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Functional Complementation Assay for 47 MUTYH Variants in a MutY-Disrupted Escherichia Coli Strain

MUTYH-associated polyposis (MAP) is an adenomatous polyposis transmitted in an autosomal-recessive pattern, involving biallelic inactivation of the MUTYH gene. Loss of a functional MUTYH protein will result in the accumulation of G:T mismatched DNA caused by oxidative damage. Although p.Y179C and p....

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Autores principales: Komine, Keigo, Shimodaira, Hideki, Takao, Masashi, Soeda, Hiroshi, Zhang, Xiaofei, Takahashi, Masanobu, Ishioka, Chikashi
Formato: Online Artículo Texto
Lenguaje:English
Publicado: John Wiley & Sons, Ltd 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4682456/
https://www.ncbi.nlm.nih.gov/pubmed/25820570
http://dx.doi.org/10.1002/humu.22794
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author Komine, Keigo
Shimodaira, Hideki
Takao, Masashi
Soeda, Hiroshi
Zhang, Xiaofei
Takahashi, Masanobu
Ishioka, Chikashi
author_facet Komine, Keigo
Shimodaira, Hideki
Takao, Masashi
Soeda, Hiroshi
Zhang, Xiaofei
Takahashi, Masanobu
Ishioka, Chikashi
author_sort Komine, Keigo
collection PubMed
description MUTYH-associated polyposis (MAP) is an adenomatous polyposis transmitted in an autosomal-recessive pattern, involving biallelic inactivation of the MUTYH gene. Loss of a functional MUTYH protein will result in the accumulation of G:T mismatched DNA caused by oxidative damage. Although p.Y179C and p.G396D are the two most prevalent MUTYH variants, more than 200 missense variants have been detected. It is difficult to determine whether these variants are disease-causing mutations or single-nucleotide polymorphisms. To understand the functional consequences of these variants, we generated 47 MUTYH gene variants via site-directed mutagenesis, expressed the encoded proteins in MutY-disrupted Escherichia coli, and assessed their abilities to complement the functional deficiency in the E. coli by monitoring spontaneous mutation rates. Although the majority of variants exhibited intermediate complementation relative to the wild type, some variants severely interfered with this complementation. However, some variants retained functioning similar to the wild type. In silico predictions of functional effects demonstrated a good correlation. Structural prediction of MUTYH based on the MutY protein structure allowed us to interpret effects on the protein stability or catalytic activity. These data will be useful for evaluating the functional consequences of missense MUTYH variants detected in patients with suspected MAP.
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spelling pubmed-46824562015-12-23 Functional Complementation Assay for 47 MUTYH Variants in a MutY-Disrupted Escherichia Coli Strain Komine, Keigo Shimodaira, Hideki Takao, Masashi Soeda, Hiroshi Zhang, Xiaofei Takahashi, Masanobu Ishioka, Chikashi Hum Mutat Research Articles MUTYH-associated polyposis (MAP) is an adenomatous polyposis transmitted in an autosomal-recessive pattern, involving biallelic inactivation of the MUTYH gene. Loss of a functional MUTYH protein will result in the accumulation of G:T mismatched DNA caused by oxidative damage. Although p.Y179C and p.G396D are the two most prevalent MUTYH variants, more than 200 missense variants have been detected. It is difficult to determine whether these variants are disease-causing mutations or single-nucleotide polymorphisms. To understand the functional consequences of these variants, we generated 47 MUTYH gene variants via site-directed mutagenesis, expressed the encoded proteins in MutY-disrupted Escherichia coli, and assessed their abilities to complement the functional deficiency in the E. coli by monitoring spontaneous mutation rates. Although the majority of variants exhibited intermediate complementation relative to the wild type, some variants severely interfered with this complementation. However, some variants retained functioning similar to the wild type. In silico predictions of functional effects demonstrated a good correlation. Structural prediction of MUTYH based on the MutY protein structure allowed us to interpret effects on the protein stability or catalytic activity. These data will be useful for evaluating the functional consequences of missense MUTYH variants detected in patients with suspected MAP. John Wiley & Sons, Ltd 2015-07 2015-06-18 /pmc/articles/PMC4682456/ /pubmed/25820570 http://dx.doi.org/10.1002/humu.22794 Text en © 2015 The Authors. **Human Mutation published by Wiley Periodicals, Inc. http://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the work is properly cited, the use is non-commercial and no modifications or adaptations are made.
spellingShingle Research Articles
Komine, Keigo
Shimodaira, Hideki
Takao, Masashi
Soeda, Hiroshi
Zhang, Xiaofei
Takahashi, Masanobu
Ishioka, Chikashi
Functional Complementation Assay for 47 MUTYH Variants in a MutY-Disrupted Escherichia Coli Strain
title Functional Complementation Assay for 47 MUTYH Variants in a MutY-Disrupted Escherichia Coli Strain
title_full Functional Complementation Assay for 47 MUTYH Variants in a MutY-Disrupted Escherichia Coli Strain
title_fullStr Functional Complementation Assay for 47 MUTYH Variants in a MutY-Disrupted Escherichia Coli Strain
title_full_unstemmed Functional Complementation Assay for 47 MUTYH Variants in a MutY-Disrupted Escherichia Coli Strain
title_short Functional Complementation Assay for 47 MUTYH Variants in a MutY-Disrupted Escherichia Coli Strain
title_sort functional complementation assay for 47 mutyh variants in a muty-disrupted escherichia coli strain
topic Research Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4682456/
https://www.ncbi.nlm.nih.gov/pubmed/25820570
http://dx.doi.org/10.1002/humu.22794
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