Whole-genome sequencing in patients with uncovers a novel recurrent tandem duplication in IFT140 Véronique Geoffroy, Corinne Stoetzel, Sophie Scheidecker, Elise Schaefer, Isabelle Perrault, Séverine Bär, Ariane Kröll, Marion Delbarre, Manuela Antin, Anne-Sophie Leuvrey, et al.

To cite this version:

Véronique Geoffroy, Corinne Stoetzel, Sophie Scheidecker, Elise Schaefer, Isabelle Perrault, etal.. Whole-genome sequencing in patients with ciliopathies uncovers a novel recurrent tandem duplication in IFT140. Human Mutation, Wiley, 2018, 39 (7), pp.983-992. ￿10.1002/humu.23539￿. ￿hal-02371583￿

HAL Id: hal-02371583 https://hal.archives-ouvertes.fr/hal-02371583 Submitted on 19 Nov 2019

HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Human Mutation

Whole Genome Sequencing in patients with ciliopathies uncovers a novel recurrent tandem duplication in IFT140

Journal: Human Mutation Manuscript ForID humu-2018-0045 Peer Review Wiley - Manuscript type: Research Article

Date Submitted by the Author: 29-Jan-2018

Complete List of Authors: geoffroy, veronique; Laboratoire de Génétique Médicale, Institut de Génétique Médicale d'Alsace, INSERM U1112, Fédération de Médecine Translationnelle de Strasbourg (FMTS), Université de Strasbourg Stoetzel, Corinne; UMR_S INSERM U1112, IGMA, Laboratoire de Génétique médicale, Scheidecker, Sophie; UMR_S INSERM U1112, IGMA, Laboratoire de Génétique médicale, SCHAEFER, Elise; Hôpital de Hautepierre, GENETICS PERRAULT, Isabelle; INSERM U781, GENETICS Bär, Séverine; Department of Molecular and Cellular Genetics, UMR7156, Centre National de Recherche Scientifique (CNRS), Université de Strasbourg, Strasbourg, France Kröll, Ariane; UMR_S INSERM U1112, IGMA, Laboratoire de Génétique médicale, Delbarre, Marion; Hôpitaux universitaire de Strasbourg, Laboratoires de diagnostic génétique Antin, Manuela; Nouvel Hopital Civi, Laboratoire de Diagnostic Génétique Jaeger, Anne-Sophie; Hôpitaux universitaire de Strasbourg, Laboratoires de diagnostic génétique Henry, Charline; Inserm U1163-institut Imagine Blanché, Hélène; Fondation Jean-Dausset-Centre d'Etude du Polymorphisme Humain (CEPH), Institut de Génétique Moléculaire, Decker, Eva; Bioscentia, Kloth, Katja; Institut für Humangenetik, Universitätsklinikum Hamburg- Eppendorf, Hamburg, Germany Klaus, Günter; University Marburg, KfH-Nierenzentrum für Kinder und Jugendliche, Marburg, Germany Mache, Christopher; Graz, Martin-Coignard, dominique; CH le Mans, Service de Génétique Médicale McGinn, Steven; CNRGH, Institut de Biologie François Jacob, DRF, CEA, Evry, France Boland, Anne; CNG Deleuze, Jean-François; CNG Friant, Sylvie; Department of Molecular and Cellular Genetics, UMR7156, Centre National de Recherche Scientifique (CNRS), Université de Strasbourg, Strasbourg, France SAUNIER, Sophie; Université René Descartes, ; INSERM U-574, Hopital Necker, Enfants Malades

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1 2 3 4 ROZET, Jean-Michel; INSERM U781, GENETICS Bergmann, Carsten; Aachen University, Department of Human Genetics 5 Dollfus, Helene; UMR_S INSERM U1112, IGMA, Laboratoire de Génétique 6 médicale, 7 Muller, Jean; Hôpitaux universitaire de Strasbourg, Laboratoires de 8 diagnostic génétique; Laboratoire de Génétique Médicale, Institut de 9 Génétique Médicale d'Alsace, INSERM U1112, Fédération de Médecine 10 Translationnelle de Strasbourg (FMTS), Université de Strasbourg, 11 Mainzer-Saldino syndrome, IFT140, Structural variation, tandem 12 Key Words: duplication, whole-genome sequencing, Alu-mediated recombination, copy 13 number variation 14 15 16 17 18 19 For Peer Review 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Human Mutation Page 2 of 52

1 2 3 Whole Genome Sequencing in patients with ciliopathies 4 5 6 IFT140 7 uncovers a novel recurrent tandem duplication in 8 9 10 Véronique Geoffroy1,*, Corinne Stoetzel1,*, Sophie Scheidecker1,2, Elise Schaefer1,3, Isabelle 11 12 Perrault4, Séverine Bär5, Ariane Kröll1, Marion Delbarre2, Manuela Antin2, AnneSophie 13 14 2 6 7 8 9 10 15 Leuvrey , Charline Henry , Hélène Blanché , Eva Decker , Katja Kloth , Günter Klaus , 16 11 12 13 13 17 Christoph Mache , Dominique MartinCoignard , Steven McGinn , Anne Boland , Jean 18 19 François Deleuze7,13,For Sylvie Peer Friant5, Sophie Review Saunier6, JeanMichel Rozet4, Carsten 20 21 Bergmann8,14, Hélène Dollfus1,15, Jean Muller1,2 22 23 24 1Laboratoire de Génétique médicale, UMR_S INSERM U1112, IGMA, Faculté de Médecine 25 26 27 FMTS, Université de Strasbourg, Strasbourg, France

28 2 29 Laboratoires de Diagnostic Génétique, Hôpitaux Universitaires de Strasbourg, Strasbourg 30 31 Cedex, France 32 33 3Service de Génétique Médicale, Hôpitaux Universitaires de Strasbourg, Strasbourg, France. 34 35 4Laboratory of Genetics in Ophthalmology (LGO), INSERM UMR1163, Institute of Genetic 36 37 Diseases, Imagine, Paris Descartes University, 75015 Paris, France. 38 39 5 40 Department of Molecular and Cellular Genetics, UMR7156, Centre National de Recherche 41 42 Scientifique (CNRS), Université de Strasbourg, Strasbourg, France. 43 6 44 INSERM, U983, Paris Descartes University, Paris, France 45 46 7Centre d’études du polymorphisme humainFondation Jean Dausset, Paris, France 47 48 8Center for Human Genetics, Bioscientia, Ingelheim, Germany 49 50 9Institut für Humangenetik, Universitätsklinikum HamburgEppendorf, Hamburg, Germany 51 52 10University Marburg, KfHNierenzentrum für Kinder und Jugendliche, Marburg, Germany 53 54 11 55 Department of Pediatrics, Medical University of Graz, Graz, Austria. 56 57 58 59 1 60 John Wiley & Sons, Inc. Page 3 of 52 Human Mutation

1 2 12 3 Service de Génétique, Centre Hospitalier, CCLAD, Le Mans, France 4 13 5 CNRGH, Institut de Biologie François Jacob, DRF, CEA, Evry, France 6 7 14Department of Medicine, University Hospital Freiburg, Freiburg, Germany 8 9 15Centre de Référence pour les affections rares en génétique ophtalmologique, CARGO, 10 11 Filière SENSGENE, Hôpitaux Universitaires de Strasbourg, 67091 Strasbourg, France. 12 13 14 15 16 17 *equal contributor 18 19 For Peer Review 20 Corresponding Author: Jean Muller 21 22 23 Email address: [email protected] 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 2 60 John Wiley & Sons, Inc. Human Mutation Page 4 of 52

1 2 3 Abstract 4 5 6 Ciliopathies represent a wide spectrum of rare diseases with overlapping phenotypes and a 7 8 high genetic heterogeneity. Among those, IFT140 is implicated in a variety of phenotypes 9 10 ranging from isolated retinis pigmentosa to more syndromic cases such as the BardetBiedl 11 12 syndrome. Using whole genome sequencing in patients with uncharacterized ciliopathies, we 13 14 15 identified a novel recurrent tandem duplication of exon 27 to 30 (6.7 kb) in IFT140, c.3454 16 17 488_4182+2588dup p.(Tyr1152_Thr1394dup), missed by whole exome sequencing. 18 19 Pathogenicity of the mutationFor was Peer assessed on Review the patients’ skin fibroblasts. Several hundreds 20 21 of patients with a phenotype were screened and biallelic mutations were identified 22 23 in 11 families representing 12 pathogenic variants of which 7 are novel. Among those 24 25 unrelated families especially with a MainzerSaldino syndrome, 8 carried the same tandem 26 27 28 duplication (2 at the homozygous state and 6 at the heterozygous state). 29 30 In conclusion, we demonstrated the implication of structural variations in IFT140 related 31 32 diseases expanding its mutation spectrum. We also provide evidences for a unique genomic 33 34 event mediated by an Alu-Alu recombination occurring on a shared haplotype. We confirm 35 36 that whole genome sequencing can be instrumental in the ability to detect structural variants 37 38 for genomic disorders. 39 40 41 42 Keywords: IFT140, MainzerSaldino syndrome, structural variation, copy number variation, 43 44 Alu 45 tandem duplication, mediated recombination, wholegenome sequencing. 46 47 48 49 50 51 52 53 54 55 56 57 58 59 3 60 John Wiley & Sons, Inc. Page 5 of 52 Human Mutation

1 2 3 4 5 Background 6 7 8 MainzerSaldino syndrome (MSS, MIM 266920) is a rare (< 1/1,000,000) autosomal 9 10 recessive ciliopathy characterized by severe earlyonset retinal dystrophy, phalangeal cone 11 12 shaped epiphyses, chronic renal failure, and mild radiographic abnormality of the proximal 13 14 15 femur (Perrault, et al., 2012), known to be caused by IFT140 (Khan, et al., 2014; Perrault, et 16 17 al., 2012; Schmidts, et al., 2013) and IFT172 mutations (Halbritter, et al., 2013). Both 18 19 have been implicated Forin several Peerother ciliopathies Review ranging from isolated Retinis Pigmentosa 20 21 (RP) to more syndromic cases such as Jeune or BardetBiedl syndromes (Bifari, et al., 2016; 22 23 Bujakowska, et al., 2015; Schaefer, et al., 2016). 24 25 (IFT) genes are involved in a bidirectional (anterograde and 26 27 28 retrograde) transport process essential for the assembly and the maintenance of the cilia 29 30 through the redistribution of ciliary between the cell body and the . In 31 32 particular, IFT140 a component of the IFT complex A (IFTA) responsible of the retrograde 33 34 IFT, is vital for both the development and the maintenance of outer segments of 35 36 photoreceptors and has a specific role in opsin transport across the connecting cilium 37 38 (Blacque, et al., 2006; Crouse, et al., 2014) of the photoreceptors. Previously, 46 different 39 40 IFT140 41 mutations in 56 families have been reported in (Additional File 1: Table S1), 42 43 encompassing missenses, essential splice sites, stop and frameshifts mutations but no 44 45 structural variations (SV) such as copy number variant (CNV). Interestingly, 10 mono allelic 46 47 variants cases have also been reported with no second pathogenic allele detected to date 48 49 (Additional File 1: Table S1). 50 51 In this study, we report for the first time a structural variation (tandem duplication) in IFT140 52 53 54 identified by Whole Genome Sequencing (WGS) and missed by Whole Exome Sequencing 55 56 (WES). The impact of this mutation was further assessed in patients’ skin fibroblasts. Given 57 58 59 4 60 John Wiley & Sons, Inc. Human Mutation Page 6 of 52

1 2 3 the difficulty to identify this mutation and the genomic context surrounding the breakpoints, 4 5 we speculated that it might have been missed by other genetic screenings and that several 6 7 other families might carry this mutation. We finally report 8 unrelated families carrying the 8 9 mutation either at the homozygous or the heterozygous state, out of 11 families identified with 10 11 IFT140 biallelic mutations in total. Moreover, the characterization of the breakpoints allowed 12 13 14 to delineate a potential molecular mechanism and to design a specific duplex PCR that will 15 16 help screening further patients (including our cohorts). 17 18 19 For Peer Review 20 21 Materials and Methods 22 23 24 25 Subjects 26 27 28 Study protocols used in each cohort have been approved by the corresponding Institutional 29 30 Review Board or equivalent committees (as an example in Strasbourg, “Comité Protection des 31 32 Personnes” EST IV, N°DC20142222), and written informed consent was given from each 33 34 participant or parents. Our research complies with the Declaration of Helsinki. Written 35 36 informed consent for openaccess publication was provided by the participants or their 37 38 parents. DNA of additional affected and unaffected family members was requested whenever 39 40 41 it was considered informative. Skin fibroblasts were obtained for family A. Clinical data for 42 43 all 11 families are presented in Additional File 1: Table S2. 44 45 46 47 48 Whole Genome Sequencing 49 50 WGS was performed for the two affected siblings from family A (AII.1 and AII.2) and their 51 52 parents (AI.1 and AI.2) by the Centre National de Génotypage (Institut de Génomique, 53 54 CEA). Genomic DNA was used to prepare a library for whole genome sequencing, using the 55 56 Illumina TruSeq DNA PCRFree Library Preparation Kit, according to the manufacturer's 57 58 59 5 60 John Wiley & Sons, Inc. Page 7 of 52 Human Mutation

1 2 3 instructions. After normalization and quality control, qualified libraries have been sequenced 4 5 on a HiSeq2000 platform from Illumina (Illumina Inc., CA, USA), as pairedend 100 bp 6 7 reads. At least 3 lanes of HiSeq2000 flow cell have been produced for each sample, in order 8 9 to reach an average sequencing depth of 30x for each sample. Sequence quality parameters 10 11 have been assessed throughout the sequencing run and standard bioinformatics analysis of 12 13 14 sequencing data was based on the Illumina pipeline to generate FASTQ file for each sample. 15 16 The sequence reads were aligned to the reference sequence of the (GRCh37) 17 18 using the BurrowsWheeler Aligner (BWA V7.12) (Li and Durbin, 2010). The 19 For Peer Review 20 UnifiedGenotyper and HaplotypeCaller modules of the Genome Analysis ToolKit (GATK) 21 22 (DePristo, et al., 2011), Platypus (http://www.well.ox.ac.uk/platypus) and Samtools (Li, et al., 23 24 2009) were used for calling both single nucleotide variations (SNV) and small 25 26 27 insertion/deletion (indel). 28 29 30 31 Bioinformatics analysis 32 33 Annotation and ranking of SNV and indel were performed by VaRank (Geoffroy, et al., 2015) 34 35 in combination with the Alamut Batch software (Interactive Biosoftware, Rouen, France). 36 37 Very stringent filtering criteria were used for excluding nonpathogenic variants, in particular: 38 39 (1) variants represented with an allele frequency of more than 1% in public variation 40 41 42 databases either dbSNP 138 (Sherry, et al., 2001), the Exome Variant Server (NHLBI GO 43 44 Exome Sequencing Project, http://evs.gs.washington.edu/EVS/), the 1000Genomes (Genomes 45 46 Project, et al., 2015), the ExAC browser database(Lek, et al., 2016) or our internal exome 47 48 database, (2) variants in 5′ UTR, 3′ UTR, downstream, upstream or intronic locations without 49 50 pathogenic prediction of local splice effect, and (3) synonymous variants without prediction 51 52 of local splice effect. Variant effect on the nearest splice site was predicted using MaxEntScan 53 54 55 (Yeo and Burge, 2004), NNSplice (Reese, et al., 1997) and Splice Site Finder (Shapiro and 56 57 58 59 6 60 John Wiley & Sons, Inc. Human Mutation Page 8 of 52

1 2 3 Senapathy, 1987). Our analysis was focused on compound heterozygous and homozygous 4 5 variants (SNV/indel/SV) consistent with a recessive mode of transmission. Structural variants 6 7 were predicted using by default the CANOES program (Backenroth, et al., 2014) and 8 9 annotated thanks to our in house script AnnotSV (manuscript in preparation, 10 11 http://www.lbgi.fr/AnnotSV/) based on the classical annotations such as the Database of 12 13 IFT140 14 Genomic Variants (DGV) (MacDonald, et al., 2014). The nomenclature is based on 15 16 the accession number NM_014714.3 from the RefSeq database (O'Leary, et al., 2016). 17 18 Genomic coordinates are defined according to GRCh37/hg19 assembly downloaded from the 19 For Peer Review 20 University of California Santa Cruz (UCSC) genome browser (Tyner, et al., 2017). 21 22 23 24 Sanger validation and segregation 25 26 27 Sanger sequencing was performed by PCR amplification with 50 ng of genomic DNA 28 29 template. The primers were designed with Primer 3 (http://frodo.wi.mit.edu/primer3) and are 30 31 detailed in Additional File 1: Table S3. Bidirectional sequencing of the purified PCR products 32 33 was performed by GATC Sequencing Facilities (Konstanz, Germany). 34 35 36 37 qPCR quantification 38 39 Absolute quantification was performed using the SyberGreen Mastermix Quantitect (Qiagen) 40 41 42 measured on the LightCycler 480 (Roche). Amplicons were designed in exon 30 of IFT140 43 44 and compared to two reference genes (HBB and HMBS). Each samples have been done in 45 46 duplicate and standard deviation was <0.2. Standard curve have been established using 5 47 48 different DNA quantity (50ng, 25ng, 10ng, 5ng and 1ng) and PCR efficiency was assessed for 49 50 each amplicon (1.9

1 2 3 control genes was calculated for each patient and plotted. The primers were designed with 4 5 Primer 3 and are detailed in Additional File 1: Table S3 6 7 8 9 RNA analysis 10 11 RNA was extracted from skin fibroblasts of individual II.1 and a healthy unrelated control 12 13 14 using Rneasy RNA kit (Qiagen) then we performed reverse transcription using the iScriptTM 15 16 cDNA Synthesis Kit (BioRad, Hercules, CA). 17 18 19 For Peer Review 20 Cell culture 21 22 Fibroblasts of patients and control individuals were obtained by skin biopsy as previously 23 24 described (Scheidecker, et al., 2014). To induce primary cilium formation, cells were deprived 25 26 27 of serum by growth for 24 hrs in DMEM with 1% PSG but only 0.1% FCS (conditions FCS) 28 29 as previously described (Stoetzel, et al., 2016). 30 31

32 33 34 Immunofluorescence 35 36 Primary fibroblasts from patients and control individuals were grown in Nunc LabTek 37 38 chamber slides (Thermo Scientific) and ciliogenesis was done as described above. Primary 39 40 cilia were labelled with an antibody directed against acetylated atubulin highlighting the 41 42 axoneme41. Pictures were taken either on a fluorescence microscope (Figure 2A) or a 43 44 confocal microscope (Figure 2C). Primary and secondary antibodies used in this study as well 45 46 47 as their dilution are indicated in Additional File 1: Table S4. 48 49 50 51 Cohort screening 52 53 A duplex PCR was designed to specifically detect the tandem duplication. Primers and 54 55 example conditions are provided in Additional File 1: Table S3 and Figure S1. Additional 56 57 58 59 8 60 John Wiley & Sons, Inc. Human Mutation Page 10 of 52

1 2 3 targeted exome sequencing datasets have been reanalyzed (see Additional File 1: 4 5 supplementary Methods). 6 7 8 9 Alu pair analysis 10 11 We obtained the annotations and sequences for the two Alu repeat in the duplication junction 12 13 14 region from the RepeatMasker track of the UCSC genome browser (Tyner, et al., 2017). We 15 16 subsequently aligned the Alu sequences and computed the identity using the online tool 17 18 EMBOSS Matcher (Rice, et al., 2000). 19 For Peer Review 20 21 22 Results 23 24 25 Identification of biallelic mutations in IFT140 26 27 28 Two affected individuals born from healthy non consanguineous parents (family A, Figure 29 30 1A), named II.1 and II.2, were referred to our lab with retinitis pigmentosa, short stature 31 32 33 (152cm and 158cm respectively at 16 and 15yo), brachydactyly, moderate renal failure and 34 35 overweight for II.1 (Additional File 1: Table S2). This BardetBiedl like phenotype prompted 36 37 us to perform an extensive first genetic analysis including Sanger sequencing of recurrent 38 39 BBS mutations (Muller, et al., 2010), targeted exome sequencing (Redin, et al., 2012) and 40 41 WES without success. Indeed no biallelic mutations including copy number variant could be 42 43 identified (see Additional File 1: Table S5). Hence, considering that affected individuals from 44 45 46 family A may harbor mutations in a region not (well) covered by the WES (i.e. deep intronic, 47 48 promotor…), we applied WGS to the 2 affected individuals and their parents. A combined 49 50 analysis including both SNV/indel and structural variant revealed biallelic mutations in 51 52 IFT140, a gene known to be responsible of several ciliopathies (ranging from isolated RP to 53 54 more syndromic cases such as Jeune or MainzerSaldino syndromes) compatible with the 55 56 phenotype of the affected patients. 57 58 59 9 60 John Wiley & Sons, Inc. Page 11 of 52 Human Mutation

