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.

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.

High resolution for single-strand conformation polymorphism analysis by capillary electrophoresis.

Since the successful completion of the Human Genome Project, increasing concern is being directed toward the polymorphic aspect of the genome and its clinical relevance. A form of single-strand DNA-conformation polymorphism analysis (SSCP) employing nondenaturing slab-gel electrophoresis (SGE) is applicable to the genetic diagnosis of bladder cancer from urine samples. To bring this technique into routine clinical practice, the use of capillary electrophoresis (CE) is naturally favorable in terms of speed and automation. However, the resolving power of SSCP, a prerequisite basis for reliability required in diagnostics, remains as a challenge for CE systems. We thus focused on this topic and conducted studies on CE instruments equipped with a single capillary or an array of multiple capillaries, using the resolution (Rs) as a quantitative scale for the resolving power. Polymer concentration and buffer are shown to be the decisive parameters. High Rs values of >2.5 are achieved for representative SNPs markers under the optimized conditions, without sacrificing such intrinsic advantages of CE over SGE as the 10-fold quicker migration time and operation that is reproducible, continuous, and automatic. The strategies presented broaden the limits of CE in both the current and related applications.

The novel germline mutation of hMSH2 gene in a case of a hereditary non-polyposis colorectal cancer (HNPCC) patient who meets the revised Amsterdam criteria.

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" have been used. In this report, we present a case of an HNPCC patient who met the revised Amsterdam criteria after the sequential history taking in which a novel germline mutation of hMSH2 gene was detected by genetic testing. The proband was a 69-year-old Japanese female who was admitted to our hospital with a diagnosis of advanced ascending colon cancer. Microsatellite instability (MSI) analysis revealed high MSI in the resected tumor tissue. PCR/direct sequencing analysis of the genomic DNA revealed the TTG(Leu) to TAG(Stop) nonsense mutation at codon 302 in exon 5 of the hMSH2 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 type of nonsense mutation is the first report in the hMSH2 gene.

Methylation profile of the MLH1 promoter region and their relationship to colorectal carcinogenesis.

Methylation of the MLH1 promoter region has been suggested to be a principal mechanism of gene inactivation in sporadic microsatellite instability (MSI)-positive colorectal carcinoma. Recently, we have shown a novel methylation profile of the MLH1 promoter region (i.e., full, partial, and no methylation), among which full methylation was strongly associated with MSI. In this study, to confirm whether methylation requires the involvement of both alleles, we studied the MLH1 promoter region concerning the methylation profile and allelic loss. Furthermore, we studied correlations of methylation profiles with genetic alternations such as loss of heterozygosity (LOH) of the TP53 locus and KRAS mutation. Eighty-eight tumors were classified as full (n = 14), partial (n = 26), and no methylation (n = 48). Full methylation was observed in 78% (14/18) of high-frequency MSI, in which all CpG sites in the promoter region were methylated. Full methylation differed significantly from partial methylation regarding absence of TP53 LOH (0/12) and KRAS mutation (0/14). In cases with full methylation, we could show biallelic methylation by use of a single-base nucleotide polymorphism in the promoter. However, this did not accompany LOH of the MLH1 locus. In contrast, there were no significant differences in molecular features between partial and no methylation, except for low frequencies of LOH of the MLH1 locus (P = 0.02). In conclusion, biallelic extensive methylation of the MLH1 promoter region plays a significant role in gene inactivation and is independent of KRAS mutation and TP53 LOH.

The novel germline mutation of hMSH2 gene in a case of a colon cancer patient without family history.

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.

A novel germline mutation of hMLH1 in a patient with hereditary non-polyposis colorectal cancer.

DNA mismatch repair genes, hMLH1 and hMSH2, assigned on chromosome 3p21-23 and 2p21-22 are involved in hereditary non-polyposis colorectal cancer (HNPCC). The heterozygous carrier of the mutated allele results in a mutator phenotype and accelerating tumorigenesis, which especially causes carcinomas in the gastrointestinal and genitourinary tracts. We screened germline mutations of mismatch repair genes hMLH1 and hMSH2 in a patient with multiple primary neoplasms (multiple stomach cancers, colon cancer and brain tumor) in a cancer clustered HNPCC family. Screening by long RT-PCR from the RNA extracted from puromycin-treated heparinized blood showed skipping of the exon 2 in hMLH1. The analysis of the genomic DNA showed a GT deletion in the splice-donor site of the exon 2, which is compatible with the splicing variant detected by long RT-PCR analysis. This is a novel germline mutation that has not been reported previously.

Concurrent mutations of K-ras oncogene at codons 12 and 22 in colon cancer.

K-ras mutation is the most common oncogenic alteration in various human cancers including colorectal carcinomas. Point mutations have the potential to activate the K-ras gene if they occur in the critical coding sequences. Almost all of these mutations have been localized in codons 12, 13 and 61. We report a case of colon cancer presenting point mutations at both codons 12 and 22 of the K-ras gene. PCR-SSCP and subsequent sequencing revealed that GGT (glycine, wild-type) to AGT (serine) substitution at codon 12 and CAG (glutamine, wild-type) to CGG (arginine) substitution at codon 22 occurred in the same allele.

Methylation status and expression of human telomerase reverse transcriptase mRNA in relation to hypermethylation of the p16 gene in colorectal cancers as analyzed by bisulfite PCR-SSCP.

BACKGROUND: The expression level of human telomerase reverse transcriptase (hTERT) is correlated with telomerase activity and is expressed at high levels in malignant tumors. It is of interest whether expression of hTERT is regulated by methylation of the CpG island in the promoter of the hTERT gene. We examined hTERT expression and methylation status of the hTERT and other genes including p16. METHODS: We analyzed methylation status by bisulfite treatment and polymerase chain reaction with single-strand conformation polymorphism analysis (PCR-SSCP) and expression of the hTERT by RT-PCR, in 13 cancer cell lines, eight white blood cell samples and 24 colorectal cancer tissues. RESULTS: In the cancer cell lines, hTERT was expressed and the CpG island of the hTERT promoter was methylated. Most colorectal cancer tissues showed similar results. The promoter of hTERT was methylated in six cases, partially methylated in 17 cases and unmethylated in one case. All cases with methylation of hMLH1 or p16 also showed methylation of hTERT; however, some of the cases lacking p16 methylation also had hTERT methylation. CONCLUSION: Increased expression of hTERT is related to hypermethylation of hTERT in colorectal cancerous tissues as well as some cancer cell lines and disconcordant with hypermethylation of p16.