Molecular screening of potential HNPCC patients using a multiplex microsatellite PCR system

doi:10.1006/mcpr.1999.0231

Molecular and Cellular Probes

Volume 13, Issue 2, April 1999, Pages 157-165

https://doi.org/10.1006/mcpr.1999.0231 Get rights and content

Abstract

Microsatellite instability (MSI) is a molecular hallmark of theHereditaryNon-PolyposisColorectalCancer (HNPCC) syndrome occurring in about 80–90% of the tumours and also in sporadic tumours of different organs, albeit at lower frequency. Highly unstable colorectal tumours (MSI-H) have different histopathological features and tend to have a better prognosis compared to neoplasms without (MSS) or with low levels of microsatellite instability (MSI-L). Since MSI classification allows the identification of potential HNPCC patients and might represent a valuable diagnostic parameter an increasing demand for high-throughput microsatellite analysis will arise. Therefore, we have adapted five diagnostic microsatellites, m(odified) ACTC, mBAT26, mD5S107, mD5S406 and mD13S153, to allow coamplification. Using this multiplex polymerase chain reaction (PCR) system 29 colorectal tumour tissues with known MSI status could be unambiguously identified as MSI-H (13 cases) or MSI-L/MSS (16 cases). Highly unstable colorectal tumour detection frequency of individual markers reached 77% (mD5S406), 85% (mACTC), 85% (mD5S107), 92% (D13S153) and 100% (mBAT26) showing similar sensitivity but improved specificity as compared with a microsatellite reference panel. In a prospective analysis of 31 colorectal tumours, the multiplex PCR system identified five MSI-H cases. Multiplex MSI PCR is a time saving and cost-effective method not restricted to specific technical equipment and applicable to a variety of microsatellite-based genotyping approaches.

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Cited by (33)

