Distinct mutations with different inheritance mode caused similar retinal dystrophies in one family: a demonstration of the importance of genetic annotations in complicated pedigrees

Distinct mutations with different inheritance mode caused similar retinal dystrophies in one... Background: Retinitis pigmentosa (RP) is the most common form of inherited retinal dystrophy presenting remarka- ble genetic heterogeneity. Genetic annotations would help with better clinical assessments and benefit gene therapy, and therefore should be recommended for RP patients. This report reveals the disease causing mutations in two RP pedigrees with confusing inheritance patterns using whole exome sequencing ( WES). Methods: Twenty-five participants including eight patients from two families were recruited and received compre - hensive ophthalmic evaluations. WES was applied for mutation identification. Bioinformatics annotations, intrafamilial co-segregation tests, and in silico analyses were subsequently conducted for mutation verification. Results: All patients were clinically diagnosed with RP. The first family included two siblings born to parents with consanguineous marriage; however, no potential pathogenic variant was found shared by both patients. Further analysis revealed that the female patient carried a recurrent homozygous C8ORF37 p.W185*, while the male patient had hemizygous OFD1 p.T120A. The second family was found to segregate mutations in two genes, TULP1 and RP1. Two patients born to consanguineous marriage carried homozygous TULP1 p.R419W, while a recurrent heterozygous RP1 p.L762Yfs*17 was found in another four patients presenting an autosomal dominant inheritance pattern. Crystal structural analysis further indicated that the substitution from arginine to tryptophan at the highly conserved residue 419 of TULP1 could lead to the elimination of two hydrogen bonds between residue 419 and residues V488 and S534. All four genes, including C8ORF37, OFD1, TULP1 and RP1, have been previously implicated in RP etiology. Conclusions: Our study demonstrates the coexistence of diverse inheritance modes and mutations affecting distinct disease causing genes in two RP families with consanguineous marriage. Our data provide novel insights into assess- ments of complicated pedigrees, reinforce the genetic complexity of RP, and highlight the need for extensive molecu- lar evaluations in such challenging families with diverse inheritance modes and mutations. Keywords: Retinitis pigmentosa, Genetic heterogeneity, Next generation sequencing, Mutation, OFD1, C8ORF37, TULP1, RP1, Consanguinity *Correspondence: yanbiao1982@hotmail.com; dr_zhaochen@163.com Xue Chen, Xunlun Sheng, Yani Liu and Zili Li contributed equally to this work Department of Ophthalmology, State Key Laboratory of Reproductive Medicine, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China Full list of author information is available at the end of the article © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/ publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Chen et al. J Transl Med (2018) 16:145 Page 2 of 12 for genomic DNA extraction. Family history and con- Background sanguineous marriages were carefully reviewed. Medical Retinitis pigmentosa (RP, MIM: 268000), the most com- records were obtained from all participants. Each par- mon form of inherited retinal degenerations, affects ticipant received general ophthalmic evaluations, while over one million individuals globally [1, 2]. Night blind- comprehensive ophthalmic examinations were selectively ness is usually the initial symptom for RP, followed by conducted on the eight included patients. Another 150 subsequent visual field constriction, and eventual vision Chinese healthy controls free of major ocular problems loss. RP is featured by great clinical heterogeneities. Its were recruited with their blood samples donated. onset age ranges from early childhood to mid-adulthood. Inter- and intra-familial phenotypic diversities caused by NGS approach and bioinformatics analyses the same RP causing mutations have also been revealed To reveal the disease causing mutation in the two fami- [3–5]. Thus, clinical diagnose for RP patients are some - lies, we selectively performed whole exome sequencing times challenged by its wide phenotypic spectrum and (WES) on three participants in family A (A-IV:3, A-VI:2 under certain conditions, like in a young patient without and A-VI:3) and two patients in family B (B-III:4 and fully onset RP phenotypes. In such situations, molecu- B-IV:1). WES was conducted with the 44.1 megabases lar testing could help to address the clinical ambiguity SeqCap EZ Human Exome Library v2.0 (Roche Nimble- in RP diagnosis. RP also shows high genetic heterogene- Gen, Madison, WI) for enrichment of 23588 genes on ity. To date, 83 RP causing genes involving hundreds of patients from family A [6], and with SureSelect Human mutations have been identified (RetNet). Next-gener - All Exon V6 60  Mb Kit (Agilent Technologies, Santa ation sequencing (NGS), enabling simultaneous paral- Clara, CA) on patients from family B [7]. Briefly, qualified lel sequencing of numerous genes with high efficiency, genomic DNA samples were randomly sheared by Cova- is an efficient tool for molecular diagnosis of RP [2, 4]. ris into 200–250  base pair (bp) fragments. Fragments Genetic annotations with NGS promote better clinical were then ligated with adapters to both ends, amplified assessments and gene therapy, and therefore should be by ligation-mediated polymerase chain reaction (LM- recommended for RP patients. However, pedigrees with PCR), purified, and hybridized. Non-hybridized frag - puzzling inheritance patterns could sometimes confuse ments were then washed out. Quantitative PCR was the genetic diagnoses. Herein, we described the geno- further applied to estimate the magnitude of enrichment typic and phenotypic findings in two complicated RP of both non-captured and captured LM-PCR products. pedigrees using NGS. Distinct inheritance patterns and Each post-capture library was then loaded on an Illumina RP causing genes/mutations were found in both families. Hiseq 2000 platform for high-throughput sequencing. Raw data were initially processed by CASAVA Soft- Methods ware 1.7 (Illumina) for image analysis and base call- Sample collection and clinical assessments ing. Sequences were generated as 90  bp pair-end reads. Our study, conformed to the Declaration of Helsinki, Reads were aligned to human h19 genome using SOAPa- was approved and prospectively reviewed by the local ligner (http://www.soap.genom ics.org.cn) and Burrows- ethics committee of People Hospital of Ningxia Hui Wheeler Aligner (BWA; http://www.bio-bwa.sourc eforg Autonomous Region (No. 10 [2017]). Eleven participants e.net/). Only mapped reads were included for subsequent from family A (Fig.  1a) and 14 