Elsevier

Experimental Eye Research

Volume 145, April 2016, Pages 93-99
Experimental Eye Research

Research article
Next-generation sequencing of ABCA4: High frequency of complex alleles and novel mutations in patients with retinal dystrophies from Central Europe

https://doi.org/10.1016/j.exer.2015.11.011Get rights and content

Highlights

  • We perform a large scale ABCA4 genotyping in patients from Central Europe.

  • We screen ABCA4 with the most advanced DNA sequencing technology.

  • The relevance of genetic variants is defined based on population evidence.

  • We identify several novel ABCA4 gene mutations and a novel complex allele.

  • The pathogenic status of several ABCA4 variants is disproved.

Abstract

Variation in the ABCA4 locus has emerged as the most prevalent cause of monogenic retinal diseases. The study aimed to discover causative ABCA4 mutations in a large but not previously investigated cohort with ABCA4-related diseases originating from Central Europe and to refine the genetic relevance of all identified variants based on population evidence. Comprehensive clinical studies were performed to identify patients with Stargardt disease (STGD, n = 76) and cone-rod dystrophy (CRD, n = 16). Next-generation sequencing targeting ABCA4 was applied for a widespread screening of the gene. The results were analyzed in the context of exome data from a corresponding population (n = 594) and other large genomic databases. Our data disprove the pathogenic status of p.V552I and provide more evidence against a causal role of four further ABCA4 variants as drivers of the phenotype under a recessive paradigm. The study identifies 12 novel potentially pathogenic mutations (four of them recurrent) and a novel complex allele p.[(R152*; V2050L)]. In one third (31/92) of our cohort we detected the p.[(L541P; A1038V)] complex allele, which represents an unusually high level of genetic homogeneity for ABCA4-related diseases. Causative ABCA4 mutations account for 79% of STGD and 31% of CRD cases. A combination of p.[(L541P; A1038V)] and/or a truncating ABCA4 mutation always resulted in an early disease onset. Identification of ABCA4 retinopathies provides a specific molecular diagnosis and justifies a prompt introduction of simple precautions that may slow disease progression. The comprehensive, population-specific study expands our knowledge on the genetic landscape of retinal diseases.

Introduction

ABCA4 gene (OMIM:601691; GenBank:NG_009073.1) mutations are among the leading causes of monogenic retinal diseases. More than 95% of cases of Stargardt's disease (STGD), the most common inherited juvenile macular degeneration, 30% of cases of cone-rod dystrophy (CRD) and about 8% of autosomal recessive retinitis pigmentosa (arRP) are caused by mutations in the ABCA4 gene (Sheffield and Stone, 2011), which encodes an ATP-binding cassette transporter. The function of ABCA4 is to translocate retinoid intermediates of the visual cycle across the photoreceptor outer segment disc membranes. Reduced activity of the ABCA4 protein results in the accumulation of retinoid intermediates, which are deleterious to photoreceptors and retinal pigment epithelium (RPE) cells (Tsybovsky et al., 2010).

The ABCA4 gene has been difficult to investigate due to its size (50 exons, over 6800-bp open reading frame) (www.ensembl.org; accessed 12/2014) and a large allelic diversity. ABCA4 is almost exclusively expressed in the retina (Allikmets et al., 1997) and absence of an established functional assay makes the assessment of the biological role of different ABCA4 variants a difficult task. Introduction of next-generation sequencing (NGS) allows an efficient and cost-effective screening of the sizable gene. An indispensable part of NGS data analysis is access to genome sequencing data with a large number of samples, which provides a basic tool in determining pathogenicity of ABCA4 variants. Identification of causal ABCA4 mutations is of practical relevance for affected families as precautions that may reduce disease progression, such as protection from excessive light exposure and avoidance of vitamin A supplementation, should be followed until therapy options become available (Haji Abdollahi and Hirose, 2013, Paskowitz et al., 2006, Radu et al., 2008, Teussink et al., 2015, Weng et al., 1999).

Currently, more than 800 different ABCA4 disease-associated variants are reported in the Human Gene Mutation Database (http://www.hgmd.cf.ac.uk/ac/index.php; accessed 08/2015). Some of them have a high prevalence in certain ethnic groups, such as p.C1490Y in South Africans (September et al., 2004), p.A1773V in Mexicans (Chacon-Camacho et al., 2013) and a number of other mutations specific for different European populations (Rivera et al., 2000, Valverde et al., 2006, Maugeri et al., 1999, Rosenberg et al., 2007), which underlines the need of genotyping patients of various ethnicities. As the prevalence of ABCA4 variants has not yet been determined for Central European patients with STGD or CRD, we have applied an amplicon-based deep sequencing strategy to investigate the ABCA4 gene. All potentially pathogenic variants identified using NGS were carefully compared with the data from several, large genomic databases to get a better insight into the prevalence of ABCA4 mutations in different populations.

Section snippets

Subjects and clinical evaluation

A total of 92 unrelated individuals with STGD (n = 76) or CRD (n = 16) from the Polish population with a mean age at disease onset (AO) of 17.3 years (CI 95%: 14.8–19.8) were recruited for the study (Supplementary Table S1). Patients and examined family members gave written consent. The study was approved by the local Ethics Committees and followed the tenets of the Declaration of Helsinki. AO was defined as the age at which visual deterioration was first observed by the patient. Next, the

Selection of ABCA4 mutations after exclusion of common variants

Deep sequencing of the ABCA4 gene in our patients (n = 92) identified a large number of allelic variants. Only novel changes and those that have already been recorded in the Human Gene Mutation Database (HGMD) as disease causing mutations (DM) were further analyzed. Thorough search of the literature and four distinct population-derived exome/genome variant databases, including a database of the Polish population showed that four of the ABCA4 variants, i.e. p.R212H, p.H423R, c.6282+7G>A and

Discussion

Collectively, it has been assumed that the most frequent disease-associated ABCA4 alleles have each been described in only approximately 10% of STGD patients (Zernant et al., 2011). Thus, it was quite unexpected to find that the most prevalent allele in Polish patients, the complex p.[(L541P; A1038V)] allele, was present in more than 33.7% (31/92) of our patients, which corresponds to at least one fourth (37/143) of all alleles with an identified mutation. The p.[(L541P; A1038V)] allele

Competing interests

The authors declare that they have no competing interests.

Acknowledgments

This work was supported by the Polish National Science Center grant no. N N402 591640 (5916/B/P01/2011/40) and the Medical University of Warsaw grant no. 1M15/PM11D/14.

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