MUTYH mutations associated with familial adenomatous polyposis: functional characterization by a mammalian cell‐based assay

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. Hum Mutat 30:1–8, 2009. © 2009 Wiley‐Liss, Inc.