participants from fam- analysis. Coverage and depth were determined based on ily B (Fig.  1b) were recruited from the People’s Hospital all mapped reads and the exome region. Atlas-SNP2 and of Ningxia Hui Autonomous Region. Written informed Atlas-Indel2 were applied for variant calling [8]. Vari- contents were obtained from all participants or their ant frequency data were obtained from the following six legal guardians before their enrollments. Peripheral single nucleotide polymorphism databases, including blood samples were collected from all 25 participants (See figure on next page.) Fig. 1 Family pedigrees and genetic annotations of identified mutations. a Pedigree of family A. Included participants are indicated by asterisk. b Pedigree of family B. Included participants are indicated by asterisk. c–f Sequence chromatograms of identified mutations, including OFD1 c.358A>G (c), C8ORF37 c.555G>A (d), TULP1 c.1255C>T (e), and RP1 c.2285_2289delTAAAT (f). g Orthologous protein sequence alignment of TULP1 from human (H. sapiens), chimpanzees (P. troglodytes), dogs (C. lupus), cows (B. taurus), rats (M. musculus), chickens (G. gallus), zebrafish (D. rerio), fruit flies (D. melanogaster), and worms (C. elegans). Conserved residues are shaded. The mutated residue 419 is boxed and indicated. h, i Crystal structural analysis of the wild type (h) and mutant (i) TULP1 protein. Hydrogen bonds between residue 419 and residues V488 and S534 were eliminated due to the substitution from arginine to tryptophan. j Conservational analysis of residues TULP1 R419, N463, V488 and S534 between TULP1 and TUB proteins Chen et al. J Transl Med (2018) 16:145 Page 3 of 12 Chen et al. J Transl Med (2018) 16:145 Page 4 of 12 Table 1 Clinical features of attainable patients Family RP causative gene Age (year)/sex Onset Night Cataract BCVA Fundus appearance ERG member age (year) blindness (logMAR) O.D. O.S. ID O.D. O.S. O.D. O.S. MD OD AA PD MD OD AA PD O.D. O.S. A-V:2 – – 10 Yes – – LP LP – – – – – – – – – – A-VI:2 C8ORF37 25/F 8 Yes No No LP LP Yes Waxy Yes Yes Yes Waxy Yes Yes D D A-VI:3 OFD1 24/M 2 Yes No No LP LP Yes Waxy Yes Yes Yes Waxy Yes Yes D D B-II:4 RP1 80/F 50 Yes Severe Severe NLP LP – – – – Yes Waxy Yes Yes – D B-III:3 RP1 59/M 30 Yes IOL IOL 0.6 0.25 Yes Waxy Yes Yes Yes Waxy Yes Yes D D B-III:5 RP1 54/F 35 Yes Mild Mild 0.3 0.3 Yes Waxy Yes Yes Yes Waxy Yes Yes D D B-IV:1 TULP1 27/M EC Yes Moderate Moderate 0.15 0.2 Yes Waxy Yes Yes Yes Waxy Yes Yes D D B-IV:2 TULP1 24/F EC Yes Moderate Moderate 0.3 0.3 Yes Waxy Yes Yes Yes Waxy Yes Yes D D B-IV:4 RP1 31/F – Yes No No 0.5 0.8 No No No Yes No No No Yes R R F female, M male, EC early childhood, BCVA best corrected visual acuity, logMAR logarithm of the minimum angle of resolution, O.D. right eye, O.S. left eye, IOL intraocular lens, LP light perception, NLP non-light perception, MD macular degeneration, OD optic disk, AA artery attenuation, PD pigment deposits, ERG electroretinography, D diminished, R reduced This patient is deceased. His clinical features are obtained based on his medical records Chen et al. J Transl Med (2018) 16:145 Page 5 of 12 dbSNP144 (http://www.hgdow nload .cse.ucsc.edu/golde angle closure glaucoma in her right eye (Fig.  2H–S). nPath /hg19/datab ase/snp13 5.txt.gz.), HapMap Pro- Noteworthy, all 6 patients presented mild to severe cata- ject (ftp://ftp.ncbi.nlm.nih.gov/hapma p), 1000 Genome racts (Table  1). Patient B-III:3 received bilateral cataract Project (ftp://ftp.1000g enome s.ebi.ac.uk/vol1/ftp), YH surgeries 2  years ago. No systemic defect was noticed in database (http://yh.genom ics.org.cn/), Exome Vari- any of the included patients. ant Server (http://www.evs.gs.washi ngton .edu/EVS/), and Exome Aggregation Consortium (http://exac.broad Genetic assessments insti tute.org/). Variants with a minor allele frequency of To identify the pathogenic mutations, WES with high over 1% in any of the above databases were discarded. quality was selectively performed on individuals A-IV:3, Sanger sequencing was employed for mutation valida- A-VI:2, and A-VI:3 from family A (mean coverage: tion and prevalence test in 150 additional controls using 98.16%; mean depth: 70.89×), and patients B-III:5 and a previously defined protocol [9]. Primer information B-IV:1 from family B (mean coverage: 98.32%; mean is detailed in Additional file  1: Table  S1 and Additional depth: 104.66×). NGS data were summarized in Addi- file 2: Table S2. tional file  3: Table  S3. Exon-specific coverage report of all known RP genes was presented in Additional file  4: In silico analysis Table  S4. For family A, patients A-VI:2 and A-VI:3 were We applied vector NTI Advance 2011 software (Invit- born to parents with consanguineous marriage, sup- rogen, Carlsbad, CA) to analyze the conservation of the porting potential autosomal recessive inheritance. WES mutated reside by aligning protein sequence of human identified 10 homozygous variants and 6 compound het - TULP1 (ENSP00000229771) with sequences of the fol- erozygous variants shared by patients A-VI:2 and A-VI:3 lowing orthologues proteins: P. troglodytes (ENSP- (Additional file  1: Table S1). However, Sanger sequencing TRP00000030898), C. lupus (ENSCAFP00000001922), revealed no variant co-segregated with the disease phe- B. taurus (ENSBTAP00000055698), M. musculus (ENS- notype. We thus hypothesized that the two patients may MUSP00000049070), G. gallus (ENSGALP00000010281), have distinct RP causing mutations. Based on WES data, D. rerio (ENSDARP00000099556), D. melanogaster patient A-VI:2 carried a recurrent homozygous C8ORF37 (FBpp0088961), and C. elegans (F10B5.4). Crystal struc- mutation c.555G>A (p.W185*; Fig. 1d and Table 2), while tural modeling of the wild type and mutant TULP1 pro- patient A-VI:3 had a novel hemizygous OFD1 mutation teins were constructed with SWISS-MODEL online c.358A>G (p.T120A; Fig. 1c and Table 2). server [10, 11], and displayed with PyMol software. As to family B, WES revealed one homozygous vari- ant and 18 compound heterozygous variants shared by Results patients B-III:4 and IV:2 (Additional file  2: Table  S2), Clinical findings while no variant was validated co-segregated with the Two patients from family A, A-VI:2 and A-VI:3, and six disease phenotype. According to the family pedigree, patients from family B, B-II:4, B-III:3, B-III:5, B-IV:1, patients B-IV:1 and B-IV:2 were born to unaffected par - B-IV:2 and B-IV:4, were included in the present study ents with consanguineous marriage, indicating a poten- with their clinical details summarized in Table  1. Oph- tial autosomal recessive inheritance pattern. However, thalmic features of patient A-V:2 were obtained accord- the RP phenotypes of patients B-III:3 and B-III:4 were ing to his medical records, and were presented in Table 1. likely inherited from the affected mother B-II:4, suggest - All patients from the two