Molecular Genetic Testing in Clinical Diagnostic Assessments That Demonstrate Correlations in Patients with Autosomal Recessive Inherited Retinal Dystrophy

Research

Original Investigation Molecular Genetic Testing in Clinical Diagnostic Assessments That Demonstrate Correlations in Patients With Autosomal Recessive Inherited Retinal Dystrophy

Xiaoxing Liu, MD; Jingjing Xiao, PhD; Hui Huang, PhD; Liping Guan, PhD; Kanxing Zhao, MD, PhD; Qihua Xu, MD; Xiumei Zhang, PhD; Xinyuan Pan, MD; Shun Gu, PhD; Yanhua Chen, PhD; Jianguo Zhang, PhD; Yulan Shen, PhD; Hui Jiang, PhD; Xiang Gao, PhD; Xiaoli Kang, PhD; Xunlun Sheng, PhD; Xue Chen, PhD; Chen Zhao, MD, PhD

Supplemental content at IMPORTANCE Inherited retinal dystrophies (IRDs) are a group of retinal degenerative diseases jamaophthalmology.com presenting genetic and clinical heterogeneities, which have challenged the genetic and clinical diagnoses of IRDs. Genetic evaluations of patients with IRD might result in better clinical assessments and better management of patients.

OBJECTIVE To determine the genetic lesions with phenotypic correlations in patients with diverse autosomal recessive IRD using next-generation sequencing.

DESIGN, SETTING, AND PARTICIPANTS A cohort of 20 Chinese families affected with autosomal recessive IRD were recruited (with data on their detailed family history and on their clinical condition). To identify disease-causing mutations in the patients, the targeted sequence capture of IRD-relevant genes using 2 in-house–designed microarrays, followed by next-generation sequencing, was performed. Bioinformatics annotation, intrafamilial cosegregation analyses, in silico analyses, and functional analyses were subsequently conducted for the variants identified by next-generation sequencing.

MAIN OUTCOMES AND MEASURES The results of detailed clinical evaluations, the identification of disease-causing mutations, and the clinical diagnosis.

RESULTS Homozygous and biallelic variants were identified in 11 of the 20 families (55%) as very likely disease-causing mutations, including a total of 17 alleles, of which 12 are novel. The 17 alleles identified here include 3 missense, 6 nonsense, 4 frameshift, and 4 splice site mutations. In addition, we found biallelic RP1 mutations in a patient with cone-rod dystrophy, which was not previously correlated with RP1 mutations. Moreover, the identification of pathogenic mutations in 3 families helped to refine their clinical diagnoses.

CONCLUSIONS AND RELEVANCE In this study, to our knowledge, many mutations identified in those known loci for autosomal recessive IRD are novel. Specific RP1 mutations may correlate with cone-rod dystrophy. Genetic evaluations with targeted next-generation sequencing might result in a better clinical diagnosis and a better clinical assessment and, therefore, should be recommended for such patients.

Author Affiliations: Author affiliations are listed at the end of this article. Corresponding Author: Chen Zhao, MD, PhD, Department of Ophthalmology, The First Affiliated Hospital of Nanjing Medical University, State Key Laboratory of Reproductive Medicine, 300 Guangzhou Rd, Nanjing, JAMA Ophthalmol. 2015;133(4):427-436. doi:10.1001/jamaophthalmol.2014.5831 Jiangsu 210029, China Published online January 22, 2015. ([email protected]).