1 2 3 First, the two affected individuals were found heterozygous for a possible splice mutation in 4 5 intron 20, c.2577+25G>A, predicted to have a local splice effect as a new strong donor site. 6 7 This variant was reported previously and is not present in any variation database (e.g. ExAC 8 9 or 1000G). Cosegregation analysis revealed the paternal inheritance (Figure 1A, primers used 10 11 are detailed in Additional File 1: Table S3). Patient’s RNA analysis confirmed that a novel 12 13 14 donor splice site within the intron 20 resulted in the incorporation of 21 additional bp 15 16 (r.2577_2578insGTGAGGGGCGCCCGCCATGGG) that are predicted to add 7 new amino 17 18 acids to the sequence (p.Leu859_Glu860insValArgGlyAlaArgHisGly) (Figure 1B). 19 For Peer Review 20 21 Second, in trans to this mutation, structural variant analysis revealed a maternally inherited 22 23 (Figure 1A) duplication of exons 27 to 30 (of sizes 6.7 kb). Analysis of the mapped reads 24 25 revealed that the duplication is occurring in tandem in direct orientation within the gene 26 27 (Additional File 1: Figure S2) with breakpoints located in intron 26 and intron 30. This 28 29 tandem duplication has not been reported previously in patients and is not present in DGV. 30 31 The results were confirmed by qPCR (Figure 1C) delineating the expected maternal 32 33 34 inheritance. The duplication is predicted to be in frame and to add 243 amino acids within the 35 36 tetratricopeptide repeat (TPR) domain of the IFT140 protein (c.3454488_4182+2588dup, 37 38 p.Tyr1152_Thr1394dup). RNA analysis revealed that the duplicated exons are transcribed 39 40 (Figure 1D). Western blot analysis revealed only a single band of the approximate size of the 41 42 wild type protein suggesting that the allele carrying the duplication is not detected as a protein 43 44 (Figure 1E). 45 46 47 Immunofluorescence analysis in patients fibroblasts revealed that IFT140 is mislocalized 48 49 compared to sex/age matched control cells. Indeed, when grown in ciliary conditions, patient 50 51 52 skin fibroblasts developed primary cilia less often (Figure 2A). Thus while 75% of control 53 54 cells developed a primary cilium, only about 5055% of patient cells did so (Figure 2B). It is 55 56 to notice that primary cilia did not seem to be altered since when cells were deprived of 57 58 59 10 60 John Wiley & Sons, Inc. Human Mutation Page 12 of 52

1 2 3 serum, fixed for immunofluorescence and labeled with an antiacetylated tubulin antibody, 4 5 their cilium as observed by fluorescence microscope and measured with image J (Schneider, 6 7 et al., 2012) was about 22,5 µm both in control and patient cells (Figure 2B). Nevertheless, a 8 9 difference in organisation could be noticed when colabelling was performed with an anti 10 11 IFT140 antibodies (Figure 2C). Then, localisation of IFT140 at the base of the cilium could 12 13 14 be observed using confocal microscopy in 82% of ciliated control cells but only in about 14% 15 16 of ciliated patient fibroblasts (Figure 2D). Thus, the mutations found in the patients lead to a 17 18 significant decrease of cilium formation and a loss of IFT140 localisation at the base of the 19 For Peer Review 20 cilium. 21 22 23 Mutation screening in a large cohort 24 25 26 To explore additional cases of this duplication possibly missed by prior analysis, we 27 28 retrospectively screened our cohorts and reanalyzed available high throughput sequencing 29 30 panels including the IFT140 gene. As mutations in IFT140 are known to cause isolated to 31 32 syndromic retinal degeneration (Bifari, et al., 2016), this included 126 patients using the 33 34 Leber panel of the Imagine institute, 117 patients using the RP panel of Strasbourg Hospital , 35 36 and 104 patients using the Ciliome panel of the Imagine institute (see Methods and Additional 37 38 39 File 1: methods online). We also setup a duplex PCR around the breakpoints (Additional File 40 41 1: Figure S1) and screened all our negative cases, including 207 from our BardetBiedl 42 43 cohort, 150 isolated RP and 40 IFT140 heterozygous patients. All together we identified 11 44 45 additional families positive for biallelic mutations in IFT140 (Figure 3 and Additional File 1: 46 47 Table S1) among which 10 families have a phenotype compatible with a Mainzer Saldino 48 49 syndrome and one patient presents with isolated retinitis pigmentosa. Among the MSS 50 51 52 patients, three patients presented a phenotype overlapping with another ciliopathy: two with 53 54 Jeune syndrome and one with Sensenbrenner syndrome. Eight of the 11 families carried the 55 56 exact same tandem duplication either at the heterozygous state (3 copies) or at the 57 58 59 11 60 John Wiley & Sons, Inc. Page 13 of 52 Human Mutation

1 2 3 homozygous state (4 copies) (Figure 3A, Additional File 1: Figure S3). Among the novel 4 5 mutations identified, we observed 2 missenses at the heterozygous state c.1319T>C 6 7 (p.Leu440Pro), c.2177C>T (p.Pro726Leu) that are both predicted deleterious (Additional File 8 9 1: supplementary Methods). Among the novel mutations we also identified another CNV, a 10 11 large deletion encompassing exon 27 to 29 (c.34541005_4040+737delinsCCC, Additional 12 13 14 File 1: Figure S4) that does not share the same breakpoints compared to the tandem 15 16 duplication. Thus to date, 120 pathogenic alleles have been reported for IFT140 of which the 17 18 tandem duplication represents 10 alleles. Interestingly this tandem duplication is the second 19 For Peer Review 20 most frequent mutation for IFT140affected patients. 21 22 23 Founder effect or recurrent mechanism? 24 25 26 Given the relatively high frequency (8/65 families) of this duplication in the patients 27 28 described with IFT140 mutations, we performed 3 additional explorations to assess whether 29 30 the duplication was an independent recurrent mechanism or the same mutational event 31 32 derived from a common ancestry suggesting a founder effect. First, we screened a French 33 34 cohort of 354 WGS of healthy individuals (FREX dataset), for which CNV analysis 35 36 (CANOES) as well as visual inspections of the bam files (IGV batch scripting 37 38 39 (Thorvaldsdottir, et al., 2013)) did not reveal any corresponding allele, as expected due to the 40 41 IFT140 diseases prevalence (e.g. MSS: 1/1,000,000). Second, we compared the haplotype at 42 43 the IFT140 from the different patients for which NGS data was available. In total 15 44 45 haplotypes, including 6 carrying the duplication, from 3 different families, were analyzed (see 46 47 Additional File 1: Table S6). We observed a shared haplotype between the duplication 48 49 carriers at the IFT140 locus, suggesting a founder effect. Third, we characterized the genomic 50 51 52 localization around the breakpoint junctions. Sanger sequencing confirmed the breakpoint’s 53 54 coordinates (Figure 1A and Additional File 1: Figure S2) and demonstrated that the exact 55 56 same breakpoint is shared among all patients tested (Family C was not investigated). The 57 58 59 12 60 John Wiley & Sons, Inc. Human Mutation Page 14 of 52

1 2 3 breakpoints overlapped two distinct repeated elements from the Alu family, AluJb and AluJr 4 5 (Figure 4A). Pairwise alignment of the two Alu sequences (Additional File 1: Figure S5) 6 7 revealed a conservation of 68% of identity that reached 81% in the junction region (Figure 8 9 4B). The tandem duplication is resulting in an Alu hybrid (Figure 4C) at both sides. Alu-Alu 10 11 recombination is a prominent mechanism underlying the formation of pathogenic SV 12 13 14 associated with distinct diseases (Boone, et al., 2014; Stankiewicz, et al., 2009). Altogether, 15 16 this led us to speculate that this tandem duplication is a rare event, which arises from a 17 18 common ancestor due to an Alu-Alu mediated genomic recombination. 19 For Peer Review 20 21 22 23 24 Discussion 25 26 27 The advent of next generation sequencing, especially Whole Exome Sequencing (WES), in 28 29 the past 10 years was a tremendous step that led to the identification of many mutations 30 31 32 accelerating the discovery of novel genes involved in human genetic diseases (Chong, et al., 33 34 2015). Nevertheless, the diagnostic yield of WES is plateauing between 25% to 50% 35 36 depending on the disease (Chong, et al., 2015; Taylor, et al., 2015) leaving many patients still 37 38 with no molecular diagnosis. The use of RNA sequencing has recently extended the 39 40 percentage by 10% to 66% (depending on the context: with or without a strong candidate by 41 42 prior DNA sequencing) by helping identifying unseen defects or interpreting variations found 43 44 45 in the WES (Cummings, et al., 2017; Kremer, et al., 2017). Lastly, Whole Genome 46 47 Sequencing (WGS) of human patients has proven to be an instrumental tool for identifying 48 49 the whole spectrum of genetic anomalies (Gilissen, et al., 2014) and will replace in a close 50 51 future other genetic screening. 52 53 In line with this, we applied a range of genetic screening including targeted sequencing, WES 54 55 and then finally WGS to unsolved patients affected with a ciliopathy. The combination of 56 57 58 59 13 60 John Wiley & Sons, Inc. Page 15 of 52 Human Mutation

1 2 3 WGS and CNV analysis was able to uncover a tandem duplication unseen by the WES and to 4 5 reconsider a distant splice site mutation. 6 7 Interestingly, the tandem duplication could be identified in family A only by using the WGS 8 9 and not the WES data. Indeed, CANOES failed to identify the event in one of the individuals 10 11 (Additional File 1: Table S7), which prevented us from considering this as a mutation of 12 13 14 interest but made us rather discard it as a false positive of the method. Thereupon, it is well 15 16 known that CNV detection from next generation sequencing (NGS) datasets is feasible and 17 18 many publication reported positive results either on gene panels or WES (de Ligt, et al., 2013; 19 For Peer Review 20 Redin, et al., 2012). However, this remains challenging (Tan, et al., 2014). WGS provides a 21 22 much more uniform distribution of sequencingquality parameters and by nature does not 23 24 have the restriction of noncontiguous regions of interests (captured exons). Therefore, it is 25 26 27 more suited for SV identification (Belkadi, et al., 2015). Having that in mind, we further 28 29 tested the CNV detection using 2 other popular programs (see Additional File 1: Table S7) 30 31 which failed to detect the tandem duplication in family A patients (Additional File 1: Table 32 33 S7). These results confirmed many efforts are still required to ensure a high quality CNV 34 35 detection from NGS data, even with the large number of programs and methods available. 36 37 Nevertheless, thanks to the split reads from WGS data, once a CNV is detected one can easily 38 39 define the CNV breakpoints and further characterize the mechanism, as done in this study 40 41 42 (Additional File 1: Figure S2). 43 44 Analysis of the breakpoint positions and in particular the split reads allowed us to identify 45 46 47 overlapping Alu elements at the breakpoint junction. Alignment of the Alu regions 48 49 surrounding the junction revealed that both elements share 81% identity and exhibit a 50 51 microhomology region of 6 nucleotides. Because the recombination occurred between 52 53 homologous sequences (e.g. imperfect match substrates), it is most likely mediated by 54 55 mechanisms other than nonallelic homologous recombination, which are mostly recurrent 56 57 58 59 14 60 John Wiley & Sons, Inc. Human Mutation Page 16 of 52

1 2 3 events (NAHR, for review see (Carvalho and Lupski, 2016)). Among the possible molecular 4 5 mechanisms, that include nonhomologous end joining (NHEJ) and replicationbased 6 7 mechanisms such as breakinduced replication (BIR), microhomologymediated BIR 8 9 (MMBIR), serial replication slippage (SRS) and fork stalling and template switching 10 11 (FoSTeS), we believe that the presence of a microhomology region points most likely to a 12 13 14 MMBIR driven recombination. Given the nonrecurrent nature of those mechanisms, the 15 16 common haplotype and the exact same breakpoint junction observed in most of our patients, 17 18 we hypothesized that the genomic event that gave rise to this tandem duplication occurred 19 For Peer Review 20 once on a common IFT140 haplotype. 21 22 23 IFT140 is a 1462aminoacid protein encoded by 31 exons and composed of 5 WD repeats 24 25 and 9 tetratricopeptide repeats (TPR) both known to act as proteinprotein interaction domains 26 27 that might be involved in IFT140 interactions within the IFTA complex (Zhu, et al., 2017). 28 29 To date, 45 different pathogenic alleles have already been described throughout the IFT140 30 31 gene with no clustering or domain preference (Figure 4A). Most of them are private mutations 32 33 34 but several are found multiple times like the c.634G>A (p.Gly212Arg) (7 alleles) which has 35 36 been proven to affect the splicing of exon 6 (Helm, et al., 2017) or the c.2399+1G>T (5 37 38 alleles) (Additional File 1: Table S1). The most frequent mutation (31 alleles) is a missense 39 40 (c.1990G>A, p.Glu664Lys) that has been observed in multiple studies and especially in 11 41 42 consanguineous families from the Arabian Peninsula sharing a common ancestor (Bifari, et 43 44 al., 2016) which might bias the allele count. Nevertheless, the tandem duplication described in 45 46 47 our study is the second most frequent cause of mutation in IFT140 representing 10 alleles. 48 49 In family A, the two mutations identified required extensive and careful analysis to be 50 51 considered. One mutation was predicted to affect the splicing of exon 20 (c.2577+25G>A) 52 53 which was confirmed at the RNA level (r.2577_2578insGTGAGGGGCGCCCGCCATGGG), 54 55 and suggested to add 7 amino acid to the protein sequence 56 57 58 59 15 60 John Wiley & Sons, Inc. Page 17 of 52 Human Mutation

1 2 3 (p.Leu859_Glu860insValArgGlyAlaArgHisGly). The tandem duplication of exons 27 to 30 4 5 (c.3454488_4182+2588dup, p.Tyr1152_Thr1394dup) is predicted to add 243 amino acids 6 7 within the TPR repeat domain. Skin fibroblast analysis revealed RNA synthesis in the patient 8 9 cells but no protein could be identified by Western Blot at the predicted size. We hypothesize 10 11 that the protein was not stable enough to maintain its 3D structure. Functional analysis on the 12 13 14 patients’ cells further revealed a reduced level of ciliated cells and mislocalization of the 15 16 IFT140 mutant away from the cilia base. 17 18 The Mainzer Saldino syndrome is a syndrome characterized by skeletal phenotype with 19 For Peer Review 20 21 phalangeal coneshaped epiphyses, chronic renal disease, and retinal dystrophy. Overlapping 22 23 phenotypes have been described with other skeletal ciliopathies such as the Jeune or 24 25 Sensenbrenner syndrome. In our cohort the diagnosis of MSS was not made in all patients 26 27 before the molecular analysis. However, all of them except one with an isolated retinitis 28 29 pigmentosa, fulfilled the criteria for this diagnosis after a new careful clinical and radiological 30 31 examination. Indeed, 11 patients presented digits anomalies, including 8 with phalangeal 32 33 34 coneshaped epiphyses. Nine patients developed renal failure among which 7 have a severe 35 36 and early onset renal disease (1.5 years old to 23 years old) as previously described 37 38 (Schmidts, et al., 2013). The retinal dystrophy is constantly reported with a highly variable 39 40 age of onset. Among our cohort, a single patient presented an isolated retinitis pigmentosa (as 41 42 previously reported by others (Hull, et al., 2016)). Two patients have thoracic dystrophy 43 44 which is a clinical criteria of Jeune syndrome and one patient has a craniosynostosis 45 46 47 compatible with a Sensenbrenner syndrome. These descriptions illustrate well the overlap 48 49 existing between the different ciliopathies associated with mutation in IFT140. On a 50 51 molecular level, no genotypephenotype correlation could be established in our cohort, 52 53 especially regarding the patients carrying the duplication at the homozygous or the 54 55 heterozygous state. 56 57 58 59 16 60 John Wiley & Sons, Inc. Human Mutation Page 18 of 52

1 2 3 Conclusions 4 5 IFT140. 6 In summary, we report here 11 novel unrelated families with mutations in Among 7 8 them, 8 families carry a recurrent tandem duplication of 4 exons either at the heterozygous 9 10 state or the homozygous state, for which we have assessed the pathogenicity in the pateints 11 12 cells. This is the first time that a structural variation is reported in IFT140 expanding the 13 14 mutation spectrum for this gene. Notably, this large duplication was missed by the WES 15 16 analysis but uncoverd thanks to the whole genome, pointing out the power of such analysis. 17 18 19 Availability of data andFor material Peer Review 20 21 22 23 Data generated or analyzed during this study are included in the published article and the 24 25 corresponding supplementary data. The raw sequencing data generated in the course of this 26 27 study are not publicly available due to the protocol and the corresponding consents used that 28 29 did not include such information. All variants have been submitted to ClinVar 30 31 (https://www.ncbi.nlm.nih.gov/clinvar/). Anonymised NGS data and genomic variant data 32 33 files will be made available upon request from qualified investigators studying the molecular 34 35 36 basis of genomic disorders. Datasets can be obtained via the corresponding author on 37 38 reasonable request. 39 40 41 Acknowledgements 42 43 44 We would like to thank the patients and their family for their participation. We thank Daniel 45 46 Backenroth and Olivier Quenez for their help in the bioinformatics setup of CANOES, 47 48 Arnaud Kress and Antony Le Béchec for informatic support, Amélie Piton, Bénédicte Gérard 49 50 and Ilia Humbert for scientific discussion. We also thank Emmanuelle Génin, Pierre 51 52 Lindenbaum and Richard Redon for giving us access to the genome dataset from the FREX 53 54 55 56 57 58 59 17 60 John Wiley & Sons, Inc. Page 19 of 52 Human Mutation

1 2 3 project and the Genomic and Bioinformatic Platform of Institut Imagine (Patrick Nitschké, 4 5 and Cécile Masson). 6 7 8 Disclosure Statment 9 10 11 Eva Decker and Carsten Bergmann are employees of Bioscientia/Sonic Healthcare. Günter 12 13 Klaus benefited from a travel grants from The Apheresis Research Institute Cologne, the 14 15 DGFF (Die Deutsche Gesellschaft zur Bekämpfung von Fettstoffwechselstörungen und ihren 16 17 Folgeerkrankungen) and is consultant to Vifor pharma, Switzerland concerning a pediatric 18 19 phosphat binder trial. For Peer Review 20 21 22 23 Funding Information 24 25 The WGS research was supported by the Laboratory of Excellence GENMED (Medical 26 27 Genomics) grant no. ANR10LABX0013 managed by the National Research Agency 28 29 30 (ANR) part of the Investment for the Future program. Whole exome sequencing was 31 32 performed by the IGBMC Microarray and Sequencing platform, a member of the ‘France 33 34 Génomique’ consortium (ANR10INBS0009) and funded by “La Fondation Maladie Rare”. 35 36 The RP panel is supported by the French program PHRC I 2013 HUS N° 5724 and by grants 37 38 from the Retina France to IP, UNADEV AVIESAN ITMO MNP to JMR. In addition, CB 39 40 holds a parttime faculty appointment at the University of Freiburg. His research lab receives 41 42 43 support from the Deutsche Forschungsgemeinschaft (DFG) Collaborative Research Centre 44 45 (SFB) KIDGEM 1140 and the Federal Ministry of Education and Research (BMBF, 46 47 01GM1515C). 48 49 50 51 52 53 54 55 56 57 58 59 18 60 John Wiley & Sons, Inc. Human Mutation Page 20 of 52

