Microarray-Based Mutation Detection and Phenotypic Characterization of Patients with Leber Congenital Amaurosis

Abstract

Microarray-based mutation detection allowed the identification of 32% of LCA sequence variants and represents an efficient first-pass screening tool. Mutations in CRB1, and to a lesser extent, in GUCY2D, underlie most LCA cases in this cohort. The present study establishes a genotype-phenotype correlation for AIPL1, CRB1, and GUCY2D.

Massively parallel sequencing for early molecular diagnosis in Leber congenital amaurosis

“…The first part of this study was mainly aimed at validation of the enrichment protocol (proof of concept, patients 1-10), whereas the second part of this study consisted of a blind screening of 12 prescreened mutation-negative patients with LCA (patients [11][12][13][14][15][16][17][18][19][20][21][22]. enrichment of LcA disease genes qPCR was used to target all exons from 16 LCA disease genes.…”

“…Moreover, p.Arg768Trp is presumably a founder mutation in the northwest of Europe. 15 This mutation was actually a missed call on the LCA chip. Second, a novel homozygous missense mutation in RDH12, c.176T>G (p.Leu59Arg), was identified in patient 13.…”

“…For the second lane, libraries were prepared from a 200-300-bp size-selected fraction, and library quantification was performed using qPCR following manufacturer's protocols (lane 2, patients [11][12][13][14][15][16][17][18][19][20][21][22]. In total, 120 µl of a pool of the 12 normalized libraries (concentration of 10 pmolar) was subjected to paired-end sequencing of 2 × 45 cycles.…”

“…This study included 17 patients in which previous Sanger sequencing of six genes and/or LCA chip analysis did not identify causal mutations (lane 1: patients 1-3, 7, and 8; lane 2: patients [11][12][13][14][15][16][17][18][19][20][21][22]. Given the exclusion of 3 of 107 known variants by the in 12.7% of the Yoruba population (1000Genomes Project, pilot_1_YRI_low_coverage_panel) points to a polymorphism rather than a mutation (rs61748445).…”

“…12 Although this technique represents a good first-pass screening, it fails in detecting new mutations, is expensive in a routine context, and has a variable, population-dependent detection rate. [13][14][15] Secondary genetic tests comprise denaturing high-performance liquid chromatography and Sanger sequencing, mostly of a subset of genes because of excessive costs and workload. 1,16,17 Hence, an urgent need exists for an effective approach able to identify mutations in all currently known LCA genes.…”