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nherited retinal dystrophies (IRDs) are a group of diverse magnetic resonance scanner (Magnetom Trio; Siemens Medi- retinal degenerative diseases with both genetic and clinical Solutions) with a transmit-receive extremity coil was used I cal heterogeneities. Clinically, IRDs manifest as isolated for an examination of the spinal cord. In addition, 150 unre- retinal degeneration or as a systemic disease with retinal dys- lated Chinese controls without IRD or other ocular diseases were trophy. Isolated IRDs include retinitis pigmentosa (RP),1 also included. Samples of venous blood (5 mL) were obtained cone-rod dystrophy (CRD),2 cone dystrophy,3 congenital sta- from each participant for genomic DNA isolation, which was per- tionary night blindness,4 Leber congenital amaurosis,5 Bietti formed using a QIAmp DNA Mini Blood Kit (Qiagen). crystalline dystrophy,6 Stargardt disease,7 Best vitelliform macular dystrophy,8 and various other comparatively rare reti- Targeted NGS nal degenerations. Typical systemic diseases that may be ac- A targeted gene approach was completed using 2 previously de- companied with IRD may include Usher syndrome (OMIM scribed capture arrays (from Roche NimbleGen). Microarray 1 276900) or Bardet-Biedl syndrome (OMIM 209900), as well as was designed to capture the targeted region of 179 IRD-related others. Clinical diagnoses of IRDs can sometimes be chal- genes and 10 candidate genes.11 Microarray 2 was designed to lenged by phenotypic overlaps among distinct diseases and capture the coding sequence region of 316 genes related to in- among certain conditions, such as a young child with a less se- herited ocular diseases.15 Sequence capture, enrichment, elu- vere clinical condition or a retinal disease that is possibly a part tion, and NGS were conducted as detailed previously.16 For bio- of a syndrome. In such situations, molecular genetic testing informatics analyses, the results of Sanger sequencing, in silico could be useful to address the clinical ambiguity in diagnosis. analyses, and the results of reverse transcription–polymerase Inherited retinal dystrophy can be inherited via all 3 meth- chain reaction, see the eAppendix in the Supplement. ods of Mendelian inheritance (ie autosomal dominant, recessive, and X-linked patterns). Digenic, mitochondrial, and incomplete dominant forms have also been reported.1,9,10 To date, 261 loci (in- Results cluding 221 identified genes) have been implicated in the etiol- Targeted NGS Approach ogy of IRDs (see the Retinal Information Network [RetNet] at Here, we only focus on the results of 11 of the 20 families inves- https://sph.uth.edu/retnet/), representing its great genetic hetero- tigated because putative disease-causing mutations were iden- geneity.Traditional approaches to the detection of mutations have tified in the 11 families. Detailed clinical data on the 11 families their limitations, resulting in low diagnostic rates. However, tar- are summarized in Table 1 and Table 2, whereas the clinical de- geted next-generation sequencing (NGS) enables parallel sequenc- tails of family pedigrees and the genetic findings of the other 9 ing of a panel of numerous candidate genes and has been proved families are presented in eFigure 1 and eTables 2 and 3 in the 11 to be an efficient tool for the molecular diagnosis of various IRDs Supplement. One or 2 family members from each family were 12 and of autosomal recessive RP. selected for NGS, and the results of NGS are detailed in eTable To develop an effective genetic diagnostic tool for IRDs, 4 in the Supplement. In brief, a total of 26 650 variants were ini- we have previously used a targeted NGS approach, by which tially detected by targeted NGS in the 11 families. Of all tested we identified disease-causing mutations in multiple types of samples, the mean call rate of the targeted region was about 11,13,14 IRDs. Herein, we have further applied this approach in 99.8%, and the mean depth was about 91.3-fold. A total of 17 the investigation of a cohort of 20 Chinese families with au- homozygous or compound heterozygous variants in the 11 fami- tosomal recessive IRD. lies passed the filtration process, of which 12 were novel and 5 were previously reported mutations (Table 3). Those novel variants were absent in 150 unrelated Chinese controls. The poten- Methods tial pathogenicity of those novel putative mutations was evaluated by multiple in silico programs and is summarized in Table 3. Participants and Clinical Investigations Our study conformed to the tenets of the Declaration of Hel- Novel Insights Into the Clinical Assessments in 4 Families sinki and was prospectively reviewed and approved by the lo- RP1 Mutations Found in Family RH15 With CRD cal institutional review boards. Written informed consent was Novel biallelic mutations in RP1 (p.[E474Gfs*11]; [K1939*]) were obtained from all participants or their legal guardians. A cohort identified in patient RH15-II:1 with CRD (Figure 1). This pa- of 20 unrelated Chinese families, including 33 patients affected tient was reported to have central visual defects since the age with IRD and 61 unaffected family members, were recruited from of 5 years, and her best-corrected visual acuity was 20/100 for multiple hospitals in China (eTable 1 in the Supplement). Fam- the right eye and 20/400 for the left eye at her last visit to our ily histories and personal medical records were carefully checked hospital. Macular degeneration was revealed in fundus pho- and reviewed. All of the participants underwent detailed oph- tographs and optical coherence tomographic images (Figure 1B thalmic examinations at the beginning of the study and sys- and C). Reduced photopic and scotopic electroretinographic temic examinations when necessary. The data obtained from responses were observed (Figure 1D). Visual field constric- these ophthalmic examinations included best-corrected visual tion and central scotoma were also indicated (Figure 1E). acuity, results of a slitlamp examination, intraocular pressure, results of a funduscopic examination, visual field, and electro- CYP4V2 Mutations Found in 3 Families With Diverse Phenotypes retinograms. Optical coherence tomography was performed for CYP4V2 mutation c.802-8_810del17insGC, a frequent mutation patients with macular degeneration. For patient RH13-II:1, a 3-T in East Asian populations,21-23 was identified in 3 families (RH6,

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Table 1. Genetic and Clinical Features of 17 Patients

BCVA Patient No./Sex/Age, y Gene Mutations Age at Onset OD OS RH1-V:1/F/18 BBS2 c.[1237C>T]; 10 y 20/40 20/32 [1237C>T] RH1-V:2/M/14 BBS2 c.[1237C>T]; 9 y 20/32 20/32 [1237C>T] RH4-II:6/M/20 ABCA4 c.[2424G>C]; 8 y 20/63 20/50 [1648del] RH6-II:1/F/24 CYP4V2 c.[802-8_810del17insGC]; 15 y 20/100 20/100 [802-8_810del17insGC] RH6-II:2/F/21 CYP4V2 c.[802-8_810del17insGC]; 18 y 20/25 20/25 [802-8_810del17insGC] RH9-V:1/M/23 PROM1 c.[1645_1648del]; 5mo LP LP [1645_1648del] RH9-V:2/F/21 PROM1 c.[1645_1648del]; 7mo CF CF [1645_1648del] RH9-V:3/M/19 PROM1 c.[1645_1648del]; 12 mo CF CF [1645_1648del] RH10-II:1/M/18 ALMS1 c.[3653C>G]; 5mo LP LP [4599C>A] RH12-II:3/M/34 CYP4V2 c.[802-8_810del17insGC]; 17 y 20/50 20/50 [802-8_810del17insGC] RH13-II:1/F/29 FLVCR1 c.[883 + 6T>C]; 4 y 20/200 20/200 [1150G>C] RH15-II:1/F/21 RP1 c.[1419_1420del]; 5 y 20/100 20/100 [5815A>T] RH17-IV:3/F/27 CRB1 c.[1816T>C]; 3 y 20/200 20/200 [1816T>C] RH18-II:2/F/39 CYP4V2 c.[802-8_807del17insGC]; 27 y CF CF [992A>C] RH18-II:4/F/37 CYP4V2 c.[802-8_807del17insGC]; 25 y CF CF [992A>C] RH18-II:6/M/30 CYP4V2 c.[802-8_807del17insGC]; 25 y 20/200 20/100 Abbreviations: BCVA, best-corrected [992A>C] visual acuity; CF, counting fingers; RH20-II:1/F/15 CNGA1 c.[1678G>A]; 4 y 20/40 20/40 LP, light perception; OD, right eye; [397del] OS, left eye.

