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Non-exomic and synonymous variants in ABCA4 are an important cause of Stargardt disease

Terry A. Braun, Robert F. Mullins, Alex Handler Wagner, Jeaneen L. Andorf, Rebecca M. Johnston, Benjamin Bakall et al. · 2013 · 180 citationsRead the paper

Mutations in ABCA4 cause Stargardt disease and other blinding autosomal recessive retinal disorders. However, sequencing of the complete coding sequence in patients with clinical features of Stargardt disease sometimes fails to detect one or both mutations. For example, among 208 individuals with clear clinical evidence of ABCA4 disease ascertained at a single institution, 28 had only one disease-causing allele identified in the exons and splice junctions of the primary retinal transcript of the gene. Haplotype analysis of these 28 probands revealed 3 haplotypes shared among ten families, suggesting that 18 of the 28 missing alleles were rare enough to be present only once in the cohort. We hypothesized that mutations near rare alternate splice junctions in ABCA4 might cause disease by increasing the probability of mis-splicing at these sites. Next-generation sequencing of RNA extracted from human donor eyes revealed more than a dozen alternate exons that are occasionally incorporated into the ABCA4 transcript in normal human retina. We sequenced the genomic DNA containing 15 of these minor exons in the 28 one-allele subjects and observed five instances of two different variations in the splice signals of exon 36.1 that were not present in normal individuals (P < 10(-6)). Analysis of RNA obtained from the keratinocytes of patients with these mutations revealed the predicted alternate transcript. This study illustrates the utility of RNA sequence analysis of human donor tissue and patient-derived cell lines to identify mutations that would be undetectable by exome sequencing.

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Working prototype

Identification of non-exomic mutations in the splice sites of a minor exon (exon 36.1) in patients with Stargardt disease. — 5 instances of 2 different variations (Patients with only one detectable disease-causing allele in exons/canonical splice junctions)
A significant portion of ABCA4 disease alleles are rare and undetectable by standard exome sequencing. — 8.2% of alleles (34/416) were undetectable (Cohort of 208 individuals with clinical evidence of ABCA4 disease)
Most undetectable ABCA4 mutations are extremely rare within the patient population. — 65% of mutations have allele frequencies of 1/400 or less (Analysis of 28 one-allele probands and their families)
Detection of minor splice variants in normal human retina that may serve as mutation susceptibility sites. — Over a dozen alternate exons (each <1% of total transcripts) (RNA extracted from 5 anatomic regions of human donor retina)

Setup: 208 probands with clinical ABCA4 disease (primary cohort of 28 one-allele subjects), 18 control trios, 300 unaffected controls, and RNA from one 91-year-old human donor eye.
How: The researchers used haplotype analysis of 60 tagged SNPs to estimate mutation frequency and next-generation RNA sequencing of human donor retina to identify alternate exons. They then sequenced genomic DNA of these alternate regions in patients and validated the resulting alternate transcripts using RNA from patient-derived keratinocytes.
Limitations: High allelic diversity of ABCA4 makes allele-specific testing less useful. · Dependence on patient-derived cell lines for functional demonstration of pathogenicity due to mutation rarity.

Summarized by Gemma 4 from the open-access full text · 9 Oct 2026. Read it