Japanese Journal of Clinical Oncology 31:346-348 (2001)
© 2001 Foundation for Promotion of Cancer Research
DNA Variants of BAT-25 in Japanese, a Locus Frequently Used for Analysis of Microsatellite Instability
1Oncogene Research Unit/Cancer Prevention Unit, Tochigi Cancer Center, Tochigi, 2Genetic Counseling Clinic, National Cancer Center Hospital, Tokyo and 3Department of Surgery, National Cancer Center Hospital, Tokyo, Japan
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ABSTRACT |
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 TOP ABSTRACT GENETICS SUMMARYCASE REPORT AND GENETIC...METHODS FOR MUTATION DETECTIONREFERENCES |
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BAT-25 is a DNA marker, intragenic to the c-kit protooncogene, assigned to 4q12, containing a polythymine tract, mostly repeats of 25 poly(T) (T25). The BAT-25 locus is frequently used in the analysis of microsatellite instability (MSI) in cancer tissues. The number of poly(T) repeats at BAT-25 is reported to be quasi-monomorphic and this property permits the easy identification of the MSI status. We report DNA variants of BAT-25, in one patient with hereditary nonpolyposis colorectal cancer presenting T21 and T25 alleles and another carrying T18 and T25 alleles in the analysis of 100 normal Japanese donors. Observed allelic frequencies were 0.5% for both T21 and T18 alleles. So far, DNA variants of BAT-25 locus have been reported in African Americans with relatively high frequencies, but not in Japanese.
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GENETICS SUMMARY |
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TOPABSTRACTGENETICS SUMMARYCASE REPORT AND GENETIC...METHODS FOR MUTATION DETECTIONREFERENCES |
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Disorder: Microsatellite instability
Gene: BAT-25 (Intron 16, c-kit oncogene)
GenBank accession number: U63834
Chromosomal assignment: 4q12
Type of DNA variant: Mononucleotide repeat
Mutation: Polythymine nucleotide repeat
Allelic frequency: No. of repeats Allelic frequency 95% C.I.
25 0.99 (200/202) 0.976-1
21 0.005 (1/202) 0-0.031
18 0.005 (1/202) 0-0.031
Method of mutation detection: PCR/fragment analysis
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CASE REPORT AND GENETIC ANALYSIS |
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TOPABSTRACTGENETICS SUMMARYCASE REPORT AND GENETIC...METHODS FOR MUTATION DETECTIONREFERENCES |
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Microsatellite instability (MSI) is a DNA aberration characterized by the appearance of the abnormal repeat numbers in mono-, di-, tri- and tetranucleotide repetitive sequences in cancer cells (1). In the analysis of colon carcinoma tissues, MSI is observed in about 10% of the samples (2). In the analysis of colon cancer from kindreds with hereditary nonpolyposis colorectal cancer (HNPCC), MSI is detected in as many as 85–95% of cases (2) and germline mutations of five mismatch repair genes, i.e. hMSH2, hMLH1, PMS1, PMS2 and hMSH6, have so far been reported to be responsible for HNPCC (2). Furthermore, methylation of the hMLH1 promoter region is reported in about 80% of sporadic colon cancer cases presenting MSI, indicating that disruption of the mismatch repair system is closely associated with MSI and tumorigenesis (3). Aaltonen et al. reported its usefulness for HNPCC screening in a Finnish study (4). Furthermore, Elsaleh et al. described that striking survival benefit was seen in MSI-positive Dukes C colon cancer in their analysis of a cohort receiving adjuvant chemotherapy as compared with MSI-negative tumors (5). Hence the detection of MSI would be useful in molecular screening of cancer with genetic or epigenetic mismatch repair defects and thus provide information for choosing diagnostic and therapeutic modalities. The selection of appropriate genetic markers is important for MSI testing and BAT-25 is the one most frequently used for this purpose (6). It contains polythymine repeats, usually repeats of 25 thymine nucleotides (T25), and is considered to be quasi-monomorphic with allelic size variation not exceeding two nucleotides (7,8). This property is an advantage in screening MSI in large numbers of samples, as there is no need to compare the allelic profiles between normal and tumor tissues and screening of the tumor tissues alone reduces the cost of MSI testing by 50%.
