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Clinical Cancer Research 13, 7515, December 15, 2007. doi: 10.1158/1078-0432.CCR-07-1028
© 2007 American Association for Cancer Research
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Cancer Susceptibility and Prevention

Analysis of hMLH1 Missense Mutations in East Asian Patients with Suspected Hereditary Nonpolyposis Colorectal Cancer

Yimei Fan1,4, Wei Wang1, Ming Zhu1, Jiji Zhou1, Jingyuan Peng1, Lizhi Xu1, Zichun Hua2, Xiang Gao3 and Yaping Wang1,4

Authors' Affiliations: 1 Department of Medical Genetics, Medical School, Nanjing University, 2 State Key Laboratory of Pharmaceutical Biotechnology, Department of Biochemistry, 3 Model Animal Research Center, Nanjing University and 4 Jiangsu Key Laboratory of Molecular Medicine, Nanjing, China

Requests for reprints: Yaping Wang, Department of Medical Genetics, Medical School, Nanjing University, Hankou Road 22, Nanjing 210093, China. Phone: 86-25-8368-6495; Fax: 86-25-8368-6559; E-mail: wangyap{at}nju.edu.cn.


   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Germ line mutations in the DNA mismatch repair gene hMLH1 are a frequent cause of hereditary nonpolyposis colorectal cancer and about one-third of these are missense mutations. Several missense mutations in hMLH1 have frequently been detected in East Asian patients with suspected hereditary nonpolyposis colorectal cancer, but their pathogenic role has not been extensively assessed. The aim of this study was to perform functional analyses of these variants and their association with gastrointestinal cancer in East Asians.

Experimental Design: Altogether, 10 hMLH1 variants were analyzed by yeast two-hybrid and coimmunoprecipitation assays.

Results: The carboxyl-terminal replacements Q542L, L549P, L574P, and P581L in hMLH1 resulted in complete loss of activity in both yeast two-hybrid and coimmunoprecipitation tests and thus might be considered as pathogenic. The amino-terminal variants S46I, G65D, G67R, and R217C did not affect complex formation with hPMS2 in coimmunoprecipitation, but partly or fully lost their activity in yeast two-hybrid assay, and we suggested that these variants might reduce the efficiency of the heterodimer to go into the nucleus and thus the mismatch repair function might be blocked or reduced. The V384D and the Q701K variant resulted in the interaction of hMLH1 with hPMS2 at reduced efficiency and might raise the gastrointestinal cancer risk of the mutation carriers.

Conclusions: This work availably evaluated the functional consequences of some missense mutations not previously determined in the hMLH1 gene and might be useful for the clinical diagnosis of hereditary gastrointestinal cancer, especially in East Asians.

The DNA mismatch repair (MMR) system ensures genomic stability in humans. The heterodimers of MSH2 and MSH6 (MutS{alpha}) or MSH2 and MSH3 (MutSβ) initiate repair by recognizing single–base pair mismatches and insertion-deletion loops. Similarly, MLH1 dimerizes with PMS2, PMS1, or MLH3 to form three kinds of MutL heterodimers: MutL{alpha}, MutLβ, and MutL{gamma}. Upon mismatch binding, the MutS heterodimers recruit MutL{alpha} (MLH1-PMS2), which plays an essential role in the MMR system (1–3). Deficiency in MMR causes a mutator phenotype and is linked to cancer, in particular, with hereditary nonpolyposis colorectal cancer (HNPCC). Approximately 50% of germ line mutations identified in patients with HNPCC are in MLH1, with about one-third of them being missense mutations. But the functional consequences of missense mutations are difficult to determine. In general, missense mutations are predicted to be pathogenic if they segregate with the disease phenotype, if they are not observed in the normal population, if they are nonconservative changes, or if they occur in evolutionarily conserved amino acids (4). However, segregation studies are not always feasible because of the unavailability of family samples, and rare polymorphisms could also result in nonconservative changes. Therefore, functional assays of missense mutations are necessary to distinguish pathogenic mutations from normal polymorphisms.

As mentioned above, the formation of a hMutL{alpha} complex is essential for MMR activity and thus the inhibition of hMLH1-hPMS2 interaction would be one of the primary causes of MMR defects in individuals with HNPCC. Therefore, we carried out an analysis of protein-protein interactions of the hMutL{alpha} complexes to assess the functional relation of the hMLH1 missense mutations with gastrointestinal cancer. We analyzed 10 mutant forms of hMLH1 which are frequently found in East Asian patients using the yeast two-hybrid system, as well as the coimmunoprecipitation assay to study such protein-protein interactions in vivo. Four of these variants have never been previously studied in functional assays. Mutation screening in a normal Chinese population was also carried out to determine whether these hMLH1 variants were clinically relevant.


