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

Frontiers in Genetics

Genetics

Lidia Fernandez-Caballero¹  Carmen Ayuso¹  Almudena Ávila-Fernández¹  Marta Del Pozo-Valero¹  Suzanne E. de Bruijn²  Susanne Roosing²  Zelia Corradi²  Frans P. M. Cremers²  Kim Rodenburg²  Jordi Corominas³  Christian Gilissen³  María Rodríguez-Hidalgo⁴  Javier Ruiz-Ederra⁵  Cristina Irigoyen⁶  Araceli Lara-López⁷  Alicia Valverde-Megías⁸ 

[1] Department of Genetics, Health Research Institute-Fundación Jiménez Díaz University Hospital, Universidad Autónoma de Madrid (IIS-FJD, UAM), Madrid, Spain;Center for Biomedical Network Research on Rare Diseases (CIBERER), Instituto de Salud Carlos III, Madrid, Spain;Department of Human Genetics, Radboud University Medical Center, Nijmegen, Netherlands;Department of Human Genetics, Radboud University Medical Center, Nijmegen, Netherlands;Radboud Institute of Molecular Life Sciences, Radboud University Medical Center, Nijmegen, Netherlands;Department of Neuroscience, Biodonostia Health Research Institute, Donostia-San Sebastián, Spain;Department of Genetic, Physical Anthropology and Animal Physiology, University of the Basque Country UPV/EHU, Leioa, Spain;Department of Neuroscience, Biodonostia Health Research Institute, Donostia-San Sebastián, Spain;Department of Ophthalmology, University of the Basque Country (UPV/EHU), San Sebastián, Spain;Department of Neuroscience, Biodonostia Health Research Institute, Donostia-San Sebastián, Spain;Ophthalmology Service, Donostia Universy Hospital, Donostia-San Sebastián, Spain;Miramoon Pharma S.L., Donostia-San Sebastián, Spain;Ophthalmology Service, San Carlos Clinical Hospital of Madrid, Madrid, Spain;

关键词: branchpoint variant;    midigene splice assay;    whole genome sequencing;    ABCA4;    Stargardt disease;   

DOI  :  10.3389/fgene.2023.1234032

 received in 2023-06-03, accepted in 2023-08-15,  发布年份 2023

来源: Frontiers

【 摘 要 】

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.

【 授权许可】

Unknown   
Copyright © 2023 Rodríguez-Hidalgo, de Bruijn, Corradi, Rodenburg, Lara-López, Valverde-Megías, Ávila-Fernández, Fernandez-Caballero, Del Pozo-Valero, Corominas, Gilissen, Irigoyen, Cremers, Ayuso, Ruiz-Ederra and Roosing.