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

Abstract

ABCA4 gene mutations are the cause of a spectrum of ABCA4 retinopathies, and the most common juvenile macular degeneration is called Stargardt disease. ABCA4 has previously been observed almost exclusively in the retina. Therefore, studying the functional consequences of ABCA4 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 ABCA4 gene splice-site variants in patients with ABCA4 retinopathies. We assessed ABCA4 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 ABCA4 transcripts and analyzed ABCA4 transcripts from three patients with Stargardt disease carrying different splice-site ABCA4 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 ABCA4 variants in patients with ABCA4 retinopathies.

Characterising splicing defects of ABCA4 variants within exons 13–50 in patient-derived fibroblasts

“…Although ABCA4 mRNA is known to be highly expressed in the human retina, it is also expressed in other human tissues at comparatively low levels (GeneCards: https://www.genecards.org/cgi-bi n/carddisp.pl?gene=ABCA4&keywords=abca4). Others have shown ABCA4 expression in choroid plexus epithelial cells (Bhongsatiern et al, 2005), capillary endothelial cells in rats brains (Ohtsuki et al, 2004), human hair follicles and fibroblasts (Albert et al, 2018;Bauwens et al, 2019;Aukrust et al, 2017;Ścieżyńska et al, 2020). In this study, we successfully amplified ABCA4 transcripts from exons 13 to 50 in STGD1 patient-derived fibroblasts.…”

“…Several studies have shown that skin fibroblasts express ABCA4 transcripts, and RNA isolated from fibroblasts has revealed splicing defects caused by mutations mainly located in the latter half of the ABCA4 gene, in particular from exon 30 onwards (Albert et al, 2018;Bauwens et al, 2019;Aukrust et al, 2017;Ścieżyńska et al, 2020). However, Sangermano et al also used patient-derived fibroblasts to investigate two ABCA4 mutations, c.769-784C > T and c.859-506G > C, located in intron 6 and 7, respectively (Sangermano et al, 2019).…”

“…Patient-derived fibroblasts have previously been used to investigate the splicing defects of several ABCA4 variants mainly located in the latter half of the ABCA4 gene (Albert et al, 2018;Bauwens et al, 2019;Aukrust et al, 2017;Ścieżyńska et al, 2020). In the present study, we characterised the ABCA4 transcripts in patient-derived dermal fibroblasts, using both in silico analysis and RT-PCR amplification (fibroblast-based splice assay).…”

Therapy Approaches for Stargardt Disease

“…Generating iPSCs can take 20-30 days, with the subsequent differentiation into retinal photoreceptor-like cells requiring a further 10-14 days of specialised culture conditions. As an alternative to this, a recent study presented evidence of ABCA4 expression in cells isolated from human skin cells and hair follicles [41]. Comparison of hair follicles from control samples to those from a STGD1 patient carrying the mutation c.5836+2T > G revealed significantly lower ABCA4 expression levels in the patient hair follicles.…”

“…Hair follicles Patient-specific genotype Some retinal gene expression may be evident, as for ABCA4 [41].…”