families were clinically diag- ing a dominant inheritance mode. Upon this hypoth- nosed with RP. In family A, all three patients had early esis, a novel homozygous TULP1 mutation c.1255C>T onset nyctalopia and rapid disease progress. Best cor- (p.R419W; Fig.  1e and Table  2) was revealed as RP rected visual acuity was light perception for both patients causative for patients B-IV:1 and B-IV:2, and a recur- A-VI:2 and A-VI:3 at their last visit to our hospital at the rent heterozygous RP1 mutation c.2285_2289delTAAAT ages of 25 and 24 respectively. Typical RP presentations (p.L762Yfs*17; Fig.  1f; Table  2) was found in patients and macular degeneration were detected upon their oph- B-II:4, B-III:3 and B-III:4. The mutated residue R419 in thalmic evaluations (Fig. 2A–G and Table 1). In family B, TULP1 was highly conserved among all tested species RP onset ages ranged from early childhood to 50  years (Fig.  1g). Crystal structures of the wild type and mutant old (Table  1). RP progression also varied among the 6 TULP1 proteins were obtained based on human TUB patients. Patients B-IV:1 and B-IV:2 reported to have protein (Protein Data Bank ID: 1S31) with a sequence nyctalopia since early childhood, while the other four identify of 75.19 and a sequence similarity of 0.54. Our patients showed RP symptoms elder than 30-year-old. data suggested that the substitution from arginine to On examination, typical RP presentations were detected tryptophan at residue 419 would lead to the elimination for all 6 patients, while patient B-II:4 also had chronic of two hydrogen bonds between residue 419 and residues Chen et al. J Transl Med (2018) 16:145 Page 6 of 12 Fig. 2 Ophthalmic presentations of included patients. A, B Fundus presentations of patient A-VI:3 (age 24, carrying OFD1 c.358A>G) indicate waxy optic disc, attenuated retinal arterioles, macular degeneration, bone spicule-like pigments and atrophy of RPE and choroid in the peripheral retina. C Fundus fluorescein angiography (FFA) of patient A-VI:3 notices a combination of speckled hypofluorescent and hyperfluorescent changes in both macular and peripheral retina. D Fundus photos of patient A-VI:2 (age 27, carrying C8ORF37 c.555G>A) show similar presentations to patient A-VI:3, but with more intensive pigmentations. E FFA of patient A-VI:2 also demonstrates intensive speckled changes of both hypofluorescence and hyperfluorescence. F OCT results of patient A-VI:3 indicate attenuated outer nuclear layer (ONL) and RPE with remarkable loss of inner segments (IS) and outer segments (OS). G OCT results of patient A-VI:2 show complete loss of IS and OS. H Patient B-III:3 (age 59, carrying RP1 c.2285_2289delTAAAT ) has a waxy optic disc, attenuated retinal arterioles, mild macular degeneration, and intensive bone spicule-like pigment deposits in the mid-peripheral retina of both eyes. I Patient B-III:5 (age 54, carrying RP1 c.2285_2289delTAAAT ) shows typical RP fundus similar to patient B-III:3, including intensive pigmentations and macular degeneration. J Fundus of patient B-IV:1 (age 27, carrying TULP1 c.1255C>T ) demonstrates attenuated retinal vessels, a waxy optic disc, remarkable macular degeneration, and diffuse pigment deposits in the periphery retina of both eyes. K Patient B-IV:2 (age 24, carrying TULP1 c.1255C>T ) shows similar fundus presentation to patient B-IV:1, presenting maculopathy and diffused pigmentations. L Slight waxy pallor of the optic disc and diffuse pigment deposits in the peripheral retina are revealed in the fundus of patient IV:4 (age 31, carrying RP1 c.2285_2289delTAAAT ). M Patient II:4 (age 80, carrying RP1 c.2285_2289delTAAAT ) shows typical RP fundus with intensive pigment deposits. N OCT results of patient B-III:3 indicate attenuated ONL and RPE with loss of IS and OS. O Thickened ONL with cystic cavities in the macular region were noticed by OCT in patient B-III:5. P OCT examinations of patient B-IV:1 demonstrate attenuated ONL and RPE with complete loss of IS and OS. Q Patient B-IV:2 shows similar OCT results to patient B-IV:1, including attenuated ONL and RPE, and loss of IS/OS. R Slightly attenuated ONL is presented in patient B-IV:4. S Typical RP presentations are revealed in patient B-II:4, demonstrating attenuated ONL and RPE with loss of IS and OS V488 and S534 (Fig.  1h, i), further supporting that this and S534 were conserved between TULP1 and TUB pro- mutation would disturb the tertiary structure of TULP1 teins (Fig.  1j). All four mutations identified in the two and interrupt its function. Residues R419, N463, V488 families segregated with the disease phenotype (Fig.  1a, Chen et al. J Transl Med (2018) 16:145 Page 7 of 12 Table 2 Mutations identified in this study Gene Variation Status Bioinformatics analysis Reported Population prevalence (allele count) or Novel Nucleotide Amino acid SIFT PolyPhen PROVEN rs no. gnomAD EXAC C8ORF37 c.555G>A p.W185* Hom NA NA NA Novel rs748014296 2/246148 1/121412 OFD1 c.358A>G p.T120A Hem 0.63 (tolerated) 0.006 (benign) − 0.616 (netural) Novel rs755625951 4/178544 1/121388 TULP1 c.1255C>T p.R419W Hom 0 (damaging) 1 (probably damaging) − 7.976 (deleterious) Novel rs775334320 12/217192 6/121222 RP1 c.2285_2289delTAAAT p.L762Yfs*17 Het NA NA NA Novel NA NA NA Hom homozygous, Hem hemizygous, Het heterozygous, NA not available SIFT: http://sift.bii.a-star.edu.sg/; PolyPhen: http://genet ics.bwh.harva rd.edu/pph2/; PROVEN: http://prove an.jcvi.org/index .php; gnomAD: http://gnoma d.broad insti tute.org/; EXAC: http://exac.broad insti tute.org/ Chen et al. J Transl Med (2018) 16:145 Page 8 of 12 Table 3 List of mutations reported in C8ORF37, OFD1 and TULP1 associated retinopathies Gene Variation Disease References Nucleotide Amino acid Domain C8ORF37 c.155+2T>C – – CRD [56] C8ORF37 c.156−2A>G – – CRD [15, 18] C8ORF37 c.243+2T>C – – RP [21] C8ORF37 c.244−2A>C – – RP [17] C8ORF37 c.374+2T>C – – EORD [20] C8ORF37 c.497>A p.L166* – RP [15, 18] C8ORF37 c.529C>T p.R177W – CRD, BBS [15, 18, 19, 22] C8ORF37 c.545A>G p.Q182R – RP [15, 18] C8ORF37 c.555G>A p.W185* – RP [17], this study C8ORF37 c.575delC p.T192Mfs*28 – EORD [20] OFD1 p.T120A – RP This study OFD1 IVS9+706A>G p.N313fs*330 Coiled coil domain RP [13] TULP1 c.3G>A p.M1I – RP [25] TULP1 c.99+1G>A – – LCA, RP [23, 26] TULP1 c.280G>T p.D94Y – LCA [27] TULP1 c.286_287delGA p.E96Gfs*77 – RP [57] TULP1 c.350−2delAGA – – RP [28] TULP1 c.394_417del p.E120_D127del – RP [29] TULP1 c.539G>A p.R180H – LCA [30] TULP1 c.627delC p.S210Qfs*27 – LCA [31] TULP1 c.629C>G p.S210* – RP [32] TULP1 c.718+2T>C – – LCA, RP [33] TULP1 c.725_728delCCAA p.P242Qfs*16 – LCA [34] TULP1 c.901C>T p.Q301* Tubby domain LCA, CRD [35, 36] TULP1 c.937delC p.Q301fs*9 Tubby domain RP [28] TULP1 c.932G>A p.R311Q Tubby domain RP [37] TULP1 c.956G>A p.G319D Tubby domain RP [38] TULP1 c.961T>G p.Y321D Tubby domain LCA [34] TULP1 c.999+5G>C – Tubby domain LCA, RP [33] TULP1 c.1025G>A p.R342Q Tubby domain RP [37] TULP1 c.1047T>G p.N349K Tubby domain RP [39] TULP1 c.1064A>T p.D355V Tubby domain LCA [34] TULP1 c.1087G>A p.G363R Tubby domain CRD [40] TULP1 c.1081C>T p.R361* Tubby domain LCA [41] TULP1 c.1102G>T p.G368W Tubby domain LCA [26] TULP1 c.1112+2T>C – Tubby domain RP [42] TULP1 c.1113–2A>C – Tubby domain LCA [34] TULP1 c.1138A>G p.T380A Tubby domain LCA, RP [43, 45, 46] TULP1 c.1145T>C p.F382S Tubby domain RP [47] TULP1 c.1198C>T p.R400W Tubby domain LCA, RP, CRD [26, 48, 49] TULP1 c.1199G>A p.A400Q Tubby domain RP [50] TULP1 c.1204G>T p.E402* Tubby domain LCA [26] TULP1 c.1224+4A>G – Tubby domain RP [29] TULP1 c.1246C > T p.R416C Tubby domain RP [25] TULP1 c.1255C>T p.R419W Tubby domain RP This study TULP1 c.1258C>A p.R420S Tubby domain RCD [51] TULP1 c.1259G>C p.R420P Tubby domain RP [23] TULP1 c.1318C>T p.R440* Tubby domain LCA [31] Chen et al. J Transl Med (2018) 16:145 Page 9 of 12 Table 3 (continued) Gene Variation Disease References Nucleotide Amino acid Domain TULP1 c.1349G>A p.W450* Tubby domain LCA [27] TULP1 c.1376T>A p.I459K Tubby domain RP [23, 24] TULP1 c.1376T>C p.I459T Tubby domain RP [42] TULP1 c.1376_1377delTA p.I459Rfs*12 Tubby domain LCA [34] TULP1 c.1381C>G p.L461V Tubby domain LCA, RP [33] TULP1 c.1444C > T p.R482W Tubby domain RP [44, 48] TULP1 c.1445G>A p.A482Q Tubby domain RP [46] TULP1 c.1466A>G p.K489R Tubby domain RP [29, 43, 52, 57] TULP1 c.1472T>C p.F491L Tubby domain RP [23] TULP1 c.1495+1G>A – Tubby domain RP [24] TULP1 c.1495+2_1495+3insT – Tubby domain RP [53] TULP1 c.1495+4A>C – Tubby domain RP [57] TULP1 c.1496−6C>A – Tubby domain RP [23, 29] TULP1 c.1511_1521del p.L504fs*140 Tubby domain RP [44] TULP1 c.1518C>A p.F506L Tubby domain LCA [31] TULP1 c.1561C>T p.P521S Tubby domain RP [57] TULP1 c.1582_1587dup p.F528_A529dup Tubby domain LCA, RP [54] TULP1 c.1604T>C p.F535S Tubby domain LCA [55] CRD cone-rod dystrophy, RP retinitis pigmentosa, EORD early-onset retinal dystrophy, BBS Bardet–Biedl syndrome, LCA Leber congenital amaurosis b), and were confirmed absent in 150 Chinese controls in modulating retinal function is not fully elucidated. In free of major ocular problems. this study, the patient carrying homozygous nonsense C8ORF37 mutation presents early onset RP with macular involvement, which is similar to previous reports [15, 17]. Discussion TULP1 mutations are implicated in autosomal recessive RP is a genetically heterogeneous disease with 83 dis- RP and LCA etiologies (Table  3) [22–57]. TULP1 pro- ease causative genes and hundreds of mutations. In tein plays crucial roles in maintaining retinal homeosta- this report, molecular test reveals the coexistence of sis. According to previous reports, TULP1 interacts and mutations affecting distinct RP causing genes in two co-localizes with F-actin in photoreceptor cells of bovine RP families, thus providing novel insights into genetic retina [58], and RPE phagocytosis ability was remarkably assessments in complicated pedigrees. Among the four −/− reduced in TULP1 mice [59]. Thus, TULP1 is required mutations identified in the two families, two were novel for maintaining regular functions of photoreceptors and (OFD1 p.T120A and TULP1 p.R419W) and two were RPE cells. We herein identified TULP1 mutations in two recurrent (C8ORF37 p.W185* and RP1 p.L762Yfs*17 siblings demonstrating RP with early onset and quick [Human Gene Mutation Database ID: CD991855]). progression. Further confirmatory functional studies are OFD1 mutations have been reported to cause X-linked still needed to better illustrated pathogenesis of the iden- recessive Joubert syndrome, orofaciodigital syndrome tified novel mutations. and isolated RP (Table 3) [12, 13]. OFD1, protein encoded by the OFD1 gene, is a crucial component of the cen- trioles. OFD1 is involved in ciliogenesis regulation and Conclusions exhibits neuroprotective roles [14]. Herein, a hemizygous In summary, we demonstrate the coexistence of diverse OFD1 missense mutation is associated with a severe form inheritance modes and mutations affecting distinct of RP presenting early onset age and fast disease progres- disease causing genes in two RP families. Our findings sion. C8ORF37 mutations correlate with a wide spectrum reinforce the genetic complexity of RP, provide novel of autosomal recessive retinopathies ranging from RP to insights into the assessments of complicated pedigrees Bardet-Biedl syndrome (Table  3) [15–22]. The encoded with consanguinity, and highlight the need for exten- C8ORF37 protein is a ciliary protein located at the base sive molecular evaluations in such challenging families of the photoreceptor connecting cilia [16], while its role involving diverse inheritance modes and mutations. Chen et al. J Transl Med (2018) 16:145 Page 10 of 12 Received: 27 November 2017 Accepted: 17 May 2018 Additional files Additional file 1: Table S1. Post-filtration variants in family A. Additional file 2: Table S2. Post-filtration variants in family B. References Additional file 3: Table S3. Overview of data production. 1. Anasagasti A, Irigoyen C, Barandika O, de Munain AL, Ruiz-Ederra J. Current mutation discovery approaches in retinitis pigmentosa. Vis Res. Additional file 4: Table S4. Coverage for all exons in all known RP genes. 2012;75:117–29. 2. Liu Y, Chen X, Xu Q, Gao X, Tam PO, Zhao K, Zhang X, Chen LJ, Jia W, Zhao Q, et al. SPP2 mutations cause autosomal dominant retinitis pigmentosa. Sci Rep. 2015;5:14867. Abbreviations 3. Hartong DT, Berson EL, Dryja TP. Retinitis pigmentosa. Lancet. RP: retinitis pigmentosa; NGS: next-generation sequencing; WES: whole exome 2006;368:1795–809. sequencing; bp: base pair; LM-PCR: ligation-mediated polymerase chain reac- 4. Liu X, Xiao J, Huang H, Guan L, Zhao K, Xu Q, Zhang X, Pan X, Gu S, Chen tion; RPE: retinal pigment epithelium. Y, et al. Molecular genetic testing in clinical diagnostic assessments that demonstrate correlations in patients with autosomal recessive inherited Authors’ contributions retinal dystrophy. JAMA Ophthalmol. 2015;133:427–36. XC, XS, YL, and ZL contributed equally to this report. All authors were involved 5. Chen X, Sheng X, Liu X, Li H, Liu Y, Rong W, Ha S, Liu W, Kang X, Zhao in managing the patients. XC, BY and CZ wrote the report. XC, XS, YL and ZL K, Zhao C. Targeted next-generation sequencing reveals novel USH2A did the genetic analysis and whole exome sequencing, and CZ reviewed the mutations associated with diverse disease phenotypes: implications for genetic results. All authors read and approved the final manuscript. clinical and molecular diagnosis. PLoS ONE. 2014;9:e105439. 6. Sheng X, Chen X, Lei B, Chen R, Wang H, Zhang F, Rong W, Ha R, Liu Y, Author details Zhao F, et al. 