Cost-effective sequence analysis of 113 genes in 1,192 probands with retinitis pigmentosa and Leber congenital amaurosis

Daan M Panneman^{1

2}, Rebekkah J Hitti-Malin^{1

2}, Lara K Holtes¹, Suzanne E de Bruijn^{1

2}, Janine Reurink^{1

2}, Erica G M Boonen¹, Muhammad Imran Khan¹, Manir Ali³, Sten Andréasson⁴, Elfride De Baere^{5

6}, Sandro Banfi^{7

8}, Miriam Bauwens^{5

6}, Tamar Ben-Yosef⁹, Béatrice Bocquet^{10

11}, Marieke De Bruyne^{5

6}, Berta de la Cerda¹², Frauke Coppieters^{5

6

13}, Pietro Farinelli¹⁴, Thomas Guignard¹⁵, Chris F Inglehearn³, Marianthi Karali^{8

16}, Ulrika Kjellström⁴, Robert Koenekoop^{17

18}, Bart de Koning¹⁹, Bart P Leroy^{20

21

22

6}, Martin McKibbin^{3

23}, Isabelle Meunier^{10

11}, Konstantinos Nikopoulos²⁴, Koji M Nishiguchi²⁵, James A Poulter³, Carlo Rivolta^{26

27

28}, Enrique Rodríguez de la Rúa²⁹, Patrick Saunders³⁰, Francesca Simonelli¹⁶, Yasmin Tatour⁹, Francesco Testa¹⁶, Alberta A H J Thiadens³¹, Carmel Toomes³, Anna M Tracewska¹, Hoai Viet Tran³², Hiroaki Ushida²⁵, Veronika Vaclavik³², Virginie J M Verhoeven^{31

33}, Maartje van de Vorst¹, Christian Gilissen^{1

34}, Alexander Hoischen^{1

34

35}, Frans P M Cremers^{1

2}, Susanne Roosing^{1

2}

Affiliations

¹ Department of Human Genetics, Radboud University Medical Center, Nijmegen, Netherlands.
² Donders Institute for Brain, Cognition and Behavior, Radboud University Medical Center, Nijmegen, Netherlands.
³ Division of Molecular Medicine, Leeds Institute of Medical Research, St. James's University Hospital, University of Leeds, Leeds, United Kingdom.
⁴ Department of Ophthalmology and Clinical Sciences Lund, Lund University, Skane University Hospital, Lund, Sweden.
⁵ Department of Biomolecular Medicine, Ghent University, Ghent, Belgium.
⁶ Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium.
⁷ Telethon Institute of Genetics and Medicine, Pozzuoli, Italy.
⁸ Department of Precision Medicine, University of Campania "Luigi Vanvitelli", Naples, Italy.
⁹ Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel.
¹⁰ National Reference Centre for Inherited Sensory Diseases, University of Montpellier, Montpellier University Hospital, Sensgene Care Network, ERN-EYE Network, Montpellier, France.
¹¹ Institute for Neurosciences of Montpellier (INM), L'Institut National de la Santé et de la Recherche Médicale, University of Montpellier, L'Institut National de la Santé et de la Recherche Médicale, Montpellier, France.
¹² Andalusian Center for Molecular Biology and Regenerative Medicine (CABIMER), Seville, Spain.
¹³ Department of Pharmaceutics, Ghent University, Ghent, Belgium.
¹⁴ Department of Computational Biology, Unit of Medical Genetics, University of Lausanne, Lausanne, Switzerland.
¹⁵ Chromosomal Genetics Unit, University Hospital of Montpellier, Montpellier, France.
¹⁶ Eye Clinic, Multidisciplinary Department of Medical, Surgical and Dental Sciences, University of Campania Luigi Vanvitelli, Naples, Italy.
¹⁷ McGill University Health Center (MUHC) Research Institute, Montreal, QC, Canada.
¹⁸ Departments of Paediatric Surgery, Human Genetics, and Adult Ophthalmology, McGill University Health Center, Montreal, QC, Canada.
¹⁹ Department of Clinical Genetics, Maastricht University Medical Center+ (MUMC+), Maastricht, Netherlands.
²⁰ Department of Ophthalmology, Ghent University Hospital, Ghent, Belgium.
²¹ Department of Head & Skin, Ghent University, Ghent, Belgium.
²² Division of Ophthalmology & Center for Cellular & Molecular Therapeutics, Children's Hospital of Philadelphia, Philadelphia, PA, United States.
²³ Department of Ophthalmology, St. James's University Hospital, Leeds, United Kingdom.
²⁴ Laboratory of molecular diagnostics, UNILABS SA, Lausanne, Switzerland.
²⁵ Department of Ophthalmology, Nagoya University Graduate School of Medicine, Nagoya, Japan.
²⁶ Institute of Molecular and Clinical Ophthalmology Basel, Basel, Switzerland.
²⁷ Department of Ophthalmology, University of Basel, Basel, Switzerland.
²⁸ Department of Genetics and Genome Biology, University of Leicester, Leicester, United Kingdom.
²⁹ Department of Ophthalmology, Retics Patologia Ocular, OFTARED, Instituto de Salud Carlos III, University Hospital Virgen Macarena, Madrid, Spain.
³⁰ Molecular Loop Biosciences Inc., Woburn, MA, United States.
³¹ Department of Ophthalmology, Erasmus, Rotterdam, Netherlands.
³² Oculogenetic Unit, University Eye Hospital Jules Gonin, Geneva, Switzerland.
³³ Department of Clinical Genetics, Erasmus, Rotterdam, Netherlands.
³⁴ Radboud Institute of Molecular Life Sciences, Radboud University Medical Center, Nijmegen, Netherlands.
³⁵ Department of Internal Medicine and Radboud Center for Infectious Diseases, Radboud University Medical Center, Nijmegen, Netherlands.

