An augmented ABCA4 screen targeting non-coding regions reveals a deep intronic founder causal variant in Belgian Stargardt patients

Abstract

Purpose: Autosomal recessive Stargardt disease (STGD1) is hallmarked by a large proportion of patients with a single heterozygous causative variant in the disease gene ABCA4. Braun et al. (2013) reported deep intronic variants of ABCA4, prompting us to perform an augmented screen in 131 Belgian STGD1 patients with one or no ABCA4 variant to uncover deep intronic causal ABCA4 variants.

Methods: All 131 prescreened patients underwent targeted resequencing of four deep intronic ABCA4 regions using next-generation sequencing (NGS) (Miseq, Illumina). Haplotype analysis was performed on patients with variant c.4539+2001G>A (known as V4) and family members. Analysis and comparison of clinical and molecular data allowed us to establish phenotype-genotype correlations of V4.<br /> Data analysis was performed with CLC Bio. Filtering of non-coding variants was based on criteria such as minor allele frequency, conservation score, splicing predictions and regulatory potential.The cohort without an identified second mutation currently undergoes targeted NGS with a customized Haloplex panel that includes the entire ABCA4 gene and its regulatory domain, and the entire genomic regions of BEST1, RDH12 and PRPH2.

Results: Our augmented screen revealed a second variant in 28.6% of cases. Twenty-six percent of these carry the same causal variant V4. Haplotyping in V4 carriers showed a common region of 63 kb, suggestive of a founder mutation. Genotype-phenotype correlations indicate a moderate-to-severe impact of V4 on the STGD1 phenotype.<br /> The remaining patients in whom no second ABCA4 mutation was identified undergo NGS of the entire genomic regions of ABCA4, BEST1, RDH12 and PRPH2, some of which are regulatory regions (~ 500 kb in total). Preliminary sequence data of five patients show that the standard filtering yields approximately 130 variants per patient that require further investigation.

Conclusions: Causal variant V4 occurs in a high fraction of Belgian STGD1 patients and represents the first deep intronic founder mutation in ABCA4. This emphasizes the importance of augmented molecular genetic testing of ABCA4 in Belgian STGD1 patients. Finally, more extensive resequencing in the remainder of the STGD1 patients without an identified second ABCA4 mutation, will allow us to identify novel deep intronic and non-coding mutations and to establish a more definite molecular diagnosis.