Comprehensive Mutation Analysis by Whole-Exome Sequencing in 41 Chinese Families with Leber Congenital Amaurosis

Genetics Comprehensive Mutation Analysis by Whole-Exome Sequencing in 41 Chinese Families With Leber Congenital Amaurosis

Yabin Chen,1 Qingyan Zhang,2 Tao Shen,1 Xueshan Xiao,1 Shiqiang Li,1 Liping Guan,2 Jianguo Zhang,2 Zhihong Zhu,2 Ye Yin,2 Panfeng Wang,1 Xiangming Guo,1 Jun Wang,2 and Qingjiong Zhang1

1State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China 2BGI-Shenzhen, Shenzhen, China

Correspondence: Qingjiong Zhang, PURPOSE. Leber congenital amaurosis (LCA) is a genetically heterogeneous disease with, to State Key Laboratory of Ophthal- date, 19 identified causative genes. Our aim was to evaluate the mutations in all 19 genes in mology, Zhongshan Ophthalmic Chinese families with LCA. Center, Sun Yat-sen University, 54 Xianlie Road, Guangzhou 510060, METHODS. LCA patients from 41 unrelated Chinese families were enrolled, including 25 China; previously unanalyzed families and 16 families screened previously by Sanger sequencing, but [email protected]. with no identified mutations. Genetic variations were screened by whole-exome sequencing YC, QZ, JW, and QZ contributed and then validated using Sanger sequencing. equally to the work presented here RESULTS. A total of 41 variants predicted to affect protein coding or splicing was detected by and should therefore be regarded as whole-exome sequencing, and 40 were confirmed by Sanger sequencing. Bioinformatic and equivalent authors. segregation analyses revealed 22 potentially pathogenic variants (17 novel) in 15 probands, Submitted: January 4, 2013 comprised of 3 of 16 previously analyzed families and 12 of 25 (48%) previously unanalyzed Accepted: April 29, 2013 families. In the latter 12 families, mutations were found in CEP290 (three probands); Citation: Chen Y, Zhang Q, Shen T, et GUCY2D (two probands); and CRB1, CRX, RPE65, IQCB1, LCA5, TULP1, and IMPDH1 (one al. Comprehensive mutation analysis proband each). Based on the results from 87 previously analyzed probands and 25 new cases, by whole-exome sequencing in 41 GUCY2D, CRB1, RPGRIP1, CEP290, and CRX were the five most frequently mutated genes, Chinese families with Leber congeni- which was similar to the results from studies in Caucasian subjects. tal amaurosis. Invest Ophthalmol Vis Sci. 2013;54:4351–4357. CONCLUSIONS. Whole-exome sequencing detected mutations in the 19 known LCA genes in DOI:10.1167/iovs.13-11606 approximately half of Chinese families with LCA. These results, together with our previous results, demonstrate the spectrum and frequency of mutations of the 19 genes responsible for LCA in Han Chinese individuals. Whole-exome sequencing is an efficient method for detecting mutations in highly heterogeneous hereditary diseases. Keywords: Leber congenital amaurosis, exome sequencing, mutation screening, genotype- phenotype, Chinese

Downloaded from jov.arvojournals.org on 10/02/2021 Mutation Analysis in Leber Congenital Amaurosis IOVS j June 2013 j Vol. 54 j No. 6 j 4352

