Japanese Journal of Clinical Oncology 31:61-64 (2001)
© 2001 Foundation for Promotion of Cancer Research
Infrequent Frameshift Mutations in the Simple Repeat Sequences of hMLH3 in Hereditary Nonpolyposis Colorectal Cancers
1Department of Molecular Oncology, Graduate School of Medicine and Dentistry, Tokyo Medical and Dental University, Tokyo and 2Department of Surgery, Hoshi General Hospital, Fukushima, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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Background: A recently identified mismatch repair gene, hMLH3, contains two simple repeat sequence regions, (A)9 and (A)8, in its coding region. To clarify the role of hMLH3 in hereditary nonpolyposis colorectal cancer (HNPCC), we searched for hMLH3 somatic and germline mutations, particularly in the repeat regions, in 41 HNPCC patient cells.
Methods: We analyzed the hMLH3 (A)9 and (A)8 repeats in 27 colorectal cancers with microsatellite instability (MSI) as well as in normal cells from 41 HNPCC patients by means of polymerase chain reaction–single-strand conformation polymorphism. hMSH3 (A)8 and hMSH6 (C)8 repeats were also examined in these cancers.
Results: Frameshift mutations in the hMLH3 (A)9 repeat were observed in 4/27 (14.8%) cancers with MSI, all of which showed the severe MSI phenotype. No mutations in the (A)8 repeat were found in any case. The mutation frequency of the hMLH3 (A)9 repeat was similar to that of the hMSH6 (C)8 repeat (5/26, 19.2%), but was significantly lower than that of the hMSH3 (A)8 repeat (16/27, 59.3%) (P < 0.001). All four cancers with hMLH3 mutations exhibited germline hMSH2 and/or somatic hMSH3 mutations. No germline mutation in the hMLH3 (A)9 or (A)8 repeat was detected in normal cells from the 41 HNPCC patients.
Conclusion: hMLH3 mutations were infrequently observed in HNPCC cancers with MSI and they may be secondary to other mismatch repair gene mutations. Hence hMLH3 may only play a small role in HNPCC tumorigenesis.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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Hereditary nonpolyposis colorectal cancer (HNPCC) is the predominant cause of familial colorectal cancer (1) and is caused by germline mutations in one of the DNA mismatch repair (MMR) genes, i.e. hMSH2, hMSH6, hMLH1, hPMS1 and hPMS2 (2–4). Among these germline mutations, those of hMSH2 and hMLH1 are the most common and those of hMSH6 are less common in HNPCC kindreds, whereas the other two MMR gene mutations are very rare (2,5). Although another MMR gene, hMSH3, participates in the hMSH2-dependent MMR system (6), its germline mutations have not yet been identified in any HNPCC kindred. Tumor DNA from HNPCC patients mostly shows microsatellite instability (MSI), which is induced by a defective MMR system (2).
hMLH3 was recently cloned and the hMLH3 protein was characterized as an hMLH1-interacting protein (7). A dominant negative hMLH3 protein induced MSI when stably overexpressed in mammalian cell culture (7). In the yeast MMR system, an MLH3/MLH1 complex seems to play a role in the repair of a subset of insertion/deletion loops recognized by an MSH2/MSH3 heterodimer (8,9). Therefore, it is likely that hMLH3 participates as an MMR protein and the function of hMLH3 may be similar to that of yeast MLH3 (7–9).