Table 2. Ophthalmic Phenotypes of 17 Patients

Appearance in Fundus Photograph Right Eye Left Eye VF, degrees Patient No. MD OD AA PD MD OD AA PDERG OD OS RH1-V:1 Moderate Waxy Yes Yes Moderate Waxy Yes Yes Reduced 15 15 RH1-V:2 Moderate Waxy Yes Yes Moderate Waxy Yes Yes Reduced 25 25 RH4-II:6 Severe Waxy Yes Yes Severe Waxy Yes Yes NA NA NA RH6-II:1 Severe Waxy Yes Yes Severe Waxy Yes Yes Reduced 30 25 RH6-II:2 Normal Normal No Yes Normal Normal No Yes Reduced NA NA RH9-V:1 Severe Waxy Yes No Severe Waxy Yes No Reduced NA NA RH9-V:2 Mild Waxy No No Mild Waxy No No NA NA NA RH9-V:3 Mild Waxy No No Mild Waxy No No NA NA NA RH10-II:1 Severe Normal Yes No Severe Normal Yes No NA NA NA RH12-II:3 Severe Waxy No Yes Severe Waxy No Yes Reduced 5 5 RH13-II:1 Severe Waxy Yes Yes Severe Waxy Yes Yes Reduced 10 10 RH15-II:1 Severe Waxy Yes No Severe Waxy Yes No Reduced CS CS RH17-IV:3 Severe Waxy Yes Yes Severe Waxy Yes Yes Reduced 10 10 RH18-II:2 Severe Waxy Yes Yes Severe Waxy Yes Yes Reduced NA NA RH18-II:4 Severe Waxy Yes Yes Severe Waxy Yes Yes Reduced NA NA RH18-II:6 Severe Waxy Yes Yes Severe Waxy Yes Yes NA 15 10 RH20-II:1 Moderate Waxy No Yes Moderate Waxy No Yes Reduced NA NA

Abbreviations: AA, artery attenuation; CS, central scotoma; ERG, electroretinography; MD, macular degeneration; NA, not available; OD, optic disc; PD, pigment deposits; VF, visual field.

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Table 3. Mutations Identified in This Study

Variation Bioinformatics Analysis Reported Gene Family Disease Nucleotide Amino AcidStatus SIFTa PolyPhenb Condelc Provend or Novel BBS2 RH1 BBS c.1237C>T p.R413* HOM NA NA NA NA Reported c.397del p.G133Vfs*28 HET NA NA NA NA Novel CNGA1 RH20 RP c.1678G>A p.R560* HET NA NA NA NA Novel CRB1 RH17 RP c.1816T>C p.C606R HOM D PD NE DE Novel RH6 BCD c.802-8_810del17insGC p.I260_N339del HOM NA NA NA NA Reported RH12 BCD c.802-8_810del17insGC p.I260_N339del HOM NA NA NA NA Reported CYP4V2 c.802-8_810del17insGC p.I260_N339del HET NA NA NA NA Reported RH18 RP c.992A>C p.H331P HET D PD DE DE Reported c.2424G>C p.Y808* HET T NA NA NA Novel ABCA4 RH4 STGD c.1648del p.V521Sfs*46 HET NA NA NA NA Novel c.3653C>G p.S1218* HET T NA NA NA Novel ALMS1 RH10 AS c.4599C>A p.Y1533* HET T NA NA NA Novel c.883 + 6T>C p.? HET NA NA NA NA Novel FLVCR1 RH13 RP c.1150G>C p.G384R HET D PD DE DE Novel PROM1 RH9 RP with c.1645_1648del p.K549Qfs*2 HOM NA NA NA NA Novel MD c.1419_1420del p.E474Gfs*11 HET NA NA NA NA Novel RP1 RH15 CRDs c.5815A>T p.K1939* HET NA NA NA NA Novel

Abbreviations: AS, Alström syndrome; BBS, Bardet-Biedl syndrome; BCD, Bietti a Kumar et al.17 Crystalline corneoretinal dystrophy; CRD, cone rod dystrophy; D, damaging; b Adzhubei et al.18 DE, deleterious; HET, heterozygous; HGMD, Human Genome Mutation Database; c González-Pérez and López-Bigas.19 HOM, homozygous; MD, macular degeneration; NA, not available; NE, neutral; PD, probably damaging; RP, retinitis pigmentosa; STGD, Stargardt disease; T, tolerated. d Choi et al.20

Figure 1. Detailed Ophthalmic Evaluations for Patient RH15-II:1

A Family RH15 Pedigree and Genotypes B Fundus Photographs C OCT Images

1 2 I MU1/+ +/MU2

1 2 II +/+ MU1/MU2

MU1 RP1 c.1419_1420del HET MU2 RP1 c.5815A>T HET

D ERG Responses E Automated Visual Field Examinations Patient RH15-II:1 (OD) Rod response OD OS OD OS

0.5-Hz white light ERG indicates electroretinographic; 30-Hz white light HET, heterozygous; OCT, optical coherence tomographic; OD, right eye; OS, left eye; and MU, mutation.