eber congenital amaurosis (LCA, MIM 204000) is the most male and 12 were female; 34 were isolated cases, while six Lsevere form of inherited retinal dystrophy, with an showed autosomal-recessive inheritance, and one showed estimated prevalence of 1:81,0001 to 1:30,000.2 This condition autosomal-dominant inheritance. Written informed consent accounts for more than 5% of all retinal dystrophies and conforming to the tenets of the Declaration of Helsinki was approximately 20% of children attending schools for the blind.3 obtained from the participants or their guardians before the LCA is inherited most frequently as an autosomal-recessive study. This study was approved by the Institutional Review trait, but also may be transmitted as an autosomal-dominant Board of the Zhongshan Ophthalmic Center. Genomic DNA trait in rare cases.4 Clinical features of LCA include profound was extracted from leukocytes of a peripheral blood sample of loss of visual function at birth or within the first year of life, each participant as described previously.37 nystagmus, oculodigital sign of Franceschetti, sluggish pupil- lary light reflex, and variable fundus changes ranging from Whole-Exome Sequencing relatively normal appearance to severe pigmentary degenera- Whole-exome sequencing was performed through a commercial tion. Markedly reduced or no identifiable rod and cone service from BGI Shenzhen (Shenzhen, China; available in the responses on electroretinogram recording in infancy are the public domain at http://www.genomics.cn/index). The methods 5 hallmark of LCA. for exome capture, exon-enriched DNA library construction, To date, mutations in at least 19 genes have been identified 6 sequencing, genotyping, and variant analysis have been reported as responsible for LCA: guanylate cyclase 2D (GUCY2D), previously.38 In brief, a NimbleGen SeqCap EZ Exome (44M; retinal pigment epithelium-specific protein 65 kDa (RPE65),7 8 Roche, Basil, Switzerland) array was used to perform the exome spermatogenesis-associated protein 7 (SPATA7), aryl hydro- capture. Subsequently, exon-enriched DNA fragments were carbon-interacting receptor protein-like 1 (AIPL1),9 Leber 10 loaded on the Illumina Genome Analyzer II (Illumina, Santiago, congenital amaurosis 5 gene (LCA5), retinitis pigmentosa CA) platform for sequencing. The mean exome coverage was set (RP) GTPase regulator-interacting protein (RPGRIP1),11 cone- 12 as 60-fold. For variant analysis, alignment of the sequencing rod homeobox-containing gene (CRX), crumbs homolog 1 reads with UCSC hg19 was performed on a SOAPaligner.39,40 (Drosophila)(CRB1),13 centrosomal protein 290 kDa 14 0 SOAPsnp was used to calculate the likelihood of possible (CEP290), inosine 5 -monophosphate dehydrogenase type genotypes in target regions.41 Variants in all 19 LCA-related 1(IMPDH1),15 retinal degeneration 3 (RD3),16 retinol dehy- 17 genes detected by whole-exome sequencing were selected for drogenase 12 (RDH12), lecithin retinol acyltransferase further verification. (LRAT),18 tubby-like protein 1 (TULP1),19 inwardly rectifying potassium channel Kir7.1 (KCNJ13),20 calcium-binding protein Sanger Sequencing 4(CABP4),21 IQ motif-containing protein B1 (IQCB1),22 orthodenticle homolog 2 (OTX2),23 and nicotinamide nucleo- Sanger sequencing was used to validate the variants found in tide adenylyltransferase 1 (NMNAT1).24 Mutations in most of whole-exome sequencing in the 19 genes. Segregation analyses the genes above are associated with autosomal-recessive LCA, were performed in patients with available relatives. Genomic whereas mutations in CRX,4 IMPDH1,15 and OTX223 usually information about the 19 genes is listed in Supplementary Table are associated with autosomal-dominant LCA. Studies of these S1. Primers used to amplify fragments harboring individual genes, based on an individual gene or a subset of genes, have variants were designed by Primer3 (available in the public identified numerous mutations.25–31 However, to our knowl- domain at http://frodo.wi.mit.edu/primer3/), and the sequenc- edge no systematic analysis of these 19 genes to evaluate the es of these primers are listed in Supplementary Table S2. full spectrum of variations in patients with LCA has been Additionally, a known mutation hot spot outside the capture reported. Exon-by-exon analysis of the 19 genes by conven- range of the exome array, c.2991 þ 1665A > GinCEP290, was tional Sanger sequencing is not suitable for mutation detection analyzed by direct Sanger sequencing in all probands. in a clinical setting, as it is labor- and time-intensive.32 Polymerase chain reaction was used to amplify the genomic Therefore, fast and reliable new techniques are needed to fragments with variants, and the sequences of the amplicons detect mutations in this disease. An LCA mutation chip (Asper were determined by Sanger sequencing using a BigDye Ophthalmics; Asper Biotech Ltd., Tartu, Estonia) was devel- Terminator cycle sequencing kit v3.1 and an ABI 3130 Genetic oped that detects all known mutations identified in LCA, but Analyzer (both from Applied Biosystems, Foster City, CA). The cannot detect novel mutations.33 A SNP-chip for LCA diagnosis resultant sequences were compared to consensus sequences may be used in patients with a family history, but most LCA using Seqman software (Lasergene 8.0; DNASTAR, Inc., cases are isolated patients born to unaffected parents.34 Madison, WI). The possible impact of amino acid substitutions Recently, with the rapid development of next-generation was predicted by SIFT (available in the public domain at http:// sequencing, whole-exome sequencing has arisen as an sift.jcvi.org/) and PolyPhen-2 (available in the public domain at 42 impressive tool for mutation screening, especially for highly http://genetics.bwh.harvard.edu/pph2/). Splice site predic- heterogeneous hereditary diseases.35 In our study, whole- tion by a neural network was used to predict the effects of exome sequencing was used to detect variants in 41 unrelated variants on splicing sites (available in the public domain at Chinese patients with LCA. Variants found in the 19 LCA- http://www.fruitfly.org/seq_tools/splice.html).43 Base-by-base related genes were verified by Sanger sequencing. conservation scores ranging from 0 to 1, with higher scores indicating the higher degrees of conservation, were obtained using PhastCons (available in the public domain at http:// MATERIALS AND METHODS varianttools.sourceforge.net/Annotation/PhastCons).44 Each Subjects novel putative disease-causing variant was evaluated further in 192 normal individuals. All probands with LCA were collected from the Pediatric and Genetic Clinic in Zhongshan Ophthalmic Center. In total, 41 RESULTS probands from unrelated Chinese families were involved in this study, including 25 patients not analyzed before and 16 Whole-exome sequencing detected 41 variants that affected patients in whom some portion of the 15 LCA-related genes either encoded residues or splicing in 15 of the 19 genes. No has been screened by Sanger sequencing previously, but such variants were detected in RD3, LRAT, KCNJ13,orOTX2 without identified mutations.36 Of the 41 patients, 29 were by exome sequencing in this LCA cohort. The 41 variants were