It is important to analyze hMLH3 germline mutations in HNPCC kindreds, because there are some HNPCC cases with no identified MMR gene mutations (5,10). However, there have been no reports on germline and somatic mutations of hMLH3 in HNPCC and sporadic cancers with MSI. It was noted that hMLH3 contains two simple repeat sequence regions, (A)9 and (A)8, in its coding region. Other MMR genes, hMSH3 and hMSH6, also contain (A)8 and (C)8, respectively, and somatic mutations in these repeat regions are frequently observed in HNPCC and sporadic cancers with MSI (11,12). Therefore, it is possible that hMLH3 may be an MSI target gene like hMSH3 and hMSH6. Moreover, germline mutations in the hMSH6 (C)8 repeat have been detected in some HNPCC kindreds (3,13,14). To clarify the role of hMLH3 in HNPCC tumorigenesis, we searched for hMLH3 somatic and germline mutations, particularly in the simple repeat regions, in HNPCC patient cells.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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Subjects
We examined hMLH3 alterations in 41 patients from 41 unrelated HNPCC kindreds, 16 satisfying the Amsterdam and 25 the Japanese clinical criteria (5). A total of 27 colorectal cancers with MSI were collected from 18 patients. Among the patients with MSI-positive cancers, seven hMSH2, one hMLH1 and one hMSH6 germline mutation were identified (5). Sixteen cancers without MSI from another 16 patients were also analyzed. The remaining seven patients were only examined for hMLH3 germline mutations using normal cell DNA. Genomic DNA was extracted from frozen tissues and peripheral blood as described previously (11).
Mutation Analysis of the Simple Repeat Sequences of hMLH3, hMSH3 and hMSH6
We amplified the regions encompassing the (A)9 and (A)8 microsatellite tracts within hMLH3 exon 2 (Genbank No. AF195658) by polymerase chain reaction (PCR). The PCR primers were 5'-GCCTTTTGCAACAACATTATGG-3' and 5'-GTGGAACATAATTTAACTCGCC-3' for the (A)9 repeat in codons 583–585 and 5'-AGACATCAAAGATTTAGCCAGC-3' and 5'-CTGTAGGTTCATTCTCTAGCC-3' for the (A)8 one in codons 672–674. Each PCR comprised 30 cycles of 94°C (1 min), 62°C (2 min) and 72°C (1 min), with a final 10 min extension at 72°C in standard solutions. Non-radioisotopic single-strand conformation polymorphism (SSCP) was performed according to the method described previously (11). The PCR products were denatured and electrophoresed on 15% non-denaturing polyacrylamide gels containing 10% glycerol in a Tris–glycine buffer (25 mM Tris–HCl and 200 mM glycine, pH 8.3). After electrophoresis, the gels were stained with silver (Dai-ichi, Tokyo, Japan).
We also examined somatic mutations in the hMSH3 (A)8 and hMSH6 (C)8 repeats by means of PCR-SSCP as described above (11).
When abnormal SSCP patterns were observed, the PCR products were purified with a QIA-quick spin PCR purification kit (QIAGEN, Chatsworth, CA) and then cloned into the pCR2.1-TOPO vector (TOPO TA Cloning kit; Invitrogen, Carlsbad, CA). Several clones were sequenced directly with a cycle sequencing kit (TaKaRa, Kyoto, Japan).
Analysis of MSI
According to the National Cancer Institute International Workshop (15), 11 microsatellite markers were selected in this study. We examined DNA for MSI using loci containing the (A)n repeat (BAT-25, BAT-26 and BAT-RII) and loci containing the (CA)n repeat (D2S119, D2S123, D3S1029, D5S346, D10S197, D13S175, D17S250 and D18S58).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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As for HNPCC cancers, abnormal SSCP bands of PCR products containing the hMLH3 (A)9 region were observed in four (14.8%) of the 27 cases with MSI (Fig. 1a), but in none of the 16 without MSI (Table 1). However, there was no significant difference in the hMLH3 mutation frequency between cancers with MSI and those without MSI (P = 0.142). All the cases with abnormal SSCP bands showed a 1 bp deletion in the (A)9 repeat region (Fig. 1b), resulting in truncated proteins up to codon 608 through frameshift mutations. No somatic mutation in the (A)8 repeat was detected in any case. Overall, the mutation frequency of the hMLH3 simple repeats was low (4/27, 14.8%) in HNPCC cancers.
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The four cancers with hMLH3 mutations exhibited severe MSI (Table 2). To clarify the effects of hMLH3 mutations further, we compared the total MSI frequencies (total number of microsatellite markers with MSI/total number of markers examined) between the hMLH3-mutated and -non-mutated cancers with MSI. The total MSI frequency in the hMLH3-mutated cases (four cases, 26/33, 78.8%) was similar to that in the non-mutated cases (24 cases, 133/183, 72.7%) (data not shown).
