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

Aneta Ścieżyńska,Anna Podgórska,Jacek P Szaflik,Jacek Malejczyk,Marta Soszyńska,Monika Ołdak,Kamil Szulborski,Aleksandra Nogowska,Natalia Krześniak,Martyna Smolińska,Michał Komorowski,Bartłomiej Noszczyk

doi:10.3390/ijms21103430

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.

Highlights

The ABCA4 gene (OMIM 601691; GenBank:NG_009073.1) encodes an ATP-binding cassette transporter which translocates retinoid intermediates of the visual cycle across the disc membranes of photoreceptor cells [1]
Using hair follicles from patients with Stargardt disease, we investigated the molecular consequences of selected ABCA4 variants on gene processing
ABCA4 Gene is Expressed in Human Hair Follicles and Skin

Summary

Introduction

The ABCA4 gene (OMIM 601691; GenBank:NG_009073.1) encodes an ATP-binding cassette transporter which translocates retinoid intermediates of the visual cycle across the disc membranes of photoreceptor cells [1]. Mutations of the ABCA4 gene are the cause of more than 95% of cases of Stargardt disease (STGD)—the most common form of inherited juvenile macular degeneration—as well as other monogenic retinal diseases, the so-called ABCA4 retinopathies [2]. The ABCA4 gene carries a high number of non-canonical splice variants and protein-truncating mutations, which constitute the second highest type of genetic aberration, after missense mutations [12,13]. Residual activity of the mutant ABCA4 protein supposedly determines the severity of the disease [14]. To date, the pathogenicity of many ABCA4 variants remains unclear, and some supposedly deleterious variants may influence the onset of Stargardt disease in different ways [15]. Identification of the ABCA4 mutations, as well as assessment of their pathogenicity, is essential for affected families and may be helpful in prediction of the disease’s severity and the introduction of precautions which may reduce the disease’s progression [21,22]

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