RH12, and RH18) (Figure 2A). This mutation, located in the flank- lacking 80 in-frame amino acids. This mutation was found to be ing intronic region (Figure 2B), has been proven to cause the skip homozygous in the patients from families RH6 and RH12 with of whole exon 7 by reverse transcription–polymerase chain Bietti crystalline dystrophy,whereas biallelic CYP4V2 mutations reaction,24 thus generating a truncated protein (p.I260_N339del) (p.[I260_N339del]; [H331P]) were found in patients from family

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Figure 2. Clinical Phenotypes and CYP4V2 Mutations Identified in Families RH6, RH12, and RH18

A Pedigrees and Mutation Distributions B Sanger Sequencing of CYP4V2 Mutations

Family RH6 Family RH12 1 2 12 MU1 I I G C G A A C G G G C C A A T G A A MU1/+ MU1/+ MU1/+

12 132 II II CYP4V2 c.802-8_810del17insGC HOM MU1/MU1 MU1/MU1 MU1/+ MU1/+ MU1/MU1 G C G A A C G G G C C A A T G A A T C A T A C A G G T C A T C G C T

Family RH18 12 I +/MU2 CYP4V2 c.802-8_810del17insGC HET

1 23 4 5 6 7 WT II T C A T A C A G G T C A T C G C T MU1/MU2MU1/+ MU1/MU2 +/+ MU1/MU2 123 4 III MU1/+ MU1/+ MU1/+

MU2 C Orthologous Protein Sequence Alignment G T A G G G G C A/C C G A T A C A A p.His331Pro

Homo sapiens 322 E V D T F M F E G H D T T A A A I N W Pan troglodytes 322 E V D T F M F E G H D T T A A A I N W Canis lupus 320 E V D T F M F E G H D T T A A A I N W CYP4V2 c.992A>C HET Bos taurus 324 E V D T F M F E G H D T T A A A I N W Sus scrofa 320 E V D T F M F E ------WT Mus musculus 322 E V D T F M F E G H D T T A A A I N W G T A G G G G C A C G A T A C A A Gallus gallus 326 E V D T F M F E G H D T T A A A M N W Danio rerio 310 E V D T F M F E G H D T T A A S M N W Drosophila melanogaster 335 E V D T F M F E G H D T T S A A I S W Caenorhabditis elegans 303 E V D T F M F A G H D T T T T S V S W

HET indicates heterozygous; HOM, homozygous; MU, mutation; and WT, wild type.

RH18 with typical RP (Figure 2A and B; Figure 3A). Conservational sentations (Figure 5). Other than ophthalmic abnormities, re- analysis also proved the high level of conservation of residue H331 sults of systemic examinations revealed polydactylism and mild in multiple orthologous protein sequences (Figure 2C). Clinical cognitive impairment in both patients. The affected sister showed evaluations were presented in Figure 3A and B and Tables 1 and no signs of menophania at her visit when she was 18 years of age. 2, and waxy optic discs and attenuated vessels are present in all Each member of this family thus received a diagnosis of Bardet- patients. Crystal deposits are present in patients RH6-II:1, RH6- Biedl syndrome based on genetic and clinical findings. II:2, and RH12-II:3, whereas bone spicule–like pigments are revealed in patients RH12-II:3, RH18-II:1, RH18-II:2, and RH18-II: Identification of PROM1 Mutations in Family RH9 3. Macular atrophy was detected on the optical coherence tomo- A novel homozygous frameshift mutation in PROM1 graphic images of patient RH6-II:1. An interesting finding is that (p.K549Qfs*2) was identified in patients from family RH9 intensive bone spicule–like pigments were observed in the fun- (Figure 4A). PROM1 mutations have been reported to be the dus photographs of patient RH12-II:3, whereas crystal deposits cause of recessive RP with macular degeneration, dominant Star- were only found in the macular region (Figure 3A). gardt disease–like macular dystrophy, dominant cone dystrophy, and dominant CRD (RetNet). All 3 patients from family RH9 Finalizing the Clinical Diagnoses in 4 Families reported having night blindness since early childhood, and at Identification of BBS2 Mutation in Family RH1 their last visit, each patient had central vision that was se- A previously reported homozygous missense variant in BBS2 verely impaired owing to severe macular degeneration (Figure 5; (p.R413* [CM033336]) was identified in family RH1 with Bardet- Tables 1 and 2). Presentations of RP and macular degeneration Biedl syndrome (Figure 4A). The 2 siblings, RH1-V:1 and RH1- were indicated in the fundus photographs of patients from this V:2, were still in their teens when they first were referred to our family. Therefore, based on the genetic and clinical findings, we clinic for having poor night vision and a constricted visual field. finalized the clinical diagnosis to autosomal recessive RP with Results of an ophthalmic examination revealed typical RP pre- macular degeneration for this family.

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Figure 3. Fundus Photographs of Patients in Families RH6 and RH18 Carrying CYP4V2 Mutations and OCT Images of Patients in Family RH6

A Fundus Photographs RH6-II:1 RH6-II:2 RH12-II:3 RH18-II:1 RH18-II:2 RH18-II:3

B OCT Images RH6-II:1 RH6-II:2

OS OS

OCT indicates optical coherence tomographic; OS, left eye.