   Materials and Methods
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 Abstract
 Materials and Methods
Results
 Discussion
 References
 
hMLH1 variants and patients with suspected HNPCC. We focused on hMLH1 missense mutations frequently detected in East Asian patients with suspected HNPCC. The variants and the phenotypic characteristics of the respective patients included in the present investigation are listed in Table 1 . Most of the variants were found in HNPCC patients fulfilling the Amsterdam criteria. Three variants selected (S46I, G65D, and P581L) are unique in Chinese patients, and five (R217C, V384D, Q542L, L549P, and L574P) have only been identified in East Asians, although the remaining one (G67R) had been reported in several ethnic patients. In addition, we included Q701K, a variant that has recently been found in 2 out of 105 Chinese gastric cancer patients with low familial recurrence (5). Available clinical data showed that six of the variants displayed high microsatellite instability (MSI-H) and four showed loss of the expression of hMLH1 protein in tumor. Age of cancer onset for most of the patients was <45 years. No second mutation in the MMR genes was detected in the patients who carried 1 of the 10 variants investigated in this work, except for S46I in a Chinese patient with HNPCC, in whom a deletion of 12 bp in exon 11 of hMLH1 was shown (Table 1).


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  		Table 1. Genetic and clinical data of variants in hMLH1 under study

 
Mutation screening of normal individuals. One hundred and twelve normal individuals from Jiangsu, China were recruited. Total genomic DNA was extracted from peripheral blood using QIAamp DNA blood mini kit (Qiagen) according to the manufacturer's instructions. Genomic DNA from samples was screened for germ line mutations by PCR of specific exons (as listed in Table 1), followed by denaturing high-performance liquid chromatography analysis done on the WAVE system (Transgenomic). The samples showing abnormal denaturing high-performance liquid chromatography profiles were sequenced on an ABI 3100-Avant automated sequencer (Applied Biosystems). Informed consent was obtained from all subjects. The study was approved by the ethics committee of the Medical School of Nanjing University.

Plasmid constructions. The wild-type hMLH1 and hPMS2 cDNAs were a gift from Prof. Robert Brown and Dr. Helen Robinson. Overlapping PCR site-directed mutagenesis was used to introduce the mutations into hMLH1 cDNA (6). The nucleotide sequences of the PCR primers used are available upon request from the authors. The mutated fragments were cloned into pGADT7 (Clontech) using EcoRI and BamHI to produce the transcriptional AD-fused hMLH1 construct pGADT7-hMLH1 for yeast two-hybrid assay. Similarly, the hPMS2 cDNA was ligated into pGBKT7 to produce pGBKT7-hPMS2, the DNA BD-fused hPMS2 construct. Then the hMLH1 cDNA (wild-type or mutant) was cloned into the XhoI and SfiI sites of pCMV-Myc vector (Clontech), whereas the wild-type hPMS2 cDNA was cloned into the SalI and SfiI sites of pCMV-HA vector (Clontech) to construct the two kinds of plasmids for coimmunoprecipitation assays. The resulting hMLH1 proteins (wild-type or mutants) were tagged at their NH2 terminus with c-Myc, whereas hPMS2 was tagged with HA. All of the plasmid constructs were verified by DNA sequencing on an ABI 3100-Avant automated sequencer (Applied Biosystems).

Yeast two-hybrid assay. Yeast two-hybrid assay was carried out with the matchmaker GAL4 two-hybrid system (Clontech). Briefly, pGBKT7-hPMS2 and pGADT7-hMLH1 (wild-type or mutant) clones were cotransformed into the Saccharomyces cerevisiae strain Y187 and selected on SD/-Leu-Trp medium. Positive protein-protein interactions were ascertained by transcription activation of the highly inducible GAL1 UAS-driving lacZ reporter gene in the host strain by quantitative liquid culture β-galactosidase activity assay with o-nitrophenyl-1-thio-β-D-galactopyranoside (Amresco) as the substrate (7).

cDNA transfections and Western blot. The human embryonic kidney fibroblast cell HEK293T has been reported to not express hMLH1 because of promoter hypermethylation, and thus lacks hPMS2 as it would not be stable without hMLH1. This allows the expression and analysis of MutL{alpha} variants in this cell line without the interference of endogenous MutL{alpha} (8, 9). The 293T cells (Cells Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Science, Shanghai, China) were transiently cotransfected with pCMV-MYC-hMLH1 and pCMV-HA-hPMS2 using a Lipofect Transfection Reagent (RM201-1; Tiangen Biotech Beijing Co. Ltd.) according to the manufacturer's instructions. The cells were lysed and separated on 10% SDS-polyacrylamide gels, followed by electroblotting, and probed with anti-MYC (mouse monoclonal; Clontech) and anti-HA (rabbit polyclonal; Clontech) antibodies and visualized with enhanced chemiluminescence reagents (Applygen Technologies, Inc.).

