ABCA4 c.6480-35A>G, a novel branchpoint variant associated with Stargardt disease

Front Genet. 2023 Sep 7:14:1234032. doi: 10.3389/fgene.2023.1234032. eCollection 2023.


Introduction: Inherited retinal dystrophies (IRDs) can be caused by variants in more than 280 genes. The ATP-binding cassette transporter type A4 (ABCA4) gene is one of these genes and has been linked to Stargardt disease type 1 (STGD1), fundus flavimaculatus, cone-rod dystrophy (CRD), and pan-retinal CRD. Approximately 25% of the reported ABCA4 variants affect RNA splicing. In most cases, it is necessary to perform a functional assay to determine the effect of these variants. Methods: Whole genome sequencing (WGS) was performed in one Spanish proband with Stargardt disease. The putative pathogenicity of c.6480-35A>G on splicing was investigated both in silico and in vitro. The in silico approach was based on the deep-learning tool SpliceAI. For the in vitro approach we used a midigene splice assay in HEK293T cells, based on a previously established wild-type midigene (BA29) containing ABCA4 exons 46 to 48. Results: Through the analysis of WGS data, we identified two candidate variants in ABCA4 in one proband: a previously described deletion, c.699_768+342del (p.(Gln234Phefs*5)), and a novel branchpoint variant, c.6480-35A>G. Segregation analysis confirmed that the variants were in trans. For the branchpoint variant, SpliceAI predicted an acceptor gain with a high score (0.47) at position c.6480-47. A midigene splice assay in HEK293T cells revealed the inclusion of the last 47 nucleotides of intron 47 creating a premature stop codon and allowed to categorize the variant as moderately severe. Subsequent analysis revealed the presence of this variant as a second allele besides c.1958G>A p.(Arg653His) in an additional Spanish proband in a large cohort of IRD cases. Conclusion: A splice-altering effect of the branchpoint variant, confirmed by the midigene splice assay, along with the identification of this variant in a second unrelated individual affected with STGD, provides sufficient evidence to classify the variant as likely pathogenic. In addition, this research highlights the importance of studying non-coding regions and performing functional assays to provide a conclusive molecular diagnosis.

Keywords: ABCA4; Stargardt disease; branchpoint variant; midigene splice assay; whole genome sequencing.

Grants and funding

The study was funded by the National Institute of Health Carlos III and co-funded by the European Union, project no. PI20/01186 (to CI) project no. PI22/00321 (to CA). This work was also supported by the Education Department of the Basque Government, grant no. PRE_2019_1_0325 (to MR-H), the Education Department of the Basque Government, grant no. 325 EP_2022_1_0060 (to MR-H), EMBO Scientific Exchange Grant, grant no 9507 (to MR-H), and University Chair UAM-IIS-FJD of Genomics Medicine (to CA). This work was also supported by the Basque Retinitis Pigmentosa Foundation (to JR-E). The work of SEdB was funded by the European Union’s Horizon 2020 Research and Innovation Programme under the EJP RD COFUND-EJP N° 825575 (to FPMC and SR). The work of KR was supported by grant awards from Fighting Blindness Ireland (FB18CRE) (to FPMC and SR). The work of KR and SR was funded by the Foundation Fighting Blindness (FFB)–career development award (CD-GE-0621-0809-RAD) (to SR).