Identification of ALMS1 Mutations in Family RH10 disks were indicated in C4/5, C5/6, and C6/7, and magnetic reso- Novel biallelic ALMS1 mutations p.[S1218*]; [Y1533*] were iden- nance imaging at C4/5 revealed the centrally herniated disk ma- tified as disease causing for family RH10 (Figure 4A; Tables 1-3). terial and the narrowing of the spinal canal (Figure 6E-G). Patient RH10-II:1 was reported to have nystagmus and photo- By use of targeted NGS, we identified novel biallelic muta- phobia since 5 months of age. His visual acuity began to de- tions in the FLVCR1 gene in patient RH13-II:1, including the crease rapidly early in the second decade of his life. At his last paternal inherited splice site mutation c.883+6T>C and the ma- visit to our clinic, he was 18 years of age when his best- ternal inherited missense variation p.G384R. The missense varia- corrected visual acuity was light perception for both eyes. Fun- tion was predicted to be deleterious by all in silico programs, and dus photographs and electroretinographic responses indi- the residue G384 was absolutely conserved through evolution cated a typical presentation of CRD in this patient (Figure 5). (Figure 1A and D). Reverse transcription–polymerase chain re- Because ALMS1 mutations have been reported to cause Al- action was then performed to determine the effect of the pater- ström syndrome, his medical records were further examined, nal inherited allele. Reverse transcription–polymerase chain re- and more biochemical tests performed. Detailed systematic ex- action products were separated by agarose gel electrophoresis, aminations revealed sensorineural hearing loss, early-onset type and 4 bands were observed in patient RH13-II:1 and her unaf- 2 diabetes mellitus, obesity, dilated cardiomyopathy, and he- fected father RH13-I:1, who carried the same allele (eFigure 2A patic dysfunction in this patient, which fully met the diagnos- in the Supplement). Sequencing of the product revealed that this tic criteria for Alström syndrome. Therefore, the genetic find- splice site mutation would cause aberrant splicing of the FLVCR1 ings for this patient helped to define the clinical diagnosis of gene by generating a mutant complementary DNA fragment Alström syndrome in this case. inserted with a 127–base pair (bp) fragment from intron 2 beginning at c.883+524 (eFigure 2B in the Supplement). FLVCR1 mu- Identification of FLVCR1 Mutations in Family RH13 tations have been implicated in the disease etiology of autoso- Patient RH13-II:1 had poor night vision since early childhood, fol- mal recessive posterior column ataxia with RP (PCARP).25-27 lowed by a rapid decrease in her visual field and central vision Therefore, this patient received a diagnosis of PCARP. (Figure 6A; Tables 1 and 2). Ophthalmic evaluations demonstrated typical RP phenotypes with macular edema in her right Putative Mutations and Clinical Manifestations eye (Figure 6B and C). Complex neural phenotypes were also in Another 3 Families noted in patient RH13-II:1, including mild ataxia since child- The novel homozygous missense mutation in CRB1 (p.C606R) hood, attenuation of deep tendon reflexes, and superficial sen- was identified in patient RH17-IV:3 with RP, whose parents were sations. Magnetic resonance imaging revealed a mild enlarge- first-degree cousins. Typical RP presentations on fundus pho- ment of the central canal from C3 to T8 vertebral levels. Herniated tographs, including waxy pallor of optic discs, attenuated ves-

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Figure 4. Clinical Phenotypes and Mutations Identified in Families RH1, RH4, RH9, RH10, RH15, RH17, and RH20

A Pedigrees and Genotypes

Family RH1 Family RH9 Family RH10 1 2 1 2 1 2 I II

1 2 3 4 1 2 3 4 1 II II II

1 2 3 4 1 2 34 MU6/MU7 III III MU6:ALMS1 c.3653C>G; p.S1218* MU7:ALMS1 c.4599C>A; p.Y1533* 1 2 1 2 IV IV MU1/+ MU1/+ MU2/+ MU2/+ 1 2 123 Family RH17 V V 1 2 MU2/MU2 MU2/MU2 MU2/MU2 I MU1/MU1 MU1/MU1 MU2:PROM1 c.1645_1648del; p.K549Qfs*2 1234 MU1:BBS2 c.1237C>T ; p.R413* II

Family RH20 Family RH4 12 III 12 1 2 I I MU3/+ MU3/+ 12 3 4 5 1 23 456 7 IV II II MU3/+ MU3/+ +/+ +/+ MU3/MU3 MU8/MU9 MU4/MU5 12 3 4 5 12 MU6:CNGA1 c.397del; p.G133Vfs*28 III MU8:CNGA1 c.1678G>A; p.R560* V +/+ MU3/+ MU3/+ MU4:ABCA4 c.2424C>G; p.Y808* MU5:ABCA4 c.1648del; p.V521Sfs*46 MU3:CRB1 c.1816T>C; p.C606R

B Orthologous Protein Sequence Alignment of CRB1 C ERG Responses CRB1 p.Cys606Arg Patient Patient RH17-IV:3 (OD) RH17-IV:3 (OS) Homo sapiens 596 P L E S D - Q S I C A F Q N S F L G G Pan troglodytes 596 P L E S D - Q S I C A F Q N S F L G G Rod response Canis lupus 596 P F E S H - R S I C A F Q N S F L G G Bos taurus 596 P F E S D G S S A C A L Q N S F L G G Sus scrofa 594 P F G S D - R S A C A L Q N S F L G G 0.5-Hz white light Mus musculus 595 P V E N H - Q S I C A L Q D S F L G G Gallus gallus 569 M I D N D - H V R L A F Q S T F L G S 30-Hz white light Danio rerio 611 V K I G A L E L E S A L L S T F V G G Drosophila melanogaster 1259 ------P N L K S

ERG indicates electroretinographic; OD, right eye; OS, left eye; and MU, mutation.