1 2 3 References 4 5 6 7 Backenroth D, Homsy J, Murillo LR, Glessner J, Lin E, Brueckner M, Lifton R, Goldmuntz 8 E, Chung WK, Shen Y. 2014. CANOES: detecting rare copy number variants from whole 9 exome sequencing data. Nucleic Acids Res 42(12):e97. 10 Belkadi A, Bolze A, Itan Y, Cobat A, Vincent QB, Antipenko A, Shang L, Boisson B, 11 Casanova JL, Abel L. 2015. Wholegenome sequencing is more powerful than wholeexome 12 sequencing for detecting exome variants. Proc Natl Acad Sci U S A 112(17):54738. 13 Bifari IN, Elkhamary SM, Bolz HJ, Khan AO. 2016. The ophthalmic phenotype of IFT140 14 related ciliopathy ranges from isolated to syndromic congenital retinal dystrophy. Br J 15 Ophthalmol 100(6):82933. 16 Blacque OE, Li C, Inglis PN, Esmail MA, Ou G, Mah AK, Baillie DL, Scholey JM, Leroux 17 MR. 2006. The WD repeatcontaining protein IFTA1 is required for retrograde intraflagellar 18 transport. Mol Biol Cell 17(12):505362. 19 For Peer Review 20 Boone PM, Yuan B, Campbell IM, Scull JC, Withers MA, Baggett BC, Beck CR, Shaw CJ, 21 Stankiewicz P, Moretti P and others. 2014. The Alurich genomic architecture of SPAST 22 predisposes to diverse and functionally distinct diseaseassociated CNV alleles. Am J Hum 23 Genet 95(2):14361. 24 Bujakowska KM, Zhang Q, Siemiatkowska AM, Liu Q, Place E, Falk MJ, Consugar M, 25 Lancelot ME, Antonio A, Lonjou C and others. 2015. Mutations in IFT172 cause isolated 26 retinal degeneration and BardetBiedl syndrome. Hum Mol Genet 24(1):23042. 27 Carvalho CM, Lupski JR. 2016. Mechanisms underlying structural variant formation in 28 genomic disorders. Nat Rev Genet 17(4):22438. 29 Chong JX, Buckingham KJ, Jhangiani SN, Boehm C, Sobreira N, Smith JD, Harrell TM, 30 McMillin MJ, Wiszniewski W, Gambin T and others. 2015. The Genetic Basis of Mendelian 31 Phenotypes: Discoveries, Challenges, and Opportunities. Am J Hum Genet 97(2):199215. 32 33 Crouse JA, Lopes VS, Sanagustin JT, Keady BT, Williams DS, Pazour GJ. 2014. Distinct 34 functions for IFT140 and IFT20 in opsin transport. Cytoskeleton (Hoboken) 71(5):30210. 35 Cummings BB, Marshall JL, Tukiainen T, Lek M, Donkervoort S, Foley AR, Bolduc V, 36 Waddell LB, Sandaradura SA, O'Grady GL and others. 2017. Improving genetic diagnosis in 37 Mendelian disease with transcriptome sequencing. Sci Transl Med 9(386). 38 de Ligt J, Boone PM, Pfundt R, Vissers LE, Richmond T, Geoghegan J, O'Moore K, de 39 Leeuw N, Shaw C, Brunner HG and others. 2013. Detection of clinically relevant copy 40 number variants with wholeexome sequencing. Hum Mutat 34(10):143948. 41 DePristo MA, Banks E, Poplin R, Garimella KV, Maguire JR, Hartl C, Philippakis AA, del 42 Angel G, Rivas MA, Hanna M and others. 2011. A framework for variation discovery and 43 genotyping using nextgeneration DNA sequencing data. Nat Genet 43(5):4918. 44 Genomes Project C, Auton A, Brooks LD, Durbin RM, Garrison EP, Kang HM, Korbel JO, 45 46 Marchini JL, McCarthy S, McVean GA and others. 2015. A global reference for human 47 genetic variation. Nature 526(7571):6874. 48 Geoffroy V, Pizot C, Redin C, Piton A, Vasli N, Stoetzel C, Blavier A, Laporte J, Muller J. 49 2015. VaRank: a simple and powerful tool for ranking genetic variants. PeerJ 3:e796. 50 Gilissen C, HehirKwa JY, Thung DT, van de Vorst M, van Bon BW, Willemsen MH, Kwint 51 M, Janssen IM, Hoischen A, Schenck A and others. 2014. Genome sequencing identifies 52 major causes of severe intellectual disability. Nature 511(7509):3447. 53 Halbritter J, Bizet AA, Schmidts M, Porath JD, Braun DA, Gee HY, McInerneyLeo AM, 54 Krug P, Filhol E, Davis EE and others. 2013. Defects in the IFTB component IFT172 cause 55 Jeune and MainzerSaldino syndromes in humans. Am J Hum Genet 93(5):91525. 56 57 58 59 19 60 John Wiley & Sons, Inc. Page 21 of 52 Human Mutation

1 2 3 Helm BM, Willer JR, Sadeghpour A, Golzio C, Crouch E, Vergano SS, Katsanis N, Davis 4 EE. 2017. Partial uniparental isodisomy of 16 unmasks a deleterious biallelic 5 mutation in IFT140 that causes MainzerSaldino syndrome. Hum Genomics 11(1):16. 6 Hull S, Owen N, Islam F, TraceyWhite D, Plagnol V, Holder GE, Michaelides M, Carss K, 7 Raymond FL, Rozet JM and others. 2016. Nonsyndromic Retinal Dystrophy due to BiAllelic 8 Mutations in the Ciliary Transport Gene IFT140. Invest Ophthalmol Vis Sci 57(3):105362. 9 Khan AO, Bolz HJ, Bergmann C. 2014. Earlyonset severe retinal dystrophy as the initial 10 presentation of IFT140related skeletal ciliopathy. J AAPOS 18(2):2035. 11 Kremer LS, Bader DM, Mertes C, Kopajtich R, Pichler G, Iuso A, Haack TB, Graf E, 12 Schwarzmayr T, Terrile C and others. 2017. Genetic diagnosis of Mendelian disorders via 13 14 RNA sequencing. Nat Commun 8:15824. 15 Lek M, Karczewski KJ, Minikel EV, Samocha KE, Banks E, Fennell T, O'DonnellLuria AH, 16 Ware JS, Hill AJ, Cummings BB and others. 2016. Analysis of proteincoding genetic 17 variation in 60,706 humans. Nature 536(7616):28591. 18 Li H, Durbin R. 2010. Fast and accurate longread alignment with BurrowsWheeler 19 transform. BioinformaticsFor 26(5):58995. Peer Review 20 Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin 21 R, Genome Project Data Processing S. 2009. The Sequence Alignment/Map format and 22 SAMtools. Bioinformatics 25(16):20789. 23 MacDonald JR, Ziman R, Yuen RK, Feuk L, Scherer SW. 2014. The Database of Genomic 24 Variants: a curated collection of structural variation in the human genome. Nucleic Acids Res 25 42(Database issue):D98692. 26 27 Muller J, Stoetzel C, Vincent MC, Leitch CC, Laurier V, Danse JM, Helle S, Marion V, 28 BennounaGreene V, Vicaire S and others. 2010. Identification of 28 novel mutations in the 29 BardetBiedl syndrome genes: the burden of private mutations in an extensively 30 heterogeneous disease. Hum Genet 127(5):58393. 31 O'Leary NA, Wright MW, Brister JR, Ciufo S, Haddad D, McVeigh R, Rajput B, Robbertse 32 B, SmithWhite B, AkoAdjei D and others. 2016. Reference sequence (RefSeq) database at 33 NCBI: current status, taxonomic expansion, and functional annotation. Nucleic Acids Res 34 44(D1):D73345. 35 Perrault I, Saunier S, Hanein S, Filhol E, Bizet AA, Collins F, Salih MA, Gerber S, Delphin 36 N, Bigot K and others. 2012. MainzerSaldino syndrome is a ciliopathy caused by IFT140 37 mutations. Am J Hum Genet 90(5):86470. 38 Redin C, Le Gras S, Mhamdi O, Geoffroy V, Stoetzel C, Vincent MC, Chiurazzi P, Lacombe 39 D, Ouertani I, Petit F and others. 2012. Targeted highthroughput sequencing for diagnosis of 40 41 genetically heterogeneous diseases: efficient mutation detection in BardetBiedl and Alstrom 42 syndromes. J Med Genet 49(8):50212. 43 Reese MG, Eeckman FH, Kulp D, Haussler D. 1997. Improved splice site detection in Genie. 44 J Comput Biol 4(3):31123. 45 Rice P, Longden I, Bleasby A. 2000. EMBOSS: the European Molecular Biology Open 46 Software Suite. Trends Genet 16(6):2767. 47 Schaefer E, Stoetzel C, Scheidecker S, Geoffroy V, Prasad MK, Redin C, Missotte I, 48 Lacombe D, Mandel JL, Muller J and others. 2016. Identification of a novel mutation 49 confirms the implication of IFT172 (BBS20) in BardetBiedl syndrome. J Hum Genet 50 61(5):44750. 51 Scheidecker S, Etard C, Pierce NW, Geoffroy V, Schaefer E, Muller J, Chennen K, Flori E, 52 Pelletier V, Poch O and others. 2014. Exome sequencing of BardetBiedl syndrome patient 53 54 identifies a null mutation in the BBSome subunit BBIP1 (BBS18). J Med Genet 51(2):1326. 55 Schmidts M, Frank V, Eisenberger T, Al Turki S, Bizet AA, Antony D, Rix S, Decker C, 56 Bachmann N, Bald M and others. 2013. Combined NGS approaches identify mutations in the 57 58 59 20 60 John Wiley & Sons, Inc. Human Mutation Page 22 of 52

1 2 3 intraflagellar transport gene IFT140 in skeletal ciliopathies with early progressive kidney 4 Disease. Hum Mutat 34(5):71424. 5 Schneider CA, Rasband WS, Eliceiri KW. 2012. NIH Image to ImageJ: 25 years of image 6 analysis. Nat Methods 9(7):6715. 7 Shapiro MB, Senapathy P. 1987. RNA splice junctions of different classes of eukaryotes: 8 sequence statistics and functional implications in gene expression. Nucleic Acids Res 9 15(17):715574. 10 Sherry ST, Ward MH, Kholodov M, Baker J, Phan L, Smigielski EM, Sirotkin K. 2001. 11 dbSNP: the NCBI database of genetic variation. Nucleic Acids Res 29(1):30811. 12 Stankiewicz P, Sen P, Bhatt SS, Storer M, Xia Z, Bejjani BA, Ou Z, Wiszniewska J, Driscoll 13 14 DJ, Maisenbacher MK and others. 2009. Genomic and genic deletions of the FOX gene 15 cluster on 16q24.1 and inactivating mutations of FOXF1 cause alveolar capillary dysplasia 16 and other malformations. Am J Hum Genet 84(6):78091. 17 Stoetzel C, Bar S, De Craene JO, Scheidecker S, Etard C, Chicher J, Reck JR, Perrault I, 18 Geoffroy V, Chennen K and others. 2016. A mutation in VPS15 (PIK3R4) causes a ciliopathy 19 and affects IFT20 releaseFor from the Peer cisGolgi. NatReview Commun 7:13586. 20 Tan R, Wang Y, Kleinstein SE, Liu Y, Zhu X, Guo H, Jiang Q, Allen AS, Zhu M. 2014. An 21 evaluation of copy number variation detection tools from wholeexome sequencing data. Hum 22 Mutat 35(7):899907. 23 Taylor JC, Martin HC, Lise S, Broxholme J, Cazier JB, Rimmer A, Kanapin A, Lunter G, 24 Fiddy S, Allan C and others. 2015. Factors influencing success of clinical genome sequencing 25 across a broad spectrum of disorders. Nat Genet 47(7):717726. 26 27 Thorvaldsdottir H, Robinson JT, Mesirov JP. 2013. Integrative Genomics Viewer (IGV): 28 highperformance genomics data visualization and exploration. Brief Bioinform 14(2):17892. 29 Tyner C, Barber GP, Casper J, Clawson H, Diekhans M, Eisenhart C, Fischer CM, Gibson D, 30 Gonzalez JN, Guruvadoo L and others. 2017. The UCSC Genome Browser database: 2017 31 update. Nucleic Acids Res 45(D1):D626D634. 32 Yeo G, Burge CB. 2004. Maximum entropy modeling of short sequence motifs with applications to 33 RNA splicing signals. J Comput Biol 11(2-3):377-94. 34 Zhu B, Zhu X, Wang L, Liang Y, Feng Q, Pan J. 2017. Functional exploration of the IFT-A complex in 35 intraflagellar transport and ciliogenesis. PLoS Genet 13(2):e1006627. 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 21 60 John Wiley & Sons, Inc. Page 23 of 52 Human Mutation

1 2 3 Figure legends 4 5 6 IFT140 7 Figure 1. Identification of 2 mutations in for a family explored by WGS. 8 9 (A) Pedigree of family A, which has 2 affected individuals. Segregation analysis of the 2 10 11 12 mutations noted M1: 2577+25G>A and M2: 3454488_4182+2588dup. Example of Sanger 13 14 sequencing profiles for individual II.1. The breakpoint junction between exon 30 and intron 15 16 26 are shown. 17 18 19 (B) M1 analysis. SangerFor sequencing Peer was performed Review on RNA extracted from fibroblasts of 20 21 individual II.1 and a healthy unrelated control amplified between exon 18 and exon 22. 22 23 24 (C) M2 analysis. Quantitative realtime PCR was performed on DNA from exon 30 in all 25 26 individuals from family A and one unrelated control. DNA quantity from exon 30 of IFT140 27 28 was compared to 2 reference genes (HBB and HMBS) using the absolute quantification 29 30 method. 31 32 33 (D) M2 RNA expression in patient’s II.1 skin fibroblasts is demonstrated by PCR 34 35 amplification (PRMT9 as a control) and by Sanger sequencing. 36 37 38 (E) IFT140 expression in skin fibroblasts was revealed by western blot using antiIFT140 39 40 antibody. 41 42 43 44 45 Figure 2. Patients’ fibroblasts have reduced number of ciliated cells and mislocalized 46 47 IFT140 48 49 50 (A) Number of ciliated cells were counted on serum deprived (24H) control and patient’s skin 51 52 fibroblasts fixed for immunofluorescence and stained with antiIFT140 (red) and anti 53 54 acetylated tubulin (green) antibodies. Nuclei were colored in blue (Dapi). 55 56 57 58 59 22 60 John Wiley & Sons, Inc. Human Mutation Page 24 of 52

1 2 3 (B) Based on 15 fields in three independent experiments (100200 cells per experiment), 4 5 mean percentages of ciliated cells are shown in a histogram together with corresponding 6 7 standard deviation and pvalues (n.s.: nonsignificant, *: p< 0,0001). 8 9 10 (C) IFT140 localisation was assessed on serum deprived (24H) control and patient’s skin 11 12 fibroblasts fixed for immunofluorescence and stained with antiIFT140 (red) and anti 13 14 acetylated tubulin (green) antibodies. Nuclei were colored in blue (Dapi). 15 16 17 (D) Primary cilia containing IFT140 were counted based on three independent experiments 18 19 (60200 ciliated cells Forper experiment), Peer mean percentagesReview are shown in a histogram together 20 21 with corresponding standard deviation and pvalues (n.s.: nonsignificant, *: p<0,0005). 22 23 24 25 26 Figure 3. Mutations in IFT140 27 28 29 (A) Pedigrees of 7 additional families with affected individuals carrying the tandem 30 31 duplication. 32 33 34 (B) Pedigrees of additional families with IFT140 pathogenic variants. 35 36 37 38 39 Figure 4. Schematic representation of IFT140 and of the tandem duplication 40 41 42 (A) IFT140 extends over 105.6 kb in chromosomal region 16p13.3 and contains 31 exons 43 44 encoding a 1462 amino acids protein containing two repeated domains, 5 WD repeats and 9 45 46 tetratricopeptide (TPR) repeats. Previously reported mutations in IFT140 are represented 47 48 using a black “asterisk” whereas novel mutations are colored in red. In particular, the two 49 50 51 mutations from family A are also shown in introns 20 (c.2577+25G>A) and 30 (c.3454 52 53 490_4182+2588dup). The tandem duplication (black horizontal bar) spans from exon 27 to 30 54 55 (6.7 kb). The breakpoints are located within Alu elements (AluJb and AluJr). 56 57 58 59 23 60 John Wiley & Sons, Inc. Page 25 of 52 Human Mutation

1 2 3 (B) Breakpoint junction sequence of affected individuals with tandem duplication. Breakpoint 4 5 junction sequence is aligned to the proximal and distal genomic references, respectively AluJr 6 7 (chr16:15655701565697) and AluJb (chr16:15712391571365), and colormatched. 8 9 Microhomology at the breakpoint is indicated in red. The aligned genomic region is 128 bp 10 11 long showing 81% of sequence identity. 12 13 14 (C) Proposed rearrangement of AluAlu mediated duplication in affected individuals. 15 16 17 18 19 For Peer Review 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 24 60 John Wiley & Sons, Inc. Human Mutation Page 26 of 52

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 For Peer Review 20 21 22 23 24 25 Figure 1. Identification of 2 mutations in IFT140 for a family explored by WGS. 26 (A) Pedigree of family A, which has 2 affected individuals. Segregation analysis of the 2 mutations noted 27 M1: 2577+25G>A and M2: 3454-488_4182+2588dup. Example of Sanger sequencing profiles for individual 28 II.1. The breakpoint junction between exon 30 and intron 26 are shown. 29 (B) M1 analysis. Sanger sequencing was performed on RNA extracted from fibroblasts of individual II.1 and 30 a healthy unrelated control amplified between exon 18 and exon 22. (C) M2 analysis. Quantitative real-time PCR was performed on DNA from exon 30 in all individuals from 31 family A and one unrelated control. DNA quantity from exon 30 of IFT140 was compared to 2 reference 32 genes (HBB and HMBS) using the absolute quantification method. 33 (D) M2 RNA expression in patient’s II.1 skin fibroblasts is demonstrated by PCR amplification (PRMT9 as a 34 control) and by Sanger sequencing. 35 (E) IFT140 expression in skin fibroblasts was revealed by western blot using anti-IFT140 antibody. 36

37 297x166mm (300 x 300 DPI) 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Page 27 of 52 Human Mutation

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 For Peer Review 20 21 22 23 24 25 26 27 28 Figure 2. Patients’ fibroblasts have reduced number of ciliated cells and mislocalized IFT140 29 (A) Number of ciliated cells were counted on serum deprived (24H) control and patient’s skin fibroblasts 30 fixed for immunofluorescence and stained with anti-IFT140 (red) and anti-acetylated tubulin (green) antibodies. Nuclei were colored in blue (Dapi). 31 (B) Based on 15 fields in three independent experiments (100-200 cells per experiment), mean percentages 32 of ciliated cells are shown in a histogram together with corresponding standard deviation and p-values (n.s.: 33 non-significant, *: p< 0,0001). 34 (C) IFT140 localisation was assessed on serum deprived (24H) control and patient’s skin fibroblasts fixed for 35 immunofluorescence and stained with anti-IFT140 (red) and anti-acetylated tubulin (green) antibodies. 36 Nuclei were colored in blue (Dapi). (D) Primary cilia containing IFT140 were counted based on three independent experiments (60-200 ciliated 37 cells per experiment), mean percentages are shown in a histogram together with corresponding standard 38 deviation and p-values (n.s.: non-significant, *: p<0,0005). 39 40 220x141mm (300 x 300 DPI) 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Human Mutation Page 28 of 52

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 For Peer Review 20 21 22 23 24 25 26 Figure 3. Mutations in IFT140 27 (A) Pedigrees of 7 additional families with affected individuals carrying the tandem duplication. 28 (B) Pedigrees of additional families with IFT140 pathogenic variants. 29 558x325mm (300 x 300 DPI) 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Page 29 of 52 Human Mutation

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 For Peer Review 20 21 22 23 24 25 26 27 28 Figure 4. Schematic representation of IFT140 and of the tandem duplication 29 (A) IFT140 extends over 105.6 kb in chromosomal region 16p13.3 and contains 31 exons encoding a 1462 30 amino acids protein containing two repeated domains, 5 WD repeats and 9 tetratricopeptide (TPR) repeats. 31 Previously reported mutations in IFT140 are represented using a black “asterisk” whereas novel mutations 32 are colored in red. In particular, the two mutations from family A are also shown in introns 20 33 (c.2577+25G>A) and 30 (c.3454-490_4182+2588dup). The tandem duplication (black horizontal bar) spans 34 from exon 27 to 30 (6.7 kb). The breakpoints are located within Alu elements (AluJb and AluJr). (B) Breakpoint junction sequence of affected individuals with tandem duplication. Breakpoint junction 35 sequence is aligned to the proximal and distal genomic references, respectively AluJr (chr16:1565570- 36 1565697) and AluJb (chr16:1571239-1571365), and color-matched. Microhomology at the breakpoint is 37 indicated in red. The aligned genomic region is 128 bp long showing 81% of sequence identity. 38 (C) Proposed rearrangement of Alu-Alu mediated duplication in affected individuals. 39 40 232x153mm (300 x 300 DPI) 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Human Mutation Page 30 of 52