PMID: 36819107
PMCID: PMC9936074
DOI: 10.3389/fcell.2023.1112270

Cost-effective sequence analysis of 113 genes in 1,192 probands with retinitis pigmentosa and Leber congenital amaurosis

Daan M Panneman et al. Front Cell Dev Biol. 2023.

. 2023 Feb 3:11:1112270.

doi: 10.3389/fcell.2023.1112270. eCollection 2023.

Authors

Affiliations

¹ Department of Human Genetics, Radboud University Medical Center, Nijmegen, Netherlands.
² Donders Institute for Brain, Cognition and Behavior, Radboud University Medical Center, Nijmegen, Netherlands.
³ Division of Molecular Medicine, Leeds Institute of Medical Research, St. James's University Hospital, University of Leeds, Leeds, United Kingdom.
⁴ Department of Ophthalmology and Clinical Sciences Lund, Lund University, Skane University Hospital, Lund, Sweden.
⁵ Department of Biomolecular Medicine, Ghent University, Ghent, Belgium.
⁶ Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium.
⁷ Telethon Institute of Genetics and Medicine, Pozzuoli, Italy.
⁸ Department of Precision Medicine, University of Campania "Luigi Vanvitelli", Naples, Italy.
⁹ Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel.
¹⁰ National Reference Centre for Inherited Sensory Diseases, University of Montpellier, Montpellier University Hospital, Sensgene Care Network, ERN-EYE Network, Montpellier, France.
¹¹ Institute for Neurosciences of Montpellier (INM), L'Institut National de la Santé et de la Recherche Médicale, University of Montpellier, L'Institut National de la Santé et de la Recherche Médicale, Montpellier, France.
¹² Andalusian Center for Molecular Biology and Regenerative Medicine (CABIMER), Seville, Spain.
¹³ Department of Pharmaceutics, Ghent University, Ghent, Belgium.
¹⁴ Department of Computational Biology, Unit of Medical Genetics, University of Lausanne, Lausanne, Switzerland.
¹⁵ Chromosomal Genetics Unit, University Hospital of Montpellier, Montpellier, France.
¹⁶ Eye Clinic, Multidisciplinary Department of Medical, Surgical and Dental Sciences, University of Campania Luigi Vanvitelli, Naples, Italy.
¹⁷ McGill University Health Center (MUHC) Research Institute, Montreal, QC, Canada.
¹⁸ Departments of Paediatric Surgery, Human Genetics, and Adult Ophthalmology, McGill University Health Center, Montreal, QC, Canada.
¹⁹ Department of Clinical Genetics, Maastricht University Medical Center+ (MUMC+), Maastricht, Netherlands.
²⁰ Department of Ophthalmology, Ghent University Hospital, Ghent, Belgium.
²¹ Department of Head & Skin, Ghent University, Ghent, Belgium.
²² Division of Ophthalmology & Center for Cellular & Molecular Therapeutics, Children's Hospital of Philadelphia, Philadelphia, PA, United States.
²³ Department of Ophthalmology, St. James's University Hospital, Leeds, United Kingdom.
