potassium-bromate and Adenomatous-Polyposis-Coli

The MUTYH DNA glycosylase counteracts mutagenesis by removing adenine misincorporated opposite DNA 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG). Biallelic germline mutations in MUTYH cause the autosomal recessive MUTYH-associated polyposis (MAP). The impact on genetic instability of the p.Tyr179Cys and p.Arg245His MUTYH variants was evaluated in lymphoblastoid cell lines (LCLs) derived from MAP patients and their relatives in comparison to wild-type LCLs. No difference in MUTYH expression was identified between wild type and LCLs with the p.Tyr179Cys, while the p.Arg245His mutation was associated with an unstable MUTYH protein. LCLs homozygous for the p.Tyr179Cys or the p.Arg245His variant contained increased DNA 8-oxodG levels and exhibited a mutator phenotype at the PIG-A gene. The extent of the increased spontaneous mutation frequency was 3-fold (range 1.6- to 4.6-fold) in four independent LCLs carrying the p.Tyr179Cys variant, while a larger increase (6-fold) was observed in two p.Arg245His LCLs. A similar hypermutability and S-phase delay following treatment with KBrO3 was observed in LCLs homozygous for either variant. When genetic instability was investigated in monoallelic p.Arg245His carriers, mutant frequencies showed an increase which is intermediate between wild-type and homozygous cells, whereas the mutator effect in heterozygous p.Tyr179Cys LCLs was similar to that in homozygotes. These findings indicate that the type of MUTYH mutation can affect the extent of genome instability associated with MUTYH inactivation. In addition, the mild spontaneous mutator phenotype observed in monoallelic carriers highlights the biological importance of this gene in the protection of the genome against endogenous DNA damage.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Adenomatous Polyposis Coli; Adult; Bromates; Cell Cycle; Cell Line; Deoxyguanosine; DNA Glycosylases; Genetic Variation; Genomic Instability; Humans; Male; Membrane Proteins; Middle Aged; Polymorphism, Single Nucleotide; Protein Stability; Young Adult

MUTYH-associated polyposis (MAP) is a colorectal cancer syndrome, due to biallelic mutations of MUTYH. This Base Excision Repair gene encodes for a DNA glycosylase that specifically mitigates the high mutagenic potential of the 8-hydroxyguanine (8-oxodG) along the DNA. Aim of this study was to characterize the biological effects, in a mammalian cell background, of human MUTYH mutations identified in MAP patients (137insIW [c.411_416dupATGGAT; p.137insIleTrp]; R171W [c.511C>T; p.Arg171Trp]; E466del [c.1395_1397delGGA; p.Glu466del]; Y165C [c.494A>G; p.Tyr165Cys]; and G382D [c.1145G>A; p.Gly382Asp]). We set up a novel assay in which the human proteins were expressed in Mutyh(-/-) mouse defective cells. Several parameters, including accumulation of 8-oxodG in the genome and hypersensitivity to oxidative stress, were then used to evaluate the consequences of MUTYH expression. Human proteins were also obtained from Escherichia coli and their glycosylase activity was tested in vitro. The cell-based analysis demonstrated that all MUTYH variants we investigated were dysfunctional in Base Excision Repair. In vitro data complemented the in vivo observations, with the exception of the G382D mutant, which showed a glycosylase activity very similar to the wild-type protein. Our cell-based assay can provide useful information on the significance of MUTYH variants, improving molecular diagnosis and genetic counseling in families with mutations of uncertain pathogenicity.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Adenomatous Polyposis Coli; Animals; Bromates; Cell Death; Cell Line; Deoxyguanosine; DNA Glycosylases; DNA Repair; Enzyme Assays; Fibroblasts; Fluorescent Antibody Technique; Gamma Rays; Genetic Predisposition to Disease; Humans; Kinetics; Mice; Mutant Proteins; Mutation; Protein Transport