1 2 3 SUPPLEMENTARY METHODS 4 5 Whole Exome Sequencing 6 7 Whole Exome Sequencing (WES) was performed for the two affected siblings from family A 8 9 (AII.1 and AII.2) by the IGBMC (Institute of Genetics and Molecular and Cellular Biology) 10 11 Microarray and Sequencing platform. Exons of DNA samples were captured with insolution 12 13 14 enrichment methodology (Agilent SureSelect All Exon XT2 50 Mb Kit) and sequenced with 15 16 an Illumina HiSeq 2500 instrument as pairedend 100 bp reads, in order to reach an average 17 18 sequencing depth of 80x for each sample. The sequence reads were aligned to the reference 19 For Peer Review 20 sequence of the human genome (GRCh37) using the BurrowsWheeler Aligner (BWA V7.12) 21 22 (Li and Durbin, 2010). The HaplotypeCaller module of the Genome Analysis ToolKit 23 24 (GATK, v.3.4.46) (DePristo, et al., 2011) was used for calling both SNV and indel. 25 26 27 28 29 Targeted Exome Sequencing 30 31 Leber panel 32 33 Targeted exome sequencing of 14 full genes related to Leber congenital amaurosis was 34 35 performed for 126 patients (including family B, C, D, E and I). Illumina compatible 36 37 precapture barcoded genomic DNA libraries were constructed according to the 38 39 manufacturer’s sample preparation protocol (Ovation Ultralow, Nugen Technologies). Exons 40 41 42 of DNA samples were captured with insolution enrichment methodology (Agilent SureSelect 43 44 custom panel) and sequenced with an Illumina HiSeq2500 (PairedEnd sequencing 2x125 45 46 bases, Rapid Mode, 80 libraries per lane). Bioinformatics analysis was performed as described 47 48 in Gerber et al (Gerber, et al., 2016) using an in house pipeline (POLYWEB). 49 50 51 52 53 54 55 56 57 58 59 1 60 John Wiley & Sons, Inc. Page 31 of 52 Human Mutation

1 2 3 RP panel 4 5 Targeted exome sequencing of 267 genes related to retinitis pigmentosa was performed for 6 7 117 patients (including family J). Exons of DNA samples were captured with insolution 8 9 enrichment methodology (Agilent QXT SureSelect custom panel) and sequenced with an 10 11 Illumina NextSeq 550 instrument (PairedEnd sequencing 2x150 bases, 48 libraries per lane). 12 13 14 SNVs and indels were called with the Genome Analysis Toolkit v.3.4.46 thanks to our in 15 16 house pipeline (STARK) and following the GATK best practice. 17 18 19 For Peer Review 20 Ciliome panel 21 22 Targeted exome sequencing of 1221 ciliary candidate genes including genes related to 23 24 ciliopathies like Jeune asphyxiating thoracic dystrophy, MainzerSaldino syndrome, Bardet 25 26 27 Biedl syndrome, Joubert syndrome and Senior Loken was performed for 364 patients 28 29 (including families C, D, I and K). Sequencing was conducted using a custom SureSelect 30 31 capture kit (Agilent Technologies) targeting 4.5 Mb of 20,168 exons, and performed on 32 33 SOLiD5500XL (Life Technologies) or HiSeq (Illumina). Bioinformatics analysis was 34 35 performed as described in Grampa et al (Grampa, et al., 2016) using an in house pipeline 36 37 (POLYWEB).The mean depth of coverage obtained was greater than 165x, with ≥89% of the 38 39 bases covered at least 15x. In order to evaluate duplication and large deletion events, for each 40 41 42 individual the relative read count for each targeted region was determined as the ratio of the 43 44 read count for that region divided by the total absolute read counts of all targeted regions of 45 46 the design. The ratio of the relative read count of a region in a given individual over the 47 48 average relative read counts in other individuals of the run resulted in the estimated copy 49 50 number for that region in that individual. 51 52

53 54 55 56 57 58 59 2 60 John Wiley & Sons, Inc. Human Mutation Page 32 of 52

1 2 3 Missense pathogenicity analysis 4 5 For the 2 novel missenses identified in this study, conservation has been assessed using the 6 7 multiple sequence alignment available from the eggnog database (KOG3717, (HuertaCepas, 8 9 et al., 2016)) and they were analyzed thanks to the PolyPhen2, SIFT and MutationTaster 10 11 softwares (Adzhubei, et al., 2010; Kumar, et al., 2009; Schwarz, et al., 2010) 12 13 14 15 16 . 17 18 19 For Peer Review 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 3 60 John Wiley & Sons, Inc. 4 Family

Family I Family

Family B Family

count count Allelic Exon 5 5 Exon 3/120 Exon 8 8 Exon 1/120 Exon 8 8 Exon 8 Exon 4/120 4/120 Exon 6 6 Exon 1/120 Exon 30 30 Exon 1/120 Exon 17 17 Exon 17 Exon 31/120 31/120

Location Location Exon 17 17 Exon 31/120 Exons 2730 2730 Exons 1/120 Introns 26 30 30 26 Introns 10/120 Exon 8 8 Exon 4/120 Exon 7 7 Exon 7/120 allele not found not found allele not found allele not found allele not found allele not found allele not found allele not found allele not found allele not found allele nd nd nd nd nd nd nd nd nd Allele 2 c.3454-1005_4040+737delinsCCC, (p.Tyr1152Aspfs*14) (p.Tyr1152Aspfs*14) c.3454-1005_4040+737delinsCCC, c.3454-488_4182+2588dup p.(Tyr1152_Thr1394dup) p.(Tyr1152_Thr1394dup) c.3454-488_4182+2588dup count count Allelic Allelic Human Mutation John Wiley & Sons, Inc. Exon 8 Exon 8 4/120 Exon 5 Exon 5 1/120 2 Exon 9 Exon 9 1/120 Exon 7 (p.Ile286Lysfs*6) c.857_860del Exon 7 7/120 2/120 (p.Ala1306Glyfs*56) c.3916dupG (p.Ile233Met) c.699T>G 29 Exon Exon 5 1/120 7 Exon 3/120 (p.Leu152Phe) c.454C>T 2/120 Exon 4 Exon 5 1/120 2/120 2 2 Exon 17 Exon 17 Exon 17 31/120 31/120 (p.Glu664Lys) c.1990G>A (p.Glu664Lys) c.1990G>A Exon 14 4/120 Exon 15 1/120 2 Exon 14 Exon 14 4/120 4/120 (p.Val292Met) c.874G>A Exon 19 (p.Val292Met) c.874G>A 2/120 2 Exon 30 Exon 19 1/120 Exon 14 1/120 2 1/120 2 2 Exon 12 Exon 12 1/120 (p.Val292Met) c.874G>A Intron 19 19 Intron 5/120 (p.Glu664Lys) c.1990G>A 19 Intron 5/120 (p.Gly212Arg) c.634G>A 19 Intron 5/120 19 Intron (p.Cys1360Arg) c.4078T>C 5/120 2 Exons 5/7 5/7 Exons 1/120 (p.Glu164*) c.490G>T Location Location

For Peer Review 2013 (Schmidts, et al., 2013) al., 2013) et 2013 (Schmidts, 2012 (Perrault, et al., 2012) and this study study this 2012) and et al., 2012 (Perrault, Allele 1 c.489C>T (p.Gly163Gly), and c.488_491del (p.Glu164Thrfs*10) (p.Glu164Thrfs*10) and c.488_491del (p.Gly163Gly), c.489C>T splice site donor additional creation of from et al et et al et Perrault Perrault MSS MSS (p.?) c.2399+1G>T MSS (p.Tyr311Cys) c.932A>G MSS (p.Glu664Lys) c.1990G>A MSS (p.Glu664Lys) c.1990G>A MSS (p.Gly212Arg) c.634G>A JATD (p.Ile233Met) c.699T>G MSS (p.?) c.2399+1G>T (p.Gly522Glu) c.1565G>A MSS MSS (p.Arg576Gln) c.1727G>A Schmidts JATD JATD (p.Asn460Lysfs*28) c.1380delC JATD (p.Gly522Glu) c.1565G>A JATD (p.Gly522Glu) c.1565G>A JATD (p.Leu152Phe) c.454C>T MSS (p.Arg759*) c.2278C>T MSS (p.?) c.2399+1G>T JATD (p.Glu267Gly) andc.800A>G (p.Gly140Arg) c.418G>A JATD (p.?) c.2399+1G>T JATD G>C (p.Pro1353Arg) c.4058 JATD T>C (p.Asp787Gly) c.2360 JATD (p.Leu514His) A>T c.1541 JATD (p.Arg110His) c.329G>A (p.Pro161Thr) c.481C>A

Disease Disease JATD/MSS (p.Val292Met) c.874G>A Page 33 of 52 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 34 of 52 5

2/120 1/120 1/120 1/120 1/120 1/120 1/120 1/120 31/120 31/120 31/120 31/120 31/120 31/120 31/120 31/120 31/120 31/120 31/120 Exon 14 14 Exon Exon 9 9 Exon Exon 4 4 Exon Exon 17 17 Exon 17 Exon 31/120 17 Exon 31/120 17 Exon 17 Exon 17 Exon 17 Exon 17 Exon 17 Exon 17 Exon 17 Exon 17 Exon 17 Exon Exon 28 28 Exon 2/120

Exon 23 23 Exon Exon 17 17 Exon allele not found not found allele nd Human Mutation John Wiley & Sons, Inc. Exon 7 Exon 7 1/120 (p.Pro71Leu) c.212C>T Exon 5 Exon 5 2/120 2 Exon 5 1/120 (p.Gly522Glu) c.1565G>A 14 Exon 4/120 Exon 14 Exon 14 2/120 (p.Leu514Gln) c.1541_1542delinsAA Exon 17 Exon 17 Exon 17 31/120 31/120 (p.Glu664Lys) c.1990G>A Exon 17 (p.Glu664Lys) c.1990G>A Exon 17 31/120 Exon 17 31/120 (p.Glu664Lys) c.1990G>A Exon 17 31/120 (p.Glu664Lys) c.1990G>A Exon 17 31/120 (p.Glu664Lys) c.1990G>A Exon 17 31/120 (p.Glu664Lys) c.1990G>A Exon 17 31/120 (p.Glu664Lys) c.1990G>A Exon 17 31/120 (p.Glu664Lys) c.1990G>A Exon 17 31/120 (p.Glu664Lys) c.1990G>A Exon 17 31/120 (p.Glu664Lys) c.1990G>A Exon 17 31/120 (p.Glu664Lys) c.1990G>A 31/120 (p.Glu664Lys) c.1990G>A (p.Glu664Lys) c.1990G>A Exon 31 Exon 28 31/120 (p.Asn633Serfs*10) c.1898_1901delATAA 1/120 Exon 21 (p.Cys663Trp) Exon 11 c.1989C>G 1/120 Exon 13 1/120 (p.Trp459*) Exon 19 c.1377G>A 3/120 in trans presumed (p.Ala974Val) c.2921C>T 1/120 Exon 28 (p.Cys329Arg) c.985T>C (p.Glu522Glyfs*6) c.1655_1656delAG 2/120 (p.Gly1276Glu) c.3827G>A 17 Exon 14 Exon 12 Exon

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2015 (Beheshtian, et al., 2015) al., et 2015 (Beheshtian, 2016 (Weisschuh, et al., 2016) 2016) al., et 2016 (Weisschuh, et al et al et 2015 (Bifari, et al., 2016) 2016) et al., 2015 (Bifari, 2014 (Khan, et al., 2014) 2014) al., et (Khan, 2014 2016 (Hull, et al., 2016) 2016) al., et 2016 (Hull, 2015 (Xu, et al., 2015) 2015) al., et (Xu, 2015 et al et et al et et al et et alet JATD JATD Khan (p.Pro161Thr) c.481C>A MSS MSS (p.Glu664Lys) c.1990G>A Bifari (p.Glu664Lys) c.1990G>A EORD EORD (p.Glu664Lys) c.1990G>A EORD (p.Glu664Lys) c.1990G>A EORD (p.Glu664Lys) c.1990G>A EORD (p.Glu664Lys) c.1990G>A EORD (p.Glu664Lys) c.1990G>A EORD (p.Glu664Lys) c.1990G>A EORD (p.Glu664Lys) c.1990G>A EORD (p.Glu664Lys) c.1990G>A EORD (p.Glu664Lys) c.1990G>A EORD (p.Glu664Lys) c.1990G>A (p.Glu664Lys) c.1990G>A EORD (p.Leu514Gln) c.1541_1542delinsAA Xu RP RP RP (p.Leu1399Pro) c.4196T>C RP (p.Gly1276Arg) c.3826G>A RP (p.Val217Glyfs*2) c.650_651delTG LCA (p.Arg871Cys) c.2611C>T LCA (p.Ala418Pro) c.1252G>C (p.Thr484Met) c.1451C>T Beheshtian (p.Glu790Lys) c.2368G>A RP Weisschuh RP (p.Gly1276Glu) c.3827G>A Hull (p.Arg158Trp) c.472C>T 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

6

) ) have analyzed et al Exon 22 22 Exon 1/120 Exon 9 9 Exon 9 Exon 4/120 4/120 Exon 11 11 Exon 1/120 K Family Introns 2630 2630 Introns 10/120 A Family Exon 3 3 Exon 1/120 2630 Introns 10/120 D Family 2630 Introns 10/120 G Family Introns 2630 2630 Introns 10/120 E Family Introns 2630 2630 Introns 10/120 H Family Introns 2630 2630 Introns 10/120 C Family 2630 Introns 10/120 F Family 31 Exon 1/120 J Family Exon 7 7 Exon 7/120 Exon 12 12 Exon 1/120

c.1319T>C (p.Leu440Pro) (p.Leu440Pro) c.1319T>C c.3454-488_4182+2588dup (p.Tyr1152_Thr1394dup) (p.Tyr1152_Thr1394dup) c.3454-488_4182+2588dup (p.Tyr1152_Thr1394dup) c.3454-488_4182+2588dup c.3454-488_4182+2588dup (p.Tyr1152_Thr1394dup) (p.Tyr1152_Thr1394dup) c.3454-488_4182+2588dup c.3454-488_4182+2588dup (p.Tyr1152_Thr1394dup) (p.Tyr1152_Thr1394dup) c.3454-488_4182+2588dup c.3454-488_4182+2588dup (p.Tyr1152_Thr1394dup) (p.Tyr1152_Thr1394dup) c.3454-488_4182+2588dup (p.Tyr1152_Thr1394dup) c.3454-488_4182+2588dup (p.Tyr1414Leufs*48) c.4236_4239dup c.3454-488_4182+2588dup (p.Tyr1152_Thr1394dup) c.3454-488_4182+2588dup Human Mutation John Wiley & Sons, Inc. Exon 3 Exon 3 1/120 Exon 9 Exon 9 Exon 9 4/120 4/120 (p.Cys333Tyr) c.998G>A (p.Cys333Tyr) c.998G>A Exon 7 7/120 Exon 7 (p.Arg759*) c.2278C>T 7/120 Exon 7 (p.Gly212Arg) c.634G>A 7/120 Exon 5 3/120 Exon 7 19 Exon 7/120 2/120 Exon 13 Exon 13 3/120 (p.Thr484Met) c.1451C>T Exon 10 1/120 Exon 24 (p.Arg475Asnfs*14) c.1422_23insAA 2/120 (p.?) c.11_6del 13 Exon 3/120 Exon 24 Exon 24 2/120 Exon 18 Exon 18 1/120 Intron 19 19 Intron 5/120 (p.Ser939Pro) c.2815T>C Intron 20 20 Intron 1/120 Introns 2630 2630 Introns 10/120 2630 Introns 10/120 IFT140. IFT140.

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2016 (Pena-Padilla, et al., 2016) 2016) al., et 2016 (Pena-Padilla, et al et . Previously reported and novel mutations in mutations and reported novel . Previously c.2577+25G>A (p.Leu859_Glu860insValArgGlyAlaArgHisGly) c.2577+25G>A (p.Tyr1152_Thr1394dup) c.3454-488_4182+2588dup (p.Tyr1152_Thr1394dup) c.3454-488_4182+2588dup (p.Pro726Leu) c.2177C>T c.1-?_147+?del, start loss loss start c.1-?_147+?del, 2017 (Bayat, et al., 2017) 2017) et al., 2017 (Bayat, 2017 (Helm, et al., 2017) 2017) al., et 2017 (Helm, et al et et al et RP RP RP RP (p.Thr484Met) c.1451C>T RP (p.?) c.2399+1G>T RP (p.Cys333Tyr) c.998G>A Pena-Padilla (p.Cys333Tyr) c.998G>A OTCS (p.Ala341Thr) c.1021G>A Bayat (p.?) c.3141+1G>T SB Helm MSS (p.Gly212Arg) c.634G>A study This (p.Gly212Arg) c.634G>A MSS MSS MSS (p.Gly212Arg) c.634G>A MSS MSS (p.Leu152Phe) c.454C>T MSS/SB RP MSS (p.Gly212Arg) c.634G>A Mutation allelic counts are reported for only one affected individual per family. It is to notice that some studies (i.e. Bifari consanguineous consanguineous families from the same asphyxiating thoracic dystrophy; region MSS: MainzerSaldino increasing Syndrome; OCTS: the Opitz trigonocephaly global C Sensenbrenner syndrome. syndrome; RP: allelic retinitis pigmentosa; count. SB: EORD: earlyonset retinal dystrophy; JATD: Jeune Mutations are reported only once per family. Those reported in this study are in bold and those described for the first time are also in italic. Table S1 (mild) (mild) JATD/MSS JATD/MSS (p.?) c.3141+1G>T Page 35 of 52 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

F No No K-II.2 K-II.2 7 yo 7 yo rods rods (3 yo) (3 yo) digits) digits) 14/02/2008 14/02/2008 MSS (mild) MSS strabismus (5 mo) (5 mo) strabismus epiphyses (second (second epiphyses Bilateral divergent divergent Bilateral Nystagmus (6 mo) (6 mo) Nystagmus Joint luxations with with Joint luxations damage of cones and cones of damage No eye pursuit (1 mo) (1 mo) pursuit eye No Page 36 of 52 phalanges of 4 2, the 3, of phalanges Coneshaped phalangeal phalangeal Coneshaped Renal ultrasound normal normal ultrasound Renal Early onset advanced RP RP advanced onset Early ERG: severe and primary primary and ERG: severe spontaneous subluxations subluxations spontaneous 7

M M No No J-II.1 J-II.1 (9 yo) (9 yo) ( 1 ( SD) RP slow slow RP evolution evolution Overweight Overweight when younger when younger

normal function Renal

M M I-II.1 I-II.1 Cone shaped shaped ERG: no no ERG: Thoracic Thoracic signal left left signal epiphyses epiphyses dystrophy dystrophy phalangeal phalangeal 16/04/1986 16/04/1986 05/06/1993 Yes (cystic) (cystic) Yes

F H H No No No No Yes Yes kidney) kidney) Neonatal Neonatal epiphyses epiphyses Transplant Transplant proteinuria proteinuria Strabismus Strabismus Nystagmus Nystagmus No seizures seizures No Short hands hands Short differentiated differentiated CKD stage V stage CKD No hematuria hematuria No Refraction +2 Refraction Hepatomegaly Hepatomegaly Craniostenosis Craniostenosis gestational age gestational No polydactyly polydactyly No Dialysis (3.8 yo) (3.8 yo) Dialysis oculomotor apraxia apraxia oculomotor MSS/Sensenbrenner MSS/Sensenbrenner Jeune/MSS RP Isolated Developmental delay delay Developmental Brachymesophalangy Brachymesophalangy End stage renal failure failure renal End stage Papillae edges not well well not edges Papillae Glomerular and tubular and tubular Glomerular Coneshaped phalangeal phalangeal Coneshaped No cerebellar ataxia, but but ataxia, cerebellar No Intracranial hypertension hypertension Intracranial Hip anomalies not known not known anomalies Hip Normal liver function tests tests function liver Normal Renal US (small cysts, small small cysts, (small US Renal Retinal dystrophy not known not known dystrophy Retinal CKD: Cystic Kidney Disease, CKD: Kidney Cystic . .