Downloaded from jov.arvojournals.org on 10/02/2021 Mutation Analysis in Leber Congenital Amaurosis IOVS j June 2013 j Vol. 54 j No. 6 j 4353

present in 28 of the 41 probands. Of the 41 variants, 40 were confirmed by Sanger sequencing, and the remaining variant (c.293G > AinCABP4) was a false-positive with low read depth (G4A2). The pathogenicity of the 40 variants was evaluated by bioinformatics and segregation analysis. Homozygous and compound heterozygous variants were initially selected. Single heterozygous variants in CRX or IMPDH1 also were selected for further analysis. Unreported variants were evaluated in 192 normal individuals. By these criteria, 22 of the 40 variants were considered putatively pathogenic for 15 of the 41 patients (Table 1, Fig. 1, Supplementary Fig. S1). Of the 22 variants, 17 were novel and five were known. All of the novel mutations were predicted to be pathogenic in silico and were absent from 384 control chromosomes. The 22 variants found in 15 probands were present in 10 of the 19 genes: CEP290, GUCY2D, CRB1, CRX, RPGRIP1, IQCB1, RPE65, IMPDH1, LCA5,andTULP1. The less likely causal variants are summarized in Supplementary Table S3. The 15 probands with identified mutations included 12 of the 25 previously unanalyzed probands and three of 16 probands who were analyzed previously, but without identified mutations. Among the 25 probands who were not analyzed previously, analysis of the 19 known genes detected 19 mutations in 12 probands, which accounted for 48% (12/25) of the cases. The 12 probands had mutations in individual FIGURE 1. Pedigree and segregation analysis in 15 LCA families with genes as follows: CEP290 (3 probands), GUCY2D (2 pro- mutations identified in this study. þ, wild-type allele. bands), CRB1 (1 proband), CRX (1 proband), RPE65 (1 proband), IQCB1 (1 proband), LCA5 (1 proband), TULP1 (1 homozygous mutation in IQCB1 was identified in one proband proband), and IMPDH1 (1 proband). For the three of 16 by whole-exome sequencing. The intronic mutation c.2991 þ probands analyzed previously by Sanger sequencing in whom 1665A > GinCEP290 was not detected in any proband. we found mutations, two homozygous mutations in RPGRIP1 Clinical features of affected individuals with mutations were identified in two probands, respectively, and one identified in this study are shown in Table 2. These patients

TABLE 1. The 22 Potentially Pathogenic Variants Identiﬁed in 15 of 41 Chinese Families With LCA

Variations Bioinformatic Analysis Reported Gene Inheritance Family ID Nucleotide Amino AcidState SIFT P/SS Phastcons or Not

