO-injected em- retinal fold and low visual function of the right eye unchanged. bryos (2 dpf) using the RNeasy Mini Kit (Qiagen) according to Family W05-220. the manufacturer’s instructions. Here 500 ng of total RNA was used to produce first-strand cDNA. Reverse-transcription was Patient IV:3. performed using the iScript cDNA Synthesis Kit (BioRad) ac- History. Visually impaired from birth. At age 44 y, cataract ex- cording to the manufacturer’s instructions. traction of the right eye. Examination at age 44 y; best corrected This was followed by nested PCR analysis. The primers used for visual acuities: the first PCR were f1 (5′-CTCTGCTTGGTCTGGAGGAG-3′) and r1 (5′-GCCTCCAGAGCTTCATCTTG-3′), and the primers Right eye: 1/300 used for the nested PCR were f2 (5′-GAGGAGCCTGGAGA- Left eye: 20/50 (with contact lens); refraction: sphere, −6. AACAATC-3′) and r2 (5′-GAGCTTCATCTTGTTCTCTG-3′). Fragments thus obtained were extracted from gel using a Nucleo- Convergent squint in right eye; horizontal nystagmus. Anterior Spin Gel Extraction Kit (Macherey-Nagel) and Sanger-sequenced segments: Nuclear cataract in left eye. Fundi. fi as described above. Right eye: No details visible due to capsular opaci cation. Left eye: Normal optic disk; vessels slightly dragged temporally; Clinical Data for Families W05-215, W05-220, and F117. temporal periphery lacking blood vessels; pigmentary abnor- malities. Family W05-215. Patient V:2. Patient III:5. History. Visually impaired from birth. Examination at age 8 y; Family D, patient IV-15 of van Nouhuys (1). best corrected visual acuities: History. Myopia and spectacles at school age. No history of + − × premature birth or ocular trauma. Retinal detachment of the left Right eye: 20/100; refraction: sphere, 1/cylinder, 0.75 70° eye at age 20 y, treated with diathermy. Examination at age 47 y; Left eye: Hand movements; refraction: sphere, −3/cylinder, best corrected visual acuity: −4.25 × 180°. Right eye: 20/100; refraction: sphere, −12/cylinder, −1.0 × 0° Microstrabismus convergens in left eye; horizontal nystagmus.

Collin et al. www.pnas.org/cgi/content/short/1220864110 2of8 Fundi. Right eye: Normal optic disk, normal vessels; absence of Left eye: 8/100; refraction noncorrectable. retinal vessels in temporal periphery. Left eye: Slightly tilted optic disk; vessels dragged to the Anterior segments: IOL in both eyes. Fundi. temporal periphery in a horizontal course; maculae displaced Both eyes: Sharply demarcated lobular atrophy of choroid temporally; temporal periphery lacking blood vessels; pigmentary and RPE in the posterior pole and midperiphery. Retinal vessels “ ” abnormalities. without the stretching often found in FEVR. Localized bone spicule pigmentations, reminiscent in appearance to those seen Family F117. in retinitis pigmentosa, within the chorioretinal atrophy. Not previously reported. Patient II:2. Family F117 History. No history of premature birth. Myopia and spectacles at school age. Examination at age 34 y; best corrected visual acuities: I:1 I:2 Right eye: 20/20; refraction: sphere, S-6/cylinder, −0.5 × 150° Ser126Asn/+ +/+ LE: 20/20; refraction: sphere, −4.5/cylinder, −1.0 × 110°. Anterior segments: No abnormalities. Fundi. II:1 II:2 Both eyes: No typical dragged disk in the posterior pole, +/+ Ser126Asn/+ but elongated disk-to-macula distances. Abnormally stretched retinal vessels with prominent ramification especially to the fi Patient I:1. temporal area, with whitish brous tissues seen between the vascular and avascular retina. A few retinal holes and exudative History. No history of premature birth or ocular trauma. Exu- dots in the avascular regions. dative retinal detachment of the left eye at age 40 y and of the fl right eye at age 48 y. Uveal effusion diagnosed in both eyes. Deformed retinal network in both fundi detected on uo- Treatment with focal photocoagulation and oral steroids. Cata- rescein angiography. Focal leakage of dye in the left eye, in- ract extraction and intraocular lens insertion on both eyes at age dicating neovascularization in the temporal periphery. Course/Treatment. 50 y. Examination at age 72; best corrected visual acuities: Laser photocoagulation in the temporal pe- ripheral avascular retina and the retinal holes in both eyes at age Right eye: 20/100; refraction: sphere, −1D 34 y.

