© 2004 Foundation for Promotion of Cancer Research
Cancer Genetics Report |
The Novel Germline Mutation of the hMLH1 Gene in a Case of Suspected Hereditary Non-polyposis Colorectal Cancer (HNPCC) in a Patient with No Family History of Cancer
1 Department of Surgery, Kansai Rosai Hospital, Amagasaki, Hyogo, 2 Department of Pathology, School of Allied Health Science, Faculty of Medicine, Osaka University, Suita, Osaka, 3 Hyogo Medical College, Nishinomiya, Hyogo, 4 Aino-Gakuen College, Ibaraki, Osaka and 5 Oncogene Research Unit/Cancer Prevention Unit, Tochigi Cancer Center Research Institute, Utsunomiya, Japan
For reprints and all correspondence: Naohiro Tomita, Department of Surgery, Kansai Rosai Hospital, 3-1-69 Inabaso, Amagasaki 660-8511, Japan. E-mail: ntomita{at}kanrou.net
Received May 7, 2004; accepted June 10, 2004
Abstract
Hereditary non-polyposis colorectal cancer (HNPCC) is a very important clinical entity in oncology. In order to identify HNPCC, the international diagnostic criteria, named ‘Amsterdam criteria’, has been used. In this report, we present a patient with HNPCC who completely lacks a family history of cancer, thus does not meet the revised Amsterdam criteria and was finally confirmed as HNPCC by genetic testing which revealed a novel germline mutation of the hMLH1 gene. The proband was a 52-year-old Japanese female with a diagnosis of advanced ascending colon cancer. She had a past history of Miles' operation for rectal cancer at the age of 40. A subtotal colectomy was performed and the subsequent microsatellite instability (MSI) analysis revealed high MSI in the resected tumor tissue. PCR/direct sequencing analysis of the genomic DNA revealed the base deletion 2006delAAAAG at codon 669 in exon 18 of the hMLH1 gene, which was considered to be a pathogenic mutation. According to the Human Mutation Database and International Collaborative Group on HNPCC (ICG-HNPCC) Database, this is the first report of this type of deletion mutation in the hMLH1 gene.
Key Words: genetic testing • hereditary non-polyposis colorectal cancer (HNPCC) • hMLH1 • microsatellite instability (MSI) • revised Amsterdam Criteria
CASE REPORT AND GENETIC ANALYSIS
A genetic summary is included in Table 1.
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Hereditary non-polyposis colorectal cancer (HNPCC ) is a very important clinical entity because of its clinical characteristics such as relatively high frequency (1.0–5.0%) among all colorectal cancers and relatively early onset of malignancies in various organs, etc. (1). In order to identify HNPCC, the international diagnostic criteria, named ‘Amsterdam minimum criteria’ was proposed in 1991 and had been used in many countries, including Japan. However, considering the variety of malignancies other than colorectal cancer occurring in HNPCC families, some proportion of true HNPCC might be missed under this criteria, which needs at least three colorectal cancer patients in the pedigree. Thus, the revised version, Amsterdam criteria II, was proposed in 1999. This revised criteria allows diagnosis of the family containing at least three HNPCC-related cancer patients as HNPCC, however, there still remains the possibility of the so-called de novo HNPCC patient, in whom the initial mutation of mismatch repair gene might occur, could be missed.
In this report, we present an HNPCC-suspected case with no family history of cancer, who, thus, does not meet the revised Amsterdam criteria, and who was finally confirmed as HNPCC by genetic testing which revealed a novel germline mutation of hMLH1.
The proband was a 52-year-old Japanese female who was admitted to Kansai Rosai Hospital with a diagnosis of advanced ascending colon cancer. The initial history taking was done at the outpatient's clinic and revealed that she had a history of surgery of Miles' operation with sigmoid colostomy for advanced rectal cancer at the age of 40. There is no family history of cancer as shown in Figure 1. The patient's past history of rectal cancer at her relatively young age of 40, together with the present occurrence of the ascending colon cancer, made us suspect that this case might be of the HNPCC kindred, even without a family history of cancer. The preoperative abdominal computed tomography revealed the existence of uterine myoma, but no other disorders, including malignancies, could be found.
