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

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.


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].
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.

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.
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).

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][15][16][19][20][21][22][23][24][25][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][20][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][28][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 Ã 13 and L2058R in cytosolic nucleotide binding domain 1 and T1519Rfs Ã 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.