Validation of Leber Congenital Amaurosis (LCA) Mutations with Capture Sequencing

LCA is a vision disorder that affects about 1 in every 30,000 to 80,000 people, yet 30% of cases are still unclear.  Currently about 16 genes are known to cause LCA. It is hypothesized that LCA may be caused by novel mutations in known LCA genes or alleles in other retinal disease genes that lead to LCA, due to different nature of alleles or genetic background. To systematically evaluate the genetic makeup of LCA patients, we performed retinal capture sequencing on a cohort of LCA patients who have been pre-screened by conventional genotyping methods but showed negative results. For each patient, the exon DNA of several retinal disease genes were enriched and sequenced on a high throughput sequencing machine. A standard sequence processing pipeline was used to process the data and identify variants, resulting in forty mutations in sporadic LCA patients. To validate each mutation, capture sequencing was performed for 32 mutations by amplification using LCA patient DNA and sequencing of specific mutated exon regions. Six mutated LCA genes revealed poor quality sequencing due to inadequate amplification. Validation could not be determined for these genes. Twenty-six mutations illustrated high-quality sequencing for validation. These target mutated regions were marked as reference points and displayed on a chromatograph using Sequencher software to characterize the gene mutation and genotype. Analysis of each chromatograph reading confirmed previously recorded mutations. Therefore, assays of different alleles and genetic background among numerous LCA families play a key role in the discovery of novel mutations for retinal disorders.