yo yo F F G G st No No No No No No No No MSS MSS 1 (23 (23 yo) Austria Austria Myopia Myopia IFT140 RP (1 yo) (1 RP yo) (+ 1,5 SD) (+ SD) 1,5 proteinuria proteinuria 21/05/1993 21/05/1993 Short hands hands Short corticomedullar corticomedullar Late onset obesity obesity onset Late of weeks birth 28 Premature Hypotonia at at birth Hypotonia echogenicity, loss of of loss echogenicity, Developmental delay delay Developmental Brachymesophalangy Brachymesophalangy Renal US (cysts, low (cysts, US Renal normal size, increased increased size, normal differentiation (11 yo) (11 yo) differentiation End stage renal failure failure renal End stage epiphyses (feet, hands) (feet, hands) epiphyses Glomerular and tubular tubular and Glomerular Coneshaped phalangeal phalangeal Coneshaped

F F F No No No No No No No No Yes Yes 4 yo 4 yo MSS MSS cysts) cysts) Austria Austria (1 SD) (1 Myopia Myopia (4.7 yo) (4.7 yo) increased increased RP (4 yo) (4 RP yo) Infections Infections anomalies anomalies 11/02/2007 11/02/2007 Proteinuria, Proteinuria, Short hands hands Short (feet, hands) hands) (feet, Coneshaped Coneshaped Moderate hips’ Moderate microhematuria microhematuria corticomedullar corticomedullar Renal US (Small (Small US Renal differentiation, no no differentiation, Recurrent respiratory respiratory Recurrent phalangeal epiphyses epiphyses phalangeal Brachymesophalangy Brachymesophalangy kidneys (<3th centile, (<3th centile, kidneys echogenicity, reduced reduced echogenicity, End stage renal failure renal failure End stage F E No No No No mo) mo) (hands) (hands) set ears) ears) set Neonatal Neonatal Caucasian Caucasian 20/09/2010 20/09/2010 Jeune/MSS Jeune/MSS Short hands hands Short Genu varum varum Genu Brachymelia Brachymelia Astigmatism Astigmatism Renal anemia anemia Renal Splenomegaly Splenomegaly Hyperlordosis Hyperlordosis Brachyphalangy Brachyphalangy Pes planovalgus planovalgus Pes Slightly enlarged enlarged Slightly Asphyxia at birth birth at Asphyxia Delayed bone age age bone Delayed Hypoplastic thorax thorax Hypoplastic Transplant (2.5 yo) yo) (2.5 Transplant No autistic features autistic features No Recurrent infections infections Recurrent Arterial hypertension Arterial hypertension No ataxia, no seizures seizures no ataxia, No Obstructive bronchitis bronchitis Obstructive Strabismus Hyperopia Strabismus Hydronephrosis (4 mo) Hydronephrosis Severe visual reduction reduction visual Severe Brachydactyly 3rd toe of Brachydactyly Peritoneal dialysis (2 yo) (2 yo) dialysis Peritoneal Slightly hyperechoic liver liver hyperechoic Slightly + disproportionate (3 SD) (3 disproportionate + Short, slightly broader ribs broader slightly Short, No premature craniosynostosis craniosynostosis premature No Truncal adiposity, breast tissue tissue breast adiposity, Truncal End stage renal failure (1.5 yo) yo) (1.5 failure renal End stage Congenital pendular nystagmus nystagmus pendular Congenital epiphyses, steep acetabular roofs) roofs) acetabular steep epiphyses, Yes (high hairline, frontal bossing, bossing, frontal hairline, (high Yes Coneshaped phalangeal epiphyses epiphyses phalangeal Coneshaped Hip anomalies (hip dysplasia, broad broad (hip dysplasia, anomalies Hip right kidney with 3 chambered cysts) cysts) 3 with chambered right kidney Renal US (small hyperechoic kidney, kidney, hyperechoic (small US Renal eyes, anteverted nares, long philtrum, philtrum, long nares, anteverted eyes, Generalized brain atrophy (CT scan 4 scan (CT atrophy brain Generalized 4 mo, sitting 8 mo, walking 24 mo, first first 24 mo, walking mo, sitting 8 4 mo, everted lower lip, prominent chin, deep lip, prominent lower everted words 18 mo, slow speech development) development) speech slow 18 mo, words Slight Developmental delay (head control (head delay Slight Developmental hypertelorism, broad nasal ridge, deepset deepset ridge, nasal broad hypertelorism, Incomplete syndactyly of digits 34 (toes) (toes) digits 34 of syndactyly Incomplete

Human Mutation digits th John Wiley & Sons, Inc.

F F No No No No No No side side MSS MSS 45 yo yo 45 D-II.1 D-II.1 France France (52 yo) (52 yo) RP (16 yo) RP yo) (16 27/12/1968 27/12/1968 + (< 5 SD) 5 SD) (< + Advanced RP RP Advanced (feet and hands) hands) and (feet Dialysis (10 yo) (10 yo) Dialysis Varus of the feet feet the of Varus Brachytelephalangy Brachytelephalangy short femoral heads) heads) femoral short Hemeralopia (10 yo) (10 yo) Hemeralopia Bilateral ulnar valgus valgus ulnar Bilateral Marked curved radius radius curved Marked End stage renal failure failure renal End stage Transplantation (16 yo) (16 yo) Transplantation VA: 2.4/10 RE 1/20 LE LE 1/20 RE 2.4/10 VA: VF: very reduced on right on reduced very VF: No coneshaped epiphyses epiphyses coneshaped No Brachymesophalangy with Brachymesophalangy Enlarged metaphyses (feet) metaphyses Enlarged due to the age of the patient the patient of to age the due Divergent strabismus (2 yo) (2 yo) strabismus Divergent clinodactyly of the 5 the of clinodactyly Hip anomalies (large pelvis, pelvis, (large anomalies Hip

F F 6 yo 6 yo MSS MSS C-II.2 C-II.2 France France (hands) (hands) Dialysis Dialysis + ( 3 SD) 3 ( SD) + 26/08/1976 26/08/1976 VA: 4/10RLE 4/10RLE VA: Night blindness blindness Night polydipsia (6 yo) (6 yo) polydipsia For Peer (7 yo) Transplant Review important arterial damage arterial damage important tubulointersticial nephritis nephritis tubulointersticial Arterial hypertension (24/12) (24/12) hypertension Arterial End stage renal failure (7 yo) (7 renal failure yo) End stage Nephronophtisis with polyria polyria with Nephronophtisis kidney's size: R55mm L59mm) L59mm) R55mm size: kidney's Metaphyseal defect (long bones) bones) (long defect Metaphyseal bones broad and Short Renal US (no cysts, echogenicity, echogenicity, (no cysts, US Renal Coneshaped phalangeal epiphyses epiphyses phalangeal Coneshaped Obsolescent/hyalinized glomerulus glomerulus Obsolescent/hyalinized

No F No No No No yo) 3 yo 3 yo MSS MSS B-II.1 B-II.1 Dialysis Dialysis L60mm) L60mm) Scoliosis Scoliosis (forearms) (forearms) Transplant Transplant Nystagmus Nystagmus 17/03/1978 17/03/1978 Micromelia Micromelia Short hands hands Short + (< 5 SD) 5 SD) (< + Altered ERG Altered Onset (3 mo) (3 mo) Onset Maculopathy Maculopathy VA: 2/10RLE 2/10RLE VA: Hip anomalies anomalies Hip Hemosiderosis Hemosiderosis Night blindness Night blindness epiphyses (hands) (hands) epiphyses Serbia, Yugoslavia Yugoslavia Serbia, Metaphyseal defect Metaphyseal Portal space fibrosis space Portal Arterial hypertension hypertension Arterial Nephronophtisis with with Nephronophtisis kidney's size: R76mm R76mm size: kidney's Coneshaped phalangeal phalangeal Coneshaped polyria polydipsia (3 yo) (3 yo) polydipsia polyria End stage renal failure (5 failure renal End stage Renal US (Several cortical cortical (Several US Renal cysts (10 yo), echogenicity, echogenicity, (10 yo), cysts

M M No No No No No No No No No No RP RP MSS MSS chin) chin) 15 yo 15 yo A-II.2 A-II.2 + (2 SD) SD) (2 + epiphyses) epiphyses) 31/08/1999 31/08/1999 Short hands hands Short and 5 digits) and 5 digits) France, Italy France, Altered ERG Altered VA: 7/10RLE 7/10RLE VA: brachymetatarsia brachymetatarsia Renal US normal US normal Renal Microalbuminuria Microalbuminuria Brachydactyly and Brachydactyly of superior femoral femoral superior of Hemeralopia (7 yo) (7 yo) Hemeralopia Yes (high forehead, forehead, (high Yes philtrum, prominent prominent philtrum, frontal bossing, long long bossing, frontal Arterial hypertension hypertension Arterial Divergent strabismus strabismus Divergent Moderate renal failure renal failure Moderate brachymetacarpia (1, 4 brachymetacarpia wings, lack of coverage coverage of lack wings, pelvis with narrow iliac narrow with pelvis Clinical Features of MSSaffected individuals harboring compoundheterozygous mutations in mutations compoundheterozygous harboring individuals of MSSaffected Features Clinical M M No No No No No No No No No No yo) MSS MSS 14 yo failure failure A-II.1 A-II.1 Asthma Asthma philtrum, philtrum, + (3 SD) (3 + 10/01/1998 10/01/1998 ptosis, long long ptosis, France, Italy France, VA: 9/10RLE 9/10RLE VA: (1, 3, 4 digits) digits) 3, 4 (1, frontal bossing, bossing, frontal prominent chin) chin) prominent Renal US normal normal US Renal Brachymetatarsia Brachymetatarsia Microalbuminuria Microalbuminuria Syndrome, R: Right, RE: Right Eye, RLE: Right and left Eye, RP: Retinitis Pigmentosa, SD: standard deviation, US: Ultrasound, VA: Visual Visual VA: Ultrasound, US: deviation, standard SD: RP: Pigmentosa, Eye, Retinitis and RLE: left Right Eye, Right RE: Right, R: Syndrome, field, old. yo: years visual VF: acuity, CT: Computed Tomography, ERG: Electroretinogram, F: Female, L: Left, LE: Left Eye, M: Male, mo: month old, MSS: MainzerSaldino MainzerSaldino MSS: month mo:old, M: Eye, Male, Left, LE: L: Left F: Female, Electroretinogram, ERG: Tomography, Computed CT: Table S2 Individual Individual Birth Sex diagnostic Suspected Onset (SD) stature Short features Skeletal bones long Broad (narrow anomalies Hip anomalies Digit Brachymetacarpia dystrophy Retinal (10 Hemeralopia involvement Renal renal Moderate involvement Hepatic fibrosis Pancreatic anomalies Cerebral features Dysmorphic hairline, (high Yes anomalies Oral Other Neurological features features Neurological 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 8 Reverse (5'-3') (5'-3') Reverse CACACCTGACAGGCACACAC CACACCTGACAGGCACACAC CGGTGTTAAAATGGGCACCT GCGTGTCCGACTTCTCGTA CAATTAGCACTTTCACCTTTGG TCTGGTTCCTCCAGGAGCA CAACTTCATCCACGTTCACC CCCAGGGTCAGGTGATCTTCCTA

Forward (5'-3') (5'-3') Forward GATGACTACTTGCCCCAGGA GATGACTACTTGCCCCAGGA TGAGCGAGTGAATGAGTGGA ATCAAAAGTGAGGCCGTCTG GTGGAACGCTGGCACTTTAT CATAGAAAGTTTCCTCCTGCCAT ACACAACTGTGTTCACTAGC CCCAAAGCGTCATTTCTGGTGTTC CACAGCACTCCCACTGACAAC CACAGCACTCCCACTGACAAC GAGGGAGGCGATAGTAGGACC Human Mutation John Wiley & Sons, Inc. Name Name IFT140ex20 IFT140ex20 IFT140ex14 IFT140RTPCRex1819F_ex22R PRMT9RTPCRex3F_ex6R IFT140QPCRex30F_ex30R BGLOBQPCRex1F_ex1R intron26R IFT140intron30F_

RefSeq RefSeq For Peer Review

intron26R IFT140intron30F_ CCGGAGTAGCTGGGATTACA CTGTCTAGCTGGGGTGGGTA IFT140RTPCRex30F_ex28R AAGCAGTGTGAGCTGCTCCT GGGCACAAGCGTCATAAAAG NM_000190 NM_000190 HMBSQPCR NM_152618 BBS12ex2F_ex2R AGTTCTCATTGAGGGTGACCT GCCAGAGATGAAGCCAGCCA NM_014714.3 NM_014714.3 NM_001304458 NM_014714.3 NM_000518 NM_014714.3 Gene Gene

HMBS HMBS BBS12 IFT140 IFT140 PRMT9 IFT140 HBB IFT140 Application Application DNA c.2577+25G>A c.2577+25G>A DNA TD breakpoint DNA c.1565G>A DNA c.2577+25G>A cDNA TD cDNA control cDNA qPCR qPCR qPCR Duplex PCR List and characteristics of primers used in this study. TD: Tandem duplication of exon 2730 exon 2730 of duplication Tandem TD: study. in used this primers of characteristics and List Table S3

Page 37 of 52 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Human Mutation Page 38 of 52

1 2 3 4 5 Name primary antibodies Applications Producer Conditions

6 Mouse antiAcetylated αTubulin WB, IF Abcam #ab24610 IF: 1/200 7 IF:1/100 Rabbit antiIFT140 WB, IF Proteintech #174601AP 8 WB: 1/1000 9 Mouse antiβTubulin WB Euromedex # TUB2A2 1/2500 10 11 Name secondary antibodies 12 13 Donkey antiMouse IgG (H+L), FITC conjugate IF Thermo Scientific #A16012 1/500 14 15 Goat antiRabbit IgG (H+L) Alexa Fluor 568 conjugate IF Thermo Scientific #A11011 1/500 16 17 Chicken antiRabbbit IgGHRP WB Santa Cruz #sc2955 1/5000 Goat anti mouse IgGHRP WB Santa Cruz #sc2060 1/5000 18 19 For Peer Review 20 Table S4. List of antibodies used in this study. IF: immunofluorescence, WB Western Blot. 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 9 60 John Wiley & Sons, Inc. Page 39 of 52 Human Mutation

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 For Peer Review 20 21 Figure S1. Duplex PCR profiles on a LabChip GX (Perkin Elmer) for a rapid detection of the 22 IFT140 tandem duplication (see Table S3 for primers details). Normal samples harbor a 23 single band (BBS12) whereas homozygous or heterozygous carriers for the tandem 24 duplication show an additional band specific for the duplication breakpoint. 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 10 60 John Wiley & Sons, Inc. Human Mutation Page 40 of 52

1 2 3 4 Patient A-II.1 A-II.2 5 Type of sequence variant SNV indel SNV indel 6 7 Total number of variants 49489 2168 49418 2133 8 After exclusion of variants with an allele frequency >1% (extracted 9 from dbSNP, the Exome Variant Server and the ExAC database or our 6528 1038 6517 1017 10 internal exome database) 11 After exclusion of variants found at the homozygous state and more 12 1159 90 1029 72 than once at the heterozygous state in 90 control exomes 13 14 After exclusion of 5'UTR, 3'UTR, downstream, upstream and intron locations without local splice effect prediction (from the 517 20 458 12 15 “localSpliceEffect” field of AlamutBatch) 16 After exclusion of synonymous variants without local splice effect 17 417 20 373 12 prediction (from the “localSpliceEffect” field of AlamutBatch) 18 19 After selection of variantsFor consistent Peer with recessive Review transmission 2 compound heterozygous in 20 (compound heterozygous, homozygous variants). PLEKHA8 21 22 23 24 Table S5 Summary of the whole exome sequencing results from family A 25 26 WES data processing and variant calling revealed respectively 51,657 and 51,551 SNV and 27 indels in individual AII.1 and AII.2. Variant filtering using stringent criteria reduced the 28 number of genetics variants to respectively 437 and 385 variants per proband. To identify 29 variants consistent with autosomal recessive inheritance, we kept only compound shared 30 heterozygous or homozygous variants, reducing the number of variants to 2 heterozygous 31 variants in the PLEKHA8 gene: NM_001197026.1:c.1517_1518insAT (p.Leu507Phefs*27) 32 33 and NM_001197026.1:c.1522del (p.Tyr508Metfs*25). Both variants being always supported 34 on the same reads (as shown in the IGV view below), they are in cis and are probably an 35 artifact due to a bad reads alignment. So, no candidate gene was found by the WES analyses. 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 11 60 John Wiley & Sons, Inc. Page 41 of 52 Human Mutation

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 For Peer Review 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 12 60 John Wiley & Sons, Inc. Human Mutation Page 42 of 52

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 For Peer Review 20 21 22 23 24 Figure S2. Next generation sequencing data from patient AII.1 displayed around IFT140, in 25 particular from exon 27 to exon 30 highlighting the tandem duplication. 26 27 (A) Comparison between whole genome sequencing (WGS) and whole exome sequencing 28 (WES) data from patient AII.1. The reads are displayed as pairs and sorted according to their 29 insert size using IGV (Thorvaldsdottir, et al., 2013). 30 31 (B) Highlight of the WGS read pairs at the breakpoint junctions showing the typical drawing 32 (read pairs pointing in opposite direction) for tandem duplications in direct orientation. 33 34 (C) Highlight of the WGS read pairs at the breakpoints including soft clipped bases. The left 35 36 breakpoint in intron 30 overlaps the AluJr while the right breakpoint in intron 26 overlaps the 37 AluJb. Analysis of the reads and the split reads that are misaligned allowed us to define the 38 39 exact position of the duplication. 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 13 60 John Wiley & Sons, Inc. Page 43 of 52 Human Mutation

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 For Peer Review 20 21 Figure S3. Homozygous duplication in IFT140 validated by quantitative realtime PCR. 22 23 Quantitative realtime PCR was performed on DNA from exon 30 in all individuals from 24 25 family A and D as well as in one unrelated control. DNA quantity from exon 30 of IFT140 26 27 was compared to 2 reference genes (HBB and HMBS) using the absolute quantification 28 29 30 method. 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 14 60 John Wiley & Sons, Inc. Human Mutation Page 44 of 52

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Figure S4. Next generationFor sequencing Peer data forReview family A and I displayed around IFT140, in 20 particular from exon 27 to exon 30. 21 22 (A) Comparison between whole genome sequencing (WGS) from patient AII.1 (tandem 23 duplication carrier) and targeted exome sequencing using the full IFT140 gene as a target 24 region from patient III.1 (deletion of exon 27 to 29). The reads are displayed as pairs and 25 sorted according to their insert size using IGV (Thorvaldsdottir, et al., 2013). The two events 26 do not share the same breakpoints. 27 28 (B) Highlight of the read pairs from patient III.1 at the breakpoint junctions showing the 29 30 typical drawing (read pairs pointing at each other) for a genomic deletion. 31 32 (C) Highlight of the read pairs from patient III.1 at the breakpoints including soft clipped 33 bases. The right breakpoint overlaps a SINE element from the Alu family (Flam_C) but not 34 35 the left breakpoint. Analysis of the split reads that are misaligned allows us to define the exact 36 position of the deletion (c.34541005_4040+737delinsCCC). 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 15 60 John Wiley & Sons, Inc. Page 45 of 52 Human Mutation