CEP290 AR Family 1 c.3361G > T p.E1121* Het – – 1.000 Novel c.2817 þ 2T > C SD Het – SSA 0.998 Novel Family 2 c.3265C > T p.Q1089* Het – – 0.999 Novel c.4090G > T p.E1364* Het – – 1.000 Novel Family 3 c.2954delT p.M985fs Het – – 1.000 Novel c.7028_7034 þ 3 dup SD Het – SSA 0.298 Novel GUCY2D AR Family 4 c.2015G > A p.C672Y Het D PrD 1.000 Novel c.2476C > T p.Q826* Het – – 0.997 Novel Family 5 c.1956 þ 1G > T SD Het – SSA 1.000 Novel c.3034A > C p.T1012P Het D PrD 1.000 Novel CRB1 AR Family 6 c.1841G > T p.G614V Hom D PrD 1.000 Novel CRX AD Family 7 c.573T > A p.Y191* Het – – 0.975 Novel RPGRIP1 AR Family 8 c.535delG p.E179fs Hom – – 0.923 Reported36 Family 9 c.2236G > A p.G746R Hom D PrD 0.978 Novel IQCB1 AR Family 10 c.1090C > T p.R364* Hom – – 0.993 Reported29 Family 11 c.994C > T p.R332* Hom – – 1.000 Novel RPE65 AR Family 12 c.200T > G p.L67R Het D PrD 1.000 Reported28 c.430T > C p.Y144H Het T PrD 1.000 Novel IMPDH1 AD Family 13 c.592G > T p.G198C Het D PrD 1.000 Novel LCA5 AR Family 14 c.795T > G p.Y265* Hom – – 0.861 Novel TULP1 AR Family 15 c.1198C > T p.R400W Het D PrD 0.998 Reported19 c.1444C > T p.R482W Het D PrD 0.994 Reported31 Of the 15 families, Family 1, Family 2, Family 3, and Family 12 had triallelic variants. The remaining variants are listed in Supplementary Table S3. Family 8, Family 9, and Family 11 were screened previously for some of the 15 LCA-related genes by Sanger sequencing, but had no identiﬁed mutations. The two mutations of RPGRIP1 in Family 8 and Family 9 most likely were omitted from the previous results, and IQCB1 had not been reported to be associated with LCA at that time, so it was not screened previously. AR, autosomal-recessive; AD, autosomal-dominant; P/SS, Polyphen-2/Splice Site Prediction; SD, splicing defect; Het, heterozygous; Hom, homozygous; D, damaging; T, tolerated; PrD, probably damaging; SSA, splicing site abolished.

Downloaded from jov.arvojournals.org on 10/02/2021 Downloaded fromjov.arvojournals.org on10/02/2021 uainAayi nLbrCneia Amaurosis Congenital Leber in Analysis Mutation TABLE 2. Clinical Features of Affected Individuals With Mutations Identiﬁed in This Study

Age, y, at ERG Visual Acuity, Fundus Patient ID Gene Mutations Sex Exam Onset Inheritance Symptoms Right; Left Change Rod Cone

