Skin cells as a model to study the consequences of mutations in genes causing hereditary retinal dystrophies

High-throughput DNA sequencing results obtained from patients with inherited retinal diseases revealed a huge number of genetic variants of unknown clinical significance. The urgent need for further molecular studies to verify the pathogenicity of these variants crosses with the lack of access to the retina, the part of the eye directly affected by the disease. In view of the new findings pointing to the expression of human retinal genes also in the skin, we believe that skin cells may be a good model for studying the processing of mutant genes responsible for the development of retinal dystrophy. In this study, we intend to focus on the ABCA4 gene, which is one of the main genes of the retina and is responsible for the pathomechanism of many of its inherited diseases. Our main goal is to determine the composition of ABCA4 transcripts and to study the expression of this gene at the mRNA and protein level in hair follicles, keratinocytes and fibroblasts. For the purpose of this task, primary cultures of keratinocytes and fibroblasts will be established from the collected skin biopsies. Our next goal will be to compare the experimental data to select and implement the most appropriate cell type to study the metabolism of the mutant ABCA4 gene in patients. In order to achieve the presented goals, a qualitative analysis of transcripts will be carried out using the technique of rapid amplification of cDNA ends (RACE) and cDNA sequencing. Quantitative expression of the ABCA4 gene at the mRNA and protein level will be measured using real-time PCR and Western Blot methods, respectively. The expected effect of this work will be the analysis of the influence of the ABCA4 mutation on its processing and verification of the pathogenicity of these changes in the intracellular environment. In addition, ABCA4 transcript sequences in various types of skin cells will be identified for the first time. We are convinced that the model proposed in these studies will be successfully implemented to analyze mutations also of other retinal genes involved in the development of a broad spectrum of posterior ocular dystrophy and may be the starting point for further research aimed at discovering the role of ABCA4 in the skin.