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A CRISPR and high-content imaging assay compliant with ACMG/AMP guidelines for clinical variant interpretation in ciliopathies

A CRISPR and high-content imaging assay compliant with ACMG/AMP guidelines for clinical variant interpretation in ciliopathies
A CRISPR and high-content imaging assay compliant with ACMG/AMP guidelines for clinical variant interpretation in ciliopathies
Ciliopathies are a broad range of inherited developmental and degenerative diseases associated with structural or functional defects in motile or primary non-motile cilia. There are around 200 known ciliopathy disease genes and whilst genetic testing can provide an accurate diagnosis, 24-60% of ciliopathy patients who undergo genetic testing do not receive a genetic diagnosis. This is partly because following current guidelines from the American College of Medical Genetics and the Association for Molecular Pathology it is difficult to provide a confident clinical diagnosis of disease caused by missense or non-coding variants, which account for more than one third of cases of disease. Mutations in PRPF31 are the second most common cause of the degenerative retinal ciliopathy autosomal dominant retinitis pigmentosa. Here we present a high-throughput high content imaging assay providing quantitative measure of effect of missense variants in PRPF31 which meets the recently published criteria for a baseline standard in vitro test for clinical variant interpretation. This assay utilizes a new PRPF31+/- human retinal cell line generated using CRISPR gene editing to provide a stable cell line with significantly fewer cilia in which novel missense variants are expressed and characterised. We show that high content imaging of cells expressing missense variants in a ciliopathy gene on a null background can allow characterisation of variants according to the cilia phenotype. We hope that this will be a useful tool for clinical characterisation of PRPF31 variants of uncertain significance and can be extended to variant classification in other ciliopathies.
CRISPR/Cas gene editing, Screening, cilia, ciliopathies, genetics
0340-6717
Nazlamova, Liliya
0cc21013-aeeb-4eef-af56-31f6fa0766fd
Thomas, N. Simon
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Cheung, Man-Kim
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Legebeke, Jelmer
f6062b8c-22ac-465c-9528-3bac881137d0
Lord, Jenny
e1909780-36cd-4705-b21e-4580038d4ec6
Pengelly, Reuben
af97c0c1-b568-415c-9f59-1823b65be76d
Tapper, William
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Wheway, Gabrielle
2e547e5d-b921-4243-a071-2208fd4cc090
Nazlamova, Liliya
0cc21013-aeeb-4eef-af56-31f6fa0766fd
Thomas, N. Simon
1a601957-288d-4f12-a9f7-4f4279b7f9b3
Cheung, Man-Kim
8553ef1d-7690-4b23-b10a-5d89eca558c1
Legebeke, Jelmer
f6062b8c-22ac-465c-9528-3bac881137d0
Lord, Jenny
e1909780-36cd-4705-b21e-4580038d4ec6
Pengelly, Reuben
af97c0c1-b568-415c-9f59-1823b65be76d
Tapper, William
9d5ddc92-a8dd-4c78-ac67-c5867b62724c
Wheway, Gabrielle
2e547e5d-b921-4243-a071-2208fd4cc090

Nazlamova, Liliya, Thomas, N. Simon, Cheung, Man-Kim, Legebeke, Jelmer, Lord, Jenny, Pengelly, Reuben, Tapper, William and Wheway, Gabrielle (2020) A CRISPR and high-content imaging assay compliant with ACMG/AMP guidelines for clinical variant interpretation in ciliopathies. Human Genetics. (doi:10.1007/s00439-020-02228-1).

Record type: Article

Abstract

Ciliopathies are a broad range of inherited developmental and degenerative diseases associated with structural or functional defects in motile or primary non-motile cilia. There are around 200 known ciliopathy disease genes and whilst genetic testing can provide an accurate diagnosis, 24-60% of ciliopathy patients who undergo genetic testing do not receive a genetic diagnosis. This is partly because following current guidelines from the American College of Medical Genetics and the Association for Molecular Pathology it is difficult to provide a confident clinical diagnosis of disease caused by missense or non-coding variants, which account for more than one third of cases of disease. Mutations in PRPF31 are the second most common cause of the degenerative retinal ciliopathy autosomal dominant retinitis pigmentosa. Here we present a high-throughput high content imaging assay providing quantitative measure of effect of missense variants in PRPF31 which meets the recently published criteria for a baseline standard in vitro test for clinical variant interpretation. This assay utilizes a new PRPF31+/- human retinal cell line generated using CRISPR gene editing to provide a stable cell line with significantly fewer cilia in which novel missense variants are expressed and characterised. We show that high content imaging of cells expressing missense variants in a ciliopathy gene on a null background can allow characterisation of variants according to the cilia phenotype. We hope that this will be a useful tool for clinical characterisation of PRPF31 variants of uncertain significance and can be extended to variant classification in other ciliopathies.

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More information

Accepted/In Press date: 13 October 2020
e-pub ahead of print date: 23 October 2020
Published date: 23 October 2020
Keywords: CRISPR/Cas gene editing, Screening, cilia, ciliopathies, genetics

Identifiers

Local EPrints ID: 444838
URI: http://eprints.soton.ac.uk/id/eprint/444838
ISSN: 0340-6717
PURE UUID: b4e4dec2-c67b-42fd-9b70-e9f1448d9fbe
ORCID for Jenny Lord: ORCID iD orcid.org/0000-0002-0539-9343
ORCID for Reuben Pengelly: ORCID iD orcid.org/0000-0001-7022-645X
ORCID for William Tapper: ORCID iD orcid.org/0000-0002-5896-1889
ORCID for Gabrielle Wheway: ORCID iD orcid.org/0000-0002-0494-0783

Catalogue record

Date deposited: 06 Nov 2020 17:31
Last modified: 26 Nov 2021 05:58

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Contributors

Author: Liliya Nazlamova
Author: N. Simon Thomas
Author: Man-Kim Cheung
Author: Jelmer Legebeke
Author: Jenny Lord ORCID iD
Author: Reuben Pengelly ORCID iD
Author: William Tapper ORCID iD

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