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Open Access

Peer-reviewed

Research Article

Identification of Genetic Defects in 33 Probands with Stargardt Disease by WES-Based Bioinformatics Gene Panel Analysis

  • Wei Xin,

    Affiliation State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China

    ⨯
  • Xueshan Xiao,

    Affiliation State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China

    ⨯
  • Shiqiang Li,

    Affiliation State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China

    ⨯
  • Xiaoyun Jia,

    Affiliation State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China

    ⨯
  • Xiangming Guo,

    Affiliation State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China

    ⨯
  • Qingjiong Zhang

    zhangqji@mail.sysu.edu.cn

    Affiliation State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China

    ⨯

Abstract

Stargardt disease (STGD) is the most common hereditary macular degeneration in juveniles, with loss of central vision occurring in the first or second decade of life. The aim of this study is to identify the genetic defects in 33 probands with Stargardt disease. Clinical data and genomic DNA were collected from 33 probands from unrelated families with STGD. Variants in coding genes were initially screened by whole exome sequencing. Candidate variants were selected from all known genes associated with hereditary retinal dystrophy and then confirmed by Sanger sequencing. Putative pathogenic variants were further validated in available family members and controls. Potential pathogenic mutations were identified in 19 of the 33 probands (57.6%). These mutations were all present in ABCA4, but not in the other four STGD-associated genes or in genes responsible for other retinal dystrophies. Of the 19 probands, ABCA4 mutations were homozygous in one proband and compound heterozygous in 18 probands, involving 28 variants (13 novel and 15 known). Analysis of normal controls and available family members in 12 of the 19 families further support the pathogenicity of these variants. Clinical manifestation of all probands met the diagnostic criteria of STGD. This study provides an overview of a genetic basis for STGD in Chinese patients. Mutations in ABCA4 are the most common cause of STGD in this cohort. Genetic defects in approximately 42.4% of STGD patients await identification in future studies.

Citation: Xin W, Xiao X, Li S, Jia X, Guo X, Zhang Q (2015) Identification of Genetic Defects in 33 Probands with Stargardt Disease by WES-Based Bioinformatics Gene Panel Analysis. PLoS ONE 10(7): e0132635. https://doi.org/10.1371/journal.pone.0132635

Editor: Michael G. Anderson, University of Iowa, UNITED STATES

Received: December 31, 2014; Accepted: June 16, 2015; Published: July 10, 2015

Copyright: © 2015 Xin et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Data Availability: All relevant data is available via Dryad (http://dx.doi.org/10.5061/dryad.0qk7f).

Funding: This study was supported by the National Natural Science Foundation of China (U1201221), Natural Science Foundation of Guangdong (S2013030012978), Guangdong Department of Science and Technology Translational Medicine Center (2011A080300002), and the Fundamental Research Funds of State Key Laboratory of Ophthalmology. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Stargardt Disease (STGD), with a world-wide prevalence of at least 1:10,000 [1], is the most common hereditary macular degeneration in juveniles. It is characterized by macular dystrophy associated with loss of central visual in the first or second decade of life, a “beaten-metal” appearance in the fovea or parafoveal region, yellowish flecks around the macula or in posterior area of the retina, progressive atrophy of the bilateral foveal retinal pigment epithelium, and the “dark choroid” sign on Fundus Fluorescein Angiography (FFA) in 80% of patients [2,3]. STGD is frequently transmitted as an autosomal recessive trait but rarely as an autosomal dominant trait [4,5].

To date, mutations in five genes have been reported to be responsible for classic STGD or “Stargardt-like” disease (S1 Table): fatty acid elongase 4 (ELOVL4, MIM: 605512) [6–8], prominin 1 (PROM1, MIM: 604365) [5,9], peripherin 2 (PRPH2, MIM: 179605) [10,11], Bestrophin 1 (BEST1, MIM: 607854) [12] and ATP-binding cassette, sub-family A, member 4 (ABCA4, MIM: 601691) [13]. Mutations in these genes are responsible for over half of STGD cases, based on analysis of individual gene or a subset of genes [14,15]. Recently, targeted exome sequencing on 144 or 213 genes detected mutations in 100% (6/6) or 68% (59/88) of families with STGD [15,16], where 13% (11/88) of the families with atypical STGD had mutations in genes other than STGD-associated genes [15]. Additional comprehensive studies are expected to enrich the understanding of the genetic defects responsible for STGD.

