A dual approach for comprehensive genetic testing of ABCA4 in Stargardt disease

Abstract

Purpose: Stargardt disease (STGD1) is one of the most frequent autosomal recessive retinal dystrophies, with a prevalence of ~1/8000. Manifestation of STGD1 occurs in the 1st-2nd decade and causes a rapid decrease in visual function, ultimately resulting in legal blindness. So far, over 600 mutations have been found in the 7 kb coding region of the disease gene ABCA4. Genetic testing is mostly limited to chip analysis or Sanger sequencing of the coding region. To overcome the high cost and limitations of this approach, we designed a comprehensive molecular test based on quantitative PCR (qPCR) and massive parallel sequencing (MPS) to screen for single nucleotide and copy number variation (CNV).

Methods: First, 50 assays were designed to cover all ABCA4 exons and intron-exon boundaries. Following qPCR amplification (LC480, Roche), ligation and shearing, amplicon pools were sequenced on a Miseq run (Illumina). Twelve STGD1 patients were included. Previous pre-screening (Asper chip and Sanger sequencing of the coding region) revealed a molecular diagnosis in 4 patients and a single heterozygous mutation in 3 patients. CNV screening comprised 50 qPCR assays performed in 48 STGD1 patients with 1 heterozygous (18) or no mutation (30) in ABCA4 following ABCA4 chip and/or sequencing, and 27 controls (qBase Plus, Biogazelle).

Results: MPS of 50 ABCA4 amplicons resulted in a minimal coverage of 20x for 98% of the amplicons. A variant allele frequency threshold of 25% allowed detection of 99% of the previously identified variants in sufficiently covered regions. In addition, a previously undetected heterozygous mutation was identified (p.L2033R), resulting in a complete molecular diagnosis for one patient. CNV analysis of ABCA4 revealed a novel heterozygous deletion in a patient heterozygous for a splice site mutation. The deletion covers minimally 1.8 kb and maximally 5.6 kb, spanning exon 20-22. Breakpoint delineation is currently ongoing.

Conclusions: MPS for ABCA4 proves to be a cost-efficient alternative to Sanger sequencing and chip analysis. In addition, qPCR screening allowed to identify a novel ABCA4 deletion. Although standard ABCA4 molecular testing does not include CNV analysis, this might be useful as in up to 30% of STGD1 patients only 1 or no coding mutation can be detected by regular ABCA4 testing. Nonetheless, the detection of only 1 CNV in 48 pre-screened cases might suggest variations in non-coding regions.

Keywords: 539 genetics • 696 retinal degenerations: hereditary • 537 gene screening