Whole genome sequencing for USH2A-associated disease reveals several pathogenic deep-intronic variants that are amenable to splice correction

Janine Reurink, Nicole Weisschuh, Alejandro Garanto, Adrian Dockery, L. Ingeborgh van den Born, Isabelle Fajardy, Lonneke Haer-Wigman, Susanne Kohl, Bernd Wissinger, G. Jane Farrar, Tamar Ben-Yosef, Fatma Kivrak Pfiffner, Wolfgang Berger, Marianna E. Weener, Lubica Dudakova, Petra Liskova, Dror Sharon, Manar Salameh, Ashley Offenheim, Elise HeonGiorgia Girotto, Paolo Gasparini, Anna Morgan, Arthur A. Bergen, Jacoline B. ten Brink, Caroline C.W. Klaver, Lisbeth Tranebjærg, Nanna D. Rendtorff, Sascha Vermeer, Jeroen J. Smits, Ronald J.E. Pennings, Marco Aben, Jaap Oostrik, Galuh D.N. Astuti, Jordi Corominas Galbany, Hester Y. Kroes, Milan Phan, Wendy A.G. van Zelst-Stams, Alberta A.H.J. Thiadens, Joke B.G.M. Verheij, Mary J. van Schooneveld, Suzanne E. de Bruijn, Catherina H.Z. Li, Carel B. Hoyng, Christian Gilissen, Lisenka E.L.M. Vissers, Frans P.M. Cremers, Hannie Kremer, Erwin van Wijk*, Susanne Roosing*

*Corresponding author for this work

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Abstract

A significant number of individuals with a rare disorder such as Usher syndrome (USH) and (non-)syndromic autosomal recessive retinitis pigmentosa (arRP) remain genetically unexplained. Therefore, we assessed subjects suspected of USH2A-associated disease and no or mono-allelic USH2A variants using whole genome sequencing (WGS) followed by an improved pipeline for variant interpretation to provide a conclusive diagnosis. One hundred subjects were screened using WGS to identify causative variants in USH2A or other USH/arRP-associated genes. In addition to the existing variant interpretation pipeline, a particular focus was put on assessing splice-affecting properties of variants, both in silico and in vitro. Also structural variants were extensively addressed. For variants resulting in pseudoexon inclusion, we designed and evaluated antisense oligonucleotides (AONs) using minigene splice assays and patient-derived photoreceptor precursor cells. Biallelic variants were identified in 49 of 100 subjects, including novel splice-affecting variants and structural variants, in USH2A or arRP/USH-associated genes. Thirteen variants were shown to affect USH2A pre-mRNA splicing, including four deep-intronic USH2A variants resulting in pseudoexon inclusion, which could be corrected upon AON treatment. We have shown that WGS, combined with a thorough variant interpretation pipeline focused on assessing pre-mRNA splicing defects and structural variants, is a powerful method to provide subjects with a rare genetic condition, a (likely) conclusive genetic diagnosis. This is essential for the development of future personalized treatments and for patients to be eligible for such treatments.

Original languageEnglish
Article number100181
JournalHuman Genetics and Genomics Advances
Volume4
Issue number2
DOIs
Publication statusPublished - 13 Apr 2023

Bibliographical note

Funding Information:
The work of J.R. was funded by the Velux Stiftung (Project number: 1129 to H.K. S.R. F.P.M.C. and E.v.W.). The work of S.R. is funded by the Foundation Fighting Blindness-career development award (CD-GE-0621-0809-RAD to S.R.). The research was also supported by the Algemene Nederlandse Vereniging ter voorkoming van Blindheid, Oogfonds, Landelijke Stichting voor Blinden en Slechtzienden, Rotterdamse Stichting Blindenbelangen, Stichting Blindenhulp, Stichting tot Verbetering van het Lot der Blinden, and Stichting Blinden-Penning (to S.R. and F.P.M.C.). The work of S.E.d.B. was funded by the European Union's Horizon 2020 Research and Innovation Program under the EJP RD COFUND-EJP N° 825575 (to F.P.M.C. and S.R.). The work of P.L. and L.D. was funded by EJP RD Solve-RET (EJPRD19-234) and GAUK (136121). We thank all individuals who participated in this study. We thank the Radboud Stem Cell Technology Center for the generation of iPSCs and the Radboud Technology Center Genomics for their assistance with whole genome sequencing. We also thank Saskia van der Velde-Visser and Marlie Jacobs-Camps for DNA sample registration and administration. The authors declare no competing interests.

Funding Information:
The work of J.R. was funded by the Velux Stiftung (Project number: 1129 to H.K., S.R., F.P.M.C., and E.v.W.). The work of S.R. is funded by the Foundation Fighting Blindness -career development award (CD-GE-0621-0809-RAD to S.R.). The research was also supported by the Algemene Nederlandse Vereniging ter voorkoming van Blindheid , Oogfonds , Landelijke Stichting voor Blinden en Slechtzienden , Rotterdamse Stichting Blindenbelangen , Stichting Blindenhulp , Stichting tot Verbetering van het Lot der Blinden , and Stichting Blinden-Penning (to S.R. and F.P.M.C.). The work of S.E.d.B. was funded by the European Union’s Horizon 2020 Research and Innovation Program under the EJP RD COFUND-EJP N° 825575 (to F.P.M.C. and S.R.). The work of P.L. and L.D. was funded by EJP RD Solve-RET (EJPRD19-234) and GAUK (136121).

Publisher Copyright:
© 2023 The Author(s)

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