In the present study, whole exome sequencing was performed on 33 probands with STGD, and potential pathogenic mutations were detected in 19 of these 33 patients.

Materials and Methods

Patients

All 33 probands from unrelated families and available family members were collected from Zhongshan Ophthalmic Center, Sun Yat-sen University. Written informed consent was obtained from participants or their guardians before the collection of their peripheral venous blood and clinical data. This study was approved by the Institutional Review Board of the Zhongshan Ophthalmic Center and was adherent to Declaration of Helsinki. Genomic DNA was extracted from the leukocytes of peripheral venous blood of each participating individual, as well as 96 unrelated healthy individuals [17] and 228 unrelated controls.

Whole exome sequencing

Genomic DNA from the 33 probands was initially analyzed by whole exome sequencing, as described previously [18]. Briefly, genomic DNA was fragmented and the fragments containing coding regions was captured by an Agilent SureSelect Human All Exon Enrichment Kit V4 array. The exome-enriched DNA fragments were sequenced with an Illumina HiSeq2000 at an average sequencing depth of 125-fold. All sequencing reads were aligned to the consensus sequence (UCSC hg19) with Burrows-Wheeler Aligner (BWA) (http://bio-bwa.sourceforge.net/) and variants were detected with SAMtools (http://samtools.sourceforge.net/). In the current study, variants for all genes related to retinal dystrophy based on RetNet (https://sph.uth.edu/RetNet/home.htm, accessed on August 14, 2014) were selected and then analyzed as follows: 1) exclusion of variants in non-coding regions or synonymous variants without affecting splicing site predicted by BDGP (http://www.fruitfly.org/seq_tools/splice.html); 2) exclusion of variants with minor allele frequency >0.01 determined with 1000 Genomes (http://browser.1000genomes.org/index.html), the Exome Variant Server (http://evs.gs.washington.edu/EVS/), ExAC (http://exac.broadinstitute.org/) and in-house controls; 3) exclusion of single heterozygous variants in genes responsible for recessive diseases; 4) exclusion of variants predicted to be benign by all three online tools (SIFT: http://sift.jcvi.org/www/SIFT_enst_submit.html; PolyPhen-2: http://genetics.bwh.harvard.edu/pph2/; and Proven: http://provean.jcvi.org/genome_submit_2.php?species=human). After the above four steps, the remaining variants were then confirmed by Sanger sequencing and were further evaluated in available family members as well as in controls.

Sanger Sequencing

For each candidate variant, primers were designed with the online tool Primer3 (http://primer3.ut.ee/) to amplify the genomic fragment harboring the variant (S2 Table). The amplicons were analyzed on an ABI 3130 genetic analyzer (Applied Biosystems, Foster City, CA, USA) with a BigDye Terminator cycle sequencing kit version 3.1. The sequencing results were aligned with consensus sequences from the Genome Bioinformatics database (http://genome.ucsc.edu/) to identify variants by SeqmanII program of DNASTAR Lasergene package (Lasergene version 7.1; DNASTAR, Madison, WI, USA).

Results

In total, 800 variants were initially detected in 163 genes by whole exome sequencing initially. After multi-bioinformatics filtering and confirmation, 28 potential pathogenic mutations were identified in 19 of the 33 probands (57.6%) (Table 1, S1 Fig). These mutations were all present in ABCA4, involving 13 novel and 15 known mutations. ABCA4 mutations were identified in 19 probands, including one proband with a homozygous mutation and another 18 probands with compound heterozygous mutations. Analysis of controls and available family members in 12 of the 19 families further support the pathogenicity of these variants (Fig 1). No potential mutations were detected in the other four STGD-associated genes (ELOVL4, PROM1, PRPH2, and BEST1) or in the 209 genes responsible for other forms of retinal dystrophy. Potential ABCA4 variants were identified in 8 probands (S3 Table), including 4 probands with two or more heterozygous variants but only one of them was predicted to be pathogenic, and 4 probands with only one heterozygous variant predicted to be pathogenic.

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Table 1. The ABCA4 causative variants in 19 Chinese probands with Stargardt disease.

https://doi.org/10.1371/journal.pone.0132635.t001

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Fig 1. The ABCA4 mutation and the pedigree.

