Molecular Diagnosis of Putative Stargardt Disease by Capture Next Generation Sequencing

et al. (2014) Molecular Diagnosis of Putative Stargardt Disease by Capture Next Generation Sequencing. PLoS ONE 9(4): e95528. doi:10.1371/journal.pone.0095528 Molecular Diagnosis of Putative Stargardt Disease by Capture Next Generation Sequencing Xiao Zhang 0 Xianglian Ge 0 Wei Shi 0 Ping Huang 0 Qingjie Min 0 Minghan Li 0 Xinping Yu 0 Yaming Wu 0 Guangyu Zhao 0 Yi Tong 0 Zi-Bing Jin 0 Jia Qu 0 Feng Gu 0 Yuk Fai Leung, Purdue University, United States of America 0 1 School of Ophthalmology and Optometry, Eye Hospital, Wenzhou Medical University, State Key Laboratory Cultivation Base and Key Laboratory of Vision Science, Ministry of Health and Zhejiang Provincial Key Laboratory of Ophthalmology and Optometry , Wenzhou, Zhejiang , China , 2 Department of Ophthalmology, Beijing Children's Hospital, Capital Medical University , Beijing , China , 3 Department of Development and Planning, Wenzhou Medical University , Wenzhou, Zhejiang , China , 4 Institute of Genomic Medicine, Wenzhou Medical University , Wenzhou, Zhejiang , China , 5 Department of Ophthalmology, The First Affiliated Hospital of Wenzhou Medical University , Wenzhou, Zhejiang , China , 6 Fuzhou Southeastern Eye Hospital , Fuzhou , China Stargardt Disease (STGD) is the commonest genetic form of juvenile or early adult onset macular degeneration, which is a genetically heterogeneous disease. Molecular diagnosis of STGD remains a challenge in a significant proportion of cases. To address this, seven patients from five putative STGD families were recruited. We performed capture next generation sequencing (CNGS) of the probands and searched for potentially disease-causing genetic variants in previously identified retinal or macular dystrophy genes. Seven disease-causing mutations in ABCA4 and two in PROM1 were identified by CNGS, which provides a confident genetic diagnosis in these five families. We also provided a genetic basis to explain the differences among putative STGD due to various mutations in different genes. Meanwhile, we show for the first time that compound heterozygous mutations in PROM1 gene could cause cone-rod dystrophy. Our findings support the enormous potential of CNGS in putative STGD molecular diagnosis. - Funding: This work was supported by grants from the Chinese National Program on Key Basic Research Project (973 Program, 2013CB967502, F.G.), the Natural Science Foundation of China (81201181/H1818, F.G.), Zhejiang provincial & Ministry of Health research fund for medical sciences (WKJ2013-2-023, F.G.), Wenzhou Medical College PI starting grant (QTJ12011, F.G.). 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. . These authors contributed equally to this work. Stargardt disease (STGD) is the most frequent cause of macular degeneration in childhood, with a prevalence of approximately 1:10000 [1]. It is usually diagnosed within the first two decades of life and leads to progressive irreversible loss of central vision, delayed dark adaptation and a poor final visual outcome. STGD is predominantly inherited as an autosomal recessive trait with mutations in ABCA4, also known as ABCR, although an autosomal dominant form has been also reported [2]. Rare cases of STGD or Stargardt-like disease phenotypes have been reported with mutations in PROM1, PRPH2, VMD2 (also known as BEST1) and ELOVL4, which are involved in various physiological pathways that are important for macular function [3]. This complex arena of genes and clinical features complicates the nomenclature in this field [3]; it is unclear how to classify individuals with classic Stargardt phenotype. Classic STGD should be restricted to only those cases caused by ABCA4 mutations and Stargardt-like or juvenile macular dystrophy should be used for other genetic etiologies. For the purposes of this study, we classify our participants with early-onset macular degeneration as putative STGD cases. Stem cell-based therapy shows great promise for the treatment of STGD [4] Accurate molecular diagnosis is therefore essential for the selection of patients for clinical trials, and is also crucial for prenatal STGD diagnosis. However, the genetic diagnosis of individuals with putative STGD is an ongoing challenge because of the relatively large sizes of some of the genes involved. ABCA4 and PROM1 are particularly large containing 50 and 26 exons, respectively. VMD2, ELOVL4 and PRPH2 have 8, 6, and 2 exons, respectively. Furthermore, although biallelic mutations in ABCA4 are found in most patients with autosomal recessive STGD, there are studies which have shown that mutations in the ABCA4 gene are responsible for a wide variety of other retinal dystrophy phenotypes, such as cone-rod dystrophy (CRD), and retinitis pigmentosa (RP) [5,6]. It has also been proposed that individuals carrying mutations in ABCA4 may have a higher risk of developing age-related macular degeneration (AMD) [1,7]. We therefore sought to investigate whether other retinal disease genes besides these reported five genes could lead to putative STGD. In this study we initially selected known retinal disease genes as a gene capture panel and applied a capture next generation sequencing (CNGS) approach to identify genetic defects in seven putative STGD patients from five independent families. This approach was used to test whether additional retinal disease genes could lead to putative STGD. Materials and Methods Patient Recruitment This study conformed to the tenets of the Declaration of Helsinki and was approved by the Ethics Committee of Eye Hospital, Wenzhou Medical University. Written informed consent was obtained from all participating individuals or their guardians. Patients from families (A, B, C, D and E, Figure 1) were recruited. Ophthalmic examination was performed for each patient. Electroretinography (ERG) and optical coherence tomography (OCT) were performed as routine retinal ophthalmic examination. A five ml venous blood sample was drawn into an ethylenediamine tetraacetic acid (EDTA) sample tube from every subject. Genomic DNA was extracted from peripheral blood leukocytes using the standard phenol/chloroform extraction protocols. Targeted Exome Illumina Library Preparation Genomic DNA was purified and quantified with Nanodrop 2000 (Thermo Fisher Scientific, DE). The generation of a targeted exome Illumina Library was performed according to the manufacturers protocol (MyGenostics, Beijing, China). A final library size of 350450 bp including adapter sequences was selected. Disease Genes Enrichment, Sequencing Data Generation and Bioinformatics Analysis A total of 144 disease genes associated with retinal diseases including the five known STGD genes (ABCA4, PROM1, PRPH2, VMD2 and ELOVL4) were selected by a gene capture strategy, using the GenCap custom enrichment kit (MyGenostics), as previously de (...truncated)