Family 1-II:1 CEP290 c.[3361G > T];[2817 þ 2T > C] M 1.2 FMB Isolated PV, RN NPL* RNA NDR NDR Family 2-II:1 CEP290 c.[3265C > T];[4090G > T] M 6.8 FMB Isolated PV, NYS FC; FC CRD, PWF NDR NDR Family 3-II:1 CEP290 c.[2954delT];[7028_7034 þ 3dup] F 1.9 FMB Isolated PV, NYS NPO AV, CRD NDR NDR Family 4-II:1 GUCY2D c.[2015G > A];[2476C > T] M 0.4 0.2 Isolated PV, NYS, ODS NPO RNA NDR NDR Family 5-II:1 GUCY2D c.[1956 þ 1G > T];[3034A > C] F 0.5 FMB Isolated PV, PA, ODS NPO AV, MD NDR NDR Family 6-II:2 CRB1 c.[1841G > T];[1841G > T] M 23 FMB AR PV, NYS FC; FC AV, PNP NDR NDR Family 6-II:3 CRB1 c.[1841G > T];[1841G > T] F 20 FMB AR PV 0.1; 0.1 AV, PNP NA NA Family 7-II:1 CRX c.[573T > A];[ ¼ ] M 0.5 0.5 Isolated PV, RN NPL AV, PD NDR NDR Family 8-II:1 RPGRIP1 c.[535delG];[535delG] F 0.5 0.3 Isolated PV, ODS NPO AV, CRD NDR NDR Family 9-II:2 RPGRIP1 c.[2236G > A];[2236G > A] M 7.5 FMB AR/Cons PV, RN HM; HM AV, CRD NA NA Family 10-II:1 IQCB1 c.[1090C > T];[1090C > T] M 28 FMB Isolated PV, NYS HM; HM AV, CRD NDR NDR Family 11-II:1 IQCB1 c.[994C > T];[994C > T] F 0.3 FMB Isolated PV, RN NPL AV, CRD NDR NDR Family 12-II:1 RPE65 c.[200T > G];[430T > C] M 2.0 FMB Isolated PV, NYS PL AV, CRD NDR NDR Family 13-III:2 IMPDH1 c.[592G > T];[ ¼ ] M 23 FMB AD PV, NYS LP; LP AV, MD, PD NDR NDR Family 14-II:1 LCA5 c.[795T > G];[795T > G] M 20 FMB AR PV, NYS HM; HM AV, MD, CRD NDR NDR Family 14-II:3 LCA5 c.[795T > G];[795T > G] M 17 FMB AR PV, NYS FC; FC AV, MD, CRD NA NA Family 15-II:1 TULP1 c.[1198C > T];[1444C > T] M 11 FMB Isolated PV, NYS 0.09; 0.05 AV, CRD NDR NDR M, male; F, female; FMB, first few months after birth; Cons, consanguineous marriage of parents; PV, poor vision; RN, roving nystagmus; NYS, nystagmus; ODS, oculodigital sign; PA, photoaversion; NPL, no pursuit of light; FC, finger counting; NPO, no pursuit of objects; HM, hand movement; PL, pursuit of light; LP, light perception; RNA, relatively normal appearance; AV, attenuated vessels; CRD, carpet-like retinal degeneration; PWF, peripheral white flecks; MD, macular dystrophy; PNP, posterior nummular pigmentation; PD, pigmentary deposit; NDR, no detectable responses. * Biocular pursuit of light or objects (such as a pen or pencil) was tested in those young children who were unable to have visual acuity measured. IOVS j ue2013 June j o.54 Vol. j o 6 No. j 4354 Mutation Analysis in Leber Congenital Amaurosis IOVS j June 2013 j Vol. 54 j No. 6 j 4355

FIGURE 2. Fundus photographs of patients from 10 families with mutations identiﬁed in our study. The corresponding patient identiﬁcation numbers and gene mutations are listed above each photo. Variable fundus changes ranging from a relatively normal appearance to obvious pigmentary degeneration were demonstrated. Further clinical information of these patients is listed in Table 2.

had a variable retinal appearance, ranging from a relatively homozygous or compound heterozygous mutations in genes normal fundus to severe pigmentary degeneration (Fig. 2). associated with recessive LCA, and two had heterozygous mutations in genes associated with dominant LCA (CRX and ). DISCUSSION IMPDH1 Additionally, triallelic variations were identified in Family 1, In our study, we tested whole-exome sequencing followed by Family 2, Family 3, and Family 12. Digenic and trigenic confirmatory Sanger sequencing as a possible diagnostic variations also were identified in our study (Supplementary approach for LCA. We identified 22 potentially pathogenic Table S3). Segregation analyses were performed on Family 2 mutations in 15 of 41 unrelated Chinese families with LCA. Of and Family 12 because of the availability of samples from family the 15 families with potentially pathogenic mutations, 13 had members (Fig. 1). The four probands with triallelic variants did

Downloaded from jov.arvojournals.org on 10/02/2021 Mutation Analysis in Leber Congenital Amaurosis IOVS j June 2013 j Vol. 54 j No. 6 j 4356

