Functional analysis of human MLH1 mutations in Saccharomyces cerevisiae.
Hereditary non-polyposis colorectal cancer (HNPCC; OMIM 120435-6) is a cancer-susceptibility syndrome linked to inherited defects in human mismatch repair (MMR) genes. Germline missense human MLH1 (hMLH1) mutations are frequently detected in HNPCC (ref. 3), making functional characterization of mutations in hMLH1 critical to the development of genetic testing for HNPCC. Here, we describe a new method for detecting mutations in hMLH1 using a dominant mutator effect of hMLH1 cDNA expressed in Saccharomyces cerevisiae. The majority of hMLH1 missense mutations identified in HNPCC patients abolish the dominant mutator effect. Furthermore, PCR amplification of hMLH1 cDNA from mRNA from a HNPCC patient, followed by in vivo recombination into a gap expression vector, allowed detection of a heterozygous loss-of-function missense mutation in hMLH1 using this method. This functional assay offers a simple method for detecting and evaluating pathogenic mutations in hMLH1.
Department of Clinical Oncology, Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan.
H
H
Shimodaira
N
N
Filosi
H
H
Shibata
T
T
Suzuki
P
P
Radice
R
R
Kanamaru
S H
SH
Friend
R D
RD
Kolodner
C
C
Ishioka
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D003123:Colorectal Neoplasms, Hereditary Nonpolyposis / Q000235:genetics*; D004260:DNA Repair / Q000235:genetics; D005799:Genes, Dominant; D014644:Genetic Variation / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D000070957:MutL Protein Homolog 1; D016296:Mutagenesis; D009154:Mutation / Q000502:physiology*; D009363:Neoplasm Proteins / Q000235:genetics*; D009687:Nuclear Proteins; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; C501486:MLH1 protein, human; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D000070957:MutL Protein Homolog 1