ABCA4-associated disease as a model for missing heritability in autosomal recessive disorders: novel noncoding splice, cis-regulatory, structural, and recurrent hypomorphic variants

Abstract

Purpose: ABCA4-associated disease, a recessive retinal dystrophy, is hallmarked by a large proportion of patients with only one pathogenic ABCA4 variant, suggestive for missing heritability.

Methods: By locus-specific analysis of ABCA4, combined with extensive functional studies, we aimed to unravel the missing alleles in a cohort of 67 patients (p), with one (p = 64) or no (p = 3) identified coding pathogenic variants of ABCA4.

Results: We identified eight pathogenic (deep-)intronic ABCA4 splice variants, of which five are novel and six structural variants, four of which are novel, including two duplications. Together, these variants account for the missing alleles in 40.3% of patients. Furthermore, two novel variants with a putative cis-regulatory effect were identified. The common hypomorphic variant c.5603A>T p.(Asn1868Ile) was found as a candidate second allele in 43.3% of patients. Overall, we have elucidated the missing heritability in 83.6% of our cohort. In addition, we successfully rescued three deep-intronic variants using antisense oligonucleotide (AON)-mediated treatment in HEK 293-T cells and in patient-derived fibroblast cells.

Conclusion: Noncoding pathogenic variants, novel structural variants, and a common hypomorphic allele of the ABCA4 gene explain the majority of unsolved cases with ABCA4-associated disease, rendering this retinopathy a model for missing heritability in autosomal recessive disorders.

Keywords: ABCA4-associated disease; AON; deep-intronic; missing heritability; noncoding.

Fig. 1

Minigene and midigene results for intronic ABCA4 splice variants. For each depicted variant (a-f) an agarose gel image is shown on the left, representing the reverse transcription polymerase chain reaction (RT-PCR) product derived from the mutant (MT) and the respective wild-type (WT) construct. On the right, the corresponding cartoon of observed sequences is shown, with mutant at the top and wild type below (exception for variant a). Above the sequence, a cartoon of the minigene or midigene construct is presented; arrows depict the location of the primers used for RT-PCR. A black “x” represents the location of the variant. “E” stands for exon, flanking Rhodopsin (RHO) exons are depicted in white, ABCA4 exons in gray, and insertions in red. “E30 M” denotes the full length exon 30, as described in NM_00350.2 (hg19), “E30 S” refers to a smaller exon 30, lacking the last 73 nucleotides p.(Cys1490Glufs*12), which was observed in several transcripts.

Fig. 2

Antisense oligonucleotide (AON)-mediated rescue for intronic ABCA4 splice variants. For three deep-intronic ABCA4 variants (c.859–540C>G, c.5197–557G>T, and c.4539+1106C>T), AON rescue experiments on transfected HEK 293-T cells were undertaken (a–c). AON experiments were also performed on the fibroblasts of a patient with c.[4539+1106C>T];[6089G>A] and control fibroblasts (d). A gel image is shown on the left, depicting the reverse transcription polymerase chain reaction (RT-PCR) product derived from mutant (MT) and wild-type (WT) midigene construct after transfection with three different AONs (HEK: untransfected HEK 293-T, MQ: negative control PCR, NT: nontransfected cells, SON: sense oligonucleotide). Rhodopsin (RHO) and actin (ACTB) were used as loading controls for RT-PCR products derived from experiments on HEK 293-T cells and fibroblasts, respectively. In fibroblasts, cycloheximide (CHX) was added. On the right, resulting RT-PCR products after AON rescue experiments are semiquantified using capillary analysis, using the ratio WT transcript/transcript including pseudo-exon (PE). If multiple PE containing transcripts were formed, they are grouped together in one PE group.

Fig. 3

Schematic overview of the ABCA4 deletions and duplications identified in this study. ABCA4 exons involved in the deletions (a-d) and duplications (e, f) identified in this study are visualized in red and blue, respectively. The localization of the primers used to perform a junction polymerase chain reaction (PCR) and to delineate the copy-number variants (CNVs) are depicted as black arrows; nucleotides on the cartoons represent microhomology at the boundaries. The orientation of the tandem duplications toward ABCA4 is the most likely one based on (non)amplification of several junction regions (data not shown). InsA: insertion of an A at a CNV junction.