Targeted sequencing and in vitro splice assays shed light on ABCA4-associated retinopathies missing heritability
Zelia Corradi,
Mubeen Khan,
Rebekkah Hitti-Malin,
Ketan Mishra,
Laura Whelan,
Stéphanie S. Cornelis,
Carel B. Hoyng,
Kati Kämpjärvi,
Caroline C.W. Klaver,
Petra Liskova,
Heidi Stöhr,
Bernhard H.F. Weber,
Sandro Banfi,
G. Jane Farrar,
Dror Sharon,
Jana Zernant,
Rando Allikmets,
Claire-Marie Dhaenens,
Frans P.M. Cremers
Affiliations
Zelia Corradi
Department of Human Genetics, Radboud University Medical Center, Nijmegen, the Netherlands; Corresponding author
Mubeen Khan
Department of Human Genetics, Radboud University Medical Center, Nijmegen, the Netherlands; Max Planck Institute for Psycholinguistics, Nijmegen, the Netherlands
Rebekkah Hitti-Malin
Department of Human Genetics, Radboud University Medical Center, Nijmegen, the Netherlands
Ketan Mishra
Department of Human Genetics, Radboud University Medical Center, Nijmegen, the Netherlands
Laura Whelan
The School of Genetics & Microbiology, Trinity College Dublin, Dublin, Ireland
Stéphanie S. Cornelis
Department of Human Genetics, Radboud University Medical Center, Nijmegen, the Netherlands
Carel B. Hoyng
Department of Ophthalmology, Radboud University Medical Center, Nijmegen, the Netherlands
Kati Kämpjärvi
Blueprint Genetics, Espoo, Finland
Caroline C.W. Klaver
Department of Ophthalmology, Radboud University Medical Center, Nijmegen, the Netherlands; Department of Epidemiology, Erasmus Medical Center, Rotterdam, the Netherlands; Department of Ophthalmology, Erasmus Medical Center, Rotterdam, the Netherlands; Institute of Molecular & Clinical Ophthalmology, Basel, Switzerland
Petra Liskova
Research Unit for Rare Diseases, Department of Paediatrics and Adolescent Medicine, First Faculty of Medicine, Charles University and General University Hospital in Prague, Prague, Czech Republic; Department of Ophthalmology, First Faculty of Medicine, Charles University and General University Hospital in Prague, Prague, Czech Republic
Heidi Stöhr
Institute of Human Genetics, University of Regensburg, Regensburg, Germany
Bernhard H.F. Weber
Institute of Human Genetics, University of Regensburg, Regensburg, Germany; Institute of Clinical Human Genetics, University Hospital Regensburg, Regensburg, Germany
Sandro Banfi
Department of Precision Medicine, University of Campania “Luigi Vanvitelli,” Naples and Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy
G. Jane Farrar
The School of Genetics & Microbiology, Trinity College Dublin, Dublin, Ireland
Dror Sharon
Department of Ophthalmology, Hadassah Medical Center, Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem, Israel
Jana Zernant
Department of Ophthalmology, Columbia University, New York, NY, USA
Rando Allikmets
Department of Ophthalmology, Columbia University, New York, NY, USA; Department of Pathology & Cell Biology, Columbia University, New York, NY, USA
Claire-Marie Dhaenens
Department of Human Genetics, Radboud University Medical Center, Nijmegen, the Netherlands; University Lille, Inserm, CHU Lille, U1172 - LilNCog - Lille Neuroscience & Cognition, 59000 Lille, France
Frans P.M. Cremers
Department of Human Genetics, Radboud University Medical Center, Nijmegen, the Netherlands
Summary: The ABCA4 gene is the most frequently mutated Mendelian retinopathy-associated gene. Biallelic variants lead to a variety of phenotypes, however, for thousands of cases the underlying variants remain unknown. Here, we aim to shed further light on the missing heritability of ABCA4-associated retinopathy by analyzing a large cohort of macular dystrophy probands. A total of 858 probands were collected from 26 centers, of whom 722 carried no or one pathogenic ABCA4 variant, while 136 cases carried two ABCA4 alleles, one of which was a frequent mild variant, suggesting that deep-intronic variants (DIVs) or other cis-modifiers might have been missed. After single molecule molecular inversion probes (smMIPs)-based sequencing of the complete 128-kb ABCA4 locus, the effect of putative splice variants was assessed in vitro by midigene splice assays in HEK293T cells. The breakpoints of copy number variants (CNVs) were determined by junction PCR and Sanger sequencing. ABCA4 sequence analysis solved 207 of 520 (39.8%) naive or unsolved cases and 70 of 202 (34.7%) monoallelic cases, while additional causal variants were identified in 54 of 136 (39.7%) probands carrying two variants. Seven novel DIVs and six novel non-canonical splice site variants were detected in a total of 35 alleles and characterized, including the c.6283-321C>G variant leading to a complex splicing defect. Additionally, four novel CNVs were identified and characterized in five alleles. These results confirm that smMIPs-based sequencing of the complete ABCA4 gene provides a cost-effective method to genetically solve retinopathy cases and that several rare structural and splice altering defects remain undiscovered in Stargardt disease cases.