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Wang J, Chen X, Wang F, Zhang J, Li P, Li Z, Xu J, Gao F, Jin C, Tian H, prospectively reviewed by the local ethics committee of People Hospital of et al. OFD1, as a ciliary protein, exhibits neuroprotective function in Ningxia Hui Autonomous Region. Written informed contents were obtained photoreceptor degeneration models. PLoS ONE. 2016;11:e0155860. from all participants or their legal guardians before their enrollments. 15. Estrada-Cuzcano A, Neveling K, Kohl S, Banin E, Rotenstreich Y, Sharon D, Falik-Zaccai TC, Hipp S, Roepman R, Wissinger B, et al. Mutations in Funding C8orf37, encoding a ciliary protein, are associated with autosomal- This work was supported by the National Natural Science Foundation of recessive retinal dystrophies with early macular involvement. Am J China (81525006, 81670864 and 81730025 to C. Z., 81700877 to X. C., and Hum Genet. 2012;90:102–9. 81760180 to X. S.); Shanghai Outstanding Academic Leaders (2017BR013 to 16. 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Distinct mutations with different inheritance mode caused similar retinal dystrophies in one family: a demonstration of the importance of genetic annotations in complicated pedigrees

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Abstract

Background: Retinitis pigmentosa (RP) is the most common form of inherited retinal dystrophy presenting remarka- ble genetic heterogeneity. Genetic annotations would help with better clinical assessments and benefit gene therapy, and therefore should be recommended for RP patients. This report reveals the disease causing mutations in two RP pedigrees with confusing inheritance patterns using whole exome sequencing ( WES). Methods: Twenty-five participants including eight patients from two families were recruited and received compre - hensive ophthalmic evaluations. WES was applied for mutation identification. Bioinformatics annotations, intrafamilial co-segregation tests, and in silico analyses were subsequently conducted for mutation verification. Results: All patients were clinically diagnosed with RP. The first family included two siblings born to parents with consanguineous marriage; however, no potential pathogenic variant was found shared by both patients. Further analysis revealed that the female patient carried a recurrent homozygous C8ORF37 p.W185*, while the male patient had hemizygous OFD1 p.T120A. The second family was found to segregate mutations in two genes, TULP1 and RP1. Two patients born to consanguineous marriage carried homozygous TULP1 p.R419W, while a recurrent heterozygous RP1 p.L762Yfs*17 was found in another four patients presenting an autosomal dominant inheritance pattern. Crystal structural analysis further indicated that the substitution from arginine to tryptophan at the highly conserved residue 419 of TULP1 could lead to the elimination of two hydrogen bonds between residue 419 and residues V488 and S534. All four genes, including C8ORF37, OFD1, TULP1 and RP1, have been previously implicated in RP etiology. Conclusions: Our study demonstrates the coexistence of diverse inheritance modes and mutations affecting distinct disease causing genes in two RP families with consanguineous marriage. Our data provide novel insights into assess- ments of complicated pedigrees, reinforce the genetic complexity of RP, and highlight the need for extensive molecu- lar evaluations in such challenging families with diverse inheritance modes and mutations. Keywords: Retinitis pigmentosa, Genetic heterogeneity, Next generation sequencing, Mutation, OFD1, C8ORF37, TULP1, RP1, Consanguinity *Correspondence: yanbiao1982@hotmail.com; dr_zhaochen@163.com Xue Chen, Xunlun Sheng, Yani Liu and Zili Li contributed equally to this work Department of Ophthalmology, State Key Laboratory of Reproductive Medicine, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China Full list of author information is available at the end of the article © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/ publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Chen et al. J Transl Med (2018) 16:145 Page 2 of 12 for genomic DNA extraction. Family history and con- Background sanguineous marriages were carefully reviewed. Medical Retinitis pigmentosa (RP, MIM: 268000), the most com- records were obtained from all participants. Each par- mon form of inherited retinal degenerations, affects ticipant received general ophthalmic evaluations, while over one million individuals globally [1, 2]. Night blind- comprehensive ophthalmic examinations were selectively ness is usually the initial symptom for RP, followed by conducted on the eight included patients. Another 150 subsequent visual field constriction, and eventual vision Chinese healthy controls free of major ocular problems loss. RP is featured by great clinical heterogeneities. Its were recruited with their blood samples donated. onset age ranges from early childhood to mid-adulthood. Inter- and intra-familial phenotypic diversities caused by NGS approach and bioinformatics analyses the same RP causing mutations have also been revealed To reveal the disease causing mutation in the two fami- [3–5]. Thus, clinical diagnose for RP patients are some - lies, we selectively performed whole exome sequencing times challenged by its wide phenotypic spectrum and (WES) on three participants in family A (A-IV:3, A-VI:2 under certain conditions, like in a young patient without and A-VI:3) and two patients in family B (B-III:4 and fully onset RP phenotypes. In such situations, molecu- B-IV:1). WES was conducted with the 44.1 megabases lar testing could help to address the clinical ambiguity SeqCap EZ Human Exome Library v2.0 (Roche Nimble- in RP diagnosis. RP also shows high genetic heterogene- Gen, Madison, WI) for enrichment of 23588 genes on ity. To date, 83 RP causing genes involving hundreds of patients from family A [6], and with SureSelect Human mutations have been identified (RetNet). Next-gener - All Exon V6 60  Mb Kit (Agilent Technologies, Santa ation sequencing (NGS), enabling simultaneous paral- Clara, CA) on patients from family B [7]. Briefly, qualified lel sequencing of numerous genes with high efficiency, genomic DNA samples were randomly sheared by Cova- is an efficient tool for molecular diagnosis of RP [2, 4]. ris into 200–250  base pair (bp) fragments. Fragments Genetic annotations with NGS promote better clinical were then ligated with adapters to both ends, amplified assessments and gene therapy, and therefore should be by ligation-mediated polymerase chain reaction (LM- recommended for RP patients. However, pedigrees with PCR), purified, and hybridized. Non-hybridized frag - puzzling inheritance patterns could sometimes confuse ments were then washed out. Quantitative PCR was the genetic diagnoses. Herein, we described the geno- further applied to estimate the magnitude of enrichment typic and phenotypic findings in two complicated RP of both non-captured and captured LM-PCR products. pedigrees using NGS. Distinct