Figure 5. Fundus Photographs of Patients From Families RH1, RH4, RH9, RH10, and RH17

RH1-V:1 RH1-V:2 RH4-II:6RH9-V:1 RH9-V:2 RH9-V3 RH10-II:1 RH17-IV:3

Typical presentations of retinitis pigmentosa on fundus photographs can be seen for patients RH1-V:1, RH1-V:2, RH4-II:6, and RH17-IV:2. Macular degeneration can been in the fundus photographs of patients RH9-V:1, RH9-V:2, RH9-V3, and RH10-II:1.

sels, and bone spicular pigmentations, were demonstrated by IV:3 (Figure 4C). According to RetNet, CRB1 mutations have been this patient (Figure 5). Significantly reduced scotopic and phot- implicated in a wide panel of IRDs, including recessive RP, re- opic electroretinographic responses are shown for patient RH17- cessive Leber congenital amaurosis, and dominant pigmented

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Figure 6. Clinical Phenotypes and FLVCR1 Mutations Identified in Patient RH13-II:1

A Family RH13 Pedigree and Genotypes B Fundus Photographs C OCT Images

1 2 I MU1/+ +/MU2

1 II

MU1/MU2

MU1 FLVCR1 c.883+6T>C HET MU2 FLVCR1 c.1150G>C HET

D Orthologous Protein Sequence Alignment of CYP4VZ MRI Scans of Spinal Cord p.Gly384Arg E F G Homo sapiens 375 L T L V V A G M V G S I L C G L W L D Pan troglodytes 375 L T L V V A G M V G S I L C G L W L D Canis lupis 375 L T L V V A G M V G S I L C G L W L D Bos taurus 376 L T L V V A G M V G S I L C G L W L D Sus scrofa 375 L T L V L A G M V G S I L C G L W L D Mus musculus 380 L T L V V A G M V G S I L C G L W L D Gallus gallus 326 L T L V V A G M V G S I I C G L W L D Danio rerio 344 L T L V V A G M F G S I L C G I W L D Drosophila melanogaster 326 L S I V L A G M L G S V V S G I V L D Caenorhabditis elegans 416 L L I V V A G M A G S V V G G F I L D

HET indicates heterozygous; OCT, optical coherence tomographic; MRI, magnetic resonance imaging; and MU, mutation. The white arrowheads indicate bone spicular pigmentation. The yellow arrowheads indicate enlargement of the central canal from C3 to T8 vertebral levels.

paravenous chorioretinal atrophy.The affected residue C606 was tation analyses for a cohort of 20 Chinese families with auto- conserved among all mammal species and is located in the first somal recessive IRD. A detection rate of 55% (11 of 20 families) laminin G–like domain of the protein Crumbs homologue 1 en- is achieved in this cohort, similar to that in our previous study coded by the CRB1 gene (Figure 4B). The nature of p.C606R, a (56%)11 and a bit higher than that in other Chinese cohorts with missense mutation, may explain why it correlates with reces- autosomal recessive IRD.12,28,29 Seventeen disease-causing mu- sive RP but not with more severe diseases such as recessive Leber tations have been identified in the 11 families investigated, congenital amaurosis or dominant retinal dystrophy. among which 12 are novel to our knowledge. Thus, our study Biallelic mutations in ABCA4 (p.[Y808*]; [V521Sfs*46]) were extends the spectrum of IRD disease-causing mutations. found to cause Stargardt disease in patient RH4-II:6 (Figure 4A; Mutations in RP1 have been reported to cause RP, account- Figure 5; Tables 1-3), and novel biallelic mutations in CNGA1 ing for 5.5% of the autosomal dominant form and 1% of the re- (p.[G133Vfs*28]; [R560*]) were revealed in patient RH20-II:1 with cessive form.30 The RP1 gene encodes a protein of 2156 amino typical RP (Figure 4A). ABCA4 mutations have been found to acids and is located in the connecting cilia of both rod and cone cause recessive Stargardt disease, recessive macular dystrophy, photoreceptors.31 The RP1 protein contains 2 doublecortin (DCX) recessive RP, recessive fundus flavimaculatus, and recessive CRD, domains (residues 36 to 118 [DCX1] and 154 to 233 [DCX2]), via whereas CNGA1 mutations were only implicated in the disease which the RP1 protein interacts with microtubules. The RP1 pro- etiology of RP (RetNet). Therefore, the correlations between the tein is thus the first identified photoreceptor-specific and mi- affected genes and the phenotypes observed in the 2 families crotubule-associated ciliary protein, which functions in the or- were previously established. ganization of the photoreceptor outer segments to ensure the exact orientation and higher-order stacking of outer segment disks along the photoreceptor axoneme.32 Close interactions be- Discussion tween RP1 and other ciliary proteins, including the Rp1-like protein (RP1L1) and the male germ cell–associated kinase, have also We have previously evaluated the efficiency of targeted NGS in been identified.32,33 RP1L1 mutations have been implicated in families with IRD, which is mainly in autosomal dominant trait occult macular dystrophy34 and RP,35 suggesting a potential di- or sporadic cases.11 In the present study, we focused on the mu- verse role of RP1 in IRDs. By far, RP1 mutations have only been

