Mutational Screening of LCA Genes Emphasizing RPE65 in South Indian Cohort of Patients Anshuman Verma 1 , Vijayalakshmi Perumalsamy 2 , Shashikant Shetty 2 , Maigi Kulm 3 , Periasamy Sundaresan 1 * 1 Department of Genetics, Dr. G. Venkataswamy Eye Research Institute, Aravind Medical Research Foundation, Aravind Eye Hospital, Madurai, Tamil Nadu, India, 2 Pediatric Eye Clinic, Aravind Eye Hospital, Madurai, Tamil Nadu, India, 3 Asper Biotech, Tartu, Estonia Abstract Background: Leber congenital amaurosis (LCA) is the most severe form of inherited retinal visual impairment in children. So far, mutations in more than 20 genes have been known to cause LCA and among them, RPE65 is a suitable candidate for gene therapy. The mutational screenings of RPE65 and other LCA genes are requisite in support of emerging gene specific therapy for LCA. Therefore, we have carried out a comprehensive LCA genes screening using a combined approach of direct sequencing and DNA microarray based Asper chip analysis. Methodology/Principal Findings: Thirty clinically diagnosed index LCA cases from Southern India were screened for coding and flanking intronic regions of RPE65 through direct sequencing. Among thirty, 25 cases excluded from RPE65 mutations were subjected to Asper chip analysis, testing 784 known pathogenic variations in 15 major LCA genes. In RPE65 screening, four different pathogenic variations including two novel (c.361insT & c.939T.A) and two known (c.394G.A & c.361delT) mutations were identified in five index cases. In the chip analysis, seven known pathogenic mutations were identified in six index cases, involving genes GUCY2D, RPGRIP1, AIPL1, CRX and IQCB1. Overall, 11 out of 30 LCA cases (36.6%) revealed pathogenic variations with the involvement of RPE65 (16.6%), GUCY2D (10%), RPGRIP1 (3.3%), AIPL1 (3.3%) and CRX & IQCB1 (3.3%). Conclusions/Significance: Our study suggests that such combined screening approach is productive and cost-effective for mutation detection and can be applied in Indian LCA cohort for molecular diagnosis and genetic counselling. Citation: Verma A, Perumalsamy V, Shetty S, Kulm M, Sundaresan P (2013) Mutational Screening of LCA Genes Emphasizing RPE65 in South Indian Cohort of Patients. PLoS ONE 8(9): e73172. doi:10.1371/journal.pone.0073172 Editor: Andreas R. Janecke, Innsbruck Medical University, Austria Received January 29, 2013; Accepted July 19, 2013; Published September 16, 2013 Copyright: ß 2013 Verma 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. Funding: This study was supported by the Indian Council of Medical Research (ICMR), New Delhi, India, (http://www.icmr.nic.in) and Aravind Medical Research Foundation (AMRF), Madurai, India. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors would also like to declare that the company Asper Biotech has no competing interests for the publication and it does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials. * E-mail: sundar@aravind.org Background Leber congenital amaurosis (LCA; [OMIM] #204000) is a congenital retinal disorder causing severe visual impairment before the age of two years [1]. Though, usually considered as a rare disorder with reported prevalence of 2 to 3 per 100,000 live births,[2] its incidence appears to be higher in consanguineous population like South India [3,4]. Clinically LCA is a pool of different phenotypes. The severe vision loss is accompanied with sensory nystagmus, sluggish eye pupil responses, high refractive errors, heterogeneous retinal changes and reduced or absent electroretinogram (ERG) responses. The other features which may or may not be present includes pressing and poking of eyes, photoaversion, nyctalopia, keratoconus and cataract [5]. LCA is caused by mutations in 23 genes meanwhile; the major contrib- uting one includes GUCY2D, CRB1, RPE65, CEP290, RPGRIP1, RDH12, AIPL1, CRX, LCA5, TULP1, LRAT, IMPDH1, RD3, MERTK, SPATA7 [6]. These genes all together account for 70% of LCA cases, and their individual mutational frequency may vary from one ethnic population to other [5,6,7]. Depending on the underlying gene mutations, some LCA phenotypes may be absent or prominent or may arise in later infancy of life [8]. LCA is typically inherited as an autosomal recessive form, although few mutations show the autosomal dominant pattern of inheritance too [9,10]. For the management of LCA patients, the most promising aspect is the gene therapy success for RPE65 LCA cases, achieved in phase 2&1 clinical trials and previous animal model studies, which have paved the pathway to cure inherited blindness [11,12]. The animal model based gene therapy trials for other LCA genes like LCA1 (GUCY2D), LCA4 (AIPL1) and LCA6 (RPGRIP1) are also in progress, which further provides a prospect for the effective treatment regime in inherited blinding retinal disorders [13,14,15]. Since, future treatment will be geared towards gene specific therapies, studies involving mutational screening of LCA genes, particularly RPE65 are prerequisites to establish a pool of patients who could be benefited by such emerging treatments. Among the available gene screening system, the DNA micro-array based Asper LCA chip (http://www.asperophthalmics.com/Leber con- genital amaurosis DNA test) offers a rapid, cost-effective and accurate genotyping method for detection of all known variations associated with LCA [16,17]. Though, the chip is limited to detect PLOS ONE | www.plosone.org 1 September 2013 | Volume 8 | Issue 9 | e73172