Applying next generation sequencing with microdroplet PCR to determine the disease-causing mutations in retinal dystrophies

Wang et al. BMC Ophthalmology (2017) 17:157 DOI 10.1186/s12886-017-0549-5 RESEARCH ARTICLE Open Access Applying next generation sequencing with microdroplet PCR to determine the diseasecausing mutations in retinal dystrophies Xinjing Wang1*, Wadih M. Zein1, Leera D’Souza1, Chimere Roberson1, Keith Wetherby1, Hong He1, Angela Villarta1, Amy Turriff1, Kory R. Johnson2 and Yang C. Fann2 Abstract Background: Inherited Retinal dystrophy (IRD) is a broad group of inherited retinal disorders with heterogeneous genotypes and phenotypes. Next generation sequencing (NGS) methods have been broadly applied for analyzing patients with IRD. Here we report a novel approach to enrich the target gene panel by microdroplet PCR. Methods: This assay involved a primer library which targeted 3071 amplicons from 2078 exons comprised of 184 genes involved in retinal function and/or retinal development. We amplified the target regions using the RainDance target enrichment PCR method and sequenced the products using the MiSeq NGS platform. Results: In this study, we analyzed 82 samples from 67 families with IRD. Bioinformatics analysis indicated that this procedure was able to reach 99% coverage of target sequences with an average sequence depth of reads at 119×. The variants detected by this study were filtered, validated, and prioritized by pathogenicity analysis. Genotypes and phenotypes were correlated by determining a consistent relationship in 38 propands (56.7%). Pathogenic variants in genes related to retinal function were found in another 11 probands (16.4%), but the clinical correlations showed inconsistencies and insufficiencies in these patients. Conclusions: The application of NGS in IRD clinical molecular diagnosis provides a powerful approach to exploring the etiology and pathology in patients. It is important for the clinical laboratory to interpret the molecular findings in the context of patient clinical presentations because accurate interpretation of pathogenic variants is critical for delivering solid clinical molecular diagnosis to clinicians and patients and improving the standard care of patients. Keywords: Retinal, Microdroplet PCR, Next-generation-sequencing, Mutation screening Background Inherited Retinal dystrophy (IRD) is a clinically and genetically heterogeneous group of disorders that affects approximately 1 in 2000 individuals worldwide [1]. Clinically IRD can be classified according to the clinical phenotype and course of the disease, as well as the underlying inheritance. However, sporadic retinal dystrophies, such as Retinitis Pigmentosa (RP), Leber Congenital Amaurosis (LCA), Cone and Cone-Rod dystrophy (CRD), Macular Dystrophy (MD), and Congenital Stationary Night * Correspondence: 1 Ophthalmic Genetics and Visual Function Branch, National Eye Institute, National Institutes of Health, 10D43, 10 Center Drive, Bethesda, MD 20892, USA Full list of author information is available at the end of the article Blindness (CSNB) can present difficulties in the clinical differential diagnosis. The heterogeneity of some syndromic conditions with retinal abnormalities (i.e., Usher syndrome, Bardet-Biedl syndrome, and Nephronophthisis) could add more complexity because these diseases are caused by multiple underlying gene defects [2, 3]. In molecular pathology, IRD may result from mutations in a variety of genes and may show different inheritance patterns, including autosomal dominant, autosomal recessive, Xlinked, and mitochondrial inheritance [3, 4]. Additionally, mutations within the same gene may be associated with different phenotypes [2, 5]. Although monogenic forms have been reported in most families, some digenic forms have also been identified [2, 3]. © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Wang et al. BMC Ophthalmology (2017) 17:157 Retinitis pigmentosa is the most frequent type of IRD and it is also highly genetic heterogeneous. More than 50 genes have been identified responsible for RP with similar clinical presentations. Typically, it presents night blindness and peripheral visual field decreasing. As part of progression in RP, nigh blindness will eventually become blindness. On the other hand, the night blindness is also a typical presentation of CSDB. While CSDB can be distinguished by electroretinogram (ERG), more than 10 genes have been identified responsible for CSDB and a portion of CSDB patients were found no mutation in the known CSDB genes. LCA, CRD, MD, and Occult Macular Dystrophy (OCMD) can be differentiated clinically in general, but the molecular pathology of malfunction in cone photoreceptors cannot be established without identifying the disease-causing gene in each patient. To date, more than 200 genes have been identified for different retinal diseases by a variety of methods (http:// www.sph.uth.tmc.edu/retnet/sum-dis.htm). Significant progress has been made in determining the molecular causes of IRD, but much more work remains to be done. Genetic testing for IRD can identify causative mutations but requires sequencing of many individual candidate genes. Next generation sequencing (NGS) has been broadly applied for analyzing patients with IRD. NGS enables rapid and cost-effective parallel sequencing of a large panel of disease genes. It offers an ideal model in a clinical diagnostic setting. Many studies investigating sequencecapture technology with selected gene targets for enrichment have been published [6–10]. A significantly higher rate of molecular diagnosis (as compared to Sanger sequencing potential candidate genes) of well above 50% was also achieved. Furthermore, NGS can be adapted to include other retinal diseases without significantly increasing the cost. In this study, we performed a comprehensive molecular screening of patients by NGS using Illumina MiSeq platform and target enrichment using a customized primer library based microdroplet PCR. We accomplished targeted gene panel sequencing of 184 genes in 74 patients with different inheritance patterns and clinical diagnoses including Choroidermia (CHM), CRD, CSNB, LCA, MD, OCMD, and RP. Careful clinical evaluation and follow-up led to the more precise clinical diagnoses and extensive phenotyping in these RD families. Collectively, this study underscores the importance of combining comprehensive molecular screening and clinical information to accurately diagnose diverse retinal disorders. Methods Subjects and (...truncated)