²⁴ Laboratory of molecular diagnostics, UNILABS SA, Lausanne, Switzerland.
²⁵ Department of Ophthalmology, Nagoya University Graduate School of Medicine, Nagoya, Japan.
²⁶ Institute of Molecular and Clinical Ophthalmology Basel, Basel, Switzerland.
²⁷ Department of Ophthalmology, University of Basel, Basel, Switzerland.
²⁸ Department of Genetics and Genome Biology, University of Leicester, Leicester, United Kingdom.
²⁹ Department of Ophthalmology, Retics Patologia Ocular, OFTARED, Instituto de Salud Carlos III, University Hospital Virgen Macarena, Madrid, Spain.
³⁰ Molecular Loop Biosciences Inc., Woburn, MA, United States.
³¹ Department of Ophthalmology, Erasmus, Rotterdam, Netherlands.
³² Oculogenetic Unit, University Eye Hospital Jules Gonin, Geneva, Switzerland.
³³ Department of Clinical Genetics, Erasmus, Rotterdam, Netherlands.
³⁴ Radboud Institute of Molecular Life Sciences, Radboud University Medical Center, Nijmegen, Netherlands.
³⁵ Department of Internal Medicine and Radboud Center for Infectious Diseases, Radboud University Medical Center, Nijmegen, Netherlands.

PMID: 36819107
PMCID: PMC9936074
DOI: 10.3389/fcell.2023.1112270

Abstract

Introduction: Retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA) are two groups of inherited retinal diseases (IRDs) where the rod photoreceptors degenerate followed by the cone photoreceptors of the retina. A genetic diagnosis for IRDs is challenging since >280 genes are associated with these conditions. While whole exome sequencing (WES) is commonly used by diagnostic facilities, the costs and required infrastructure prevent its global applicability. Previous studies have shown the cost-effectiveness of sequence analysis using single molecule Molecular Inversion Probes (smMIPs) in a cohort of patients diagnosed with Stargardt disease and other maculopathies. Methods: Here, we introduce a smMIPs panel that targets the exons and splice sites of all currently known genes associated with RP and LCA, the entire RPE65 gene, known causative deep-intronic variants leading to pseudo-exons, and part of the RP17 region associated with autosomal dominant RP, by using a total of 16,812 smMIPs. The RP-LCA smMIPs panel was used to screen 1,192 probands from an international cohort of predominantly RP and LCA cases. Results and discussion: After genetic analysis, a diagnostic yield of 56% was obtained which is on par with results from WES analysis. The effectiveness and the reduced costs compared to WES renders the RP-LCA smMIPs panel a competitive approach to provide IRD patients with a genetic diagnosis, especially in countries with restricted access to genetic testing.

Keywords: cost-effective; high-throughput; inherited retinal diseases; smMIPs; targeted gene sequencing.