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Frameshift mutations in the hMSH3 (A)8 and hMSH6 (C)8 regions were detected in 16/27 (59.3%) and 5/26 (19.2%) cancers with MSI, respectively, but in none of the 16 cancers without MSI (Table 1). In the cancers with MSI, the mutation frequency of hMLH3 was similar to that of hMSH6, but was significantly lower than that of hMSH3 (P < 0.001, Fisher’s exact test) (Table 1).
As shown in Table 2, three of the four patients with hMLH3-mutated cancers also exhibited hMSH2 germline mutations (5). Moreover, somatic hMSH3 mutations were also detected in three cancers with hMLH3 mutations.
By means of PCR–SSCP, we searched for germline mutations in both the hMLH3 (A)9 and (A)8 repeat regions in normal cells from 41 HNPCC patients. However, no germline mutation of hMLH3 was detected in these regions in any case (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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hMLH3, hMSH3 and hMSH6 contain mononucleotide repeat sequences in their coding regions (7,11,12). In this study, somatic frameshift mutations in the hMLH3 (A)9 repeat were observed in 4/27 (14.8%) of HNPCC cancers with MSI but not in 16 cancers without MSI. All the frameshift mutations in the hMLH3 (A)9 repeat predict truncated proteins without the C-terminal region that functionally interacts with hMLH1 (7), resulting in a defective hMLH3/hMLH1 complex. When we compared the mutation frequencies among the three MMR genes examined, the mutation frequencies of hMLH3 and hMSH6 were lower than that of hMSH3. These data imply that hMLH3 may also be involved in some HNPCC cancers with MSI, even though its mutation frequency was low.
All the four cancers with hMLH3 mutations exhibited germline hMSH2 and/or somatic hMSH3 mutations, indicating two or more MMR gene alterations in these cancers. According to the idea that ‘a mutator mutates another mutator’ (16), hMLH3 mutations may be secondary to other MMR gene mutations.
The four cancers with hMLH3 mutations showed the severe MSI phenotype. When we examined MSI using 11 microsatellite markers and compared the total MSI frequencies between hMLH3-mutated (four cases, 26/33, 78.8%) and non-mutated (24 cases, 133/183, 72.7%) cancers with MSI, their MSI frequencies were similar. Functional studies on yeast MLH3 indicated that both MLH3 single-mutant and MLH3/MSH3 double-mutant cells induced a weak MSI phenotype (8,9). Although MSH2/MLH3 double-mutant cells showed higher mutability, this phenotype of the double-mutant cells was similar to that of MSH2 single-mutant cells (8,9). Taken together, it is likely that hMLH3 mutation alone might not induce severe MSI in HNPCC and hence MSI in the four cancers with hMLH3 mutations may have been caused by other defective MMR genes.
Germline mutations in the hMSH6 (C)8 repeat have been identified in some HNPCC kindreds (3,13,14), suggesting that hMLH3 may also be associated with HNPCC. We accordingly searched for germline mutations in the hMLH3 (A)9 and (A)8 repeats in 41 HNPCC kindreds, but none were found in any case. These results, together with the MSI data above, imply that hMLH3 may not segregate with most HNPCC kindreds, even though we did not examine sequences outside of the repeat regions.
In conclusion, somatic frameshift mutations of hMLH3 were infrequently observed in HNPCC cancers with MSI. Therefore, hMLH3 may only play a small role in HNPCC tumorigenesis.
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Acknowledgments |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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We thank the doctors who provided useful samples. This work was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports and Culture of Japan and by the Foundation for Promotion of Cancer Research in Japan.
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FOOTNOTES |
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+ For reprints and all correspondence: Yasuhito Yuasa, Department of Molecular Oncology, Graduate School of Medicine and Dentistry, Tokyo Medical and Dental University, 1–5–45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan. E-mail: yuasa.monc@tmd.ac.jp
Abbreviations: HNPCC, hereditary nonpolyposis colorectal cancer; MMR, mismatch repair; MSI, microsatellite instability; PCR, polymerase chain reaction; SSCP, single-strand conformation polymorphism
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REFERENCES |
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Received August 10, 2000; accepted November 17, 2000.
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