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We gave a precise explanation to the patient and her family members, including her husband, about her possibility of HNPCC kindred and the choice of surgical procedures of either right hemicolectomy or total colectomy, and also about the optional surgery for uterus and ovary. Under her informed consent, total colectomy with ileostomy and simple total hysterectomy with bilateral salpingo-oophorectomy were carried out with the cooperation of the doctors of the gynecological department in May 2003. In the operation, direct invasion of the ascending colon cancer to the jejunum was found and part of the jejunum was resected, together with the main tumor. The laterally spreading tumor, 2.5 cm in size, was found in the cecum in the resected specimen and the existence of well differentiated adenocarcinoma localized in the mucosal layer in the tubular adenoma was later diagnosed histologically. A histopathological diagnosis of the main tumor was moderately differentiated adenocarcinoma without lymph node metastasis, therefore the clinical stage was determined as Dukes' B.
Microsatellite instability (MSI) analysis revealed that among five microsatellite markers used, four markers, D2S136, D3S1067, D18S51 and BAT26, were positive, resulting in high MSI in the tumor tissue (Fig. 2). This result, taken together with the patient's history, further suggested that the patient should be of the HNPCC kindred. Under written informed consent, genetic testing was carried out using DNA extracted from the proband's peripheral lymphocytes. As a result, the five base deletion 2006delAAAAG at codon 669 in exon 18 of the hMLH1 gene was detected by long RT–PCR/direct sequencing analysis and was confirmed by PCR/direct sequencing analysis. This could lead to the stop codon of codon 672, and is thus considered to be a functional pathologic mutation. There was no mutation detected in the hMSH2 gene. The representative profiles of germline DNA analysis are shown in Figures 3 and 4.
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Our search of the Human Mutation Database and International Collaborative Group on HNPCC (ICG-HNPCC ) Database, found that the base deletion 2006delAAAAG at codon 669 in exon 18 of the hMLH1 gene had not been reported previously and this is considered to be the first case. The mutation detected in the proband was likely to be a de novo occurrence, which includes the possibility of the parent's gonadal mosaicism and that of the proband's post-zygotic mutation. Another possibility was that either the father or mother of the proband might be an asymptomatic carrier of the same mutation. In order to confirm that the mutation detected in this report had truly occurred de novo, further genetic analysis of the family members, especially the proband's parents, without history of cancer, was needed. And the confirmation that this mutation is truly the disease-causing mutation should be obtained by some functional assay. Also, career diagnosis in this family might be clinically useful and beneficial for each family member. However, since the proband did not want to approach other family members at the time of diagnosis, the further genetic analysis has not yet been undertaken.
One of the disadvantages of the present Amsterdam criteria (Amsterdam criteria II) is that this criteria could not diagnose the de novo HNPCC patient due to the lack of a family cancer history, which might be the case in this report. Further extensive study on large numbers of HNPCC-suspected cases, with or without family history, coupled with their genetic analyses, are needed.
METHODS FOR MUTATION DETECTION
Fluorescence-based PCR was performed with the following conditions and parameters for identification of MSI and loss of heterozygosity (LOH) on four dinucleotide markers, as described previously (2,3).
PCR primers for chromosome 2p (D2S136 locus): forward, 5'-AGCTTGAGACCTCTTGTGTCC-3'; reverse, 5'-ATTCAGAAGAAACAGTGATGGT-3'; size of PCR product, 95 bp.
PCR primers for chromosome 3p (D3S1067 locus): forward, 5'-TCATCTATCTCCCAACTGTTGAG-3'; reverse, 5'-GAGCACTACCTGTTTAAGATAGG-3'; size of PCR product, 95 bp.
