Supplementary table S1. Pathogenic potential of identified DYNC2H1 mutations Family Nucleotide Change Mutation GERP PhyloP SIFT PolyPhen-2 Mutation Taster JATD-1 c. 90443A>G p.d3015g 6.17 5.3 Tolerated Prob damaging (0.995) Disease causing (0.997) (Case 4 A ) c. 1306G>T p.e436* - - - - - JATD-2 c.9044a>g p.d3015g 6.17 5.3 Tolerated Prob damaging (0.995) Disease causing (0.997) (Case 5 A ) c.3459-1g>a Exon 24 splice acceptor 5.56 6.0 Splicing defect - Splicing defect JATD-3 c.9817c>t p.e3273* - - - - - (Case 6, 7 A ) c.7442g>a p.r2481q 5.8 3.9 Tolerated Benign (0.040) Polymorphism (0.78) JATD-4 c.1306g>t p.e436* - - - - - UCL47 c.8457a>g p.i2819m -0.39 0.04 Deleterious Prob damaging (0.954) Disease causing (0.97) JATD-5 c.3682c>a p.l1228i -1 0.3 Tolerated Prob damaging (0.973) Disease causing (0.8) UCL61 c.7663g>a p.v2555m 5.73 3.0 Tolerated Poss damaging (0.476) Polymorphism (0.8) c.7718a>g p.y2573c 6.0 4.2 Tolerated Prob damaging (0.917) Disease causing (0.995) JATD-6 14kb deletion (g.103191405-103204921) p.g3891_q4020del - - - - - c.9044a>g p.d3015g 6.17 5.3 Tolerated Prob damaging (0.995) Disease causing (0.997) c.11437c>t p.r3813c 5.69 4.3 Damaging Prob damaging (1.0) Disease causing (0.999) JATD-7 c.10163c>t p.p3388l 4.78 5.9 Deleterious Prob damaging (0.961) Disease Causing (0.997) UCL19 c.12480_13556del (g.103325916-103350592) p.n4160_q4314del - - - - - JATD-8 c.3719t>c p.i1240t 4.32 5.0 Deleterious Poss damaging (0.803) Disease causing (0.964) UCL 63 c.12716t>g p.l4239r 4.47 4.3 Damaging (low confidence) Prob damaging (0.846) Disease causing (0.998) JATD-9 c. 11560T>G p.w3854g 4.9 4.4 Tolerated Prob damaging (0.994) Disease causing (0.978) 57809 JATD-10 c.6910g>a p.a2304t 4.13 4.3 Tolerated Benign (0.047) Disease Causing (0.93) UCL15 c.8389_8397 delccagctttg p.p2797_l2799 del - - - - - JATD-11 c.195g>t p.t65t 4.37 5.3 Synonymous; splicing defect - Splicing defect UCL90 c.4135a>g p.m1379v 4.32 2.3 Deleterious Benign (0.127) Polymorphism (0.04), splice donor gained JATD-12 c.312_313delta p.p104pfsx*2 - - - - - UCL48 c.5972t>a p.m1991k 4.47 5.0 Deleterious benign (0.429) Disease Causing (0.99) JATD-13 c.4325g>a p.g1442d 3.65 4.3 Deleterious Prob damaging (0.999) Disease causing (0.997) UCL39 c.1953g>a p.k651k 4.47 2.7 Synonymous; splicing defect - Splicing defect JATD-14 c.988c>t p.r330c 4.04 3.5 Deleterious Unknown Disease causing (0.99) UCL80 JATD-15 c. 7594C>T p.r2532w 4.11 1.2 Tolerated Poss. damag (0.761) Disease causing (0.58) UCL58 JATD-16 c.7437+3a>g Exon 45 splice donor 2.11 1.9 Splicing defect - Splicing defect UCL81 c.7539a>t p.g2513g 3.16 1.9 Synonymous; splicing defect - Splicing defect JATD-17 c.6679a>g p.m2227v -0.83 0.47 Deleterious Benign (0.002) Polymorphism, splice donor gained UCL95 c.12538delc p.l4177ffs*29 - - - - - JATD-18 c.2346-5t>g Exon 17 splice acceptor 2.88 1.0 Splicing defect - Splicing defect UCL62 c.7085a>g p.n2362s 4.13 3.3 Deleterious Poss damaging (0.86) Polymorphism (0.66) JATD-19 UCL109 c.7919t>c p.i2640t 4.77 3.7 Deleterious benign (0.255) Disease causing (0.84) c.2612t>c p.l871p 4.05 4.5 Deleterious Prob damaging (0.987) Disease causing (0.99) A Patient described in J. De Vries et al. 2010. Eur J Pediatr 169:77-88. Prediction software used for missense and splicing defects only, evolutionary conservation is scored at nucleotide level by GERP and PhyloP (HG19 values). A Mutation Taster polymorphism classification indicates a change will be tolerated, rather than a known SNP. 1