1. van Nouhuys CE (1982) Dominant exudative vitreoretinopathy and other vascular 5. Gilissen C, et al. (2010) Exome sequencing identifies WDR35 variants involved in developmental disorders of the peripheral retina. Doc Ophthalmol 54(1-4):1–414. Sensenbrenner syndrome. Am J Hum Genet 87(3):418–423. 2. Miller SA, Dykes DD, Polesky HF (1988) A simple salting out procedure for extracting 6. Kantardzhieva A, et al. (2005) MPP5 recruits MPP4 to the CRB1 complex in DNA from human nucleated cells. Nucleic Acids Res 16(3):1215. photoreceptors. Invest Ophthalmol Vis Sci 46(6):2192–2201. 3. Hoffmann K, Lindner TH (2005) easyLINKAGE-Plus—automated linkage analyses using 7. Arts HH, et al. (2007) Mutations in the gene encoding the basal body protein RPGRIP1L, large-scale SNP data. Bioinformatics 21(17):3565–3567. a nephrocystin-4 interactor, cause Joubert syndrome. Nat Genet 39(7):882–888. 4. Hoischen A, et al. (2010) De novo mutations of SETBP1 cause Schinzel–Giedion syndrome. 8. Kimmel CB, Ballard WW, Kimmel SR, Ullmann B, Schilling TF (1995) Stages of embryonic Nat Genet 42(6):483–485. development of the zebrafish. Dev Dyn 203(3):253–310.

Collin et al. www.pnas.org/cgi/content/short/1220864110 3of8 Fig. S1. Pedigree structure, linkage analysis and, segregation analysis of PASK and ZNF408 variants in autosomal dominant (ad) FEVR families. (A) Pedigrees of part of adFEVR family W05-215 and of family W05-220. Individuals of family W05-215 designated with an asterisk were selected for Illumina 700K SNP genotypingfollowedby linkage analysis, whereas two individuals designated with # were selected for exome sequencing. Genealogical studies in families W05-215 and W05-220 revealed common ancestors born in the late 17th century, two generations above generation I of both families. For two candidate variants in PASK and ZNF408 that remained after linkage analysis and exome sequencing, the segregation analysis is shown for all available relatives. M/+ indicates heterozygous carriers of the mutation, and +/+ indicates individuals with two WT alleles. No clinical information was available for the deceased individuals, except for IV:8. (B) Genome-wide LOD score calculations, using the 700K SNP array genotyping data from the individuals indicated with an asterisk in A. The two highest peaks, with LOD scores of 2.7, are indicated with arrows. In each of the two peaks, one candidate variant from the exome sequencing was present: in PASK on chromosome 2 and in ZNF408 on chromosome 11.

Collin et al. www.pnas.org/cgi/content/short/1220864110 4of8 Fig. S2. Pedigrees and chromosome 11p11.2 haplotype analysis of FEVR families W05-215 and W05-220. CA-marker and SNP analysis of chromosome 11p11.2 revealed identical haplotypes (in orange) encompassing a 9.9-Mb genomic region in FEVR patients from families W05-215 and W05-220. This haplotype carries the ZNF408 c.1363T variant. Genealogic studies revealed common ancestors born in the late 17th century, two generations above generation 1 of both families. Question marks in the haplotypes denote inconclusive or untested markers. The question mark for individual IV:2 in family W05-220 indicates that this individual was not studied in sufficient detail to establish or rule out FEVR. Nonpenetrance (individuals V:1 and V:4 of family W05-215) is not uncommon in autosomal dominant FEVR.