Coimmunoprecipitation. Total cell protein extracts were incubated with anti-MYC monoclonal antibody (mouse monoclonal; Clontech) followed by immunoprecipitation with protein A-agarose (Invitrogen). The protein-agarose complexes were then collected and resolved through SDS-PAGE. Immunoblots were done as described in the previous section. The densities of the desired protein bands were quantified with the Quantity One-4.5.0 program (Bio-Rad) and the relative amount of hPMS2 was determined (10).


   Results
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 Materials and Methods
 Results
Discussion
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Sequence alignment and functional domain analysis. As indicated in Fig. 1 , the 10 variants now studied cover different regions of the hMLH1 gene (11, 12). The variants S46I, G65D, G67R, and R217C are located in the conserved NH2-terminal region of hMLH1 which is considered important for ATP-binding. Two NH2-terminal missense mutations, G65D and G67R, affect conserved amino acid residues in the second motif of the four ATP-binding pockets. The S46I variant resides near the first ATP-binding pocket, but this codon is not conserved. R217C is located near the domain interface. V384D is located at the boundary between the NH2-terminal region and the COOH-terminal domain. The remaining mutations are in the COOH-terminal region of the polypeptide which is important for hPMS2 interaction.


Figure 1
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  		Fig. 1. Location of hMLH1 variants studied in this work.

 
Identification of mutations in 112 normal individuals. Out of the 10 single-nucleotide variants examined, only one (c.1151T>A, V384D) appeared in the normal population examined. The allele frequency of this variant was 2.67% (6 of 224) in the Chinese normal individuals checked. None of the other nine variants were detected in this population [although we still found the c.655 A>G (p.Ile219Val) in 3 of the 112 samples, this variant is not contained in the current study].

Yeast two-hybrid assay. Liquid cultures were assayed quantitatively for β-galactosidase activity to verify and compare the relative strength of the protein-protein interactions. R659P and the blank plasmid pGADT7 were used as the pathologic missense mutation control and the blank control, respectively. As expected, the five missense mutations (Q542L, L549P, L574P, P581L, and Q701K) located in the COOH-terminal of hMLH1 necessary for interaction with hPMS2 showed extremely low levels of β-galactosidase activities in comparison with wild-type hMLH1. Their activities are similar to the pathologic missense mutation control R659P. Interestingly, the two NH2-terminal missense mutations, G67R and R217C, and the V384D variant in the region between the NH2-terminal and the COOH-terminal also displayed very weak β-galactosidase activities, whereas the remaining two NH2-terminal missense mutations S46I and G65D retained ~40% of their activities (Table 2 ).


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  		Table 2. Yeast two-hybrid assay of hMLH1 variants

 
Western blot and coimmunoprecipitation assays. To clarify whether the low levels of β-galactosidase activities in the two-hybrid assay were caused by defects in the physical interaction between hMLH1 variants and a wild-type hPMS2, we next performed a combined coimmunoprecipitation/Western blot analysis (Fig. 2 ).


Figure 2
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  		Fig. 2. Interaction studies between hMLH1 variants and hPMS2. A and B, Western blot of total protein extracts (TE; 35 µg each) of 293T cells transfected with pCMV-HA-PMS2-wt with either wild-type pCMV-MYC-MLH1 (hMLH1-wt) or pCMV-MYC-MLH1 variants (top). Immunoprecipitates (IP; 500 µg of TE in each) obtained with anti-MYC antibody (middle). Following transfer on the membrane, the proteins were visualized with a mixture of the MYC and HA antibodies. Relative quantity of hPMS2 coprecipitated with different hMLH1 proteins (bottom). The densities of the desired protein bands in immunoprecipitation were quantified with the Quantity One-4.5.0 program. The relative amount of hPMS2, which bound to wild-type or mutant hMLH1, was determined by normalizing the intensity of hPMS2 band to the intensity of the corresponding hMLH1 band. The amount of hPMS2 bound to wild-type hMLH1 is set to 1. Columns, mean; bars, SD.

 
Immunoblotting of total cell extracts confirmed that mutant and wild-type hMLH1 proteins were expressed equally well (Fig. 2A and B, top). The expression levels of hPMS2 were also similar (Fig. 2A and B, top).