inheritance patterns and Each post-capture library was then loaded on an Illumina RP causing genes/mutations were found in both families. Hiseq 2000 platform for high-throughput sequencing. Raw data were initially processed by CASAVA Soft- Methods ware 1.7 (Illumina) for image analysis and base call- Sample collection and clinical assessments ing. Sequences were generated as 90  bp pair-end reads. Our study, conformed to the Declaration of Helsinki, Reads were aligned to human h19 genome using SOAPa- was approved and prospectively reviewed by the local ligner (http://www.soap.genom ics.org.cn) and Burrows- ethics committee of People Hospital of Ningxia Hui Wheeler Aligner (BWA; http://www.bio-bwa.sourc eforg Autonomous Region (No. 10 [2017]). Eleven participants e.net/). Only mapped reads were included for subsequent from family A (Fig.  1a) and 14 participants from fam- analysis. Coverage and depth were determined based on ily B (Fig.  1b) were recruited from the People’s Hospital all mapped reads and the exome region. Atlas-SNP2 and of Ningxia Hui Autonomous Region. Written informed Atlas-Indel2 were applied for variant calling [8]. Vari- contents were obtained from all participants or their ant frequency data were obtained from the following six legal guardians before their enrollments. Peripheral single nucleotide polymorphism databases, including blood samples were collected from all 25 participants (See figure on next page.) Fig. 1 Family pedigrees and genetic annotations of identified mutations. a Pedigree of family A. Included participants are indicated by asterisk. b Pedigree of family B. Included participants are indicated by asterisk. c–f Sequence chromatograms of identified mutations, including OFD1 c.358A>G (c), C8ORF37 c.555G>A (d), TULP1 c.1255C>T (e), and RP1 c.2285_2289delTAAAT (f). g Orthologous protein sequence alignment of TULP1 from human (H. sapiens), chimpanzees (P. troglodytes), dogs (C. lupus), cows (B. taurus), rats (M. musculus), chickens (G. gallus), zebrafish (D. rerio), fruit flies (D. melanogaster), and worms (C. elegans). Conserved residues are shaded. The mutated residue 419 is boxed and indicated. h, i Crystal structural analysis of the wild type (h) and mutant (i) TULP1 protein. Hydrogen bonds between residue 419 and residues V488 and S534 were eliminated due to the substitution from arginine to tryptophan. j Conservational analysis of residues TULP1 R419, N463, V488 and S534 between TULP1 and TUB proteins Chen et al. J Transl Med (2018) 16:145 Page 3 of 12 Chen et al. J Transl Med (2018) 16:145 Page 4 of 12 Table 1 Clinical features of attainable patients Family RP causative gene Age (year)/sex Onset Night Cataract BCVA Fundus appearance ERG member age (year) blindness (logMAR) O.D. O.S. ID O.D. O.S. O.D. O.S. MD OD AA PD MD OD AA PD O.D. O.S. A-V:2 – – 10 Yes – – LP LP – – – – – – – – – – A-VI:2 C8ORF37 25/F 8 Yes No No LP LP Yes Waxy Yes Yes Yes Waxy Yes Yes D D A-VI:3 OFD1 24/M 2 Yes No No LP LP Yes Waxy Yes Yes Yes Waxy Yes Yes D D B-II:4 RP1 80/F 50 Yes Severe Severe NLP LP – – – – Yes Waxy Yes Yes – D B-III:3 RP1 59/M 30 Yes IOL IOL 0.6 0.25 Yes Waxy Yes Yes Yes Waxy Yes Yes D D B-III:5 RP1 54/F 35 Yes Mild Mild 0.3 0.3 Yes Waxy Yes Yes Yes Waxy Yes Yes D D B-IV:1 TULP1 27/M EC Yes Moderate Moderate 0.15 0.2 Yes Waxy Yes Yes Yes Waxy Yes Yes D D B-IV:2 TULP1 24/F EC Yes Moderate Moderate 0.3 0.3 Yes Waxy Yes Yes Yes Waxy Yes Yes D D B-IV:4 RP1 31/F – Yes No No 0.5 0.8 No No No Yes No No No Yes R R F female, M male, EC early childhood, BCVA best corrected visual acuity, logMAR logarithm of the minimum angle of resolution, O.D. right eye, O.S. left eye, IOL intraocular lens, LP light perception, NLP non-light perception, MD macular degeneration, OD optic disk, AA artery attenuation, PD pigment deposits, ERG electroretinography, D diminished, R reduced This patient is deceased. His clinical features are obtained based on his medical records Chen et al. J Transl Med (2018) 16:145 Page 5 of 12 dbSNP144 (http://www.hgdow nload .cse.ucsc.edu/golde angle closure glaucoma in her right eye (Fig.  2H–S). nPath /hg19/datab ase/snp13 5.txt.gz.), HapMap Pro- Noteworthy, all 6 patients presented mild to severe cata- ject (ftp://ftp.ncbi.nlm.nih.gov/hapma p), 1000 Genome racts (Table  1). Patient B-III:3 received bilateral cataract Project (ftp://ftp.1000g enome s.ebi.ac.uk/vol1/ftp), YH surgeries 2  years ago. No systemic defect was noticed in database (http://yh.genom ics.org.cn/), Exome Vari- any of the included patients. ant Server (http://www.evs.gs.washi ngton .edu/EVS/), and Exome Aggregation Consortium (http://exac.broad Genetic assessments insti tute.org/). Variants with a minor allele frequency of To identify the pathogenic mutations, WES with high over 1% in any of the above databases were discarded. quality was selectively performed on individuals A-IV:3, Sanger sequencing was employed for mutation valida- A-VI:2, and A-VI:3 from family A (mean coverage: tion and prevalence test in 150 additional controls using 98.16%; mean depth: 70.89×), and patients B-III:5 and a previously defined protocol [9]. Primer information B-IV:1 from family B (mean coverage: 98.32%; mean is detailed in Additional file  1: Table  S1 and Additional depth: 104.66×). NGS data were summarized in Addi- file 2: Table S2. tional file  3: Table  S3. Exon-specific coverage report of all known RP genes was presented in Additional file  4: In silico analysis Table  S4. For family A, patients A-VI:2 and A-VI:3 were We applied vector NTI Advance 2011 software (Invit- born to parents with consanguineous marriage, sup- rogen, Carlsbad, CA) to analyze the conservation of the porting potential autosomal recessive inheritance. WES mutated reside by aligning protein sequence of human identified 10 homozygous variants and 6 compound het - TULP1 (ENSP00000229771) with sequences of the fol- erozygous variants shared by patients A-VI:2 and A-VI:3 lowing orthologues proteins: P. troglodytes (ENSP- (Additional file  1: Table S1). However, Sanger sequencing TRP00000030898), C. lupus (ENSCAFP00000001922), revealed no variant co-segregated with the disease phe- B. taurus (ENSBTAP00000055698), M. musculus (ENS- notype. We thus hypothesized that the two patients may MUSP00000049070), G. gallus (ENSGALP00000010281), have distinct RP causing mutations. Based on WES data, D. rerio (ENSDARP00000099556), D. melanogaster patient A-VI:2 carried a recurrent homozygous C8ORF37 (FBpp0088961), and C. elegans (F10B5.4). Crystal struc- mutation c.555G>A (p.W185*; Fig. 1d and Table 2), while tural modeling of the wild type and mutant TULP1 pro- patient A-VI:3 had a novel hemizygous OFD1 mutation teins were constructed with SWISS-MODEL online c.358A>G (p.T120A; Fig. 1c and Table 2). server [10, 11], and displayed with PyMol software. As to family B, WES revealed one homozygous vari- ant and 18 compound