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

Abstract

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.

A Report on Molecular Diagnostic Testing for Inherited Retinal Dystrophies by Targeted Genetic Analyses

“…Whereas, molecular diagnostic testing for ABCA4-associated diseases was requested for a majority (113 of 150) of patients. The rate of detection of causative mutations in these patients varies between 50% to 80%, depending on testing methods and patient ethnicity (Braun et al, 2013;Fujinami et al, 2015). In a previous study, we observed causative mutations in 48% of patients diagnosed with ABCA4-associated disease, whereas mutations were found in 59% of the probands in the current study by screening the coding region of the ABCA4 gene (Downs et al, 2007).…”

“…Furthermore, failure to detect certain mutation types due to methodology limitations may also contribute to the low detection rate (Amano et al, 2009;Lee and Garg, 2015). Clinically significant intronic mutations have been described in patients with IRDs (den Hollander et al, 2006;Braun et al, 2013). Similarly, copy number variations have also been observed in IRD patients (Schrider and Hahn, 2010).…”

Comprehensive genotyping reveals RPE65 as the most frequently mutated gene in Leber congenital amaurosis in Denmark

“…Our screening also does not cover deep-intronic regions, and thus variants in this part of the genome will not be identified. 15,47 Moreover, variants in regulatory elements located far away from the respective genes cannot be ruled out. Finally, the aggregate carrier frequency of variants in the most intensively studied genes in this study (AIPL1, CEP290, CRB1, GUCY2D, LRAT and RPE65) is estimated 1 in 25 individuals (Supplementary Table S3), meaning that three or four probands could carry a variant in one of these genes in this cohort of 86 families, without pathologic consequences.…”

Corticosteroids and Anti-Complement Therapy in Retinal Diseases

“…Rare autosomal dominant forms of Stargardt macular dystrophy STGD3 and STGD4 are linked to ELOVL4 (elongation of very long chain fatty acids protein 4) and PROM1 (prominin1), respectively (Yanoff and Duker 2008). More than 400 sequence variations in the ABCA4 gene have been identified (Allikmets et al 1997;Sun and Nathans 2000;Kaminski et al 2006;Braun et al 2013). While there is no clear genotype-phenotype relationship, Stone's group has suggested that the overall pathogenicity of ABCA4 alleles for a patient can be calculated as the sum of the pathogenicity of each risk allele (Schindler et al 2010).…”