Previously, we reported a case of Japanese HNPCC kindreds with 33 bp deletion in exon 7 of the hMSH2 (9), presenting MSI in tumor tissue (10). Analysis of the BAT-25 locus in this case showed aberrant DNA bands in colon carcinoma tissue suggestive of MSI (Fig. 1, panel 2c), but another allele was found in the analysis of normal colonic mucosa comprising T21 and T25 repeats (Fig. 1, panel 2b). The same profile was observed in the analysis of peripheral blood lymphocytes (PBL) from this patient (Fig. 1, panel 2a). It was not clear whether this result indicated de novo mismatch repair insufficiency in normal cells or the polymorphic DNA variant of BAT-25 allele, so we examined the allelic profiles of the BAT-25 locus in 100 anonymous DNA samples from PBL of normal Japanese. As a result, 99 samples showed monomorphic BAT-25, homozygous for T25 allele, but one case was found to be heterozygous comprising T25 and T18 alleles (Fig. 1, panel 3). From this result, we concluded that BAT-25 is polymorphic in Japanese, although its observed allelic frequency is 0.5% (95% C.I., 0-0.031; modified Wald method) for both T21 and T18 alleles. Pyatt et al. reported DNA variants of BAT-25 in African Americans (11). In the analysis of 103 African Americans, the BAT-25 allelic frequencies were 90.77, 6.80 and 2.43% for T25, T19 and T17 alleles, respectively (11), which seems much higher than those of the Japanese in this study. They also reported DNA variants of BAT-26, a locus containing monoadenine repeats, which is also frequently used in MSI testing. However, we did not find any variant allele at the BAT-26 locus in our analysis of Japanese cases. To conclude, the observed frequencies of variant alleles at the BAT-25 locus show an ethnic difference and those showing genotypes other than T25 is as low as 1% in the Japanese population. Analysis of tumor tissue alone would be acceptable in routine screening of MSI using BAT-25 and BAT-26 loci, but comparison with normal tissue is required when abnormal alleles appear in the analysis of tumor tissue.
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METHODS FOR MUTATION DETECTION |
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TOPABSTRACTGENETICS SUMMARYCASE REPORT AND GENETIC...METHODS FOR MUTATION DETECTIONREFERENCES |
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PCR/fragment analysis was performed under the following conditions and parameters:
PCR primer, forward: 5'TCG CCT CCA AGA ATG TAA GT3'
PCR primer, reverse: 5'TCT GCA TTT TAA CTA TGG CTC3'
Size of PCR product: 125 bp for A25 repeat
Thermal cycle profile:
Initial denaturation: 95°C, 10 min
30 cycles of 94 °C, 45 s/58 °C, 45 s/74 °C, 45 sc
Final extension: 74 °C, 7 min
The 5' end of the forward primer was labeled with FAM fluorescent dye and PCR product was electrophoresed with an ABI PRISM 310 genetic analyzer equipped with a 47 cm length capillary and POP4 polymer. Sizes of the DNA fragments were determined by comparison with molecular size standards.
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Acknowledgement |
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This work was supported in part by Grants-in-Aid for Cancer Research and for the 2nd Term Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health, Labor and Welfare, Japan.
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FOOTNOTES |
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+ For reprints and all correspondence: Kokichi Sugano, Oncogene Research Unit/Cancer Prevention Unit, Tochigi Cancer Center Research Institute, 4–9 Yohnan 13-chome, Utsunomiya, Tochigi 320-0834, Japan. E-mail: ksugano@tcc.pref.tochigi.jp
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REFERENCES |
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4 Aaltonen LA, Salovaara R, Kristo P, Canzian F, Hemminki A, Peltomaki P, et al. Incidence of hereditary nonpolyposis colorectal cancer and the feasibility of molecular screening for the disease. N Engl J Med 1998;338:1481–7.
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9 Nomura S, Sugano K, Kashiwabara H, Taniguchi T, Fukayama N, Fujita S, et al. Enhanced detection of deleterious and other germline mutations of hMSH2 and hMLH1 in Japanese hereditary nonpolyposis colorectal cancer kindreds. Biochem Biophys Res Commun 2000;271:120–9.[Web of Science][Medline]
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Received February 19, 2001; accepted March 12, 2001.
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