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reported to cause RP, whereas, in the present study, we have PCARP.25-27 In our study, compound heterozygous mutations identified biallelic mutations in RP1 (p.E474Gfs*11 and p.K1939*) in FLVCR1, c.[883+6T>C]; [1150G>C] (p.[?]; [Gly384Arg]), are in patient RH15-II:1 with CRD. Similar to our findings, most iden- found in a Chinese patient with PCARP. Similar to previous find- tified RP1 mutations are protein-truncating mutations result- ings, these 2 mutations were also located in the transmem- ing in premature termination codons, which would lead to RP1 brane domains, indicating a pathogenesis similar to that of the defects via the generation of truncated proteins or nonsense- previously investigated mutations.43 Thus, we hypothesize that mediated messenger RNA decay.36,37 Thus, we have identified mutant hFLVCR with p.Gly384Arg would also misfold in the en- RP1 mutations in patients with CRD, suggesting a potential re- doplasmic reticulum, partially degrade in lysosomes, lose heme lationship between RP1 defects and the pathogenesis of CRD. export activity, cause intracellular accumulation, and lead to Bietti crystalline dystrophy, also referred to as Bietti crys- apoptosis.43 Furthermore, the splice site mutation, c.883+6T>C, talline corneoretinal dystrophy, is inherited in an autosomal is predicted to generate a splice donor site and a splice recep- recessive fashion with only 1 disease-causing gene, CYP4V2.38 tor site within the intronic sequence between exons 2 and 3, Patients with Bietti crystalline dystrophy often present with thus leading to the insertion of a 127-bp fragment. This inser- corneal crystals, yellow deposits in the retina, and progres- tion would probably generate an altered protein with an ir- sive retinal and choroidal atrophy.39 Other than Bietti crystal- regular function or cause messenger RNA decay. Of note, fur- line dystrophy, biallelic mutations in CYP4V2 have been re- ther investigation of this inserted fragment has revealed its ported in Chinese families with recessive RP,12,23 one of which existence as the second exon of the coding sequence of an- shows complicated phenotypes, including RP, thin corneas, other FLVCR1 transcript (ENST00000579295); however, no pro- congenital cataracts, and high myopia.23 In our study, we have tein product has been annotated for this transcript. Because the identified biallelic CYP4V2 mutations c.802-8_810del7insGC specific mechanism underlying the association between FLVCR1 (p.I260_N339del) and c.992A>C (p.H331P) in a Chinese family gene deficits and symptoms of PCARP is still unclear, we hy- with RP. These mutations are frequently seen in East Asian pothesize a potential linkage between hFLVCR protein dys- populations. The ages at onset of disease for all 3 patients in function and neurological problems. this family are similar to those for patients in the previously Therefore, our study demonstrates that the targeted NGS reported families.12,23 No other ophthalmic abnormalities were approach might help to generate a molecular diagnosis for au- observed. Cytochrome P450 4V2, the protein encoded by the tosomal recessive IRDs. However, this approach also has limi- CYP4V2 gene containing 525 amino acids, plays a crucial role tations. A mean mismatch rate of 0.234% was reached in our in the metabolism of fatty acids and steroids in the eye,40 par- study,indicating the potential existence of false-positive or false- ticularly in the hydroxylation of the omega-3 polyunsatu- negative variants. In addition, deep intronic variations and copy rated fatty acids, including docosahexaenoic acid and eicos- number variations could not be detected by this approach. No apentaenoic acid.41 Polyunsaturated fatty acids function in the disease-causing mutations were identified in the remaining 9 renewal of disk membranes in the outer segments of photo- families. These 9 families may carry mutations in unknown loci, receptor cells and demonstrate a much higher level of expres- deep intronic mutations, or copy number variations, all of which sion in the retina when compared with other tissues.42 Thus, are not detectable by our targeted NGS approach. CYP4V2 defects would probably lead to disease via the dysfunction or deficiency of polyunsaturated fatty acids. Di- etary supplementation of polyunsaturated fatty acids in such Conclusions patients would possibly help to slow down the progression of disease and might be recommended by clinicians. In conclusion, by means of targeted NGS, we have revealed 15 The human feline leukemia virus subgroup C receptor 1 disease-causing mutations, including 12 novel mutations found (hFLVCR) protein, encoded by the FLVCR1 gene, is a heme ex- in 11 of 20 Chinese families with autosomal recessive IRD. Our porter protein crucial for maintaining the intracellular concen- finding reaches a detection rate of 55% in the investigated co- tration of heme. As a cell surface receptor, hFLVCR mainly func- hort, which demonstrates the efficiency of targeted NGS in ana- tions in heme export and erythroid maturation. Inhibition of lyzing the etiology for autosomal recessive IRDs. In addition, hFLVCR will cause apoptosis of erythroid cells. Six mutations based on all our findings, we believe that the genetic evalua- in the FLVCR1 gene have been previously reported to cause tions would help with clinical assessments.

