Molecular Analysis of the ABCA4 Gene Mutations in Patients with Stargardt Disease Using Human Hair Follicles

Abstract

<i>ABCA4</i> gene mutations are the cause of a spectrum of <i>ABCA4</i> retinopathies, and the most common juvenile macular degeneration is called Stargardt disease. <i>ABCA4</i> has previously been observed almost exclusively in the retina. Therefore, studying the functional consequences of <i>ABCA4</i> variants has required advanced molecular analysis techniques. The aim of the present study was to evaluate whether human hair follicles may be used for molecular analysis of the <i>ABCA4</i> gene splice-site variants in patients with <i>ABCA4</i> retinopathies. We assessed <i>ABCA4</i> expression in hair follicles and skin at mRNA and protein levels by means of real-time PCR and Western blot analyses, respectively. We performed cDNA sequencing to reveal the presence of full-length <i>ABCA4</i> transcripts and analyzed <i>ABCA4</i> transcripts from three patients with Stargardt disease carrying different splice-site <i>ABCA4</i> variants: c.5312+1G>A, c.5312+2T>G and c.5836-3C>A. cDNA analysis revealed that c.5312+1G>A, c.5312+2T>G variants led to the skipping of exon 37, and the c.5836-3C>A variant resulted in the insertion of 30 nucleotides into the transcript. Our results strongly argue for the use of hair follicles as a model for the molecular analysis of the pathogenicity of <i>ABCA4</i> variants in patients with <i>ABCA4</i> retinopathies.