As shown in Fig. 2, the four hMLH1 variants carrying missense mutations in the amino-terminal exons 2 (S46I, G65D, and G67R) and exon 8 (R217C) did not exhibit significant changes in their physical interaction with hPMS2 (Fig. 2A, middle and bottom, lanes 2-5). The results are consistent with other previous observations that mutations in the NH2 terminus of hMLH1 are unlikely to affect the formation of MutL{alpha} heterodimer. The missense mutation V384D in exon 12 retained half of its interaction with hPMS2 (Fig. 2A, middle and bottom, lanes 6). For the five hMLH1 variants carrying mutations at the carboxyl end, the missense mutation in exon 14 (Q542L L549P) and exon 15 (L574P) tended to display defects in their physical interactions with hPMS2 as R659P, the mutant chotrol, in exon 17. The P581L missense mutation in exon 16 yielded a polypeptide that interacted only very weakly with hPMS2 (~16% compared with the wild-type of hMLH1), whereas the missense mutation Q701K in exon 18 retained ~70% interaction with hPMS2 (Fig. 2B, middle and bottom, lanes 2-7). In those cases, the antibody against MYC-hMLH1 precipitated MYC-hMLH1 but not HA-hPMS2, or only partly, from the total protein extracts.

Evaluation of pathogenicity. The evaluation of pathogenicity of the 10 variants based on this study and on literature data is summarized in Table 3 .


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  		Table 3. Summary of functional analysis results

 

   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
In this study, the functional consequences of 10 hMLH1 missense variants detected frequently in East Asians with suspected HNPCC were evaluated mainly by yeast two-hybrid and coimmunoprecipitation, four of the variants have thus far never been tested. The R659P variant was used as a control because the mutation is located in the essential hPMS2-interaction domain and was already predicted to be pathogenic from different laboratory works (refs. 6, 13–16). All variants, except for the V384D variant, were absent from any of the 112 Chinese normal controls.

Our data showed that complex formation between any one of the four hMLH1 protein variants carrying mutations at the carboxyl end (Q542L L549P, L574P, and P581L) and hPMS2 were not detectable or were only weakly detectable, similar to the R659P variant in both assays (Table 2; Fig. 2B, middle and bottom, lanes 2-5). All four variants were detected in patients from HNPCC families fulfilling the Amsterdam criteria, and available data showed that the probands were young patients with colorectal cancer (CRC; Table 1; refs. 17–20). Out of these variants, P581L in hMLH1 was evaluated, for the first time, for its functional consequence. Proline is an amino acid with a closed ring, whereas leucine is a hydrophobic amino acid. This change might affect the structure of the protein, and thus, its interaction with hPMS2. Our data suggested that this variant was pathogenic due to the functional impairment of the hMLH1 protein in its interaction with hPMS2 by coimmunoprecipitation analysis, and the extremely low level of β-galactosidase activities in comparison with wild-type hMLH1 in yeast two-hybrid assay. Actually, clinical data showed that this variant displayed MSI-H and loss of the expression of hMLH1 protein in tumor (17). No consistent results from previous functional studies have been obtained on Q542L. It was considered to be a loss-of-function mutation by in vivo DNA MMR in the yeast assay (14) and showed ~40% reduction in the β-galactosidase activity in yeast two-hybrid assay by Kondo et al. (15). However, it was suggested to be a normal polymorphism in the GST-IVTT assay (16) and a mutator effect in the yeast assay (13). Our results suggested that Q542L in hMLH1 causes a complete inactivation of this gene product either with yeast two-hybrid assay or with coimmunoprecipitation. Hence, we refer to it as a disease-causing mutation. Wanat et al. suggested that L549P was pathogenic in humans, as L549P (corresponding to yeast mutant allele L559P) showed significant MMR defects through examination of the MMR proficiency in the S288c strain of S. cerevisiae (21). L574P was previously referred to as pathogenic in mutator effect assay in yeast (13), GST-IVTT assays (15, 16), and yeast two-hybrid assay (15). Our results, obtained by yeast two-hybrid assay and coimmunoprecipitation analysis in human cells, also showed the causative role of these two variants in HNPCC.

Thus far, few functional assays have been done on the amino terminal variants of hMLH1. No work has been reported on the functional consequence of the variants S46I and G65D. G67R was shown in MMR analysis in yeast to be a loss-of-function mutation (13, 14, 22). Kondo's work showed that this NH2-terminal missense variant, G67R, in hMLH1 displayed low levels of β-galactosidase activity in a yeast two-hybrid assay, but did not exhibit any change in their physical interaction with hPMS2 in the GST-IVTT assay (15). However, they did not give a clear explanation for this discrepancy. Ellison et al. referred to R217C as an efficiency polymorphism as it functioned in DNA MMR at a reduced efficiency (14). However, it restored MMR efficiency at ~80% in 293T cells and was classified as a rare polymorphism by Trojan et al. (8).

