Department of Genetics, Dr. G. Venkataswamy Eye Research Institute, Aravind Medical Research Foundation, Aravind Eye Hospital, Madurai, Tamil Nadu, India.
PLoS One. 2013 Sep 16;8(9):e73172. doi: 10.1371/journal.pone.0073172. eCollection 2013.
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.
莱伯先天性黑矇（LCA）是儿童遗传性视网膜视觉障碍中最严重的形式。到目前为止，已经知道超过 20 个基因的突变会导致 LCA，其中 RPE65 是基因治疗的合适候选者。RPE65 和其他 LCA 基因的突变筛查是支持新兴的 LCA 基因特异性治疗所必需的。因此，我们使用直接测序和基于 DNA 微阵列的 Asper 芯片分析的组合方法对 LCA 基因进行了全面筛查。
方法/主要发现：从印度南部筛选了 30 例临床诊断为 LCA 的指数病例，通过直接测序筛选 RPE65 的编码和侧翼内含子区域。在 30 例中，排除 RPE65 突变的 25 例病例进行了 Asper 芯片分析，测试了 15 个主要 LCA 基因中的 784 个已知致病性变异。在 RPE65 筛选中，在 5 个指数病例中发现了包括两个新的（c.361insT 和 c.939T>A）和两个已知的（c.394G>A 和 c.361delT）突变在内的四个不同的致病性突变。在芯片分析中，在 6 个指数病例中发现了 7 个已知的致病性突变，涉及基因 GUCY2D、RPGRIP1、AIPL1、CRX 和 IQCB1。总的来说，在 30 例 LCA 病例中（36.6%）有 11 例发现了致病性变异，涉及 RPE65（16.6%）、GUCY2D（10%）、RPGRIP1（3.3%）、AIPL1（3.3%）和 CRX&IQCB1（3.3%）。
我们的研究表明，这种组合筛查方法对于突变检测是富有成效且具有成本效益的，并且可以应用于印度 LCA 队列进行分子诊断和遗传咨询。