not manifest a more severe phenotype than other patients, precise molecular diagnosis for highly heterogeneous heredi- which suggests that the third allele does not always aggravate tary diseases, such as LCA. As additional genes are implicated in retinal dysfunction (Table 2). On the other hand, the parents the pathogenesis of LCA and more patients become available who carried digenic variants in Family 2 and Family 12 had over time, it should be possible to explore further the normal visual acuity, without any detectable abnormalities on underlying pathogenesis of the remaining probands. fundus examination. A similar phenomenon also was described in a previous LCA case report.45 Although digenic and triallelic inheritance patterns have been established in probands with Acknowledgments RP46 and BBS,47 respectively, further studies are needed to The authors thank all patients and their family members for their determine whether digenic variations are causative and participation. whether triallelic mutations result in more severe retinal Supported by the National Natural Science Foundation of China dysfunction. Because these types of variants would be (81170881, U1201221), Guangdong Translational Medicine Public expected to be detected with increasing frequency with the Platform (4202037), the ‘‘985 project’’ of Sun Yat-sen University, rapid development of high-throughput sequencing, rigorous and the Fundamental Research Funds of the State Key Laboratory investigation should be undertaken before reaching a final of Ophthalmology. conclusion regarding pathogenicity. Disclosure: Y. Chen, None; Q. Zhang, None; T. Shen, None; X. The detection rate of 48% (12 of 25 families) in newly Xiao, None; S. Li, None; L. Guan, None; J. Zhang, None; Z. Zhu, recruited probands was higher than that in our prior study, None; Y. Yin, None; P. Wang, None; X. Guo, None; J. Wang, which had a detection rate of 35.6% when screening exons None; Q. Zhang, None reported previously to contain mutations by Sanger sequencing.36 Given that the number of newly recruited probands was limited and the other 16 families had been included in a References previous study,36 the analyses of frequency and spectrum of mutated genes were based on 112 families, combining the 1. Stone EM. Leber congenital amaurosis - a model for efficient previous 87 probands with 25 new cases. The most frequently genetic testing of heterogeneous disorders: LXIV Edward Jackson Memorial Lecture. . 2007;144:791– mutated genes in our LCA cohorts were GUCY2D (10%), CRB1 Am J Ophthalmol 811. (7%), RPGRIP1 (5%), CEP290 (4%), and CRX (3%). The five most frequently mutated genes are similar to those in other 2. Koenekoop RK. An overview of Leber congenital amaurosis: a studies conducted in Caucasian populations.4 Potential geno- model to understand human retinal development. Surv type-phenotype correlations could not be extracted for Ophthalmol. 2004;49:379–398. mutations in most of these patients because of variable 3. Schappert-Kimmijser J, Henkes HE, Van Den Bosch J. manifestation and limited number of cases, except for the Amaurosis congenita (Leber). AMA Arch Ophthalmol. 1959; association of nummular pigmentation with CRB1 mutations 61:211–218. (Table 2, Fig. 2). CRB1 mutations were identified in two 4. den Hollander AI, Roepman R, Koenekoop RK, Cremers FP. patients in Family 6 in this study and five probands out of 87 Leber congenital amaurosis: genes, proteins and disease LCA families previously,36 all were associated with similar mechanisms. Prog Retin Eye Res. 2008;27:391–419. fundus change of nummular pigmentation. 5. Franceschetti A, Dieterle P. [Diagnostic and prognostic Compared to conventional sequencing methods, whole- importance of the electroretinogram in tapetoretinal degener- exome sequencing is a rapid, reliable, and less labor-intensive ation with reduction of the visual field and hemeralopia]. way to establish a precise molecular diagnosis for genetically Confin Neurol. 1954;14:184–186. heterogeneous diseases. Most mutations identified in the 41 6. Perrault I, Rozet JM, Calvas P, et al. Retinal-specific guanylate LCA probands were novel (77.3%, 17/22); therefore, they cyclase gene mutations in Leber’s congenital amaurosis. Nat could not be detected by an LCA mutation chip.33 Considering Genet. 1996;14:461–464. the heterogeneous nature of LCA, whole-exome sequencing 7. Marlhens F,Bareil C, Griffoin JM, et al. Mutations in RPE65 cause also reveals a broader picture of the molecular background of Leber’s congenital amaurosis. Nat Genet. 1997;17:139–141. LCA and provides an opportunity to identify new genes 8. Wang H, den Hollander AI, Moayedi Y, et al. Mutations in responsible for the disease. Given that the numerous variants SPATA7 cause Leber congenital amaurosis and juvenile retinitis identified by exome sequencing may easily lead researchers pigmentosa. Am J Hum Genet. 2009;84:380–387. astray, it would be reasonable to extend this study to include 9. Sohocki MM, Bowne SJ, Sullivan LS, et al. Mutations in a new other RP-related genes because it can be difficult to distinguish photoreceptor-pineal gene on 17p cause Leber congenital LCA from early-onset severe RP by clinical examination, and amaurosis. Nat Genet. 2000;24:79–83. because some genes have been implicated in LCA and RP 10. den Hollander AI, Koenekoop RK, Mohamed MD, et al. (available in the public domain at https://sph.uth.edu/retnet/). Mutations in LCA5, encoding the ciliary protein lebercilin, Among the 26 LCA families without identified mutations, 18 cause Leber congenital amaurosis. Nat Genet. 2007;39:889–895. variants that affected either encoded residues or splicing in 11. Dryja TP, Adams SM, Grimsby JL, et al. Null RPGRIP1 alleles in known RP genes were detected (Supplementary Table S4). patients with Leber congenital amaurosis. Am J Hum Genet. However, none of these variants could be confirmed to be 2001;68:1295–1298. pathogenic mutations after bioinformatic and segregation 12. Freund CL, Wang QL, Chen S, et al. De novo mutations in the analyses. Extensive additional analysis may be required to CRX homeobox gene associated with Leber congenital identify new genes responsible for LCA in patients without amaurosis. Nat Genet. 1998;18:311–312. mutations identified in the known genes. 13. den Hollander AI, Heckenlively JR, van den Born LI, et al. In conclusion, our study suggests that it should be possible Leber congenital amaurosis and retinitis pigmentosa with to identify mutations in approximately half of Han Chinese Coats-like exudative vasculopathy are associated with muta- families with LCA by screening the 19 known LCA genes. tions in the crumbs homologue 1 (CRB1) gene. Am J Hum Together with our previous studies, these results delineate Genet. 2001;69:198–203. further the mutation spectrum and mutation frequencies of 14. den Hollander AI, Koenekoop RK, Yzer S, et al. Mutations in LCA in Han Chinese individuals. Our study also shows that the CEP290 (NPHP6) gene are a frequent cause of Leber whole-exome sequencing is an efficient method to establish a congenital amaurosis. Am J Hum Genet. 2006;79:556–561.