Supplementary figure S1 Segregation analysis of DYNC2H1 mutations in JATD families Variants identified in the families screened in this study, with double line indicating consanguineous marriages, black filled symbols indicating affected individuals. All identified variants were consistent with affection status and recessive inheritance pattern. 2
Supplementary figure S2 Depth of sequence coverage across DYNC2H1 using different exome sequencing kits Representative samples of coverage achieved using the different exome sequencing kits (A) Agilent v1 38Mb (DYNC2H1 not targeted by this kit) (B) Agilent v2 50Mb, processed in the Netherlands (C) Nimblegen v3 (D) Agilent v2 50Mb, processed in the UK. The y axis shows the coverage, on a scale of 0-100x density of sequence reads. The heterozygously inherited 3 exon deletion in patient from family JATD-7 is boxed. 3
Supplementary figure S3 Genomic deletion present in family JATD-7 patient Heterozygous deletion of 3 exons (exon 87-90) in DYNC2H1 gene identified by ExomeDepth from exome sequence data. The x-axis shows the chromosome position and the y-axis the ratio between the observed and the expected number of reads mapping to each exon. The grey area shows the 99% confidence interval for this ratio. Exons are marked by red crosses, joined by red lines spanning non-sequenced (nonexonic) regions. 4
Supplementary figure S4 Cross-species amino acid alignment in DYNC2H1 for 21 JATD missense mutations and PAL deletion Sequences used: NP_001073932.1 (H.sapiens), XP_863187.1 (C.lupus), XP_589775.4 (B.taurus), NP_084127.2 (M.musculus), NP_075413.1 (R.norvegicus), XP_417173.2 (G.gallus), XP_002664761.1 (D.rerio), NP_492221.2 (C.elegans). Selected protein sequence are shown only. 5
Supplementary figure S5 DYNC2H1 missense mutations are enriched in functional domains similarly to DNAH5 The DYNC2H1 (upper) and DNAH5 (lower) missense mutation count per window is shown (red line) using a sliding window of non-overlapping 100 amino acids. Fishers exact test was used to calculate the enrichment for mutations per 100 amino acid window, the associated log10 p value is shown by the open circles. The lower dotted line indicates p=0.05 and the higher dotted line indicates p<0.01. A single 100 amino acid window between AAA3 and AAA4 is statistically enriched for DYNC2H1 mutations, with similar findings for DNAH5; DNAH5 also shows a non-significant trend of enrichment at 3 other domains in the N- terminus and the MT-binding stalk domain. However, after correcting for multiple hypothesis testing using the Benjamini-Hochberg False Discovery Rate correction, these values are no longer significant for either protein (corrected p values per window shown by filled triangles). DNAH5 mutations taken from previous reports referred to in the main paper. 6
Supplementary figure S6 Skeletal hallmarks of JATD in DYNC2H1 patients (A-K) Hallmarks of JATD: Representative examples of brachydactyly type D (B, JATD-2 in their 20s has solely short and broad thumbs) and cone shaped epiphyses (D, JATD-9; E, JATD-14) were observed. These were found to be absent in some patients (G, JATD-1 when newborn and A, JATD-1 in their 20s; F, JATD-16), and much less pronounced or certain in others (C, JATD-3 in their 20s has possible short fingers). Typical handlebar clavicles were observed at the newborn stage (H, JATD-1; I, JATD-16) while patients in their 20s displayed a much milder thorax phenotype (J, JATD-2; K, JATD-3). 7