1 2 3

4 WGS TES WGS TES Chr 16 A-I.1 A-1.2 A-II.1* A-II.2* C-II.1* B-II.2* B-I.1 B-I.2 D-II.1* Control 1Control 2Control 3Control 4Control 5Control 6 5 rsID ref alt position Dup Dup Dup Dup Dup Dup rs149678731 1560882 TC T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/C 6 rs1053730 1560886 AGA/AA/GA/AA/A A/A A/A A/G A/A G/G A/AA/GA/A A/AA/GA/A rs2667688 1561446 AGA/AA/GA/AA/A A/A A/A A/G A/A G/G A/AA/GA/A A/AA/GA/A 7 rs572130902 1561488 G C G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G rs8048290 1562026 T C T/T T/C T/T T/T T/T T/T T/C T/T C/C T/T T/C T/T T/T T/C T/T 8 rs12918013 1562195 AGA/AA/GA/AA/A A/A A/A A/G A/A G/G A/AA/AA/A A/AA/GA/A rs10083822 1562252 C T C/C C/C C/C C/C C/C C/C C/T C/C T/T C/C C/T C/C C/C C/T C/C 9 rs117143480 1562399 C T C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/T C/C C/C C/C C/C rs8048980 1562460 T C T/T T/C T/T T/T T/T T/T T/C T/T C/C T/T T/C T/T T/T T/C T/T 10 rs1966171 1562804 G A G/G G/A G/G G/G G/G G/G G/A G/A A/A G/G A/A G/G G/A G/A G/G rs58369664 1562915 C G C/C C/C C/C C/C C/C C/C C/C C/G C/C C/CC/GC/C C/G C/C C/C 11 rs112450135 1562987 G C G/G G/C G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G rs7189700 1563582 G A G/G G/A G/G G/G G/G G/G G/A G/G A/A G/G G/G G/G G/G G/A G/G 12 rs10558385 1563667 ACTTT A ACTTT/ACTTTACTTT/ACTTTACTTT/ACTTTACTTT/ACTTTACTTT/ACTTTACTTT/ACTTT ACTTT/A ACTTT/ACTTT A/A ACTTT/ACTTT ACTTT/A ACTTT/ACTTTACTTT/ACTTT ACTTT/A ACTTT/ACTTT rs113508951 1563672 C CT C/C C/C C/C C/C C/C C/C C/C C/CT C/C C/C C/C C/C C/CT C/C C/C 13 rs7191239 1563672 C T C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C rs67974359 1563705 T C T/T T/C T/T T/T T/T T/T T/C T/C C/C T/T C/C T/T T/C T/C T/T 14 rs184085046 1563799 C T C/C C/T C/C C/C C/C C/C C/T C/C T/T C/C C/T C/C C/C C/T C/C rs73497522 1563800 AGA/AA/GA/AA/A A/A A/A A/G A/G G/G A/A G/G A/A A/GA/GA/A 15 rs35773761 1563865 G A G/G G/G G/G G/G G/G G/G G/G G/A G/G G/G G/G G/G G/A G/A G/G rs551416387 1563876 GA G/G G/G G/G G/G G/G G/G G/G G/A G/G G/G G/G G/G G/A G/G G/G 16 rs74430146 1563886 AGA/AA/AA/AA/A A/A A/A A/G A/G G/G A/G G/G A/A A/GA/GA/A rs62012850 1563896 C A,G C/C C/G C/C C/C C/C C/C C/G C/A G/G C/C C/C C/C C/A G/G C/C 17 rs62012851 1563916 T C T/T T/C T/T T/T T/T T/T T/C T/T C/C T/T T/T T/T T/T T/C T/T rs62012852 1563920 C T C/C C/T C/C C/C C/C C/C C/T C/C T/T C/C C/C C/C C/C C/T C/C 18 rs2745111 1564823 G A G/G G/G G/G G/G G/G G/G G/G G/A G/G G/G G/A G/G G/A G/G G/G rs12597753 1565116 C A C/C C/C C/C C/C C/C C/C C/C C/A C/C C/CC/AC/C C/A C/C C/C 19 rs11643891 1565540 C T C/C C/T C/C C/C C/C C/C C/T C/T T/T C/C T/T C/C C/T C/T C/C rs79602039 1565893 ForATA/AA/AA/AA/A Peer ReviewA/A A/A A/A A/T A/A A/AA/TA/A A/TA/AA/A 20 rs6600149 1566177 AC A/AA/AA/AA/A A/A A/A A/A A/A A/A A/AA/AA/A A/AA/AA/A rs6600150 1566178 AC A/AA/AA/AA/A A/A A/A A/A A/A A/A A/AA/AA/A A/AA/AA/A 21 rs752259914 1566178 A ACC A/AA/AA/AA/A A/A A/A A/ACC A/ACC ACC/ACC A/AA/AA/A A/ACC A/ACC A/A rs8051374 1566284 T C T/T T/C T/T T/T T/T T/T T/C T/C C/C T/T C/C T/T T/C T/C T/T 22 rs112878182 1566444 AGA/AA/GA/AA/AA/AA/AA/AA/AA/AA/AA/AA/AA/AA/AA/A rs9932727 1566586 C T C/C C/T C/C C/C C/C C/C C/T C/C T/T C/C C/T C/C C/C C/T C/C 23 rs2745185 1566597 T C T/T T/C T/T T/T T/T T/T T/C T/C C/C T/T C/C T/T T/C T/C T/T rs71385704 1566826 G A G/G G/A G/G G/G G/G G/G G/A G/G A/A G/G G/A G/G G/G G/A G/G 24 rs150027092 1567367 AAG A AAG/AAG AAG/AAG AAG/AAG AAG/AAG AAG/AAG AAG/AAG AAG/AAG AAG/AAG AAG/AAG AAG/AAG AAG/AAG AAG/AAG AAG/AAG AAG/AAG AAG/AAG rs147652844 1567368 AGA AG/AG AG/AG AG/AG AG/AG AG/AG AG/AG AG/AG AG/AG A/A AG/AG AG/AG AG/AG AG/AG AG/A AG/AG 25 rs4787262 1567368 AG A AG/AG AG/A AG/AG AG/AG AG/AG AG/AG AG/AG AG/AG AG/AG AG/AG AG/AG AG/AG AG/AG AG/AG AG/AG rs76268857 1567453 GAGA G GAGA/GAGAGAGA/GAGAGAGA/GAGAGAGA/GAGAGAGA/GAGAGAGA/GAGAGAGA/GAGA GAGA/G GAGA/GAGAGAGA/GAGA GAGA/G GAGA/GAGA GAGA/G GAGA/GAGAGAGA/GAGA 26 IFT140 Duplication rs2072990 1567651 AGA/AA/GA/AA/A A/A A/A A/G A/G G/G A/A G/G A/A A/GA/GA/A rs2235637 1567694 G A G/G G/A G/G G/G G/G G/G G/A G/A A/A G/G A/A G/G G/A G/A G/G 27 rs2076434 1568402 AGA/AA/GA/AA/A A/A A/A A/G A/A G/G A/AA/GA/A A/AA/GA/A rs71385705 1569003 ATA/AA/TA/AA/A A/AA/AA/AA/AA/T A/AA/AA/A A/AA/AA/A 28 rs117190102 1569186 GA G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G rs3784819 1569202 C T C/C C/C C/C C/C C/C C/C C/C C/T C/C C/C C/T C/C C/T C/C C/C rs3784820 1569252 G A G/G G/A G/G G/G G/G G/G G/A G/G A/A G/G G/A G/G G/G G/A G/G 29 rs3784821 1569301 G A G/G G/G G/G G/G G/G G/G G/G G/A G/G G/G G/A G/G G/A G/G G/G rs3784822 1569308 C T C/C C/C C/C C/C C/C C/C C/C C/T C/C C/C C/T C/C C/T C/C C/C 30 rs2667665 1569524 ACA/AA/CA/AA/A A/AA/AA/CA/CC/C A/A C/C A/A A/CA/CA/A rs199887622 1570059 GA G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G 31 rs113734226 1570078 C T C/C C/C C/C C/C C/T C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C rs932391 1570857 T C T/T T/C T/T T/T T/T T/T T/C T/C C/C T/T C/C T/T T/C T/C T/T 32 rs932392 1571120 T G T/T T/G T/T T/T T/T T/T T/G T/T G/G T/T T/G T/T T/T T/G T/T rs73497551 1571811 T C T/T T/C T/T T/T T/T T/T T/C T/C C/C T/T C/C T/T T/C T/C T/T 33 rs111953232 1571862 G GA G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G rs9328919 1572282 C T C/C C/T C/C C/C C/C C/C C/T C/C T/T C/C C/T C/C C/C C/C C/C 34 rs4787271 1572314 T C T/T T/C T/T T/T T/T T/T T/C T/T C/C T/T C/C T/T T/T T/C T/T rs9673971 1572393 G A G/G G/A G/G G/G G/G G/G G/G G/G A/A G/G G/A G/G G/G G/A G/G 35 rs8055979 1572569 T C T/T T/C T/T T/T T/T T/T T/C T/C C/C T/T C/C T/T T/C T/C T/T rs76393829 1572594 C T C/C C/C C/C C/C C/C C/C C/C C/T C/C C/C C/T C/C C/T C/C C/C 36 rs554375875 1573219 CAC CA/CA CA/CA CA/CA CA/CA CA/CA CA/C CA/CA CA/C C/C CA/CA CA/CA CA/CA CA/C CA/C CA/CA rs11361072 1573219 CA C CA/CA CA/C CA/CA CA/CA CA/CA CA/CA CA/CA CA/CA CA/CA CA/CA CA/C CA/CA CA/CA CA/CA CA/CA 37 rs34819117 1573485 G A G/G G/G G/G G/G G/G G/G G/G G/A G/G G/G G/A G/G G/A G/G G/G rs78028801 1573754 C T C/C C/C C/C C/C C/C C/C C/T C/C C/T C/C C/T C/C C/C C/T C/C 38 rs2745176 1573810 A G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G rs2235638 1573890 G A G/G G/G G/G G/G G/G G/G G/G G/A G/G G/G G/A G/G G/A G/G G/G 39 rs2076437 1574030 GC G/G G/G G/G G/G G/G G/C G/G G/C G/G G/G G/G G/G G/G G/G G/G rs141171038 1574170 AG A/AA/AA/AA/A A/A A/A A/A A/A A/A A/AA/AA/A A/AA/AA/A 40 rs2235639 1574252 AGA/GA/GA/AA/A A/A A/A A/G A/G G/G A/G G/G A/A G/G A/G A/A rs2235640 1574863 G A G/G G/A G/G G/G G/G G/G G/G G/G A/A G/G G/A G/G G/G G/A G/G 41 rs2745178 1575704 T C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C rs112977910 1576858 C T C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/T C/C C/C C/C 42 rs2745181 1577056 AGA/AA/GA/AA/A A/G A/A A/G A/G G/G A/A G/G A/G A/GA/GA/A rs12599859 1577184 AGA/AA/AA/AA/A A/A A/A A/A A/G A/A A/AA/GA/A A/GA/AA/A 43 rs9939655 1577963 AGA/AA/GA/AA/A A/G A/A A/G A/G G/G A/A G/G A/G A/GA/GA/A rs192452222 1578295 C T C/C C/C C/C C/C C/C C/C C/T C/C C/C C/C C/C C/C C/C C/T C/C 44 rs140400369 1578373 C G C/C C/C C/C C/C C/C C/C C/G C/C C/C C/C C/C C/C C/CC/GC/C rs149176109 1578434 G GCA G/G G/GCA G/G G/G G/GCA G/G G/GCA G/GCA GCA/GCA G/G GCA/GCA G/GCA G/GCA G/GCA G/G 45 rs1983600 1578514 ACA/AA/AA/AA/A A/A A/A A/A A/C A/A A/AA/CA/A A/CA/AA/A rs1983601 1578725 G A G/G G/G G/G G/G G/G G/G G/G G/A G/G G/G G/A G/G G/A G/G G/G 46 rs3784825 1578914 AGA/AA/AA/AA/A A/A A/A A/A A/G A/A A/AA/GA/A A/GA/AA/A rs138773419 1578999 GA G/G G/G G/G G/G G/G G/A G/G G/A G/G G/G G/G G/G G/G G/G G/G 47 rs2064291 1579011 T C T/T T/T T/T T/T T/T T/T T/T T/C T/T T/T T/C T/T T/C T/T T/T rs3784828 1579151 C T C/C C/C C/C C/C C/C C/C C/C C/T C/C C/C C/T C/C C/T C/C C/C 48 rs148131225 1579244 TC T/T T/T T/T T/T T/T T/T T/T T/T T/C T/T T/T T/T T/T T/T T/T rs9934455 1579483 C T C/C C/T C/C C/C C/T C/C C/T C/C T/T C/C C/T C/T C/C C/T C/C 49 rs4786275 1580025 AGA/GA/AA/AA/A A/A A/A A/A A/G A/A A/AA/GA/A A/GA/AA/A rs113201692 1580708 GA G/G G/G G/G G/G G/A G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G 50 rs142700555 1580752 G T G/G G/G G/G G/G G/G G/G G/G G/G G/G G/T G/G G/G G/G G/G G/G rs2745183 1581770 AGA/GA/AA/AA/A A/A A/A A/A A/G A/A A/AA/GA/A A/GA/AA/A 51 rs67832788 1582870 C T C/T C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C rs3809586 1583201 C T C/C C/C C/C C/C C/C C/C C/C C/T C/C C/C C/T C/C C/T C/C C/C 52 rs118152236 1583238 C A C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/A rs8048206 1583634 T C T/C T/T T/T T/T T/T T/T T/T T/C T/T T/T T/C T/T T/C T/T T/T 53 54 55 56 57 58 59 16 60 John Wiley & Sons, Inc. Human Mutation Page 46 of 52

1 2 rs1057612 1584446 G C G/C G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G 3 rs2076438 1584618 T C T/C T/C T/T T/T T/C T/T T/C T/C C/C T/T C/C T/C C/C T/C T/T rs12446975 1584628 G A G/G G/G G/G G/G G/G G/G G/G G/A G/G G/G G/A G/G G/A G/G G/G 4 rs143833421 1584847 G A G/G G/A G/A G/A G/G G/A G/A G/G G/G G/G G/G G/G G/G G/G G/A rs2235642 1584866 T C T/C T/C T/T T/T T/C T/T T/C T/C C/C T/T C/C T/C C/C T/C T/T rs116903565 1584867 G A G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/A G/G G/G G/G G/G 5 rs2235643 1585115 G A G/G G/A G/G G/G G/A G/G G/A G/G A/A G/G G/A G/A G/A G/A G/G rs7196649 1585456 AGA/GA/AA/AA/AA/AA/AA/AA/AA/AA/AA/AA/AA/AA/AA/A 6 rs141460785 1585650 AGA/GA/AA/GA/GA/AA/AA/AA/AA/AA/AA/AA/AA/AA/AA/A rs72761105 1585928 C T C/T C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C 7 rs2294624 1586255 G A G/A G/G G/G G/G G/G G/G G/G G/A G/G G/G G/A G/G G/A G/G G/G rs190265545 1586275 C T C/C C/C C/C C/C C/C C/C C/C C/C C/T C/C C/C C/C C/C C/C C/C 8 rs2294625 1586596 C T C/C C/C C/C C/C C/C C/C C/C C/T C/C C/C C/T C/C C/T C/C C/C rs2294626 1586717 C G C/C C/C C/C C/C C/G C/C C/G C/C G/G C/C C/G C/G C/GC/GC/C 9 rs2294627 1586821 G A G/G G/G G/G G/G G/G G/G G/G G/A G/G G/G G/A G/G G/A G/G G/G rs12923143 1587067 TC T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/C T/T 10 rs2076439 1587277 C T C/T C/C C/C C/C C/T C/C C/T C/T T/T C/C T/T C/T T/T C/T C/C rs2076440 1587322 G A G/A G/G G/G G/G G/A G/G G/A G/A A/A G/G A/A G/A A/A G/A G/G 11 rs2066937 1587511 G A G/G G/G G/G G/G G/A G/G G/A G/G A/A G/G G/A G/A G/A G/A G/G rs141085747 1587842 C T C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/T C/C C/C C/C C/C 12 rs78140717 1587938 C T C/C C/C C/C C/C C/C C/C C/C C/T C/C C/C C/T C/C C/T C/C C/C rs8051295 1588087 AGA/GA/AA/AA/A A/G A/A A/G A/G G/G A/A G/G A/G G/G A/G A/A 13 rs28668520 1588800 G A G/G G/G G/G G/G G/A G/G G/A G/G A/A G/G G/A G/A G/A G/A G/G rs35521135 1588849 C T C/C C/C C/C C/C C/T C/C C/T C/C T/T C/C C/T C/T C/T C/T C/C 14 rs189807580 1589671 T C T/T T/T T/T T/T T/T T/T T/T T/T T/T T/C T/T T/T T/T T/T T/T rs80113230 1590168 G A G/G G/G G/G G/G G/G G/G G/G G/A G/G G/G G/A G/G G/A G/G G/G 15 rs116941519 1590480 GA G/G G/G G/G G/G G/A G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G rs2076441 1590576 C G C/G C/G C/G C/G C/G C/G G/G C/G G/G C/C G/G C/G G/G G/G C/G 16 rs6600152 1590672 G A G/A G/G G/G G/G G/G G/G G/G G/A G/G G/G G/A G/G G/A G/G G/G rs2076442 1591112 G T G/T G/G G/G G/G G/T G/G G/T G/T T/T G/G T/T G/T T/T G/T G/G 17 rs117189215 1591284 GA G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G rs2050117 1591592 G A G/G G/G G/G G/G G/A G/G G/A G/G A/A G/G G/A G/A G/A G/A G/G 18 rs2076443 1591962 G A G/G G/G G/G G/G G/A G/G G/A G/G A/A G/G G/A G/A G/A G/A G/G rs2076444 1592087 T C T/T T/T T/T T/T T/C T/T T/C T/T C/C T/T T/C T/C T/C T/C T/T 19 rs2076445 1592266 ATA/TA/AA/AA/A A/T A/A A/T A/T T/T A/A T/T A/T T/T A/T A/A rs11649407 1592367 ForG A G/A Peer G/G G/G G/G ReviewG/G G/G G/G G/A G/G G/G G/A G/G G/A G/G G/G 20 rs2076446 1592524 C T C/C C/C C/C C/C C/T C/C C/T C/C T/T C/C C/T C/T C/T C/T C/C rs3784830 1592857 G C G/C G/G G/G G/G G/G G/G G/G G/C G/G G/G G/C G/G G/C G/G G/G 21 rs2050118 1593004 G A G/G G/G G/G G/G G/A G/G G/A G/G A/A G/G G/A G/A G/A G/A G/G rs3784832 1593108 G C G/C G/G G/G G/G G/G G/G G/G G/C G/G G/G G/C G/G G/C G/G G/G 22 rs2143285 1593171 AGA/GA/AA/AA/A A/G A/A A/G A/G G/G A/A G/G A/G G/G A/G A/A rs8049972 1593606 C T C/C C/C C/C C/C C/C C/C C/C C/T C/C C/C C/T C/C C/T C/C C/C 23 rs2064289 1593645 C T C/T C/C C/C C/C C/T C/C C/T C/T T/T C/T T/T C/T T/T C/T C/C rs4558418 1593843 G A G/G G/G G/G G/G G/A G/G G/A G/G A/A G/G G/A G/A G/A G/A G/G 24 rs8053605 1594158 G A G/A G/G G/G G/G G/G G/G G/A G/A A/A G/G A/A G/A A/A G/A G/G rs8053608 1594161 G T G/G G/G G/G G/G G/T G/G G/G G/G T/T G/G G/T G/T G/T G/T G/G 25 rs569784189 1594162 GCACCTGC GGCACCTGC/GCACCTGCGCACCTGC/GCACCTGCGCACCTGC/GCACCTGCGCACCTGC/GCACCTGCGCACCTGC/GCACCTGCGCACCTGC/GCACCTGCGCACCTGC/GCACCTGCGCACCTGC/GGCACCTGC/GCACCTGCGCACCTGC/GCACCTGCGCACCTGC/GCACCTGCGCACCTGC/GCACCTGCGCACCTGC/GGCACCTGC/GCACCTGCGCACCTGC/GCACCTGC rs150672825 1594218 T A T/A T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T 26 rs6600153 1594248 AGA/AA/AA/AA/AA/AA/AA/AA/AA/AA/AA/GA/GA/AA/AA/A rs35055895 1594314 AGA/AA/AA/AA/AA/AA/AA/AA/AA/AA/AA/GA/AA/AA/AA/A rs140189706 1594376 T C T/C T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T 27 rs192068259 1594672 T C T/C T/T T/C T/C T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T rs183277603 1594683 C T C/T C/C C/T C/T C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C 28 rs187549062 1594688 G C G/C G/G G/C G/C G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G rs8056892 1594926 AGA/GA/AA/AA/AA/AA/AA/AA/AA/AA/AA/AA/AA/AA/AA/A 29 rs12445282 1594944 C T C/C C/C C/C C/C C/C C/C C/C C/T C/C C/C C/T C/C C/T C/C C/C rs56342298 1595600 C T C/T C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C 30 rs2294622 1596259 C T C/C C/C C/C C/C C/C C/C C/T C/C C/T C/C C/T C/C C/C C/T C/C rs2294623 1597210 C T C/T C/C C/C C/C C/C C/C C/C C/T C/C C/C C/T C/C C/T C/C C/C 31 rs2235644 1597814 G A G/A G/G G/G G/G G/G G/G G/G G/A G/G G/G G/A G/G G/A G/G G/G rs11864966 1597877 C T C/T C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C 32 rs2235645 1597924 C T C/C C/C C/C C/C C/T C/C C/T C/C T/T C/C C/T C/T C/T C/T C/C rs368948845 1599590 GA G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G 33 rs2281227 1599913 G A G/G G/G G/G G/G G/A G/G G/A G/G A/A G/G G/A G/A G/A G/A G/G rs2281228 1600137 C T C/C C/C C/C C/C C/T C/C C/T C/C T/T C/C C/T C/T C/T C/T C/C 34 rs189219366 1600216 C G C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/G C/C C/C rs74605356 1600272 T C T/T T/T T/T T/T T/T T/T T/T T/C T/T T/T T/C T/T T/C T/T T/T 35 rs2281229 1600738 G A G/G G/G G/G G/G G/G G/G G/G G/A G/G G/G G/A G/G G/A G/G G/G rs2235646 1601287 AGA/GA/AA/AA/A A/G A/A A/G A/G G/G A/A G/G A/G G/G A/G A/A 36 rs2235647 1601331 C A C/A C/C C/C C/C C/A C/C C/A C/C A/A C/C C/A C/A C/AC/AC/C rs2667664 1601458 A G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G 37 rs2281230 1601767 T C T/C T/T T/T T/T T/C T/T T/C T/T C/C T/T T/C T/C T/C T/C T/T rs80150449 1601816 C T C/T C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C 38 rs2281231 1601879 AGA/GA/AA/AA/A A/G A/A A/G A/G G/G A/A G/G A/G G/G A/G A/A rs79038790 1602035 C T C/C C/C C/C C/C C/C C/T C/C C/T C/C C/C C/C C/C C/C C/C C/C 39 rs2281232 1602114 C A C/A C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C rs8050274 1602260 C T C/C C/C C/C C/C C/C C/C C/C C/T C/C C/C C/T C/C C/T C/C C/C 40 rs2281233 1602323 T C T/C T/T T/T T/T T/T T/T T/T T/C T/T T/T T/C T/T T/C T/T T/T rs8051229 1602427 ACA/AA/AA/AA/A A/A A/A A/A A/C A/A A/AA/CA/A A/CA/AA/A 41 rs8049566 1602594 G A G/G G/G G/G G/G G/G G/G G/G G/A G/G G/G G/A G/G G/A G/G G/G rs11409894 1602649 C CT C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C 42 rs559777878 1602649 C CT C/C C/C C/C C/C C/CT C/C C/CT C/CT C/CT C/C C/C C/C C/CT C/CT C/C rs111621426 1602738 C T C/C C/C C/C C/C C/C C/C C/C C/C C/T C/C C/C C/C C/C C/C C/C 43 rs8051895 1602811 AGA/GA/AA/AA/A A/G A/A A/G A/G G/G A/A G/G A/G G/G A/G A/A rs8051909 1602845 AGA/GA/AA/AA/A A/G A/A A/G A/G G/G A/A G/G A/G G/G A/G A/A 44 rs2281234 1602938 AGA/GA/AA/AA/A A/G A/A A/G A/G G/G A/A G/G A/G G/G A/G A/A rs2281235 1603535 T C T/C T/T T/T T/T T/C T/T T/C T/C C/C T/T C/C T/C C/C T/C T/T 45 rs2281236 1603576 G A G/A G/G G/G G/G G/A G/G G/A G/A A/A G/G A/A G/A A/A G/A G/G rs79005075 1603716 C A C/C C/C C/C C/C C/C C/C C/C C/A C/C C/CC/AC/C C/A C/C C/C 46 rs66798554 1604388 G A G/G G/G G/G G/G G/A G/G G/A G/G A/A G/G G/A G/A G/A G/A G/G rs2272976 1604701 T C T/T T/T T/T T/T T/T T/T T/T T/C T/T T/T T/C T/T T/C T/T T/T 47 rs9932984 1605051 AGA/GA/AA/AA/A A/G A/A A/G A/G G/G A/A G/G A/G G/G A/G A/A rs12443939 1605053 C T C/C C/C C/C C/C C/C C/C C/C C/T C/C C/C C/T C/C C/T C/C C/C 48 rs9922661 1605163 C A C/C C/C C/C C/C C/A C/C C/A C/C A/A C/C C/A C/A C/AC/AC/C rs114022362 1605234 T C T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/C T/T T/T T/T 49 rs1057610 1605537 T C T/C T/T T/T T/T T/C T/T T/C T/C C/C T/T C/C T/C C/C T/C T/T rs17135391 1605606 C T C/C C/C C/C C/C C/T C/C C/T C/C T/T C/C C/T C/T C/T C/T C/C rs8048382 1605975 C T C/T C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C 50 rs8053335 1606167 AGA/GA/AA/AA/AA/AA/AA/AA/AA/AA/AA/AA/AA/AA/AA/A 51 rs138001290 1606336 C T C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C 52 53 54 55 56 57 58 59 17 60 John Wiley & Sons, Inc. Page 47 of 52 Human Mutation

