Basic ResearchFunctional analysis of hMLH1 variants and HNPCC-related mutations using a human expression system*,**
Section snippets
hMLH1 variants
The majority of hMLH1 missense mutations associated with HNPCC lead to structural changes within the amino- or the carboxy-terminal regions containing the ATPase and the hPMS2-interaction domains, respectively (Figure 1).
Characterization of MMR efficiency in 293T cells
To establish a suitable human expression system for hMLH1 and its variants, we screened different cell lines for hMLH1 expression. The human embryonic kidney fibroblast cell line 293T was found to lack hMLH1 as well as hPMS2, whereas hMSH2 and hMSH6 were normally expressed (Figure 2A).
Discussion
Germline mutations of hMLH1 and hMSH2 account for the majority of MMR gene mutations identified in HNPCC families.23 Many of these mutations result in truncated proteins and are predicted to predispose their carriers to colon cancer. However, more than 30% of the reported HNPCC-linked hMLH1 mutations are missense, and, in this case, single amino-acid changes may not impair protein function or may be only partially inactivating. Functional testing of the mutant hMLH1 alleles is therefore
Acknowledgements
The authors thank Dr. Markus Raeschle for discussion.
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Address requests for reprints to: Joerg Trojan, M.D., Medizinische Klinik II, Klinikum der Johann Wolfgang Goethe-Universität, Theodor-Stern-Kai 7, D-60590 Frankfurt, Germany. e-mail: [email protected]; fax: (49) 69-6301-6448.
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Supported in part by grants from the University of Frankfurt (F15/01) and the Swiss National Science Foundation. J.T. is supported by a Harald-Goebel scholarship of the Deutsche Gesellschaft für Verdauungs- und Stoffwechselerkrankungen.