An AugmentedABCA4Screen Targeting Noncoding Regions Reveals a Deep Intronic Founder Variant in Belgian Stargardt Patients

Abstract

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. () reported deep intronic variants of ABCA4 in STGD1 patients with one coding variant, 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. This revealed a second variant in 28.6% of cases. Twenty-six percent of these carry the same causal variant c.4539+2001G>A (V4). Haplotyping in V4 carriers showed a common region of 63 kb, suggestive of a founder mutation. Genotype-phenotype correlations suggest a moderate-to-severe impact of V4 on the STGD1 phenotype. In conclusion, 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.

Expanding the Mutation Spectrum in ABCA4: Sixty Novel Disease Causing Variants and Their Associated Phenotype in a Large French Stargardt Cohort

“…responsible for overlapping phenotypes may help solving such cases; (2) Large rearrangements within the ABCA4 locus have been shown to occur in ~0.5% to ~2% of STGD1 cases in previous reports and they would not have been detected by our mutational screening [22,24]; (3) Mutations in noncoding regions of the ABCA4 gene locus have been proposed as a common source for a second causative mutation in patients with typical Stargardt phenotype. In particular, the occurrence of deep intronic variants in subjects with a single ABCA4 mutations may range from ~2% to ~18% in different reports [19,20,21,22,27,59]. This may have led not only to an underestimation of bi-allelic cases, but also of the number of novel variants found in the cohort, whose report was the main aim of this study.…”

“…Indeed, we cannot exclude the possibility that this variant may affect splicing in vivo since it has already been proven that ABCA4 can have non-canonical splice sites variant with important effects on protein [19,20,21,22,23,59]. …”

“…Nevertheless, homozygous or compound heterozygous mutations are regularly detected in no more than 70–75% of STGD1 patients, while a significant number of patients carry only a single ABCA4 mutation or none at all [15,16,17,18,19]. On the other hand, several deep intronic variants in non-coding regions were reported to segregate with STGD1 affecting correct splicing mechanisms [19,20,21,22,23]. …”

Application of targeted exome and whole‐exome sequencing for Chinese families with Stargardt disease

“…Possible reasons for these results are as follows: (1) targeted exome sequencing and WES cannot detect gross deletions, gross insertions, or complex rearrangement variants (Broadgate, Yu, Downes, & Halford, 2017), which might have been present in these families; (2) the sequence depth and coverage in this study was insufficient to call all variants accurately; (3) novel STGD-associated genes may have been filtered out during our raw data analysis; (4) deep-intronic variants in ABCA4 that are potentially associated with autosomal recessive STGD could not be captured through targeted exome sequencing and WES (Albert et al, 2018;Bauwens et al, 2015Bauwens et al, , 2019Bax et al, 2015;Braun et al, 2013;Sangermano et al, 2014;Zernant et al, 2014); and (5) diseasecausing variants with high minor allele frequency, which had not been reported previously may have been filtered out, for example, c.5603A > T, p.(Asn1868Ile) (Cremers, Cornelis, Runhart, & Astuti, 2018;Runhart et al, 2018;Zernant et al, 2017). Possible reasons for these results are as follows: (1) targeted exome sequencing and WES cannot detect gross deletions, gross insertions, or complex rearrangement variants (Broadgate, Yu, Downes, & Halford, 2017), which might have been present in these families; (2) the sequence depth and coverage in this study was insufficient to call all variants accurately; (3) novel STGD-associated genes may have been filtered out during our raw data analysis; (4) deep-intronic variants in ABCA4 that are potentially associated with autosomal recessive STGD could not be captured through targeted exome sequencing and WES (Albert et al, 2018;Bauwens et al, 2015Bauwens et al, , 2019Bax et al, 2015;Braun et al, 2013;Sangermano et al, 2014;Zernant et al, 2014); and (5) diseasecausing variants with high minor allele frequency, which had not been reported previously may have been filtered out, for example, c.5603A > T, p.(Asn1868Ile) (Cremers, Cornelis, Runhart, & Astuti, 2018;Runhart et al, 2018;Zernant et al, 2017).…”

The current status of molecular diagnosis of inherited retinal dystrophies

“…Deep intronic mutations were identified in an additional 27/114 (23.7%); no second mutations were identified in 36/114 (31.6%) and intronic variants of unknown significance were identified in the remaining 51/114 (44.7%) patients. The increase in mutation detection by incorporating these five deep intronic mutations was confirmed in two other studies [9,10]. However, the prediction and confirmation of pathogenicity for the additional intronic variants has proven difficult, especially when the variant is rare [8 && ].…”