1 2 rs138001290 1606336 C T C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C 3 rs55817051 1606444 AGA/GA/AA/AA/A A/G A/A A/G A/G G/G A/A G/G A/G G/G A/G A/A rs12599207 1606445 AGA/GA/AA/AA/A A/G A/A A/G A/G G/G A/A G/G A/G G/G A/G A/A 4 rs55963259 1606479 C T C/T C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C rs12927247 1606739 AGA/AA/AA/AA/A A/G A/A A/G A/A G/G A/AA/GA/G A/GA/GA/A rs112810668 1607113 GC G GC/G GC/GC GC/GC GC/GC GC/GC GC/GC GC/GC GC/G GC/GC GC/GC GC/G GC/GC GC/G GC/GC GC/GC 5 rs116905776 1607242 C T C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C rs11642907 1607284 G T G/G G/G G/G G/G G/G G/G G/G G/T G/G G/G G/T G/G G/T G/G G/G 6 rs78753725 1607286 G C G/C G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G rs11639523 1607564 T C T/T T/T T/T T/T T/T T/T T/T T/C T/T T/T T/C T/T T/C T/T T/T 7 rs2076435 1607574 C T C/T C/C C/C C/C C/T C/C C/T C/T T/T C/C T/T C/T T/T C/T C/C rs2076436 1608082 AGA/AA/AA/AA/A A/G A/A A/G A/A G/G A/AA/GA/G A/GA/GA/A 8 rs2235641 1608177 AGA/AA/AA/AA/A A/G A/A A/G A/G G/G A/A G/G A/G G/G A/G A/A rs12446290 1608364 G A G/G G/G G/G G/G G/G G/G G/G G/A G/G G/G G/A G/G G/A G/G G/G 9 rs74821318 1608729 G A G/G G/G G/G G/G G/G G/G G/G G/A G/G G/G G/A G/G G/A G/G G/G rs540739250 1608904 C T C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/T C/C C/C 10 rs56289115 1608934 AGA/GA/AA/AA/A A/G A/A A/G A/G G/G A/A G/G A/G G/G A/G A/A rs74381546 1609135 T C T/T T/T T/T T/T T/T T/T T/T T/C T/T T/T T/C T/T T/C T/T T/T 11 rs145464572 1609624 C A C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C rs3784835 1609843 G C G/G G/G G/G G/G G/G G/G G/G G/C G/G G/G G/C G/G G/C G/G G/G 12 rs34316191 1609944 C T C/C C/T C/T C/T C/C C/T C/T C/C C/C C/C C/C C/C C/C C/T C/T rs3784836 1610176 G A G/G G/G G/G G/G G/G G/G G/G G/A G/G G/G G/A G/G G/A G/G G/G 13 rs3784837 1610215 C G C/G C/C C/C C/C C/C C/C C/C C/G C/C C/CC/GC/C C/G C/C C/C rs2003343 1610578 GA G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/A 14 rs1016499 1610885 AGA/GA/AA/AA/A A/A A/A A/A A/G A/A A/AA/GA/A A/GA/AA/A rs748546374 1612145 GT G GT/GT GT/GT GT/GT GT/GT GT/GT GT/G GT/GT GT/G GT/GT GT/GT GT/GT GT/GT GT/GT GT/G GT/GT 15 rs188815635 1612459 C T C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C rs3784838 1612498 C G C/G C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C 16 rs12935364 1613348 AGA/GA/AA/AA/A A/A A/A A/A A/G A/A A/AA/GA/A A/GA/AA/A rs12446060 1613365 G A G/G G/G G/G G/G G/G G/G G/G G/A G/G G/G G/A G/G G/A G/G G/G 17 rs184721812 1613428 TC T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T rs28714146 1613793 ACA/AA/AA/AA/A A/CA/AA/CA/AC/C A/AA/CC/C A/CA/CA/A 18 rs778960409 1614093 GA G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G rs8048410 1614097 AGA/GA/AA/AA/A A/G A/A A/G A/G G/G A/A G/G G/G G/G A/G A/A 19 rs11248883 1614240 C G C/C C/C C/C C/C C/C C/C C/C C/G C/C C/CC/GC/C C/G C/C C/C rs17135407 1615189 ForG A G/A Peer G/G G/G G/G Review G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G 20 rs11648609 1616201 C T C/C C/C C/C C/C C/C C/C C/C C/T C/C C/C C/T C/C C/T C/C C/C rs116908844 1616528 T A T/T T/T T/T T/T C/C C/C C/C C/T C/C T/A T/T T/T C/T C/C C/C 21 rs11648819 1616663 C T C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/T C/C C/C C/C C/C rs8059442 1616722 GA G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/A 22 rs77486198 1616838 C T C/C C/C C/C C/C C/C C/C C/C C/T C/C C/C C/T C/C C/T C/C C/C rs7186358 1617688 C G C/G C/C C/C C/C C/C C/C C/C C/G C/C C/CC/GC/C C/G C/C C/C 23 rs11641175 1618007 C A C/C C/C C/C C/C C/C C/C C/C C/A C/C C/CC/AC/C C/A C/C C/C rs11646462 1618112 ACA/AA/AA/AA/A A/A A/A A/A A/C A/A A/AA/CA/A A/CA/AA/A 24 rs72761114 1618995 T C T/T T/T T/T T/T T/T T/T T/T T/T T/T T/C T/T T/T T/T T/T T/T rs180759409 1619066 C T C/T C/C C/T C/T C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C 25 rs9927187 1619074 C T C/T C/C C/C C/C C/C C/C C/C C/C C/C C/C C/T C/C C/T C/C C/C rs9939899 1619082 T C T/C T/T T/T T/T T/T T/T T/T T/C T/T T/T T/C T/T T/C T/T T/T 26 rs9927511 1619431 C T C/T C/C C/C C/C C/C C/C C/C C/T C/C C/C C/T C/C C/T C/C C/C rs9927529 1619488 C T C/T C/C C/C C/C C/T C/C C/T C/T T/T C/C T/T C/T T/T C/C C/C rs181102139 1619703 TA T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T 27 rs113900211 1619758 ATTTTTTTTTTTTATTTTTTTTTTTTTTATTTTTTTTTTTT/ATTTTTTTTTTTTATTTTTTTTTTTT/ATTTTTTTTTTTTATTTTTTTTTTTT/ATTTTTTTTTTTTATTTTTTTTTTTT/ATTTTTTTTTTTTATTTTTTTTTTTT/ATTTTTTTTTTTTATTTTTTTTTTTT/ATTTTTTTTTTTTATTTTTTTTTTTT/ATTTTTTTTTTTTATTTTTTTTTTTT/ATTTTTTTTTTTTATTTTTTTTTTTT/ATTTTTTTTTTTTATTTTTTTTTTTT/ATTTTTTTTTTTTATTTTTTTTTTTT/ATTTTTTTTTTTTTTATTTTTTTTTTTT/ATTTTTTTTTTTTATTTTTTTTTTTT/ATTTTTTTTTTTTATTTTTTTTTTTT/ATTTTTTTTTTTTATTTTTTTTTTTT/ATTTTTTTTTTTT rs57236076 1619758 AATA/ATA/AA/AA/AA/AA/AA/AA/AA/AA/AA/AA/AA/AA/AA/A 28 rs551270839 1619758 A AT,ATT A/AA/AA/AA/A A/A A/A A/A A/ATT A/AT A/AA/AA/A A/ATT A/A A/A rs117401189 1619761 T G T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/G T/T T/T T/T 29 rs141314600 1619825 GC G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G rs7200291 1619896 ATA/TA/AA/AA/AA/AA/AA/AA/AA/AA/AA/AA/AA/AA/AA/A 30 rs149487053 1620307 C T C/C C/T C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C rs118102263 1620424 G C G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/C G/G G/G G/G 31 rs76069945 1620468 G C G/G G/G G/G G/G G/G G/G G/G G/C G/G G/G G/C G/G G/C G/G G/G rs72761116 1620550 C T C/T C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C 32 rs12448197 1621119 G C G/G G/G G/G G/G G/G G/G G/G G/C G/G G/G G/C G/G G/C G/G G/G rs199826737 1621495 C T C/C C/C C/C C/C C/C C/T C/C C/T C/C C/C C/C C/C C/C C/C C/C 33 rs72481037 1621702 C T C/C C/C C/C C/C C/C C/C C/C C/T C/C C/C C/T C/C C/T C/C C/C rs59963862 1621967 T C T/T T/T T/T T/T T/T T/T T/T T/C T/T T/T T/C T/T T/C T/T T/T 34 rs17135411 1622008 AGA/GA/AA/AA/A A/A A/A A/A A/G A/A A/AA/GA/A A/GA/AA/A rs58856196 1622092 C T C/T C/C C/C C/C C/C C/C C/C C/T C/C C/C C/T C/C C/T C/C C/C 35 rs58334533 1622108 G A G/A G/G G/G G/G G/G G/G G/G G/A G/G G/G G/A G/G G/A G/G G/G rs190238492 1622539 C T C/C C/T C/T C/T C/C C/T C/T C/C C/C C/C C/C C/C C/C C/C C/T 36 rs722565 1623251 G A G/A G/G G/G G/G G/G G/G G/G G/A G/G G/G G/A G/G G/A G/G G/G rs137940418 1623473 CTTGTATT C CTTGTATT/CCTTGTATT/CTTGTATTCTTGTATT/CTTGTATTCTTGTATT/CTTGTATTCTTGTATT/CTTGTATTCTTGTATT/CTTGTATTCTTGTATT/CTTGTATTCTTGTATT/CCTTGTATT/CTTGTATTCTTGTATT/CTTGTATTCTTGTATT/CCTTGTATT/CTTGTATTCTTGTATT/CCTTGTATT/CTTGTATTCTTGTATT/CTTGTATT 37 rs77960132 1623638 C T C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C rs13380687 1623852 AGA/GA/AA/AA/A A/A A/A A/A A/G A/A A/AA/GA/A A/GA/AA/A 38 rs147054463 1624106 T C T/C T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T rs10903010 1624142 T C T/C T/T T/T T/T T/T T/T T/T T/C T/T T/T T/C T/T T/C T/T T/T 39 rs12596975 1624493 G A G/A G/G G/G G/G G/G G/G G/G G/A G/G G/G G/A G/G G/A G/G G/G rs112369751 1624528 C T C/T C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C 40 rs138310451 1624771 CCCCT C CCCCT/C CCCCT/CCCCTCCCCT/CCCCTCCCCT/CCCCTCCCCT/CCCCTCCCCT/CCCCTCCCCT/CCCCT CCCCT/C CCCCT/CCCCTCCCCT/CCCCT CCCCT/C CCCCT/CCCCT CCCCT/C CCCCT/CCCCTCCCCT/CCCCT rs8060906 1624784 T G T/T T/T T/T T/T T/G T/T T/G T/T G/G T/T T/G T/G T/G T/G T/T 41 rs8045127 1625244 T G T/T T/T T/T T/T T/G T/T T/G T/T G/G T/T T/G T/G T/G T/G T/T rs13380456 1625890 T C T/C T/T T/T T/T T/T T/T T/T T/C T/T T/T T/C T/T T/C T/T T/T 42 rs111924003 1626058 G A G/A G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G rs12443870 1626079 AGA/GA/AA/AA/A A/A A/A A/A A/G A/A A/AA/GA/A A/GA/AA/A 43 rs35262072 1626287 T TA T/TA T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T rs12444529 1626318 T C T/C T/T T/T T/T T/T T/T T/T T/T T/T T/T T/C T/T T/T T/T T/T 44 rs34888852 1626592 C CA C/C C/C C/C C/C C/CA C/CA C/CA C/CA C/C C/C C/C C/CA C/CA C/CA C/C rs59783459 1626607 C A C/C C/C C/C C/C C/C C/C C/C C/C C/C C/CC/AC/C C/C C/C C/C 45 rs148769752 1626652 TCAGA T TCAGA/TCAGATCAGA/TCAGATCAGA/TCAGATCAGA/TCAGATCAGA/TCAGATCAGA/TCAGATCAGA/TTCAGA/TCAGATCAGA/TTCAGA/TCAGATCAGA/TTCAGA/TCAGATCAGA/TCAGATCAGA/TTCAGA/TCAGA rs117108136 1626702 GA G/G G/G G/G G/G G/A G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G 46 rs8182161 1626705 T A T/A T/T T/T T/T T/T T/T T/T T/A T/T T/T T/A T/T T/A T/T T/T rs117592744 1626884 GC G/G G/G G/G G/G G/C G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G 47 rs71385707 1627426 TC T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/C T/T rs8052868 1627508 AGA/GA/AA/AA/A A/A A/A A/A A/G A/A A/AA/GA/A A/GA/AA/A 48 rs11248884 1627618 G A G/G G/G G/G G/G G/G G/G G/G G/A G/G G/G G/A G/G G/A G/G G/G rs8182166 1627755 C T C/T C/C C/C C/C C/C C/C C/C C/T C/C C/C C/T C/C C/T C/C C/C 49 rs539219664 1627856 TA T TA/TA TA/TA TA/TA TA/TA TA/T TA/TA TA/TA TA/TA TA/TA TA/TA TA/TA TA/TA TA/T TA/T TA/TA rs35320694 1627973 C CAA C/CAA C/C C/C C/C C/C C/C C/C C/CAA C/C C/C C/CAA C/C C/CAA C/C C/C 50 rs11644174 1628225 T G T/T T/T T/T T/T T/T T/T T/T T/G T/T T/T T/G T/T T/G T/T T/T rs11647721 1628295 G A G/G G/G G/G G/G G/G G/G G/G G/A G/G G/G G/A G/G G/A G/G G/G 51 rs36091619 1628579 TA T TA/TA TA/TA TA/TA TA/TA TA/TA TA/TA TA/T TA/T TA/TA TA/TA T/T TA/TA TA/TA TA/T TA/TA rs36091619 1628579TAAAAAAAAAAAAAAAATAAAAAAAAAAAAAAAA/TAAAAAAAAAAAAAAAATAAAAAAAAAAAAAAATAAAAAAAAAAAAAAAA/TAAAAAAAAAAAAAAAATAAAAAAAAAAAAAAAA/TAAAAAAAAAAAAAAAATAAAAAAAAAAAAAAAA/TAAAAAAAAAAAAAAAATAAAAAAAAAAAAAAAA/TAAAAAAAAAAAAAAAATAAAAAAAAAAAAAAAA/TAAAAAAAAAAAAAAAATAAAAAAAAAAAAAAAA/TAAAAAAAAAAAAAAAATAAAAAAAAAAAAAAAA/TAAAAAAAAAAAAAAAATAAAAAAAAAAAAAAAA/TAAAAAAAAAAAAAAAATAAAAAAAAAAAAAAAA/TAAAAAAAAAAAAAAAATAAAAAAAAAAAAAAAA/TAAAAAAAAAAAAAAAATAAAAAAAAAAAAAAAA/TAAAAAAAAAAAAAAAATAAAAAAAAAAAAAAAA/TAAAAAAAAAAAAAAAATAAAAAAAAAAAAAAAA/TAAAAAAAAAAAAAAAATAAAAAAAAAAAAAAAA/TAAAAAAAAAAAAAAAA 52 53 54 55 56 57 58 59 18 60 John Wiley & Sons, Inc. Human Mutation Page 48 of 52