Downloaded from jov.arvojournals.org on 10/02/2021 Mutation Analysis in Leber Congenital Amaurosis IOVS j June 2013 j Vol. 54 j No. 6 j 4357

15. Bowne SJ, Sullivan LS, Mortimer SE, et al. Spectrum and 31. den Hollander AI, van Lith-Verhoeven JJ, Arends ML, Strom frequency of mutations in IMPDH1 associated with autosomal TM, Cremers FP, Hoyng CB. Novel compound heterozygous dominant retinitis pigmentosa and leber congenital amaurosis. TULP1 mutations in a family with severe early-onset retinitis Invest Ophthalmol Vis Sci. 2006;47:34–42. pigmentosa. Arch Ophthalmol. 2007;125:932–935. 16. Friedman JS, Chang B, Kannabiran C, et al. Premature 32. Coppieters F, De Wilde B, Lefever S, et al. Massively parallel truncation of a novel protein, RD3, exhibiting subnuclear sequencing for early molecular diagnosis in Leber congenital localization is associated with retinal degeneration. Am J Hum amaurosis. Genet Med. 2012;14:576–585. Genet. 2006;79:1059–1070. 33. Zernant J, Kulm M, Dharmaraj S, et al. Genotyping microarray 17. Perrault I, Hanein S, Gerber S, et al. Retinal dehydrogenase 12 (disease chip) for Leber congenital amaurosis: detection of (RDH12) mutations in leber congenital amaurosis. Am J Hum modifier alleles. Invest Ophthalmol Vis Sci. 2005;46:3052– Genet. 2004;75:639–646. 3059. 18. Senechal A, Humbert G, Surget MO, et al. Screening genes of 34. Pomares E, Riera M, Permanyer J, et al. Comprehensive SNP- the retinoid metabolism: novel LRAT mutation in leber chip for retinitis pigmentosa-Leber congenital amaurosis congenital amaurosis. Am J Ophthalmol. 2006;142:702–704. diagnosis: new mutations and detection of mutational founder 19. Hanein S, Perrault I, Gerber S, et al. Leber congenital effects. Eur J Hum Genet. 2010;18:118–124. amaurosis: comprehensive survey of the genetic heterogene- 35. Singleton AB. Exome sequencing: a transformative technology. ity, refinement of the clinical definition, and genotype- Lancet Neurol. 2011;10:942–946. phenotype correlations as a strategy for molecular diagnosis. 36. Li L, Xiao X, Li S, et al. Detection of variants in 15 genes in 87 Hum Mutat. 2004;23:306–317. unrelated Chinese patients with Leber congenital amaurosis. 20. Sergouniotis PI, Davidson AE, Mackay DS, et al. Recessive PLoS One. 2011;6:e19458. mutations in KCNJ13, encoding an inwardly rectifying 37. Wang Q, Wang P, Li S, et al. Mitochondrial DNA haplogroup potassium channel subunit, cause leber congenital amaurosis. distribution in Chaoshanese with and without myopia. Mol Am J Hum Genet. 2011;89:183–190. Vis. 2010;16:303–309. 21. Aldahmesh MA, Al-Owain M, Alqahtani F, Hazzaa S, Alkuraya 38. Li Y, Vinckenbosch N, Tian G, et al. Resequencing of 200 FS. A null mutation in CABP4 causes Leber’s congenital human exomes identifies an excess of low-frequency non- amaurosis-like phenotype. Mol Vis. 2010;16:207–212. synonymous coding variants. Nat Genet. 