Under each individual, + indicates a wild type allele, and M indicates a mutant allele.

https://doi.org/10.1371/journal.pone.0132635.g001

Clinical data of the 19 probands with ABCA4 mutations were listed in Table 2. Most of the probands had complaint of poor vision in their first decades. Ophthalmoscope examination of the fundus revealed one or more of typical clinic features, including a “beaten-metal” appearance in the foveal or parafoveal macula, yellowish flecks around the macula or in posterior retina, retinal pigment epithelium (RPE) atrophy and pigment disorder to different degrees, and a “dark” or “silent” choroid on Fundus Fluorescein Angiography (FFA) (Fig 2).

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Table 2. Clinical features of Stargardt disease probands with the ABCA4 mutations identified in this study.

https://doi.org/10.1371/journal.pone.0132635.t002

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Fig 2. Fundus photographs and Fundus Fluorescein Angiography from QT058 and QT1137 respectively.

The ABCA4 mutations were listed above each photograph identified in this study. OD and OS represent right and left eyes, respectively. Fundus photographs showed yellowish flecks in all posterior of the retina and macular atrophy. FFA showed “dark choroid” sign. More clinical information about these patients is listed in Table 2.

https://doi.org/10.1371/journal.pone.0132635.g002

Discussion

In this study, we identified 28 mutations of ABCA4 in 19 of 33 (57.6%) unrelated probands with STGD. No potential pathogenic variations were detected in other four STGD-related genes and 209 genes responsible for other forms of retinal dystrophies listed in RetNet (S3 Table).

Previously, different sequencing technologies have been used to detect genetic defects of STGD patients in different populations [14–16,19–26]. Mutations in ABCA4 are the most common cause of STGD in previous studies [15,19,21,25]. Similarly, mutations ABCA4 have been detected in 57.6% STGD probands in our Chinese cohort. Several specific alleles have been reported in different populations [19–21], but no specific alleles were found in Chinese population in the present study due to the limited number of patients. The 15 known ABCA4 mutations were also found in the present study. Different patients with known mutations have different phenotypes, previously reports revealed that c.4773+1G>T and c.5196+1G>A associated with AMD (Age-related Macular Degeneration) and c.164A>G (p.H55R) associated with for cone-rod dystrophy [27–29], while homozygous c.4773+1G>T and compound heterozygous c.5196+1G>A and c.164A>G were associate with STGD in our study. Such varied phenotypes may be determined by the second mutation [30]. Three novel ABCA4 mutations were identified in three small families (QT085, QT302, and QT358) where family members was not available for analysis. These mutations occur in different protein domains, including K1978Qfs*13 and L2058R in cytosolic nucleotide binding domain 1 and T1519Rfs*7 in exocytoplasmic domains 2.

Except for ABCA4 mutations in 19 of 33 probands, potential mutations have not been detected in the other 14 probands after analysis of other 213 genes in which mutations were responsible for different forms of retinal dystrophy based on comprehensive whole exome analysis. No mutations identified in other genes except for ABCA4 may partly because of the limited number of probands analyzed. A previous study [15] also reported no pathogenic mutations in about 30% of patients with STGD. This suggests that as yet unidentified genes may also contribute to STGD. Therefore, further bioinformatics analysis on data resulted from whole exome sequencing may lead to find new genes responsible for STGD in the remaining probands without mutations. In addition, mutations in the noncoding regions may be missed by whole exome sequencing. Additional analysis using different strategies are expected to find such mutations, especially for those probands with single pathogenic variants in recessive genes.

Supporting Information

S1 Fig. Sequence chromatography.

38 sequence changes that were detected in 19 probands with Stargardt disease are shown (left column) and compared with corresponding normal sequences (right column).

https://doi.org/10.1371/journal.pone.0132635.s001

(PDF)

S1 Table. Genomic information of the five known genes responsible for STGD.

https://doi.org/10.1371/journal.pone.0132635.s002

(XLSX)

S2 Table. Primers used in Sanger sequencing.

https://doi.org/10.1371/journal.pone.0132635.s003

(XLSX)

S3 Table. Potential pathogenic ABCA4 variants in other 8 probands with Stargardt disease.

https://doi.org/10.1371/journal.pone.0132635.s004

(XLSX)

Acknowledgments

The authors are grateful to the patients and their family members for their participation in this study.