1 2 rs368082114 1628604 C T C/C C/C C/C C/C C/C C/C C/T C/C C/C C/C C/C C/C C/C C/T C/C 3 rs138003160 1629061 TC T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T rs573551047 1629964 CAC CA/CA CA/CA CA/CA CA/CA CA/CA CA/CA CA/C CA/CA CA/CA CA/CA CA/CA CA/CA CA/CA CA/CA CA/CA 4 rs11867080 1630185 T C T/C T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T rs3743507 1630381 G A G/G G/G G/G G/G G/A G/G G/A G/G A/A G/G G/A G/A G/A G/A G/G rs56218869 1631294 AGA/AA/AA/AA/AA/AA/AA/AA/AA/AA/GA/AA/AA/AA/AA/A 5 rs114623241 1631536 G A G/A G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G rs140950029 1631591 A ACT A/ACT A/A A/A A/A A/A A/A A/A A/ACT A/A A/A A/ACT A/A A/ACTA/AA/A 6 rs187641585 1631762 G A A/A G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G rs117406049 1631809 T G G/G T/T T/T T/T T/T T/T T/T T/G T/T T/T T/T T/T T/G T/T T/T 7 rs72761124 1632448 C T C/T C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C rs76835012 1632901 C T C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C 8 rs9934342 1632965 G A G/A G/G G/G G/G G/G G/G G/G G/A G/G G/G G/A G/G G/A G/G G/G rs9927202 1633100 AGA/GA/AA/AA/AA/AA/AA/AA/AA/AA/AA/AA/AA/AA/AA/A 9 rs147086637 1633446 ACA/CA/AA/CA/CA/AA/AA/AA/AA/AA/AA/AA/AA/AA/AA/A rs8055710 1633872 G A G/A G/G G/G G/G G/G G/G G/G G/A G/G G/G G/A G/G G/A G/G G/G 10 rs28406883 1633946 G C G/C G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G rs34762152 1634385 C T C/T C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C 11 rs76705317 1635137 G A G/G G/G G/G G/G G/G G/G G/A G/G G/A G/G G/A G/G G/G G/A G/G rs55695276 1635161 AGA/GA/AA/AA/AA/AA/AA/AA/AA/AA/AA/AA/AA/AA/AA/A 12 rs28647246 1635752 T C T/C T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T rs28608068 1635872 C T C/T C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C 13 rs11640303 1635908 GAA/AA/AA/AA/A A/A A/A A/A A/A A/A A/AA/AA/A A/AA/AA/A rs2236268 1636549 C A C/A C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C 14 rs71860883 1636628 GCA G GCA/G GCA/GCA GCA/GCA GCA/GCA GCA/G GCA/G GCA/G GCA/G GCA/GCA GCA/GCA G/G GCA/GCA GCA/GCA GCA/G GCA/GCA rs76842429 1636896 C T C/C C/C C/C C/C C/C C/C C/C C/T C/C C/C C/T C/C C/T C/C C/C 15 rs113143675 1636908 AGA/GA/AA/AA/AA/AA/AA/AA/AA/AA/AA/AA/AA/AA/AA/A rs77750249 1636935 T G T/T T/T T/T T/T T/T T/T T/T T/G T/T T/T T/G T/T T/G T/T T/T 16 rs574261663 1637667 AG A/AA/AA/AA/A A/A A/A A/A A/A A/A A/AA/AA/A A/AA/AA/A rs431905521 1637962 C T C/C C/C C/C C/C C/C C/C C/C C/C C/T C/C C/C C/C C/C C/C C/C 17 rs35972580 1638323 AGA/AA/AA/AA/A A/A A/A A/A A/G A/A A/AA/GA/A A/GA/AA/A rs180758804 1638450 C T C/C C/C C/C C/C C/C C/T C/C C/T C/C C/C C/C C/C C/C C/C C/C 18 rs145693119 1638528 AAATAATAATAATAATAATA A/AA/AA/AA/AA/AAATAATAATAATAATAATA/AAATAATAATAATAATAATA/A A/A A/A A/AA/AA/A A/AAATAATAATAATAATAATA/A A/A rs145693119 1638528 AAATAATAATAATAATAATAATA A/AA/AA/AA/A A/AA/AA/AA/AAATAATAATAATAATAATAATA/A A/AA/AA/A A/AA/AA/A 19 rs72761127 1638820 C T C/T C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C rs9924141 1638989 ForT C T/C Peer T/T T/T T/T Review T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T 20 rs142607578 1639029 GA G/G G/G G/G G/G G/G G/A G/G G/A G/G G/G G/G G/G G/G G/G G/G rs12447357 1639793 C G C/G C/C C/C C/C C/C C/C C/C C/G C/C C/CC/GC/C C/G C/C C/C 21 rs112431294 1639860 AT A/AA/AA/AA/A A/T A/A A/A A/A A/A A/AA/AA/A A/AA/AA/A rs72761128 1640035 T C T/C T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T 22 rs758621945 1640201 C CA C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/CA C/C C/C rs11859318 1640301 C G C/G C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C 23 rs11641248 1641093 C T C/C C/C C/C C/C C/C C/C C/C C/T C/C C/C C/T C/C C/T C/C C/C rs536399679 1641983 GA G/G G/G G/G G/G G/G G/G G/A G/G G/G G/G G/G G/G G/G G/A G/G 24 rs116859784 1642026 G A G/A G/G G/A G/A G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G rs201188361 1642177 C T C/C C/C C/C C/C C/T C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C 25 rs2273679 1642423 C T C/T C/C C/C C/C C/C C/C C/C C/T C/C C/C C/T C/C C/T C/C C/C rs112565100 1642660 T C T/C T/T T/T T/T T/T T/T T/T T/C T/T T/T T/C T/T T/C T/T T/T 26 rs9927226 1642679 G A G/A G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G rs80064098 1642906 T C T/T T/T T/T T/T T/T T/T T/T T/C T/T T/T T/C T/T T/C T/T T/T 27 rs796687743 1643316 CT C C/T C/T C/T C/T CT/C CT/CT CT/CT CT/CT CT/CT C/T C/T C/T CT/CT CT/C CT/CT rs187540964 1643455 GA G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G rs12446250 1643552 C T C/C C/C C/C C/C C/C C/C C/C C/T C/C C/C C/T C/C C/T C/C C/C 28 rs12449265 1643606 ATA/AA/AA/AA/A A/A A/A A/A A/T A/A A/AA/TA/A A/TA/AA/A rs116910642 1644032 C T C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/T 29 rs536466763 1644227 G GT G/G G/G G/G G/G G/GT G/G G/GT G/GT G/G G/G G/G G/G G/G G/GT G/GT rs57833692 1644227 G GT G/G G/G G/G G/G G/G G/G G/G G/G G/G G/GT G/G G/G G/G G/G G/G 30 rs2755183 1644312 C G C/G C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C rs2941956 1644764 AGA/GA/AA/AA/AA/AA/AA/AA/AA/AA/AA/AA/AA/AA/AA/A 31 rs9933780 1644808 C G C/G C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C rs112191146 1645058 G A G/A G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G 32 rs2755184 1645264 T C T/C T/T T/T T/T T/C T/T T/C T/C C/C T/T C/C T/C C/C T/C T/T rs548658471 1645604 G GA G/G G/G G/G G/G G/G G/G G/G G/GA G/GA G/G G/G G/G G/GA G/G G/GA 33 rs17135416 1645745 T C T/C T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T rs2755185 1645827 AGA/GA/AA/AA/A A/G A/A A/G A/G G/G A/A G/G A/G G/G A/G A/A 34 rs35576736 1646018 C T C/C C/C C/C C/C C/T C/C C/T C/C T/T C/C C/T C/T C/T C/T C/C rs2667669 1646543 G A G/A G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G 35 rs193299834 1646787 C T C/C C/C C/C C/C C/T C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C rs13334027 1646934 ACA/CA/AA/AA/AA/AA/AA/AA/AA/AA/AA/AA/AA/AA/AA/A 36 rs143684159 1646973 C A C/C C/A C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C rs6600156 1648126 ACA/CA/AA/AA/AA/AA/AA/AA/AA/AA/AA/CA/CA/AA/AA/A 37 rs6600156 1648126 AC A/AA/AA/AA/A A/CA/AA/CA/AC/C A/AA/AA/A A/CA/CA/A rs2974845 1648162 T C T/C T/T T/T T/T T/C T/T T/C T/C C/C T/T C/C T/C C/C T/C T/T 38 rs4786368 1648638 G C G/C G/G G/G G/G G/C G/G G/C G/C C/C G/C C/C G/C C/C G/C G/G rs78305748 1648685 G C G/G G/G G/G G/G G/G G/G G/G G/C G/G G/G G/C G/G G/C G/G G/G 39 rs186995221 1649287 AGA/AA/AA/AA/AA/AA/AA/AA/AA/AA/AA/GA/AA/AA/AA/A rs201340184 1649393 T C T/T T/T T/T T/T T/T T/T T/T T/T T/T T/C T/T T/C T/T T/T T/T 40 rs180744609 1649476 G T G/G G/T G/T G/T G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G rs769942411 1650461 ATA AT/AT AT/AT AT/AT AT/AT AT/AT AT/AT AT/AT AT/AT AT/A AT/AT AT/AT AT/AT AT/AT AT/AT AT/AT 41 rs137922934 1650463 TTA T TTA/TTA TTA/TTA TTA/TTA TTA/TTA TTA/TTA TTA/TTA TTA/TTA TTA/TTA TTA/TTA TTA/TTA TTA/TTA TTA/TTA TTA/TTA TTA/TTA TTA/TTA rs137922934 1650463 TTA T TTA/TTA TTA/TTA TTA/TTA TTA/TTA TTA/TTA TTA/TTA TTA/TTA TTA/T TTA/T TTA/TTA TTA/T TTA/TTA TTA/T TTA/TTA TTA/TTA 42 rs34665250 1650464 TA T TA/T TA/TA TA/TA TA/TA TA/T TA/T TA/T TA/T TA/T TA/TA TA/TA TA/TA TA/TA TA/T TA/T rs34665250 1650464TAAAAAAAAAAAAAATAAAAAAAAAAAAAA/TAAAAAAAAAAAAAATAAAAAAAAAAAAATAAAAAAAAAAAAAA/TAAAAAAAAAAAAAATAAAAAAAAAAAAAA/TAAAAAAAAAAAAAATAAAAAAAAAAAAAA/TAAAAAAAAAAAAAATAAAAAAAAAAAAAA/TAAAAAAAAAAAAAATAAAAAAAAAAAAAA/TAAAAAAAAAAAAAATAAAAAAAAAAAAAA/TAAAAAAAAAAAAAATAAAAAAAAAAAAAA/TAAAAAAAAAAAAAATAAAAAAAAAAAAAA/TAAAAAAAAAAAAAATAAAAAAAAAAAAAA/TAAAAAAAAAAAAAATAAAAAAAAAAAAA/TAAAAAAAAAAAAATAAAAAAAAAAAAA/TAAAAAAAAAAAAATAAAAAAAAAAAAAA/TAAAAAAAAAAAAAATAAAAAAAAAAAAAA/TAAAAAAAAAAAAAATAAAAAAAAAAAAAA/TAAAAAAAAAAAAAA 43 rs570261391 1650464 T TA T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T rs760111992 1650894 CAC CA/CA CA/CA CA/CA CA/CA CA/C CA/C CA/C CA/C CA/C CA/CA CA/CA CA/CA CA/C CA/C CA/C 44 rs2667684 1650946 AGA/GA/AA/AA/A A/G A/A A/G A/G G/G A/G G/G A/G G/G A/G A/A rs35379437 1651258 C T C/C C/C C/C C/C C/C C/C C/C C/T C/C C/C C/T C/C C/T C/C C/C 45 rs142390870 1651373 GC G/G G/G G/G G/G G/C G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G rs146128830 1652418 C T C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/T C/C C/C 46 rs142257947 1652546 GA G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/A G/G rs2236269 1652614 T A T/A T/T T/T T/T T/A T/T T/A T/A A/A T/T A/A T/A A/A T/A T/T 47 rs59000332 1652621 AGA/AA/AA/AA/A A/A A/A A/A A/G A/A A/AA/GA/A A/GA/AA/A rs140148755 1652680 C A C/C C/C C/C C/C C/A C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C 48 rs2667681 1653206 C T C/T C/C C/C C/C C/T C/C C/T C/T T/T C/C T/T C/T T/T C/T C/C rs75640119 1653223 C G C/C C/C C/C C/C C/C C/C C/C C/G C/C C/CC/GC/C C/G C/C C/C 49 rs537190496 1653487 C CT C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C rs1570210 1654265 G A G/G G/G G/G G/G G/G G/G G/G G/A G/G G/G G/A G/G G/A G/G G/G 50 rs552736270 1654275 CCGT,CGTGT,CGTGTGTC/C C/C C/C C/C C/C C/CGT C/C C/CGTGT C/CGT C/C C/C C/C C/CGTGT C/CGTGTGT C/CGT rs75276217 1654319 G T G/G G/G G/G G/G G/G G/G G/G G/G G/G G/T G/G G/G G/G G/G G/G 51 rs10701133 1654336 T TGAGG T/TGAGG T/T T/T T/T T/TGAGG T/T T/TGAGG T/TGAGGTGAGG/TGAGG T/T TGAGG/TGAGGT/TGAGGTGAGG/TGAGGT/TGAGG T/T 52 53 54 55 56 57 58 59 19 60 John Wiley & Sons, Inc. Page 49 of 52 Human Mutation

1 2 rs138669173 1654525 C G C/C C/C C/C C/C C/C C/C C/G C/C C/C C/C C/C C/C C/C C/G C/C 3 rs7197288 1654753 C A C/A C/C C/C C/C C/A C/C C/A C/A A/A C/C A/A C/A A/A C/A C/C rs2667679 1654946 G A G/A G/G G/G G/G G/A G/G G/A G/G A/A G/G G/A G/A G/A G/A G/G 4 rs56359342 1654948 G A G/A G/G G/G G/G G/A G/G G/A G/G A/A G/G G/A G/A G/A G/A G/G rs117745556 1655073 T C T/T T/T T/T T/T T/T T/T T/T T/C T/T T/T T/C T/T T/C T/T T/T rs35220693 1655439 C T C/C C/C C/C C/C C/C C/C C/C C/T C/C C/C C/T C/C C/T C/C C/C 5 rs6600157 1656321 G A G/A G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G rs142169797 1656564 C T C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C 6 rs2859310 1656765 A G A/G A/A A/A A/A A/G A/A A/G A/G G/G A/A G/G A/G G/G A/G A/A rs542474562 1656927 GCACA G GCACA/GCACAGCACA/GCACAGCACA/GCACAGCACA/GCACAGCACA/GCACAGCACA/GCACAGCACA/GCACAGCACA/GCACAGCACA/GCACAGCACA/GCACAGCACA/GCACAGCACA/GCACAGCACA/GCACAGCACA/GCACAGCACA/G 7 rs7192480 1657012 C T C/C C/C C/C C/C C/T C/C C/T C/C T/T C/C C/T C/T C/T C/T C/C rs34935994 1657034 CAT C CAT/C CAT/CAT CAT/CAT CAT/CAT CAT/CAT CAT/CAT CAT/CAT CAT/CAT CAT/CAT CAT/CAT CAT/CAT CAT/CAT CAT/CAT CAT/CAT CAT/CAT 8 rs1894649 1657284 C G C/G C/C C/C C/C C/G C/C C/G C/G G/G C/C G/G C/G G/G C/G C/C rs743963 1657350 A T A/T A/A A/A A/A A/T A/A A/T A/A T/T A/AA/TA/T A/T A/T A/A 9 rs3784840 1657754 C T C/C C/C C/C C/C C/T C/C C/T C/C T/T C/C C/T C/T C/T C/T C/C rs743964 1657853 C T C/C C/C C/C C/C C/C C/C C/C C/T C/C C/C C/T C/C C/T C/C C/C 10 rs763152 1658056 T C T/C T/T T/T T/T T/C T/T T/C T/C C/C T/T C/C T/C C/C T/C T/T rs763153 1658086 G C G/G G/G G/G G/G G/G G/G G/G G/C G/G G/G G/C G/G G/C G/G G/G 11 rs35888283 1658101 TAG T TAG/T TAG/TAG TAG/TAG TAG/TAG TAG/T TAG/TAG TAG/T TAG/TAG T/T TAG/TAG TAG/T TAG/T TAG/T TAG/T TAG/TAG rs9937922 1658154 C G C/G C/C C/C C/C C/G C/C C/G C/G G/G C/C G/G C/G G/G C/G C/C 12 rs115729443 1658454 C T C/C C/C C/C C/C C/C C/C C/C C/T C/C C/C C/T C/C C/T C/C C/C rs11438070 1658632 C CA C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C 13 rs28519681 1659128 T C T/C T/T T/T T/T T/C T/T T/C T/C C/C T/T C/C T/C C/C T/C T/T rs28633318 1659723 T G T/G T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T T/T 14 rs1057983 1660274 A G A/G A/A A/A A/A A/G A/A A/G A/G G/G A/A G/G A/G G/G A/G A/A rs150781872 1661491 C CA C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C 15 rs2859314 1661701 A G A/G A/A A/A A/A A/A A/A A/G A/G A/G A/A G/G A/A A/G A/G A/A 16 rs553272330 1661814 CA C CA/CA CA/CA CA/CA CA/CA CA/CA CA/C CA/C CA/C CA/C CA/CA CA/CA CA/CA CA/C CA/CA CA/C 17 Table S6. Haplotype analysis for ~100kb around IFT140 on . 18 19 SNP analysis at the IFT140For locus Peer for carrier Review individuals of the tandem duplication and in 20 controls sequenced either by WGS or Target Exome Sequencing (TES, by the Leber panel 21 which include the full IFT140 gene). In total, 6 carrier individuals from 3 families were 22 analysed (family A, B and C) and indicated. Affected patients are depicted with an “*”. Six 23 24 control samples are also shown. For each individual, SNP detected in IFT140 were extracted 25 and listed. Homozygous SNP are shown in green (reference allele) or in blue (alternative 26 allele) and heterozygous SNP in orange. The duplication region is highlighted in grey. 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 20 60 John Wiley & Sons, Inc. Human Mutation Page 50 of 52

1 2 3 4 5 6 7 8 9 10 11 12 13

14 15 Figure S5 AluJb and AluJr alignment 16 17 AluJb and AluJr sequences were extracted from IGV (Thorvaldsdottir, et al., 2013) at the 18 breakpoints of the tandem duplications. Pairwise alignment of the nucleotide sequences and 19 percent identity has beenFor computed Peer using Jalview Review (Waterhouse, et al., 2009). 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 21 60 John Wiley & Sons, Inc. Page 51 of 52 Human Mutation

1 2 3 Technique Individuals CANOES Pindel CNVnator 4 5 Ciliome panel D-II.1 DUP, intron26-intron30 67 DEL, 13 INV, 35 SI NA 2 DEL, 3DUP 6 Ciliome panel K-II.2 36 DEL, 1 INV, 6 SI, 1 TD NA (DUP, intron27-exon31) 7 DUP, intron26-intron30 Leber panel B-I.1 DUP, intron26-intron30 NA 8 (675 DEL, 70 INV, 233 SI, 5 TD) 9 Leber panel B-I.2 0 (688 DEL, 62 INV, 211 SI, 2 TD) NA 10 DUP, intron26-intron30 Leber panel B-II.1 DUP, intron26-intron30 NA 11 (595 DEL, 55 INV, 193 SI, 2 TD) DUP, intron26-intron30 12 Leber panel C-II.1 DUP, intron26-intron30 NA 13 (842 DEL, 120 INV, 417 SI, 5 TD) 14 Leber panel I-II.1 1 DEL (659 DEL, 115 INV, 339 SI, 2 TD) NA 15 WES A-II.1 0 10 DEL, 3 INV, 10 SI, 1 TD NA 16 17 WES A-II.2 DUP, intron27-intron30 13 DEL, 3 INV, 9 SI NA 18 19 WGS A-II.1For DUP,Peer exon26-intron31 Review 14 DEL, 3 INV, 4 SI 0 20 WGS A-II.2 DUP, exon26-intron31 10 DEL, 2 INV, 3 SI 0 21 22 WGS A-I.2 DUP, exon26-intron31 17 DEL, 1 INV, 2 SI 0 23 WGS A-I.1 0 53 DEL, 1 INV, 1 SI 0 24 25 26 Table S7. Summary of the CNV detected in IFT140 by using 3 different tools in 13 27 individuals. 28 29 CNV detection has been performed on 13 individuals sequenced with 4 different approaches: 30 the Ciliome panel, the Leber panel, the WES and the WGS. Three tools have been run with 31 their default options: CANOES (v1.0), Pindel (v0.2.5b9) and CNVnator (v0.3.3) (Abyzov, et 32 al., 2011; Backenroth, et al., 2014; Ye, et al., 2009). The number of CNV detected in IFT140 33 are reported here, as well as the presence or not of the tandem duplication of exons 26 to 30. It 34 35 is to notice that Pindel simultaneously calls small indel and structural variations, increasing 36 thus the number of detected variants. NA, not applicable; DUP, duplication; DEL, deletion; 37 INV, inversion; SI, short insertion; TD, tandem duplication. 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 22 60 John Wiley & Sons, Inc. Human Mutation Page 52 of 52

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