Copyright © 2023 Panneman, Hitti-Malin, Holtes, de Bruijn, Reurink, Boonen, Khan, Ali, Andréasson, De Baere, Banfi, Bauwens, Ben-Yosef, Bocquet, De Bruyne, Cerda, Coppieters, Farinelli, Guignard, Inglehearn, Karali, Kjellström, Koenekoop, de Koning, Leroy, McKibbin, Meunier, Nikopoulos, Nishiguchi, Poulter, Rivolta, Rodríguez de la Rúa, Saunders, Simonelli, Tatour, Testa, Thiadens, Toomes, Tracewska, Tran, Ushida, Vaclavik, Verhoeven, van de Vorst, Gilissen, Hoischen, Cremers and Roosing.

PubMed Disclaimer

Conflict of interest statement

PS is an employee of Molecular Loop Biosciences Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

**FIGURE 1**
Average read coverage per smMIP in the RP-LCA panel.

**FIGURE 2**
Average coverage per nucleotide. The average number of reads per nucleotide for each individual gene are indicated by the black horizontal line within the box, the range is indicated by the whiskers. The horizontal dashed line depicts the average overall coverage (374x).

**FIGURE 3**
Number of solved cases per individual gene. For all genes, the inheritance mode is depicted in either black (autosomal recessive), white (autosomal dominant), or dashed (X-linked inheritance).

**FIGURE 4**
Minigene analysis of the splicing effect of *RPE65*:c.675C>A. **(A)** A minigene construct containing either wild-type sequence or the mutant c.675C>A variant in exon 7 was generated spanning exons 6-10 of *RPE65* and was flanked by exons 3 and 5 of the *RHO* gene in the pCI-NEO-RHO vectors **(B)** After gel electrophoresis of the RT-PCR product, we observed two fragments in the wild-type situation. One fragment of 907 nt and one fragment of 825 nt corresponding to the skipping of the 82 nt exon 7. For 675C>A, we exclusively observed the 825 nt fragment, suggesting complete skipping of exon 7.

See this image and copyright information in PMC

References

1. Abu-Safieh L., Alrashed M., Anazi S., Alkuraya H., Khan A. O., Al-Owain M., et al. (2013). Autozygome-guided exome sequencing in retinal dystrophy patients reveals pathogenetic mutations and novel candidate disease genes. Genome Res. 23 (2), 236–247. 10.1101/gr.144105.112 - DOI - PMC - PubMed
1. Al-Khuzaei S., Broadgate S., Foster C. R., Shah M., Yu J., Downes S. M., et al. (2021). An overview of the genetics of ABCA4 retinopathies, an evolving story. Genes 12 (8), 1241. 10.3390/genes12081241 - DOI - PMC - PubMed
1. Black G. C., Sergouniotis P., Sodi A., Leroy B. P., Van Cauwenbergh C., Liskova P., et al. (2021). The need for widely available genomic testing in rare eye diseases: An ERN-EYE position statement. Orphanet J. Rare Dis. 16 (1), 142. 10.1186/s13023-021-01756-x - DOI - PMC - PubMed
1. Cartegni L., Wang J., Zhu Z., Zhang M. Q., Krainer A. R. (2003). ESEfinder: A web resource to identify exonic splicing enhancers. Nucleic Acids Res. 31 (13), 3568–3571. 10.1093/nar/gkg616 - DOI - PMC - PubMed
1. Cornelis S. S., Runhart E. H., Bauwens M., Corradi Z., De Baere E., Roosing S., et al. (2022). Personalized genetic counseling for Stargardt disease: Offspring risk estimates based on variant severity. Am. J. Hum. Genet. 109 (3), 498–507. 10.1016/j.ajhg.2022.01.008 - DOI - PMC - PubMed

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Cost-effective sequence analysis of 113 genes in 1,192 probands with retinitis pigmentosa and Leber congenital amaurosis

Affiliations

Cost-effective sequence analysis of 113 genes in 1,192 probands with retinitis pigmentosa and Leber congenital amaurosis

Authors

Affiliations

Abstract

Conflict of interest statement

Figures

References

LinkOut - more resources

Full Text Sources

Research Materials