heterozygous variants shared by Results patients B-III:4 and IV:2 (Additional file  2: Table  S2), Clinical findings while no variant was validated co-segregated with the Two patients from family A, A-VI:2 and A-VI:3, and six disease phenotype. According to the family pedigree, patients from family B, B-II:4, B-III:3, B-III:5, B-IV:1, patients B-IV:1 and B-IV:2 were born to unaffected par - B-IV:2 and B-IV:4, were included in the present study ents with consanguineous marriage, indicating a poten- with their clinical details summarized in Table  1. Oph- tial autosomal recessive inheritance pattern. However, thalmic features of patient A-V:2 were obtained accord- the RP phenotypes of patients B-III:3 and B-III:4 were ing to his medical records, and were presented in Table 1. likely inherited from the affected mother B-II:4, suggest - All patients from the two families were clinically diag- ing a dominant inheritance mode. Upon this hypoth- nosed with RP. In family A, all three patients had early esis, a novel homozygous TULP1 mutation c.1255C>T onset nyctalopia and rapid disease progress. Best cor- (p.R419W; Fig.  1e and Table  2) was revealed as RP rected visual acuity was light perception for both patients causative for patients B-IV:1 and B-IV:2, and a recur- A-VI:2 and A-VI:3 at their last visit to our hospital at the rent heterozygous RP1 mutation c.2285_2289delTAAAT ages of 25 and 24 respectively. Typical RP presentations (p.L762Yfs*17; Fig.  1f; Table  2) was found in patients and macular degeneration were detected upon their oph- B-II:4, B-III:3 and B-III:4. The mutated residue R419 in thalmic evaluations (Fig. 2A–G and Table 1). In family B, TULP1 was highly conserved among all tested species RP onset ages ranged from early childhood to 50  years (Fig.  1g). Crystal structures of the wild type and mutant old (Table  1). RP progression also varied among the 6 TULP1 proteins were obtained based on human TUB patients. Patients B-IV:1 and B-IV:2 reported to have protein (Protein Data Bank ID: 1S31) with a sequence nyctalopia since early childhood, while the other four identify of 75.19 and a sequence similarity of 0.54. Our patients showed RP symptoms elder than 30-year-old. data suggested that the substitution from arginine to On examination, typical RP presentations were detected tryptophan at residue 419 would lead to the elimination for all 6 patients, while patient B-II:4 also had chronic of two hydrogen bonds between residue 419 and residues Chen et al. J Transl Med (2018) 16:145 Page 6 of 12 Fig. 2 Ophthalmic presentations of included patients. A, B Fundus presentations of patient A-VI:3 (age 24, carrying OFD1 c.358A>G) indicate waxy optic disc, attenuated retinal arterioles, macular degeneration, bone spicule-like pigments and atrophy of RPE and choroid in the peripheral retina. C Fundus fluorescein angiography (FFA) of patient A-VI:3 notices a combination of speckled hypofluorescent and hyperfluorescent changes in both macular and peripheral retina. D Fundus photos of patient A-VI:2 (age 27, carrying C8ORF37 c.555G>A) show similar presentations to patient A-VI:3, but with more intensive pigmentations. E FFA of patient A-VI:2 also demonstrates intensive speckled changes of both hypofluorescence and hyperfluorescence. F OCT results of patient A-VI:3 indicate attenuated outer nuclear layer (ONL) and RPE with remarkable loss of inner segments (IS) and outer segments (OS). G OCT results of patient A-VI:2 show complete loss of IS and OS. H Patient B-III:3 (age 59, carrying RP1 c.2285_2289delTAAAT ) has a waxy optic disc, attenuated retinal arterioles, mild macular degeneration, and intensive bone spicule-like pigment deposits in the mid-peripheral retina of both eyes. I Patient B-III:5 (age 54, carrying RP1 c.2285_2289delTAAAT ) shows typical RP fundus similar to patient B-III:3, including intensive pigmentations and macular degeneration. J Fundus of patient B-IV:1 (age 27, carrying TULP1 c.1255C>T ) demonstrates attenuated retinal vessels, a waxy optic disc, remarkable macular degeneration, and diffuse pigment deposits in the periphery retina of both eyes. K Patient B-IV:2 (age 24, carrying TULP1 c.1255C>T ) shows similar fundus presentation to patient B-IV:1, presenting maculopathy and diffused pigmentations. L Slight waxy pallor of the optic disc and diffuse pigment deposits in the peripheral retina are revealed in the fundus of patient IV:4 (age 31, carrying RP1 c.2285_2289delTAAAT ). M Patient II:4 (age 80, carrying RP1 c.2285_2289delTAAAT ) shows typical RP fundus with intensive pigment deposits. N OCT results of patient B-III:3 indicate attenuated ONL and RPE with loss of IS and OS. O Thickened ONL with cystic cavities in the macular region were noticed by OCT in patient B-III:5. P OCT examinations of patient B-IV:1 demonstrate attenuated ONL and RPE with complete loss of IS and OS. Q Patient B-IV:2 shows similar OCT results to patient B-IV:1, including attenuated ONL and RPE, and loss of IS/OS. R Slightly attenuated ONL is presented in patient B-IV:4. S Typical RP presentations are revealed in patient B-II:4, demonstrating attenuated ONL and RPE with loss of IS and OS V488 and S534 (Fig.  1h, i), further supporting that this and S534 were conserved between TULP1 and TUB pro- mutation would disturb the tertiary structure of TULP1 teins (Fig.  1j). All four mutations identified in the two and interrupt its function. Residues R419, N463, V488 families segregated with the disease phenotype (Fig.  1a, Chen et al. J Transl Med (2018) 16:145 Page 7 of 12 Table 2 Mutations identified in this study Gene Variation Status Bioinformatics analysis Reported Population prevalence (allele count) or Novel Nucleotide Amino acid SIFT PolyPhen PROVEN rs no. gnomAD EXAC C8ORF37 c.555G>A p.W185* Hom NA NA NA Novel rs748014296 2/246148 1/121412 OFD1 c.358A>G p.T120A Hem 0.63 (tolerated) 0.006 (benign) − 0.616 (netural) Novel rs755625951 4/178544 1/121388 TULP1 c.1255C>T p.R419W Hom 0 (damaging) 1 (probably damaging) − 7.976 (deleterious) Novel rs775334320 12/217192 6/121222 RP1 c.2285_2289delTAAAT p.L762Yfs*17 Het NA NA NA Novel NA NA NA Hom homozygous, Hem hemizygous, Het heterozygous, NA not available SIFT: http://sift.bii.a-star.edu.sg/; PolyPhen: http://genet ics.bwh.harva rd.edu/pph2/; PROVEN: http://prove an.jcvi.org/index .php; gnomAD: http://gnoma d.broad insti tute.org/; EXAC: http://exac.broad insti tute.org/ Chen et al. J Transl Med (2018) 16:145 Page 8 of 12 Table 3 List of mutations reported in C8ORF37, OFD1 and TULP1 associated retinopathies Gene Variation Disease References Nucleotide Amino acid Domain C8ORF37 c.155+2T>C – – CRD [56] C8ORF37 c.156−2A>G – – CRD [15, 18] C8ORF37 c.243+2T>C – – RP [21] C8ORF37 c.244−2A>C – – RP [17] C8ORF37 c.374+2T>C – – EORD [20] C8ORF37 c.497>A p.L166* – RP [15, 18] C8ORF37 c.529C>T p.R177W – CRD, BBS [15, 18, 19, 22] C8ORF37 c.545A>G p.Q182R – RP [15, 18] C8ORF37 c.555G>A p.W185* – RP [17], this study C8ORF37 c.575delC p.T192Mfs*28 – EORD [20] OFD1 p.T120A – RP This study OFD1 IVS9+706A>G p.N313fs*330 Coiled coil domain RP [13] TULP1 c.3G>A p.M1I – RP [25] TULP1 c.99+1G>A – – LCA, RP [23, 26] TULP1 c.280G>T p.D94Y – LCA [27] TULP1 c.286_287delGA p.E96Gfs*77 – RP [57] TULP1 c.350−2delAGA – – RP [28] TULP1 c.394_417del p.E120_D127del – RP [29] TULP1 c.539G>A p.R180H – LCA [30] TULP1 c.627delC p.S210Qfs*27 – LCA [31] TULP1 c.629C>G p.S210* – RP [32] TULP1 c.718+2T>C – – LCA, RP [33] TULP1 c.725_728delCCAA p.P242Qfs*16 – LCA [34] TULP1 c.901C>T p.Q301* Tubby domain LCA, CRD [35, 36] TULP1 c.937delC p.Q301fs*9 Tubby domain RP [28] TULP1 c.932G>A p.R311Q Tubby domain RP [37] TULP1 c.956G>A p.G319D Tubby domain RP [38] TULP1 c.961T>G p.Y321D Tubby domain LCA [34] TULP1 c.999+5G>C – Tubby domain LCA, RP [33] TULP1 c.1025G>A p.R342Q Tubby domain RP [37] TULP1 c.1047T>G p.N349K Tubby domain RP [39] TULP1 c.1064A>T p.D355V Tubby domain LCA [34] TULP1 c.1087G>A p.G363R Tubby domain CRD [40] TULP1 c.1081C>T p.R361* Tubby domain LCA [41] TULP1 c.1102G>T p.G368W Tubby domain LCA [26] TULP1 c.1112+2T>C – Tubby domain RP [42] TULP1 c.1113–2A>C – Tubby domain LCA [34] TULP1 c.1138A>G p.T380A Tubby domain LCA, RP [43, 45, 46] TULP1 c.1145T>C p.F382S Tubby domain RP [47] TULP1 c.1198C>T p.R400W Tubby domain LCA, RP, CRD [26, 48, 49] TULP1 c.1199G>A p.A400Q Tubby domain RP [50] TULP1 c.1204G>T p.E402* Tubby domain LCA [26] TULP1 c.1224+4A>G – Tubby domain RP [29] TULP1 c.1246C > T p.R416C Tubby domain RP [25] TULP1 c.1255C>T p.R419W Tubby domain RP This study TULP1 c.1258C>A p.R420S Tubby domain RCD [51] TULP1 c.1259G>C p.R420P Tubby domain RP [23] TULP1 c.1318C>T p.R440* Tubby domain LCA [31] Chen et al. J Transl Med (2018) 16:145 Page 9 of 12 Table 3 (continued) Gene Variation Disease References Nucleotide Amino acid Domain TULP1 c.1349G>A p.W450* Tubby domain LCA [27] TULP1 c.1376T>A p.I459K Tubby domain RP [23, 24] TULP1 c.1376T>C p.I459T Tubby domain RP [42] TULP1 c.1376_1377delTA p.I459Rfs*12 Tubby domain LCA [34] TULP1 c.1381C>G p.L461V Tubby domain LCA, RP [33] TULP1 c.1444C > T p.R482W Tubby domain RP [44, 48] TULP1 c.1445G>A p.A482Q Tubby domain RP [46] TULP1 c.1466A>G p.K489R Tubby domain RP [29, 43, 52, 57] TULP1 c.1472T>C p.F491L Tubby domain RP [23] TULP1 c.1495+1G>A – Tubby domain RP [24] TULP1 c.1495+2_1495+3insT – Tubby domain RP [53] TULP1 c.1495+4A>C – Tubby domain RP [57] TULP1 c.1496−6C>A – Tubby domain RP [23, 29] TULP1 c.1511_1521del p.L504fs*140 Tubby domain RP [44] TULP1 c.1518C>A p.F506L Tubby domain LCA [31] TULP1 c.1561C>T p.P521S Tubby domain RP [57] TULP1 c.1582_1587dup p.F528_A529dup Tubby domain LCA, RP [54] TULP1 c.1604T>C p.F535S Tubby domain LCA [55] CRD cone-rod dystrophy, RP retinitis pigmentosa, EORD early-onset retinal dystrophy, BBS Bardet–Biedl syndrome, LCA Leber congenital amaurosis b), and were confirmed absent in 150 Chinese controls in modulating retinal function is not fully elucidated. In free of major ocular problems. this study, the patient carrying homozygous nonsense C8ORF37 mutation presents early onset RP with macular involvement, which is similar to previous reports [15, 17]. Discussion TULP1 mutations are implicated in autosomal recessive RP is a genetically heterogeneous disease with 83 dis- RP and LCA etiologies (Table  3) [22–57]. TULP1 pro- ease causative genes and hundreds of mutations. In tein plays crucial roles in maintaining retinal homeosta- this report, molecular test reveals the coexistence of sis. According to previous reports, TULP1 interacts and mutations affecting distinct RP causing genes in two co-localizes with F-actin in photoreceptor cells of bovine RP families, thus providing novel insights into genetic retina [58], and RPE phagocytosis ability was remarkably assessments in complicated pedigrees. Among the four −/− reduced in TULP1 mice [59]. Thus, TULP1 is required mutations identified in the two families, two were novel for maintaining regular functions of photoreceptors and (OFD1 p.T120A and TULP1 p.R419W) and two were RPE cells. We herein identified TULP1 mutations in two recurrent (C8ORF37 p.W185* and RP1 p.L762Yfs*17 siblings demonstrating RP with early onset and quick [Human Gene Mutation Database ID: CD991855]). progression. Further confirmatory functional studies are OFD1 mutations have been reported to cause X-linked still needed to better illustrated pathogenesis of the iden- recessive Joubert syndrome, orofaciodigital syndrome tified novel mutations. and isolated RP (Table 3) [12, 13]. OFD1, protein encoded by the OFD1 gene, is a crucial component of the cen- trioles. OFD1 is involved in ciliogenesis regulation and Conclusions exhibits neuroprotective roles [14]. Herein, a hemizygous In summary, we demonstrate the coexistence of diverse OFD1 missense mutation is associated with a severe form inheritance modes and mutations affecting distinct of RP presenting early onset age and fast disease progres- disease causing genes in two RP families. Our findings sion. C8ORF37 mutations correlate with a wide spectrum reinforce the genetic complexity of RP, provide novel of autosomal recessive retinopathies ranging from RP to insights into the assessments of complicated pedigrees Bardet-Biedl syndrome (Table  3) [15–22]. The encoded with consanguinity, and highlight the need for exten- C8ORF37 protein is a ciliary protein located at the base sive molecular evaluations in such challenging families of the photoreceptor connecting cilia [16], while its role involving diverse inheritance modes and mutations. Chen et al. J Transl Med (2018) 16:145 Page 10 of 12 Received: 27 November 2017 Accepted: 17 May 2018 Additional files Additional file 1: Table S1. Post-filtration variants in family A. Additional file 2: Table S2. Post-filtration variants in family B. References Additional file 3: Table S3. Overview of data production. 1. Anasagasti A, Irigoyen C, Barandika O, de Munain AL, Ruiz-Ederra J. Current mutation discovery approaches in retinitis pigmentosa. Vis Res. Additional file 4: Table S4. Coverage for all exons in all known RP genes. 2012;75:117–29. 2. Liu Y, Chen X, Xu Q, Gao X, Tam PO, Zhao K, Zhang X, Chen LJ, Jia W, Zhao Q, et al. SPP2 mutations cause autosomal dominant retinitis pigmentosa. Sci Rep. 2015;5:14867. Abbreviations 3. Hartong DT, Berson EL, Dryja TP. Retinitis pigmentosa. Lancet. RP: retinitis pigmentosa; NGS: next-generation sequencing; WES: whole exome 2006;368:1795–809. sequencing; bp: base pair; LM-PCR: ligation-mediated polymerase chain reac- 4. Liu X, Xiao J, Huang H, Guan L, Zhao K, Xu Q, Zhang X, Pan X, Gu S, Chen tion; RPE: retinal pigment epithelium. Y, et al. 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Journal of Translational MedicineSpringer Journals

Published: May 29, 2018

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