Our results, using coimmunoprecipitation analysis, revealed the normal formation of a complex between the amino-terminal hMLH1 variants (S46I, G65D, G67R, and R217C) and hPMS2 (Fig. 2A, middle and bottom, lanes 2-5). However, they showed more or less reduced β-galactosidase activities in the yeast two-hybrid assay (Table 2), which was unexpected as they were not located in the regions of hPMS2 interaction (7, 16). How to interpret the above results?

According to Ban et al., the NH2-terminal region of MutL in Escherichia coli contains four putative ATP-binding motifs. The authors further stated that MutL is actually an ATPase and that ATP binding causes conformational changes in MutL, and is likely to regulate the interactions of MutL with other components in the DNA repair machinery (11). This ATPase domain in MutL is highly conserved from bacteria to humans, and in fact, hMutL{alpha} has recently been shown to have ATPase activity and this ATPase activity is essential for the MMR system (12, 23). According to structure-based sequence alignment of the conserved NH2-terminal region in the MutL family, G65 and G67 are located in motif II of four highly conserved ATP-binding motifs of MLH1 and both are conserved in the GHL (DNA gyrase B, Hsp90, and MutL) superfamily (11). S46 is not a conserved residue in the MutL family and is not located in the conserved ATP-binding motifs (11). R217 is not a conserved residue and neither is it located in or around the ATP-binding motifs, but it is located near the domain interface. It seems to locate on the exposed surface on the side of the molecule opposite the ATPase activity site. As shown in Table 3, the four variants had all undergone changes in charge or polarity. Charge and polarity are important in the protein's structure and function, especially in the formation of a secondary structure. Thus, the alteration of the amino acids at those positions might affect the proteins' interaction between the helices and the capacity of those mutant proteins to bind and/or hydrolyze ATP, and thus, the ATPase activity of hMLH1.

It has been known that DNA MMR takes place in the nucleus. Nuclear import and export of MMR proteins is determined by nuclear localization signals and nuclear export sequences, respectively (24). Both nuclear localization signals and nuclear export sequences are present in hMLH1 and hPMS2, yet when expressed alone, hMLH1 and hPMS2 fail to localize into the nucleus. On the contrary, coexpression of hMLH1 and hPMS2 resulted in their nuclear localization. Although some NH2-terminal mutants in hMLH1 did not affect the formation of MutL{alpha}, and were also unable to translocate into the nucleus (25–27). These phenomena suggest that COOH-terminal dimerization of hMLH1 and hPMS2 is necessary for nuclear import, but MutL{alpha} also undergoes a conformational transformation upon ATP binding that includes NH2-terminal dimerization. The NH2-terminal dimerization-defective mutants might fail to localize in the nucleus despite the fact that they are dimerized by the COOH-terminal domains.

The above hypothesis could be one explanation for the discrepancy in the results of the two assays. The COOH-terminal dimerization of hMLH1 and hPMS2 may not be sustained in the nucleus because the NH2-terminal dimerization-defective hMLH1 mutant (S46I, G65D, G67R, and R217C) proteins binds normally to hPMS2 in coimmunoprecipitation analysis, but the dimers were not shown or only partly shown in a yeast two-hybrid assay that only detects protein interactions in the nucleus in vivo. The present results suggest that those kinds of variants might act similarly in human cells as in the yeast cells, that is, reducing the efficiency of the heterodimer to enter the nucleus and thus block the MMR process. Available clinical data showed that all the four variants displayed MSI-H, and three showed loss of the hMLH1 protein expression in tumor. These variants occurred either in patients from HNPCC families fulfilling the Amsterdam criteria, or in patients with familial recurrence for CRC but did not meet the Amsterdam criteria, or in sporadic CRC patients with multiple primary cancers. The reported age of cancer onset was young. No second mutation in the MMR genes was detected in the patients with one of the three variants (G65D, G67R, and R217C) except for S46I (Table 1; refs. 17, 18, 28–36). With both of the functional analysis results and clinical data, we suggest that the three NH2-terminal variants (G65D, G67R, and R217C) might affect the MMR process at different levels and might have functional relation to gastrointestinal cancer. It is interesting to refer to the variant S46I in a Chinese HNPCC patient who carried the second mutation in hMLH1, a deletion of 12 bp in exon 11 (Table 1; ref. 28). In the current yeast two-hybrid assay, this NH2-terminal missense mutation retained 44.2% of the activities and showed normal interaction with hPMS2 in coimmunoprecipitation analysis (Table 2; Fig. 2). We classify this variant as partly loss of function, but its relation to cancer is uncertain, on account of the fact that the proband also carried another mutation in the hMLH1 gene. Additional analyses are needed to determine the relationship between the structural abnormalities at the NH2 terminus of hMLH1 and the exact localization of the heterodimer of hMLH1 and hPMS2, and thus, their roles in the MMR process.

