DHPLC mutation analysis of the hereditary nonpolyposis colon cancer (HNPCC) genes hMLH1 and hMSH2

Holinski‐Feder, Elke; Müller-Koch, Y; Friedl, Waltraut; Möeslein, Gabriela; Keller, Gisela; Plaschke, Jens; Ballhausen, Wolfgang G.; Gross, M.; Baldwin-Jedele, K; Jungck, Matthias; Mangold, Elisabeth; Vogelsang, H.; Schackert, Hans K.; Lohsea, P; Murken, Jan; Meitinger, Thomas

doi:10.1016/s0165-022x(00)00148-2

Cited by 87 publications

(48 citation statements)

References 8 publications

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“…Since no further nucleotide variations were detected with direct sequencing in the five DHPLC-negative probands, DHPLC revealed a100% sensitivity compared to that for direct sequencing, which is considered the gold standard in mutational analysis (Xiao and Oefner, 2001); however, DHPLC is much less laborious, time-consuming and expensive. Our results agree with data reported by other groups on DHPLC analysis performed on many other genes, such as BRCA (Gross et al, 1999), NF1 (De Luca et al, 2004), MLH1 and MSH2 (Holinski-Feder et al, 2001). Furthermore, our prospective DHPLC mutation detection rate (89.3%) is very similar to that obtained by direct sequencing on ENG and ALK1 by other groups (Letteboer et al, 2005;Schulte et al, 2005).…”

Section: Discussionsupporting

confidence: 92%

DHPLC-based mutation analysis of ENG and ALK-1 genes in HHT Italian population

et al. 2006

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Section: Discussionsupporting

confidence: 92%

DHPLC-based mutation analysis of ENG and ALK-1 genes in HHT Italian population

et al. 2006

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“…29 Microsatellite analyses and immunohistochemical staining MSI-analysis and IHC-staining was done as described. 4,30 Mutation and deletion screening of the MLH1 gene and promoter Mutation screening of the MLH1 gene was done by DHPLC analyses 31 or by direct sequencing. Deletion or duplication analysis of the MLH1 and MSH2 gene with an MLPA assay was published previously.…”

Section: Patients and Materialsmentioning

confidence: 99%

Further evidence for heritability of an epimutation in one of 12 cases with MLH1 promoter methylation in blood cells clinically displaying HNPCC

et al. 2008

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Germline mutations in mismatch repair (MMR) genes, tumours with high microsatellite instability (MSI-H) and loss of MMR protein expression are the hallmarks of HNPCC (Lynch syndrome). While somatic MLH1 promoter hypermethylation is generally accepted in the tumorigenesis of sporadic tumours, abnormal MLH1 promoter methylation in normal body cells is controversially discussed as a mechanism predisposing patients to HNPCC. In all 94 patients suspected of HNPCC-syndrome with a mean age of onset of 45.5 years, MLH1-deficiency in their tumours but no germline mutation, underwent methylation-specific PCRscreening for MLH1 promoter methylation. In peripheral blood cells of 12 patients an MLH1 promoter methylation, in seven informative cases allele-specific, was found. Normal colonic tissue, buccal mucosa, and tumour tissue available from three patients also presented abnormal methylation in the MLH1 promoter. The heredity of aberrant methylation is questionable. Pro: MLH1 promoter methylation was found in a patient and his mother giving evidence for a familial predisposition for an epimutation in MLH1. Contra: a de novo set-up of methylation in one patient, a mosaic or incomplete methylation pattern in six patients, and no evidence for inheritance of MLH1 promoter methylation in the remaining families. Our findings provide strong evidence that MLH1 promoter methylation in normal body cells mimics HNPCC and constitutes a pathogenic pre-lesion in MLH1. The identification of hypermethylation as an epigenetic defect has important implications for surveillance recommendations, as these patients should be treated like Lynch syndrome patients, whereas the heritability of methylation is still under investigation.

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“…The MSH2 and MLH1 genes were screened for mutations by SSCP and DHPLC. Exons 1 -16 of MSH2 (GenBank accessions: U03911, version U03911.1, CDS 4..2808 and AH003235, version U41206.1 to U41220.1) and exons 1 -19 of MLH1 (GenBank accessions: U07343, version U07343.1, CDS 22..2292 and U17857, version U40960.1 to U40977.1) were PCR amplified using specific exon primers described by Kolodner et al (1994) and by Holinski-Feder et al (2001). For the SSCP analysis, PCR products were separated on 6% polyacrylamide gels (acrylamide 59: bisacrylamide 1) at 4 ºC following denaturation.…”

Section: Methodsmentioning

confidence: 99%