PCR primers for chromosome 17p (TP53 locus): forward, 5'-ACTGCCACTCCTTGCCCCATTC-3'; reverse, 5'-AGGGATACTATTCAGCCCGAG-3'; size of PCR product, 118 bp.
PCR primers for chromosome 18q (D18S51 locus): forward, 5'-CCGACTACCAGCAACAACAC-3'; reverse, 5'-CATGCCACTGCACTTCACTC-3'; size of PCR product, 278 bp.
Reverse primers were labelled with 6-FAM (2p), TET (3p), HEX (17p) or TAMRA (18q) and non-labelled forward primers for the above four regions were synthesized.
Thermal cycle profile: initial denaturation, 94°C, 3 min; amplification, 35 cycles of 94°C, 45 s/58°C, 1 min/72°C, 1.5 min; final extention, 72°C, 5 min.
Fluorescence-based PCR was performed with the following conditions and parameters for identification of MSI on mononucleotide marker, BAT26. PCR primers for chromosome 2p (BAT26): forward, 5'-CTACTTTTGACTTCAGCC-3'; reverse, 5'-ACCAATCAACATTTTTAACCC-3'; size of PCR product, 117 bp. Forward primer was labelled with HEX (2p) and non-labelled reverse primer was synthesized. Thermal cycle profile was the same as that for the four dinucleotide markers except that the denaturation temperatue was 95°C. PCR products were then 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).
Analysis using long RT–PCR and PCR using genomic DNA was performed as described previously (4).
Long RT–PCR was performed with the following conditions and parameters for identification of the hMLH1 gene containing exon 18. Forward primer, 5'-CATCTAGACGTTTCCTTGGCTCTTC-3'; reverse primer, 5'-TAAAGGAATACTATCAGAAGGCAAGTATA-3'. Thermal cycle profile: initial denaturation, 94°C, 1 min; amplification, 40 cycles of 94°C, 15 s/65°C, 4 min; final extention, 65°C, 10 min.
Reverse transcription was carried out with 200 U of MMLV reverse transcriptase SuperScriptTM II (Life Technologies, Inc., MD), 0.5 mM oligo (dT) primer and 1 µg of the total RNA in 20 µl of 50 mM Tris–HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2 and 10 mM dithiothreitol and incubated for 50 min at 42°C. Primers used for long RT–PCR are RT-Lf1 and RT-Lr1 for hMLH1. Long PCR for cDNA was carried out using AdvantageTM cDNA PCR kit (Clontech Laboratories, Inc., CA). Reagent conditions were subjected to the manufacturer's recommendation with a minor modification of adding 10% glycerol in the PCR mixture.
PCR using genomic DNA was performed with the following conditions and parameters for identification of the hMLH1 gene exon 18. Forward primer, 5'-AATTCGTACCTATTTTGAGG-3'; reverse primer, 5'-ATTGTATAGGCCTGTCCTAG-3'. Thermal cycle profile: initial denaturation, 95°C, 12 min; amplification, 40 cycles of 95°C, 30 s/50°C, 30 s/72°C, 1 min; final extension: 72°C, 5 min.
Acknowledgments
This work was supported in part by Grant-in-Aid for Medical Research from Kansai Rosai Hospital, Grant-in-Aid from the Human Science Research Project and Grant-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.
References
1 Baba S (ed.). New strategies for treatment of hereditary colorectal cancer. Tokyo: Churchill Livingston, 1996.
2 Ishida H, Furukawa H, Tatsuta M, et al. The novel germline mutation of hMSH2 gene in a case of a colon cancer patient without family history. Jpn J Clin Oncol 2002;32:266–9.
3 Tomita N, Fukunaga M, Ohzato H, et al. The novel germline mutation of hMSH2 gene in a case of hereditary non-polyposis colorectal cancer (HNPCC) patient who meets the revised Amsterdam criteria. Jpn J Clin Oncol 2003;33:486–9.
4 Nomura S, Sugano K, Kashiwabara H, 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.[CrossRef][Web of Science][Medline]
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