V384 is located in the central region of the hMLH1 protein between the NH2-terminal ATP-binding domain and the COOH-terminal hPMS2 interaction domain. The V384D mutation results in charge differences (the neutral hydrophobic amino acid valine is replaced by aspartate, a weak acidic amino acid with a negative charge) and is likely to disrupt the structure formation, and hence, the stability of the hMLH1 protein. V384D has been frequently detected in the East Asian population (Table 1). Additional investigations in Chinese cohorts showed that a higher frequency of this variant was detected in young CRC patients (<45 years) or GC patients with a family history when compared to that in normal controls (37). In a newly reported work, V384D showed ~65% in vitro MMR activity (38). Our current assay with a yeast two-hybrid assay detected low levels of β-galactosidase activity, although hPMS2 interaction was still half-retained in coimmunoprecipitation analyses. It is possible that this variant partially reduced the interaction between hMLH1 and hPMS2, and the interaction was not strong enough for the BD and AD domains of the Gal4 system to come close to activating the reporter gene lacZ. Combined with the above results, we suggest that this is partly a loss of function variant and might raise the risk of gastrointestinal cancer in the mutation carriers.

We have identified the Q701K variant in 2 out of 105 Chinese gastric cancer cases, but not in 82 patients with CRC (5). This variant was not detected in 112 normal individuals. Both patients were from families with low GC familial recurrence, which is the presence of one additional affected family member (two GC cases in the family). Unfortunately, samples from patients with the familial disease phenotype were unavailable, and we were not able to perform a cosegregation analysis of the mutation. Glutamine and lysine belong to different polarity groups. Our result first showed that this hMLH1 variant largely retained the interaction with hPMS2 from coimmunoprecipitation analyses, whereas the β-galactosidase activity was significantly reduced in yeast two-hybrid assays. The reason for the contradiction between the two assays might be the similar V384D variants used, which has been stated in the previous section. Therefore, this variant is also classified as partly loss of function and it might have a role in the occurrence of gastric cancer.

In conclusion, our data provide, for the first time, the functional evaluation of the variants S46I, G65D, P581L, and Q701K which is frequently detected in East Asians with suspected HNPCC. Our results are consistent with previous analyses on the functional consequences of such variants as G67R, L549P, and L574P, suggesting that they are pathogenic in humans. Moreover, we provide information on the consequences of the variants R217C, V384D, and Q542L which have not received consistent results in previous studies. This work evaluates the real contribution of germ line missense mutations in the hMLH1 gene to gastrointestinal cancer predisposition, at least, in East Asians. This information might be useful for mutation screening and clinical diagnosis of East Asians at high risk for gastrointestinal cancer.


   Acknowledgments
 
We thank Prof. Robert Brown and Dr. Helen Robinson (Department of Medical Oncology, Glasgow University, Glasgow, United Kingdom) for the gift of hMLH1 and hPMS2 cDNA; Dr. Waltraut Friedl and Prof. Peter Propping (Institute of Human Genetics, University of Bonn, Germany) for their valuable advice on this work; Drs. Lei Fang (State Key Laboratory of Pharmaceutical Biotechnology, Department of Biochemistry, Nanjing University, Nanjing, China) and Jianming Li (Department of Cell Biology and Medical Genetics, School of Basic Medical Science, Nanjing Medical University, Nanjing, China) for their assistance in the yeast two-hybrid assay.


   Footnotes
 
Grant support: National Natural Science Foundation of China, grant no. 30470959.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 4/30/07; revised 8/13/07; accepted 9/27/07.


