PURPOSE. We hypothesize that somatic mutations accumulate in cells of the human lens and may contribute to the development of cortical or posterior sub-capsular cataracts. Here, we used a Next-generation sequencing (NGS) strategy to screen for low-allelic frequency variants in DNA extracted from human lens epithelial samples. METHODS. Next-Generation sequencing of 151 cancer-related genes (WUCaMP2 panel) was performed on DNA extracted from post-mortem or surgical specimens obtained from 24 individuals. Usually, pairwise comparisons were made between two or more ocular samples from the same individual, allowing putative somatic variants detected in lens samples to be differentiated from germline variants. RESULTS. Use of a targeted hybridization approach enabled high sequence coverage (>1000- fold) of the WUCaMP2 genes. In addition to high-frequency variants (corresponding to homozygous or heterozygous SNPs and Indels), somatic variants with allelic frequencies of 1- 4% were detected in the lens epithelial samples. The presence of one such variant, a T > C point substitution at position 32907082 in BRCA2, was verified subsequently using droplet digital PCR. CONCLUSIONS. Low-allelic fraction variants are present in the human lens epithelium, at frequencies consistent with the presence of millimeter-sized clones.
- Cataract risk factors
- Somatic mutation