Nature Genetics
19, 384 - 389 (1998)
doi:10.1038/1277
Functional analysis of human MLH1 mutations in Saccharomyces
cerevisiaeHideki Shimodaira1, Nicole Filosi2, Hiroyuki Shibata1, Takao Suzuki1, Paolo Radice3, Ryunosuke Kanamaru1, Stephen H. Friend4, Richard D. Kolodner2, 5
& Chikashi Ishioka11
Department of Clinical Oncology, Institute of Development, Aging and Cancer, Tohoku University, Sendai 980-8575, Japan. 2
Charles A. Dana Division of Human Cancer Genetics, Dana-Farber Cancer Institute, Boston, Massachusetts 02115, USA. 3
Division of Experimental Oncology, Istituto Nazionale Tumori, Milano 20133, Italy. 4
The Seattle Project, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA. 5
Present address: Ludwig Institute for Cancer Research, University of California San Diego School of Medicine, La Jolla, California 92093, USA.
Correspondence should be addressed to Richard D. Kolodner rkolodner@UCSD.edu or Chikashi Ishioka chikashi@idac.tohoku.ac.jpHereditary non-polyposis colorectal cancer (HNPCC; OMIM 120435-6)
is a cancer-susceptibility syndrome1 linked to inherited defects
in human mismatch repair (MMR) genes2. 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.
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