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

  1. Drummond JT, Li GM, Longley MJ, Modrich P. Isolation of an hMSH2-160 heterodimer that restores DNA mismatch repair to tumor cells. Science 1995;268:1909–12.[Abstract/Free Full Text]
  2. Li GM, Modrich P. Restoration of a mismatch repair to nuclear extracts of H6 colorectal tumor cells by a heterodimer of human MutL homologs. Proc Natl Acad Sci U S A 1995;92:1950–4.[Abstract/Free Full Text]
  3. Palombo F, Iaccario I, Nakajima E, Ikejima M, Shimada T, Jiricny J. hMutSβ, a heterodimer of hMSH2 and hMSH3, binds to insertion/deletion loops in DNA. Curr Biol 1996;6:1181–4.[CrossRef][Medline]
  4. Peltomäki P, Vasen HF. Mutations predisposing to hereditary nonpolyposis colorectal cancer: database and results of a collaborative study. The International Collaborative Group on Hereditary Nonpolyposis Colorectal Cancer. Gastroenterol 1997;113:1146–58.[CrossRef][Medline]
  5. Fan YM, Liu XR, Zhang H, et al. Variations in exon 7 of the MSH2 gene and susceptibility to gastrointestinal cancer in a Chinese population. Cancer Genet Cytogenet 2006;170:121–8.[CrossRef][Medline]
  6. Nyström-Lahti M, Perrera C, Räschle M, et al. Functional analysis of MLH1 mutations linked to hereditary nonpolyposis colon cancer. Genes Chromosomes Cancer 2002;33:160–7.[CrossRef][Medline]
  7. Kondo E, Horli A, Fukushige S. The interacting domains of three MutL heterodimers in man: hMLH1 interacts with 36 homologous amino acid residues within hMLH3, hPMS1 and hPMS2. Nucl Acid Res 2001;29:1695–702.[Abstract/Free Full Text]
  8. Trojan J, Zeuzem S, Randolph A, et al. Functional analysis of hMLH1 variants and HNPCC-related mutations using a human expression system. Gastroenterology 2002;122:211–9.[CrossRef][Medline]
  9. Plotz G, Raedle J, Brieger A, Trojan J, Zeuzem S. N-terminus of hMLH1 confers interaction of hMutL{alpha} and hMutLβ with hMutS{alpha}. Nucleic Acids Res 2003;31:3217–26.[Abstract/Free Full Text]
  10. Sun XM, Zheng L, Shen BH. Functional alterations of human exonuclease 1 mutants identified in atypical hereditary nonpolyposis colorectal cancer syndrome. Cancer Res 2002;62:6026–30.[Abstract/Free Full Text]
  11. Ban C, Yang W. Crystal structure and ATPase activity of MutL: implications for DNA repair and mutagenesis. Cell 1998;95:541–52.[CrossRef][Medline]
  12. Räschle M, Dufner P, Marra G, Jiricny J. Mutations within the hMLH1 and hPMS2 subunits of the human MutL mismatch repair factor affect its ATPase activity, but not its ability to interact with hMutS. J Biol Chem 2002;277:21810–20.[Abstract/Free Full Text]
  13. Shimodaira H, Filosi N, Shibata H, et al. Functional analysis of human MLH1 mutations in Saccharomyces cerevisiae. Nat Genet 1998;19:384–9.[CrossRef][Medline]
  14. Ellison AR, Lofing J, Bitter GA. Functional analysis of human MLH1 and MSH2 missense variants and hybrid human-yeast MLH1 proteins in Saccharomyces cerevisiae. Hum Mol Genet 2001;10:1889–900.[Abstract/Free Full Text]
  15. Kondo E, Suzuki H, Horii A, Fukushige S. A yeast two-hybrid assay provides a simple way to evaluate the vast majority of hMLH1 germ-line mutations. Cancer Res 2003;63:3302–8.[Abstract/Free Full Text]
  16. Guerrette S, Acharya S, Fishel R. The interaction of the human MutL homologues in hereditary nonpolyposis colon cancer. J Biol Chem 1999;274:6336–41.[Abstract/Free Full Text]
  17. Wang CF, Zhou XY, Sun MH, et al. Two novel germline mutations of MLHl in hereditary nonpolyposis colorectal cancer families. Chin J Pathol 2006;35:68–72.
  18. Han HJ, Yuan Y, Ku JL, et al. Germline mutations of hMLHl and hMSH2 genes in Korean hereditary nonpolyposis colorectal cancer. J Natl Cancer Inst 1996;88:1317–9.[Free Full Text]
  19. Han HJ, Maruyama M, Baba S, Park JG, Nakamura Y. Genomic structure of human mismatch repair gene, hMLH1, and its mutation analysis in patients with hereditary non-polyposis colorectal cancer (HNPCC). Hum Mol Genet 1995;4:237–42.[Abstract/Free Full Text]
  20. Shin YK, Heo SC, Shin JH, et al. Germline mutations in MLH1, MSH2 and MSH6 in Korean hereditary non-polyposis colorectal cancer families. Hum Mutat 2004;24:351–8.[Medline]