Author Contributions

Conceived and designed the experiments: QZ. Performed the experiments: WX XX SL XJ. Analyzed the data: WX QZ. Contributed reagents/materials/analysis tools: XX SL XJ XG QZ. Wrote the paper: WX XX SL XJ XG QZ.

References

  1. 1. Walia S, Fishman GA. Natural History of Phenotypic Changes in Stargardt Macular Dystrophy. Ophthalmic Genet. 2009;30:63–68. pmid:19373676
  2. 2. Burke TR, Tsang SH. Allelic and Phenotypic Heterogeneity in ABCA4 Mutations. Ophthalmic Genet. 2011;32:165–174. pmid:21510770
  3. 3. Fishman GA, Farber M, Patel BS, Derlacki DJ. Visual acuity loss in patients with Stargardt's macular dystrophy. Ophthalmology. 1987;94:809–814. pmid:3658351
  4. 4. Yi J, Li S, Jia X, Xiao X, Wang P, Guo X, et al. Evaluation of the ELOVL4, PRPH2 and ABCA4 genes in patients with Stargardt macular degeneration. Mol Med Rep. 2012;6:1045–1049. pmid:22948568
  5. 5. Yang Z, Chen Y, Lillo C, Chien J, Yu Z, Michaelides M, et al. Mutant prominin 1 found in patients with macular degeneration disrupts photoreceptor disk morphogenesis in mice. J Clin Invest. 2008;118:2908–2916. pmid:18654668
  6. 6. Logan S, Anderson RE. Dominant Stargardt Macular Dystrophy (STGD3) and ELOVL4. Adv Exp Med Biol. 2014;801:447–453. pmid:24664730
  7. 7. Vasireddy V, Wong P, Ayyagari R. Genetics and molecular pathology of Stargardt-like macular degeneration. Prog Retin Eye Res. 2010;29:191–207. pmid:20096366
  8. 8. Zhang K, Kniazeva M, Han M, Li W, Yu Z, Yang Z, et al. A 5-bp deletion in ELOVL4 is associated with two related forms of autosomal dominant macular dystrophy. Nature genetics. 2001;27:89–93. pmid:11138005
  9. 9. Kniazeva M, Chiang MF, Morgan B, Anduze AL, Zack DJ, Han M, et al. A new locus for autosomal dominant stargardt-like disease maps to chromosome 4. Am J Hum Genet. 1999;64:1394–1399. pmid:10205271
  10. 10. Poloschek CM, Bach M, Lagreze WA, Glaus E, Lemke JR, Berger W, et al. ABCA4 and ROM1: implications for modification of the PRPH2-associated macular dystrophy phenotype. Invest Ophthalmol Vis Sci. 2010;51:4253–4265. pmid:20335603
  11. 11. Coco RM, Telleria JJ, Sanabria MR, Rodriguez-Rua E, Garcia MT. PRPH2 (Peripherin/RDS) mutations associated with different macular dystrophies in a Spanish population: a new mutation. Eur J Ophthalmol. 2010;20:724–732. pmid:20213611
  12. 12. Petrukhin K, Koisti MJ, Bakall B, Li W, Xie G, Marknell T, et al. Identification of the gene responsible for Best macular dystrophy. Nat Genet. 1998;19:241–247. pmid:9662395
  13. 13. Koenekoop RK. The gene for Stargardt disease, ABCA4, is a major retinal gene: a mini-review. Ophthalmic Genet. 2003;24:75–80. pmid:12789571
  14. 14. Strom SP, Gao YQ, Martinez A, Ortube C, Chen Z, Nelson SF, et al. Molecular diagnosis of putative Stargardt Disease probands by exome sequencing. BMC Med Genet. 2012;13:67. pmid:22863181
  15. 15. Zaneveld J, Siddiqui S, Li H, Wang X, Wang H, Wang K, et al. Comprehensive analysis of patients with Stargardt macular dystrophy reveals new genotype-phenotype correlations and unexpected diagnostic revisions. Genet Med. 2014; 17:262–270. pmid:25474345
  16. 16. Zhang X, Ge X, Shi W, Huang P, Min Q, Li M, et al. Molecular diagnosis of putative Stargardt disease by capture next generation sequencing. PLoS one. 2014;9:e95528. pmid:24763286