ARTICLE INFORMATION Y. Chen, J. Zhang, Shen, Jiang); Tianjin Medical Clinical Medical College of Nanjing Medical Submitted for Publication: August 20, 2014; final University, Tianjin Eye Hospital, Tianjin Key University, Nanjing Medical University, Nanjing, revision received November 26, 2014; accepted Laboratory of Ophthalmology and Visual Science, China (X. Chen); State Key Laboratory of November 30, 2014. Tianjin, China (K. Zhao); Department of Ophthalmology, Zhongshan Ophthalmic Center, Ophthalmology, The Affiliated Jiangyin Hospital of Sun Yat Sen University, Guangzhou, China (C. Zhao). Published Online: January 22, 2015. Southeast University Medical College, Jiangyin, doi:10.1001/jamaophthalmol.2014.5831. Author Contributions: Dr C. Zhao had full access to China (Xu); Department of Ophthalmology, School all of the data in the study and takes responsibility Author Affiliations: Department of of Medicine, Henan Polytechnic University, Henan, for the integrity of the data and the accuracy of the Ophthalmology, The First Affiliated Hospital of China (X. Zhang, Gao); Department of data analysis. Drs Liu, Xiao, Huang, Guan, and X. Nanjing Medical University, State Key Laboratory of Ophthalmology, Xinhua Hospital, Shanghai Jiao Chen contributed equally to this article. Reproductive Medicine, Nanjing, China (Liu, Xu, Tong University School of Medicine, Shanghai, Study concept and design: K. Zhao, X. Chen, C. Zhao. Pan, Gu, C. Zhao); BGI-Shenzhen, Shenzhen, China (Kang); Ningxia Eye Hospital, Ningxia Acquisition, analysis, or interpretation of data: Liu, Guangdong Province, China (Xiao, Huang, Guan, People’s Hospital, Ningxia, China (Sheng); The First

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Xiao, Huang, Guan, Xu, X. Zhang, Pan, Gu, Y. Chen, 11. Chen X, Zhao K, Sheng X, et al. mutations in the FLVCR1 gene. Int J Neurosci.2015; J. Zhang, Shen, Jiang, Gao, Kang, Sheng. Targeted sequencing of 179 genes associated with 125(1):43-49. Drafting of the manuscript: Liu, Xiao, Huang, Guan, hereditary retinal dystrophies and 10 candidate 28. Chen Y, Zhang Q, Shen T, et al. Comprehensive Gu, Y. Chen, J. Zhang, Shen, Jiang, X. Chen. genes identifies novel and known mutations in mutation analysis by whole-exome sequencing in 41 Critical revision of the manuscript for important patients with various retinal diseases. Invest Chinese families with Leber congenital amaurosis. intellectual content: Liu, K. Zhao, Xu, X. Zhang, Pan, Ophthalmol Vis Sci. 2013;54(3):2186-2197. Invest Ophthalmol Vis Sci. 2013;54(6):4351-4357. Gao, Kang, Sheng, X. Chen, C. Zhao. 12. Fu Q, Wang F, Wang H, et al. Next-generation Statistical analysis: Liu, Gu. 29. Huang L, Zhang Q, Li S, et al. sequencing-based molecular diagnosis of a Chinese Exome sequencing of 47 Chinese families with Obtained funding: C. Zhao. patient cohort with autosomal recessive retinitis Administrative, technical, or material support: All cone-rod dystrophy: mutations in 25 known pigmentosa. Invest Ophthalmol Vis Sci. 2013;54(6): causative genes. PLoS One. 2013;8(6):e65546. authors. 4158-4166. Study supervision: C. Zhao. 30. Hartong DT, Berson EL, Dryja TP. Retinitis 13. Chen X, Liu Y, Sheng X, et al. PRPF4 mutations pigmentosa. Lancet. 2006;368(9549):1795-1809. Conflict of Interest Disclosures: None reported. cause autosomal dominant retinitis pigmentosa. Funding/Support: This work was supported by the Hum Mol Genet. 2014;23(11):2926-2939. 31. Liu Q, Zhou J, Daiger SP, et al. Identification and subcellular localization of the RP1 protein in human National Key Basic Research Program of China 14. Sheng X, Chen X, Zhao K, Liu Y, Vollrath D, (program 973, grant 2013CB967500), the National and mouse photoreceptors. Invest Ophthalmol Vis Sci. Zhao C. A novel homozygous BEST1 mutation 2002;43(1):22-32. Natural Science Foundation of China (grants correlates with complex ocular phenotypes. 81222009, 81170856, 81260154, and 81170867), Ophthalmology. 2013;120(7):1511-1512. 32. Yamashita T, Liu J, Gao J, et al. Essential and the Thousand Youth Talents Program of China (to Dr synergistic roles of RP1 and RP1L1 in rod C. Zhao), the Jiangsu Outstanding Young 15. Rong W, Chen X, Zhao K, et al. Novel and photoreceptor axoneme and retinitis pigmentosa. Investigator Program (grant BK2012046), the recurrent MYO7A mutations in Usher syndrome J Neurosci. 2009;29(31):9748-9760. type 1 and type 2. PLoS One. 2014;9(5):e97808. Jiangsu Province’s Key Provincial Talents Program 33. Omori Y, Chaya T, Katoh K, et al. Negative (grant RC201149), the Fundamental Research Funds 16. WangJL,YangX,XiaK,etal.TGM6 identified as regulation of ciliary length by ciliary male germ of the State Key Laboratory of Ophthalmology (to a novel causative gene of spinocerebellar ataxias cell-associated kinase (Mak) is required for retinal Dr C. Zhao), the Jiangsu Province’s Scientific using exome sequencing. Brain. 2010;133(pt 12): photoreceptor survival. ProcNatlAcadSciUSA. Research Innovation Program for Postgraduates 3510-3518. 2010;107(52):22671-22676. (grant CXZZ13_0590), and a project funded by the 17. Kumar P, Henikoff S, Ng PC. Predicting the 34. Akahori M, Tsunoda K, Miyake Y, et al. Priority Academic Program Development of Jiangsu effects of coding non-synonymous variants on Higher Education Institutions. Dominant mutations in RP1L1 are responsible for protein function using the SIFT algorithm. Nat Protoc. occult macular dystrophy. 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