Supplementary figure S7 IFT88 concentrates in distal tips of cilia in DYNC2H1 JATD patient fibroblasts Antibody stainings of fibroblasts derived from patients from JATD-1 and JATD-2 families show IFT-B protein IFT88 often accumulates in ciliary tips, suggesting IFT-A transport is disrupted due to DYNC2H1 mutations in these patients. RPGRIP1L (red) is a marker for the ciliary base, and acetylated α-tubulin (cyan) is a marker of the cilia axoneme. The merged images at the left show IFT88 (green), RPGRIP1L and acetylated α-tubulin; right panels display only IFT88 in the same field. Scale bar, 10 μm. Cilia in Figure 3 (main article) are derived from the whole-field images presented here. 8
Supplementary figure S8 IFT57 concentrates in distal tips of cilia in DYNC2H1 JATD patient fibroblasts Antibody stainings of fibroblasts derived from patients from JATD-1 and JATD-2 families show IFT-B protein IFT57 often accumulates in ciliary tips, suggesting IFT-A transport is disrupted due to DYNC2H1 mutations in these patients. RPGRIP1L (red) is a marker for the ciliary base, and acetylated α-tubulin (purple) is a marker of the cilia axoneme. The merged images at the left show IFT57 (green), RPGRIP1L and acetylated α-tubulin; right panels display only IFT57 in the same field. Scale bar, 10 μm. Cilia in Figure 3 (main article) are derived from the whole-field images presented here. 9
UK10K RARE Consortium Saeed Al-Turki 1, 2 Carl Anderson 1 Dinu Antony 3 Inês Barroso 1 Phil Beales 3 Jamie Bentham 4 Shoumo Bhattacharya 4 Keren Carss 1 Krishna Chatterjee 5 Sebhattin Cirak 6 Catherine Cosgrove 4 Petr Danecek 1 Richard Durbin 1 David Fitzpatrick 7 Jamie Floyd 1 Reghan A. Foley 6 Chris Franklin 1 Marta Futema 8 Steve E. Humphries 8 Matt Hurles 1 Chris Joyce 1 Shane McCarthy 1 Hannah M. Mitchison 3 Dawn Muddyman 1 Francesco Muntoni 6 Stephen O'Rahilly 5 Alexandros Onoufriadis 3 Felicity Payne 1 Vincent Plagnol 9 Lucy Raymond 10 David B. Savage 5 Peter J. Scambler 3 Miriam Schmidts 3 Nadia Schoenmakers 5 Robert Semple 5 Eva Serra 1 Jim Stalker 1 Margriet van Kogelenberg 1 Parthiban Vijayarangakannan 1 Klaudia Walter 1 Ros Whittall 8 Kathy Williamson 7 1 The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton CB10 1HH, Cambridge, UK. 2 Department of Pathology, King Abdulaziz Medical City, Riyadh, Saudi Arabia. 3 Molecular Medicine Unit and Birth Defects Research Centre, UCL Institute of Child Health, London, WC1N 1EH, UK. 4 Department of Cardiovascular Medicine and Wellcome Trust Centre for Human Genetics, Roosevelt Drive, Oxford, OX3 7BN, UK. 5 University of Cambridge Metabolic Research Laboratories, and NIHR Cambridge Biomedical Research Centre, Institute of Metabolic Science, Addenbrooke's Hospital, Cambridge, CB2 0QQ, UK. 6 Dubowitz Neuromuscular Centre, UCL Institute of child health & Great Ormond street Hospital, London, WC1N 3JH, UK. 7 MRC Human Genetics Unit, MRC Institute of Genetic and Molecular Medicine, at the University of Edinburgh, Western General Hospital, Edinburgh, EH4 2XU, UK. 8 Cardiovascular Genetics, BHF Laboratories, Rayne Building, Institute Cardiovascular Sciences, University College London, London WC1E 6JJ, UK. 9 University College London (UCL) Genetics Institute (UGI) Gower Street, London, WC1E 6BT, UK. 10 Dept. of Medical Genetics, Cambridge Institute for Medical Research, University of Cambridge, CB2 2XY, UK. 10