  21. Wanat JJ, Singh N, Alani E. The effect of genetic background on the function of Saccharomyces cerevisiae mlh1 alleles that correspond to HNPCC missense mutations. Hum Mol Genet 2007;16:445–52.[Abstract/Free Full Text]
  22. Shcherbakova PV, Kunkel TA. Mutator phenotypes conferred by MLH1 overexpression and by heterozygosity for mlh1 mutations. Mol Cell Biol 1999;19:3177–83.[Abstract/Free Full Text]
  23. Tomer G, Buermeyer AB, Nguyen MM, Liskay RM. Contribution of human Mlh1 and Pms2 ATPase activities to DNA mismatch repair. J Biol Chem 2002;277:21801–9.[Abstract/Free Full Text]
  24. Mattaj IW, Englmeier L. Nucleocytoplasmic transport: the soluble phase. Annu Rev Biochem 1998;67:265–306.[CrossRef][Medline]
  25. Wu XS, Platt JL, Cascalho M. Dimerization of MLH1 and PMS2 limits nuclear localization of MutL{alpha}. Mol Cell Biol 2003;23:3320–8.[Abstract/Free Full Text]
  26. Brieger A, Plotz G, Raedle J, et al. Characterization of the nuclear import of human MutL{alpha}. Mol Carcinog 2005;43:51–8.[CrossRef][Medline]
  27. Raevaara TE, Korhonen MK, Lohi H, et al. Functional significance and clinical phenotype of nontruncating mismatch repair variants of MLH1. Gastroenterology 2005;129:537–49.[CrossRef][Medline]
  28. Cai Q, Sun MH, Fu G, et al. Mutation analysis of hMSH2 and hMLHl in Chinese hereditary nonpolyposis colorectal cancer families. Chin J Pathol 2003;32:323–8.
  29. Yuan Y, Huang YQ, Cai SR, Song YM, Zheng S, Zhang SZ. Genetic characterization of Chinese hereditary non-polyposis colorectal cancer by dHPLC and multiplex PCR. Jpn J Clin Oncol 2004;34:660–6.[Abstract/Free Full Text]
  30. Sasaki S, Tokino T, Miyatsu T, Muto T, Nakamura Y. Mutational analysis of the hMLH1 gene using an automated two-dimensional DNA typing system. Hum Mutat 1997;9:164–71.[CrossRef][Medline]
  31. Tannergård P, Lipford JR, Kolodner R, Frödin JE, Nordenskjöld M, Lindblom A. Mutation screening in the hMLH1 gene in Swedish hereditary nonpolyposis colon cancer families. Cancer Res 1995;55:6092–6.[Abstract/Free Full Text]
  32. Heinimann K, Scott RJ, Buerstedde JM, et al. Influence of selection criteria on mutation detection in patients with hereditary nonpolyposis colorectal cancer. Cancer 1999;85:2512–8.[CrossRef][Medline]
  33. Hutter P, Couturier A, Membrez V, Joris F, Sappino AP, Chappuis PO. Excess of hMLH1 germline mutations in Swiss families with hereditary non-polyposis colorectal cancer. Int J Cancer 1998;78:680–4.[CrossRef][Medline]
  34. Herfarth KKF, Kodner IJ, Whelan AJ, et al. Mutations in MLH1 are more frequent than in MSH2 in sporadic colorectal cancers with microsatellite instability. Genes Chromosomes Cancer 1997;18:42–9.[CrossRef][Medline]
  35. Yuan Y, Ye J, Zheng S. Clinical and genetic features of International Collaborative Group—hereditary nonpolyposis colorectal cancer families and suspected hereditary nonpolyposis colorectal cancer families. Chin Med J (Engl) 2004b;117:748–52.[Medline]
  36. Miyaki M, Konishi M, Muraoka M, et al. Germ line mutations of hMSH2 and hMLH1 genes in Japanese families with hereditary nonpolyposis colorectal cancer (HNPCC): usefulness of DNA analysis for screening and diagnosis of HNPCC patients. J Mol Med 1995;73:515–20.[Medline]
  37. Wang YP, Zhou JN, Li ZY, et al. One of the etiological factors of digestive tract cancers in Chinese: the missense mutation Val384Asp in the hMLH1 gene. Chin J Med Genet 2000;17:82–6.
  38. Takahashi M, Shimodaira H, Andreutti-Zaugg C, Iggo R, Kolodner RD, Ishioka C. Functional analysis of human MLH1 variants using yeast and in vitro mismatch repair assays. Cancer Res 2007;67:4595–604.[Abstract/Free Full Text]
  39. Wang Y, Friedl W, Lamberti C, Nothen MM, Kruse R, Propping P. A novel missense mutation in the DNA mismatch repair gene hMLH1 present among East Asians but not among Europeans. Hum Hered 1998;48:87–91.[CrossRef][Medline]
  40. Lee SC, Guo JY, Lim R, et al. Clinical and molecular characteristics of hereditary nonpolyposis colorectal cancer families in Southeast Asia. Clin Genet 2005;68:137–45.[CrossRef][Medline]




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