Japanese Journal of Clinical Oncology 32:266-269 (2002)
© 2002 Foundation for Promotion of Cancer Research
The Novel Germline Mutation of hMSH2 Gene in a Case of a Colon Cancer Patient Without Family History
1 Department of Surgery, Sakai Municipal Hospital, Osaka, 2 Oncogene Research Unit/Cancer Prevention Unit, Tochigi Cancer Center Research Institute, Tochigi and 3 Department of Surgery, Kansai Rosai Hospital, Hyogo, Japan
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ABSTRACT |
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 TOP ABSTRACT GENETIC SUMMARYCASE REPORT AND GENETIC...METHODS FOR MUTATION DETECTIONREFERENCES |
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Hereditary non-polyposis colorectal cancer (HNPCC) is generally found from the patient’s family history. The functional disorder of mismatch repair genes has been reported to be responsible for HNPCC. The proband was a 28-year-old Japanese female who was admitted to our hospital with a diagnosis of descending colon cancer. Although there was no previous or family history of malignant disorders within the first- and second-degree relatives, the early onset of colon cancer prompted genetic analysis with suspicion of HNPCC. PCR analysis of the primary tumor showed DNA replication errors at the six microsatellite regions. PCR/direct sequential analysis of the peripheral lymphocytes revealed a germline frameshift mutation due to deletion of TTCAA at nt. position from 650 to 654 in exon 4 of the hMSH2 gene. According to the Human Mutation Database and International Collaborative Group on HNPCC Database, this type of the frameshift mutation is the first report in the hMSH2 gene.
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GENETIC SUMMARY |
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TOPABSTRACTGENETIC SUMMARYCASE REPORT AND GENETIC...METHODS FOR MUTATION DETECTIONREFERENCES |
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Disorder: Hereditary non-polyposis colorectal cancer
Ethnicity of patients: Japanese
Gene: hMSH2
GenBank accession number: U04045
Chromosomal assignment: 2p21
Type of DNA variant: A germline frameshift mutation
Mutation: Deletion of TTCAA at nt. position from 650 to 654 in exon 4 of the hMSH2 gene
Method of mutation detection: PCR/direct sequencing
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CASE REPORT AND GENETIC ANALYSIS |
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TOPABSTRACTGENETIC SUMMARYCASE REPORT AND GENETIC...METHODS FOR MUTATION DETECTIONREFERENCES |
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Hereditary non-polyposis colorectal cancer (HNPCC) is seen in 1.0–3.2% of all colorectal cancers (1). The Amsterdam minimum criterion was proposed for the identification of HNPCC and the Japanese clinical criterion was proposed for use in the screening of possible patients with HNPCC (2,3). The functional disorder of mismatch repair genes, such as hMSH2, hMLH1, hPMS1, hPMS2 or hMSH6 in germlines, have been reported to be responsible for HNPCC (1). Here, we report a case of a colon cancer without family history in which the novel germline mutation of hMSH2 gene was detected.
The proband was a 28-year-old Japanese female who was admitted to our hospital with a diagnosis of descending colon cancer. There was no previous or family history of malignant disorders within the first- and second-degree relatives. The proband’s great-grandmother and granduncle died of malignant disorders, but details were unknown (Fig. 1). Left hemicolectomy was carried out in August 2000. A histopathological diagnosis of moderately differentiated adenocarcinoma of clinical stage Dukes’ B was made. A follow-up CT scan in February 2001 revealed a new low-density area in the liver. With a diagnosis of metastatic tumor, partial hepatectomy was carried out in March 2001. The histopathological diagnosis was metastatic colon cancer of the liver. The proband made an excellent recovery and is free of disease as of February 2002.
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The early onset of the colon cancer prompted genetic analysis with suspicion of HNPCC. PCR analysis of the primary tumor showed DNA replication errors at the following six microsatellite regions: D2S136 locus at chromosome 2p, D3S1067 locus at chromosome 3p, TP53 locus at chromosome 17p, D18S51 locus at chromosome 18q, BAT25 locus at chromosome 4q and BAT26 locus at chromosome 2p (Figs 2, 3 and 4). PCR/direct sequential analysis of the peripheral lymphocytes revealed a germline frameshift mutation at nt. position 650 in exon 4 of the hMSH2 gene. PCR/SSCP analysis of the hMSH2 gene exon 4 showed a heteroduplex DNA band (Fig. 5). Finally, PCR/direct sequential analysis of that mutated DNA band revealed a deletion of TTCAA at nt. position from 650 to 654 in exon 4 of the hMSH2 gene (Fig. 6). As a consequence of the five base deletion, a stop codon was found downstream from the deletion site. This is considered to be a significant functional mutation in the disease. PCR/direct sequential analysis of her peripheral lymphocytes revealed no obvious mutation in the hMLH1 gene.
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In this case, no family history was found within the first- and second-degree relatives. This suggests the possibility that the mutation was a new germline mutation arising in the proband. According to the Human Mutation Database and International Collaborative Group on HNPCC (ICG-HNPCC) Database, this is the first report of a frameshift mutation due to a deletion of TTCAA at nt. position from 650 to 654 in exon 4 of the hMSH2 gene. In order to exclude the possibility that the parents of the proband are carriers, further genetic analysis of the proband’s family is under way.
