TY - JOUR
T1 - Identification and characterisation of spontaneous mutations causing deafness from a targeted knockout programme
AU - Lewis, Morag
AU - Ingham, Neil
AU - Chen, Jing
AU - Pearson, Selina
AU - Di Domenico, Francesca
AU - Rekhi, Sohinder
AU - Allen, Rochelle
AU - Drake, Matthew
AU - Willaert, Annelore
AU - Rook, Vicky
AU - Pass, Johanna C.
AU - Keane, Thomas M
AU - Adams, David J.
AU - Tucker, Abigail
AU - White, Jacqueline K.
AU - Steel, Karen
N1 - Funding Information:
This work was supported by Wellcome (098051, 100669), Medical Research Council (MC_qA137918; G0300212 to KPS.), the European Commission (EUMODIC contract No. LSHG-CT-2006–037188 to KPS) and the BBSRC (BB/M02069X/1 to KPS) and King’s College London. AST is funded by the Wellcome Trust (102889/Z/13/Z). TK was supported by the Medical Research Council (MR/L007428/1) and BBSRC (BB/M000281/1). AW was supported by a grant from the Research Foundation Flanders (1700317 N). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.
Funding Information:
We are grateful to Cassandra Whelan for assistance with the MYO7A staining on the Atp2b2 Tkh and Tbx1 ttch mutants, Seham Ebrahim for additional analysis on the Atp2b2 Tkh mutants, Elysia James for protein modelling of KLHL18, Maria Lachgar-Ruiz for assistance with genotyping, Samoela Rexhaj for help with the mapping of the rhme mutation, Hannah Thompson for initial analysis of the Tbx1 ttch mutants, Zahra Hance for her work on the vthr mutant, Rosalind Lacey and James Bussell for assistance with mouse colony management, and the Mouse Genetics Project for initial phenotyping of all these mutants. Scanning electron microscopy was carried out at the Wellcome Sanger Institute and King’s College London Centre for Ultrastructural Imaging.
Funding Information:
This research was funded in whole, or in part, by the Wellcome Trust [089051, 100,669, 102,889/Z/13/Z]. For the purpose of open access, the author has applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission. According to UK research councils’ Common Principles on Data Policy, and Wellcome Trust’s Policy on data, software and materials management and sharing, all data supporting this study will be openly available at https://www.ebi.ac.uk/ena/browser/view/PRJEB2585 , https://www.ebi.ac.uk/ena/browser/view/PRJEB5221 , and https://www.ebi.ac.uk/ena/browser/view/PRJEB45713 .
Publisher Copyright:
© 2022, The Author(s).
PY - 2022/12
Y1 - 2022/12
N2 - Background: Mice carrying targeted mutations are important for investigating gene function and the role of genes in disease, but off-target mutagenic effects associated with the processes of generating targeted alleles, for instance using Crispr, and culturing embryonic stem cells, offer opportunities for spontaneous mutations to arise. Identifying spontaneous mutations relies on the detection of phenotypes segregating independently of targeted alleles, and having a broad estimate of the level of mutations generated by intensive breeding programmes is difficult given that many phenotypes are easy to miss if not specifically looked for. Here we present data from a large, targeted knockout programme in which mice were analysed through a phenotyping pipeline. Such spontaneous mutations segregating within mutant lines may confound phenotypic analyses, highlighting the importance of record-keeping and maintaining correct pedigrees. Results: Twenty-five lines out of 1311 displayed different deafness phenotypes that did not segregate with the targeted allele. We observed a variety of phenotypes by Auditory Brainstem Response (ABR) and behavioural assessment and isolated eight lines showing early-onset severe progressive hearing loss, later-onset progressive hearing loss, low frequency hearing loss, or complete deafness, with vestibular dysfunction. The causative mutations identified include deletions, insertions, and point mutations, some of which involve new genes not previously associated with deafness while others are new alleles of genes known to underlie hearing loss. Two of the latter show a phenotype much reduced in severity compared to other mutant alleles of the same gene. We investigated the ES cells from which these lines were derived and determined that only one of the 8 mutations could have arisen in the ES cell, and in that case, only after targeting. Instead, most of the non-segregating mutations appear to have occurred during breeding of mutant mice. In one case, the mutation arose within the wildtype colony used for expanding mutant lines. Conclusions: Our data show that spontaneous mutations with observable effects on phenotype are a common side effect of intensive breeding programmes, including those underlying targeted mutation programmes. Such spontaneous mutations segregating within mutant lines may confound phenotypic analyses, highlighting the importance of record-keeping and maintaining correct pedigrees.
AB - Background: Mice carrying targeted mutations are important for investigating gene function and the role of genes in disease, but off-target mutagenic effects associated with the processes of generating targeted alleles, for instance using Crispr, and culturing embryonic stem cells, offer opportunities for spontaneous mutations to arise. Identifying spontaneous mutations relies on the detection of phenotypes segregating independently of targeted alleles, and having a broad estimate of the level of mutations generated by intensive breeding programmes is difficult given that many phenotypes are easy to miss if not specifically looked for. Here we present data from a large, targeted knockout programme in which mice were analysed through a phenotyping pipeline. Such spontaneous mutations segregating within mutant lines may confound phenotypic analyses, highlighting the importance of record-keeping and maintaining correct pedigrees. Results: Twenty-five lines out of 1311 displayed different deafness phenotypes that did not segregate with the targeted allele. We observed a variety of phenotypes by Auditory Brainstem Response (ABR) and behavioural assessment and isolated eight lines showing early-onset severe progressive hearing loss, later-onset progressive hearing loss, low frequency hearing loss, or complete deafness, with vestibular dysfunction. The causative mutations identified include deletions, insertions, and point mutations, some of which involve new genes not previously associated with deafness while others are new alleles of genes known to underlie hearing loss. Two of the latter show a phenotype much reduced in severity compared to other mutant alleles of the same gene. We investigated the ES cells from which these lines were derived and determined that only one of the 8 mutations could have arisen in the ES cell, and in that case, only after targeting. Instead, most of the non-segregating mutations appear to have occurred during breeding of mutant mice. In one case, the mutation arose within the wildtype colony used for expanding mutant lines. Conclusions: Our data show that spontaneous mutations with observable effects on phenotype are a common side effect of intensive breeding programmes, including those underlying targeted mutation programmes. Such spontaneous mutations segregating within mutant lines may confound phenotypic analyses, highlighting the importance of record-keeping and maintaining correct pedigrees.
UR - http://www.scopus.com/inward/record.url?scp=85126383004&partnerID=8YFLogxK
U2 - https://doi.org/10.1186/s12915-022-01257-8
DO - https://doi.org/10.1186/s12915-022-01257-8
M3 - Article
SN - 1741-7007
VL - 20
JO - BMC Biology
JF - BMC Biology
IS - 1
M1 - 67
ER -