Collin et al. www.pnas.org/cgi/content/short/1220864110 5of8 Fig. S3. ZNF408 mRNA expression analysis. (A) Quantitative RT-PCR analysis of RNAs derived from several adult human tissues. The relative expression of the tissue with the least abundant ZNF408 expression was set at 1.0. (B) Similar to A, the relative ZNF408 expression was tested in fetal tissues as well. Total RNA was isolated from several adult and fetal tissues, and cDNA synthesis and quantitative RT-PCR analysis were performed as described previously (1). ZNF408 cDNA was amplified using the following primers: forward, 5′-GCTGTGCCACCTAAAGAAGC-3′; reverse, 5′-GGGCTTTGAAGCTCTCCTCT-3′. GUSB was amplified to serve as a reference.

1. Banka S, et al. (2011) Identification and characterization of an inborn error of metabolism caused by dihydrofolate reductase deficiency. Am J Hum Genet 88(2):216–225.

Fig. S4. Sequence comparison of ZNF408 proteins from several vertebrate species surrounding the Ser126 residue identified as altered in one additional individual with FEVR. The alignment contains the mutated amino acid (in bold type) and 10 flanking amino acids on each side. Identical residues are black on a white background, whereas nonidentical residues are black on a gray background. The serine at position 126 is conserved up to mouse.

Collin et al. www.pnas.org/cgi/content/short/1220864110 6of8 Fig. S5. Position and functional characterization of znf408 MOs. (Upper) Structure of the zebrafish znf408 gene. The positions of the ATG and splice-blocking MOs are indicated by a yellow box and blue box, respectively. The positions of the primers used in the RT-PCR analysis are indicated (f1, r1, f2, and r2). (Lower) Characterization of znf408 splice morphants (2 dpf) by nested RT-PCR analysis. Skipping of exon 2 led to a 65-bp alteration, resulting in a PCR fragment of 236 bp instead of 301 bp.

Table S1. Variant calling and data filtering in exome analysis of two individuals with FEVR from family W05-215 No. of identical variants V:2 III:5 in both samples

Total variants called 23,644 24,180 — Exonic + splice site (±2 nt intronic) variants 12,695 12,450 — In-house database frequency <1% (dbSNP131) 452 391 — Nonsynonymous + splice site variants + frameshifts 282 240 — Quality filters applied (at least five variant reads; at least 15% variant reads) 219 182 19 Changes in line with autosomal dominant mode of inheritance 205 176 19

The largest reduction in candidate sequence variants is observed after exclusion of variants previously found in more than 1% of 1,151 exomes performed in-house. After all quality filters were applied, and all but variants that were present heterozygously in both individuals were excluded, only 19 candidate variants remained.

Collin et al. www.pnas.org/cgi/content/short/1220864110 7of8 Table S2. Overview of candidate variants present in FEVR cases III:5 and V:2 from family W05-215 Reference Variant Occurrence of Genomic Reference Variant amino amino PhyloP in-house Chr position allele allele Gene Gene ID acid acid score dbSNP ID exome data

2 179,615,844 C G TTN NM_133379 K N 1.42 0 2 240,981,387 G A PRR21 NM_001080835 A V −0.21 0 2 242,077,452 A AA PASK NM_015148 V VX — 1 4 41,015,942 A G APBB2 NM_004307 Y H 2.75 rs34875102 3 5653,145C GCEP72 NM_018140 S R 0.07 rs62000998 8 5 140,308,137 G A PCDHAC1 NM_199509 V I 1.39 4 6 31,733,466 T C C6ORF27 NM_025258 T A −0.03 rs114810088 4 6 132,891,756 A G TAAR6 NM_175067 Y C 3.00 rs41298395 10 8 70,744,856 G A SLCO5A1 NM_030958 P L −1.30 rs61730155 6 11 46,726,613 C T ZNF408 NM_024741 H Y 5.99 0 11 46,903,329 C T LRP4 NM_002334 G E 1.54 0 11 51,516,191 G A OR4C46 NM_001004703 D N −0.01 10 11 58,477,400 T C GLYAT NM_201648 T A −0.71 0 11 108,160,416 T C ATM NM_000051 Y H 5.01 2 19 5,733,931 A C TMEM146 NM_152784 Q P −1.64 5 19 33,183,489 C T NUDT19 NM_001105570 T I −0.88 0 20 20,033,223 A T CRNKL1 NM_016652 S T 0.06 rs2273056 9 20 60,883,191 C T ADRM1 NM_007002 A V 4.22 rs45576934 8 20 62,200,738 G A PRIC285 NM_001037335 A V −1.02 rs6089924 8