2010;42:969–972. 22. Estrada-Cuzcano A, Koenekoop RK, Coppieters F, et al. IQCB1 39. Li R, Yu C, Li Y, et al. SOAP2: an improved ultrafast tool for mutations in patients with leber congenital amaurosis. Invest short read alignment. Bioinformatics. 2009;25:1966–1967. Ophthalmol Vis Sci. 2011;52:834–839. 40. Li R, Li Y, Kristiansen K, Wang J. SOAP: short oligonucleotide 23. Henderson RH, Williamson KA, Kennedy JS, et al. A rare de alignment program. Bioinformatics. 2008;24:713–714. novo nonsense mutation in OTX2 causes early onset retinal 41. Li R, Li Y, Fang X, Yang H, Wang J, Kristiansen K. SNP dystrophy and pituitary dysfunction. Mol Vis. 2009;15:2442– detection for massively parallel whole-genome resequencing. 2447. Genome Res. 2009;19:1124–1132. 24. Falk MJ, Zhang Q, Nakamaru-Ogiso E, et al. NMNAT1 42. Flanagan SE, Patch AM, Ellard S. Using SIFT and PolyPhen to mutations cause Leber congenital amaurosis. Nat Genet. predict loss-of-function and gain-of-function mutations. Genet 2012;44:1040–1045. Test Mol Biomarkers. 2010;14:533–537. 25. Li Y, Wang H, Peng J, et al. Mutation survey of known LCA 43. Liu L, Ho YK, Yau S. Prediction of primate splice site using genes and loci in the Saudi Arabian population. Invest inhomogeneous Markov chain and neural network. DNA Cell Ophthalmol Vis Sci. 2009;50:1336–1343. Biol. 2007;26:477–483. 26. Sohocki MM, Daiger SP, Bowne SJ, et al. Prevalence of 44. Siepel A, Haussler D. Phylogenetic estimation of context- mutations causing retinitis pigmentosa and other inherited dependent substitution rates by maximum likelihood. Mol retinopathies. Hum Mutat. 2001;17:42–51. Biol Evol. 2004;21:468–488. 27. Lotery AJ, Jacobson SG, Fishman GA, et al. Mutations in the 45. Li L, Xiao X, Li S, Jiao X, Hejtmancik JF, Zhang Q. Lack of CRB1 gene cause Leber congenital amaurosis. Arch Ophthal- phenotypic effect of triallelic variation in SPATA7 in a family mol. 2001;119:415–420. with Leber congenital amaurosis resulting from CRB1 muta- 28. Xu F, Dong Q, Liu L, et al. Novel RPE65 mutations associated tions. Mol Vis. 2011;17:3326–3332. with Leber congenital amaurosis in Chinese patients. Mol Vis. 46. Kajiwara K, Berson EL, Dryja TP. Digenic retinitis pigmentosa 2012;18:744–750. due to mutations at the unlinked peripherin/RDS and ROM1 29. Khanna H, Davis EE, Murga-Zamalloa CA, et al. A common loci. Science. 1994;264:1604–1608. allele in RPGRIP1L is a modifier of retinal degeneration in 47. Badano JL, Kim JC, Hoskins BE, et al. Heterozygous mutations ciliopathies. Nat Genet. 2009;41:739–745. in BBS1, BBS2 and BBS6 have a potential epistatic effect on 30. Ahmed E, Loewenstein J. Leber congenital amaurosis: disease, Bardet-Biedl patients with two mutations at a second BBS genetics and therapy. Semin Ophthalmol. 2008;23:39–43. locus. Hum Mol Genet. 2003;12:1651–1659.

Downloaded from jov.arvojournals.org on 10/02/2021