  17. 17. Wang Q, Wang P, Li S, Xiao X, Jia X, Guo X, et al. Mitochondrial DNA haplogroup distribution in Chaoshanese with and without myopia. Mol Vis. 2010;16:303–309. pmid:20208987
  18. 18. Jiang D, Li J, Xiao X, Li S, Jia X, Sun W, et al. Detection of Mutations in LRPAP1, CTSH, LEPREL1, ZNF644, SLC39A5, and SCO2 in 298 Families With Early-Onset High Myopia by Exome Sequencing. Invest Ophthalmol Vis Sci. 2015;56:339–345. pmid:25525168
  19. 19. Fujinami K, Zernant J, Chana RK, Wright GA, Tsunoda K, Ozawa Y, et al. ABCA4 gene screening by next-generation sequencing in a British cohort. Invest Ophthalmol Vis Sci. 2013;54:6662–6674. pmid:23982839
  20. 20. Chacon-Camacho OF, Granillo-Alvarez M, Ayala-Ramirez R, Zenteno JC. ABCA4 mutational spectrum in Mexican patients with Stargardt disease: Identification of 12 novel mutations and evidence of a founder effect for the common p.A1773V mutation. Exp Eye Res. 2013;109:77–82. pmid:23419329
  21. 21. Maia-Lopes S, Aguirre-Lamban J, Castelo-Branco M, Riveiro-Alvarez R, Ayuso C, Silva ED. ABCA4 mutations in Portuguese Stargardt patients: identification of new mutations and their phenotypic analysis. Mol Vis. 2009;15:584–591. pmid:19365591
  22. 22. Oldani M, Marchi S, Giani A, Cecchin S, Rigoni E, Persi A, et al. Clinical and molecular genetic study of 12 Italian families with autosomal recessive Stargardt disease. Genet Mol Res. 2012;11:4342–4350. pmid:23096905
  23. 23. Hargitai J, Zernant J, Somfai GM, Vamos R, Farkas A, Salacz G, et al. Correlation of clinical and genetic findings in Hungarian patients with Stargardt disease. Invest Ophthalmol Vis Sci. 2005;46:4402–4408. pmid:16303926
  24. 24. Utz VM, Chappelow AV, Marino MJ, Beight CD, Sturgill-Short GM, Pauer GJ, et al. Identification of three ABCA4 sequence variations exclusive to African American patients in a cohort of patients with Stargardt disease. Am J Ophthalmol. 2013;156:1220–1227, e2. pmid:24011517
  25. 25. Valverde D, Riveiro-Alvarez R, Bernal S, Jaakson K, Baiget M, Navarro R, et al. Microarray-based mutation analysis of the ABCA4 gene in Spanish patients with Stargardt disease: evidence of a prevalent mutated allele. Mol Vis. 2006;12:902–908. pmid:16917483
  26. 26. September AV, Vorster AA, Ramesar RS, Greenberg LJ. Mutation spectrum and founder chromosomes for the ABCA4 gene in South African patients with Stargardt disease. Invest Ophthalmol Vis Sci. 2004;45:1705–1711. pmid:15161829
  27. 27. Thiadens AA, Phan TM, Zekveld-Vroon RC, Leroy BP, van den Born LI, Hoyng CB, et al. Clinical course, genetic etiology, and visual outcome in cone and cone-rod dystrophy. Ophthalmology. 2012;119:819–826. pmid:22264887
  28. 28. Pang CP, Lam DS. Differential occurrence of mutations causative of eye diseases in the Chinese population. Hum Mutat. 2002;19:189–208. pmid:11857735
  29. 29. Allikmets R, Shroyer NF, Singh N, Seddon JM, Lewis RA, Bernstein PS, et al. Mutation of the Stargardt disease gene (ABCR) in age-related macular degeneration. Science. 1997;277:1805–1807. pmid:9295268
  30. 30. Riveiro-Alvarez R, Lopez-Martinez MA, Zernant J, Aguirre-Lamban J, Cantalapiedra D, Avila-Fernandez A, et al. Outcome of ABCA4 disease-associated alleles in autosomal recessive retinal dystrophies: retrospective analysis in 420 Spanish families. Ophthalmology. 2013;120:2332–2337. pmid:23755871