Overview of high-confident variants in candidate genes present in both affected individuals from family W05-215 and occurring with a frequency of <1% in our in-house exome database of 1,151 individuals. Columns are (from left to right) chromosome (Chr), genomic position of the variant nucleotide basedon hg19, reference allele on the plus strand, variant allele on the plus strand, gene in which the variant is located, RefSeq gene identification number, reference amino acid, variant amino acid, and PhyloP log odds ratio for evolutionary conservation (PhyloP score). For variants that have been reported in dbSNP132, their SNP_ID is presented. The rightmost column indicates the occurrence of the variant in our in-house database of 1,151 FEVR-unrelated exome analyses. Gray shading indicates the six variants located within the two linkage peaks with the highest LOD scores (Fig. S1B).

Table S3. Missense variants within linkage intervals and evaluation of pathogenicity Predictions of pathogenicity

Gene Variant, cDNA Variant, protein PhyloP score PolyPhen SIFT Align GVGD

PRR21 c.1013C>T p.Ala338Val −0.21 Benign Not tolerated Class C0 ZNF408 c.1369C>T p.His455Tyr 5.99 Probably damaging Not tolerated Class C65 LRP4 c.2738G>A p.Gly913Glu 1.54 Benign Tolerated Class C0 ORC4C6 c.910G>A p.Asp304Asn −0.01 Benign Tolerated Class C0 GLYAT c.730A>G p.Thr244Ala −0.71 Benign Tolerated Class C0

Assessment of pathogenicity for the five missense variants that were present within the two linkage intervals and present in the exome data of both affected individuals III:5 and V:2 from family W05-215. PhyloP score indicates the evolutionary conservation at the nucleotide level. The software tools PolyPhen, SIFT, and Align GVGD were used to assess the pathogenicity of the amino acid sub- stitution. For Align GVGD, class C0 indicates that a change is unlikely to be pathogenic, whereas class C65 represents the highest likelihood of a change to be pathogenic. Only the change in ZNF408 (gray shading) has a high PhyloP score (a score >2 is considered potentially pathogenic) ánd is predicted to be pathogenic by all three software tools.

Table S4. Pathogenicity assessment of ZNF408 missense changes in FEVR probands Predictions of pathogenicity Mutation Mutation Frequency in ethnically (cDNA level) (protein level) matched control alleles PhyloP score PolyPhen SIFT Align GVGD c.377G>A p.Ser126Asn 0/382 1.65 Probably damaging Not tolerated Class C45 c.1363C>T p.His455Tyr 0/220 5.99 Probably damaging Not tolerated Class C65

Assessment of pathogenicity for the two ZNF408 missense variants identified on sequence analysis of family W05-215, family W05-220, and 132 additional individuals with FEVR. The c.377G>A and c.1363C>T changes were analyzed in Japanese and Dutch controls, respectively. PhyloP score indicates the evolu- tionary conservation at the nucleotide level. The software tools PolyPhen, SIFT, and Align GVGD were used to assess the pathogenicity of the amino acid substitution. For Align GVGD, class C0 indicates that a change is unlikely to be pathogenic, class C65 represents the highest likelihood of a change to be pathogenic, and class C45 indicates a moderate likelihood for the change to be pathogenic.

Collin et al. www.pnas.org/cgi/content/short/1220864110 8of8