Identification and functional characterization of a novel MUTYH gene mutation.

e12026 Background: Biallelic germline mutations in MUTYH result in the autosomal recessive syndrome of MUTYH associated polyposis (MAP).Three well-known, common mutations account for the vast majority of identifiable germline mutations, and serve as the basis for current genetic testing strategies. Comprehensive sequencing of MUTYH often identifies variants of uncertain pathologic significance, and studies to determine the pathogenicity of newly identified variants may offer valuable clinical information and mechanistic insights. In the present study we seek to describe the base-excision repair function of a novel MUTYH (p.C306W) mutation identified in a patient with multiple colon polyps and a family history of colon cancers. Methods: A 50 year old patient with >50 adenomas underwent clinical and laboratory evaluation to assess for germline genetic mutations. We performed Sanger sequencing of tumor and germline DNA together with targeted restriction enzyme digest of germline DNA and fragment DNA sequencing of the alleles. We prepared MUTYH proteins with protein liquid chromatography and assessed their mismatched adenine excision repair capacity employing a glycosylase assay. Results: Analysis of the patient's germline DNA revealed an absence of APC mutations, and the presence of the previously well characterized p.G396D MUTYH mutation as well as a novel p.C306W mutation. Targeted restriction enzyme digest demonstrated trans configuration of the p.G396D and p.C306W MUTYH mutations. Mismatched adenine excision functionality of wildtype MUTYH, known mutant controls p.G396D and p.Y179C, and putative mutant p.C306W were assessed in the glycosylase assay. Consistent with previous experimental observations, relative to wildtype MUTYH, the p.G396D and p.Y179C MUTYH mutants demonstrated attenuated adenine excision activities of 43% and 0%, respectively. Comparable to the activity of the pY179C mutant, the novel p.C306D mutant demonstrated 0% adenine excision activity. Subsequent tumor analysis demonstrated G:C to T:A transversion in the APC gene in somatic DNA derived from an adenoma. Conclusions: Experimental and clinical data demonstrate that p.C306D MUTYH is a pathogenic mutation contributing to the phenotype of MAP.