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METHODS FOR MUTATION DETECTION |
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TOPABSTRACTGENETIC SUMMARYCASE REPORT AND GENETIC...METHODS FOR MUTATION DETECTIONREFERENCES |
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Fluorescence-based PCR was performed with the following conditions and parameters for identification of MSI and LOH:
PCR primer, forward: 5'-AGCTTGAGACCTCTGTGTCC-3'
PCR primer, reverse: 5'-ATTCAGAAGAAACAGTGATGGT-3' for the chromosome 2p (D2S136 locus)
Size of PCR product: 95 bp
PCR primer, forward: 5'-TCATCTATCTCCCAACTGTTGAG-3'
PCR primer, reverse: 5'-GAGCACTACCTGTTTAAGATAGG-3' for the chromosome 3p (D3S1067 locus)
Size of PCR product: 95 bp
PCR primer, forward: 5'-ACTGCCACTCCTTGCCCCATTC-3'
PCR primer, reverse: 5'-AGGGATACTATTCAGCCCGAGGTG-3' for the chromosome 17p (TP53 locus)
Size of PCR product: 118 bp
PCR primer, forward: 5'-CCGACTACCAGCAACAACAC-3'
PCR primer, reverse: 5'-CATGCCACTGCACTTCACTC-3' for the chromosome 18q (D18S51 locus)
Size of PCR product: 278 bp
Reverse primers were labeled with 6-FAM (2p), TET (3p), HEX (17p) or TAMRA (18q) and non-labeled forward primers for the four regions were synthesized.
Thermal cycle profile:
Initial denaturation: 94°C, 3 min
35 cycles of 94°C, 45 s/58°C, 1 min/72°C, 1.5 min
Final extension: 72°C, 5 min
Fluorescence-based PCR was performed with the following conditions and parameters for identification of BAT25 and BAT26:
PCR primer, forward: 5'-TCGCCTCCAAGAATGTAAGT-3'
PCR primer, reverse: 5'-TCTGCTTTTAACTATGGCTC-3' for the chromosome 4q (BAT25)
Size of PCR product: 127 bp
PCR primer, forward: 5'-CTACTTTTGACTTCAGCC-3'
PCR primer, reverse: 5'-ACCAATCAACATTTTTAACCC-3' for the chromosome 2p (BAT26)
Size of PCR product: 117 bp
Forward primers were labeled with TET (4q) or HEX (2p) and non-labeled reverse primers were synthesized.
Thermal cycle profile:
Initial denaturation: 95°C, 3 min
35 cycles of 95°C, 1 min/58°C, 1 min/72°C, 1.5 min
Final extension: 72°C, 5 min
PCR products were denatured for 5 min at 95°C in formamide dye and electrophoresed in 6% acrylamide gel (5.7% acrylamide, 0.3% N,N'-methylenebisacrylamide) containing 6 M urea using a ABI PRISM 377 DNA sequencer (Applied Biosystems, Perkin-Elmer).
Long RT-PCR was performed with the following conditions and parameters for identification of the hMSH2 gene exon 4:
PCR primer, forward: 5'-TTCCTTTTCTCATAGTAGTTTAAAC-3'
PCR primer, reverse: 5'-TTGTAATTCACATTTATAATCCATG-3'
10 µM forward primer: 1.25 µl (final 0.5 µM)
10 µM reverse primer: 1.25 µl (final 0.5 µM)
10x buffer: 2.5 µl (final 1x)
AmpliTaq Gold: 0.125 µl (1.25 units)
Template DNA (0.1 µg/µl): 1 µl
DW: 13 875 µl
Total: 25 µl
Thermal cycle profile:
Initial denaturation: 95°C, 12 min
40 cycles of 95°C, 30 s/50°C, 30 s/72°C, 60 s
Final extension: 72°C, 5 min
The yield and quality of the PCR product were ascertained by electrophoresis in 0.8% agarose gel.
Sequencing primer: the same as the PCR primer (forward).
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Acknowledgements |
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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 and a Grant-in-Aid from the Vehicle Racing Commemorative Foundation.
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FOOTNOTES |
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+ For reprints and all correspondence: Hideyuki Ishida, Department of Surgery, Sakai Municipal Hospital, 1–1–1 Minami-Yasui-Cho, Sakai, Osaka 591-0064, Japan. E-mail: h.ishida@hospital.sakai.osaka.jp
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REFERENCES |
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TOPABSTRACTGENETIC SUMMARYCASE REPORT AND GENETIC...METHODS FOR MUTATION DETECTIONREFERENCES |
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1 Utsunomia J, Miyaki M. Studies of hereditary nonpolyposis colorectal cancer in Japan. Int J Clin Oncol 1998;3:53–74.
2 Vasen HFA, Mecklin J-P, Meera Khan P, Lynch HT. The International Collaborative Group on Hereditary Non-polyposis Colorectal Cancer (ICG-HNPCC). Dis Colon Rectum 1991;34:424–5.[Web of Science][Medline]
3 Kunitomo K, Terashima Y, Sasaki K, Komi N, Yoshikawa R, Utsunomiya J, et al. HNPCC in Japan. Anticancer Res 1992;12:1856–7.
4 Bennet RL, Steinhaus KA, Uhrich SB, O’Sullivan CK, Resta RG, Lochner-Doyle, et al. Recommendations for standardized human pedigree nomenclature. Am J Hum Genet 1995;56:745–52.[Web of Science][Medline]
Received February 12, 2002; accepted April 12, 2002
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