Sensitivity and polymorphism of Bethesda panel markers in Chinese population
2020, Bulletin du Cancer
Citation Excerpt :
Buhard et al. [10] reported that BAT25 and BAT26 showed instability in 98% and 100%, respectively, of 60 MSI-H tumor samples from Hong Kong population. BAT25,BAT26 D2S123,D17S250 and D5S546 of MSI-H tumors showed instability in100%, 100%, 85%, 77% and 69% in German [7], while 87%, 88%, 62%, 38% and 44% in Seoul[8], respectively. Among 18 MSI-H patients, instability of BAT25, BAT26, D2S123, D17S250 and D5S546 occurred in 83.3%, 72.2%, 61.1%, 61.1%, 44.4% cases in an Iranian population [25].
This study aims to analyze sensitivities and polymorphisms of the Bethesda panel markers (BAT25, BAT26, D2S123, D5S346 and D17S250) for microsatellite instability testing in Chinese from Jiangsu Province and their clinical implication.
MSI, sensitivity and polymorphism analysis in 541 colorectal cancer (CRC) patients were detected by fragment analysis.
Five hundred and twenty-five tissue samples and 541 blood samples of the 541 sample pairs were successfully amplified. Thirty-four (6.5%) cases were MSI-high (MSI-H) while 33 (6.3%) and 458 (87.2%) were MSI-low (MSI-L) and microsatellite stable (MSS), respectively. BAT26 (85.3%) exhibited the highest instability followed by BAT25 (82.4%), D2S123 (67.6%), D17S250 (64.7%) and D5S346 (50.0%) in MSI-H cases. The median ages of CRC patients with LS, MSI-H, MSI-L and MSS status were 38–43, 48, 60 and 63, respectively. 75.0%, 44.1%, 12.1% and 7.0% CRC cases were mucinous carcinomas in LS, MSI-H, MSI-L and MSS group, respectively. For D2S123, D17S250 and D5S346, heterozygosity was 80.8%, 74.1% and 57.7% and sizes of polymorphic variation range (PVR) were 207 bp to 234 bp, 140 bp to 169 bp and 109 bp to 137 bp, respectively. For D2S123 and D5S346, there was a bimodal distribution distinguishing the D17S250 from an indistinct trimodal or tetramodal distribution.
MSI-H cases showed earlier onset and higher proportion of mucinous carcinomas. Mononucleotide BAT26 and BAT25 exhibited higher sensitivity than dinucleotides D2S123, D17S250 and D5S346 in the Chinese population. The dinucleotide markers were highly polymorphic with high percent of heterozygosity, great variation in repeat length and non-normal distribution in Chinese population from Jiangsu Province.
Microsatellites instability in colon cancer
2006, Immuno-Analyse et Biologie Specialisee
L'instabilité des microsatellites (MSI) se caractérise par la variation anormale du nombre de séquences répétées dans l'ADN tumoral comparé à l'ADN du même patient provenant de tissu sain. L'instabilité des microsatellites a d'abord été décrite dans un cancer héréditaire du côlon, le syndrome de Lynch ou syndrome HNPCC (hereditary nonpolyposis colon cancer) où elle est présente dans 95 % des cas. Elle est aussi présente dans environ 15 % des cancers du côlon non héréditaires (ou sporadiques). La recherche de MSI est effectuée par PCR de cinq marqueurs microsatellites répartis dans le génome des cellules tumorales. Associée à des critères cliniques comme les critères d'Amsterdam ou de Bethesda, la présence de MSI oriente vers le syndrome HNPCC qui sera confirmé par la caractérisation des mutations dans les gènes MMR (mismatch repair) impliqués dans cette pathologie. Si le syndrome HNPCC est confirmé, un conseil génétique, une enquête familiale voire des mesures prophylactiques sont entreprises. Dans les cancers sporadiques, la présence de MSI est liée aux modifications épigénétiques du promoteur du gène codant pour hMLH1, gène de la famille MMR. La présence de MSI est corrélée à une meilleure survie et permettrait d'adapter les traitements adjuvants, ce dernier point étant objet de discussions.
Characteristics of microsatellites instability (MSI) is abnormal variation of the number of repeated sequences in DNA from the tumor compared to DNA of healthy tissue from the same individual. MSI has been first described in a rare hereditary colon cancer Lynch syndrome or HNPCC syndrome (hereditary nonpolyposis colon cancer) caused by MMR (mismatch repair) genes defects. Later, MSI was described in 15% of sporadic colon cancer. To confirm the diagnosis of MSI, a panel of five markers are used, amplified by multiplex PCR. MSI associated with clinical criteria (Amsterdam or Bethesda criteria) are recommended for MMR genes molecular testing to confirm HNPCC. Genetic counselling, familial investigation and possibly prophylactic measures are then taken. In sporadic cancers, MSI is the consequence of MLH1 silencing by epimutation in the promotor and is correlated with a better prognosis and maybe a resistance to fluorouracile, an adjuvant chemotherapy of colon cancer.
Evaluation of tumor microsatellite instability using five quasimonomorphic mononucleotide repeats and pentaplex PCR
2002, Gastroenterology
Background & Aims: The microsatellite instability (MSI) phenotype is a characteristic of the hereditary nonpolyposis colorectal cancer syndrome as well as approximately 15% of sporadic colon and gastric tumors. It is a valuable diagnostic marker for the identification of hereditary nonpolyposis colorectal cancer cases and may be a molecular predictive marker for the identification of colon cancer patients who benefit from chemotherapy. To evaluate MSI, a reference panel was proposed at an international consensus meeting, comprised of 2 mononucleotide (BAT-25, BAT-26) and 3 dinucleotide repeats. Analysis of BAT-26 is sufficient for detecting the MSI phenotype in most, but not all, cases. Additional results with dinucleotide markers can sometimes lead to incorrect classification of MSI tumors. Methods: We describe here a single fluorescent multiplex system comprising 5 quasimonomorphic mononucleotide repeats for the detection of MSI tumors. Results: None of 184 germline DNA samples, including 56 from African subjects, was found to contain allelic size variations in more than 2 of these markers. In contrast, all MSI tumors showed allelic size variations in 3 or more of the microsatellites. Using this assay, we confirmed (or reclassified in 6 cases) the MSI status of 124 colon and 50 gastric primary tumors and 16 colon cell lines. Conclusions: We propose that using a pentaplex polymerase chain reaction system allows accurate evaluation of tumor MSI status of DNA with 100% sensitivity and specificity without the need to match normal DNA. This assay is simpler to use than those involving dinucleotides and is more specific than using BAT-26 alone.
GASTROENTEROLOGY 2002;123:1804-1811
Endometrial abnormalities in three sisters from a family with hereditary non-polyposis colorectal cancer syndrome
2002, BJOG: An International Journal of Obstetrics and Gynaecology
Improved microsatellite instability detection in colorectal cancer patients by a combination of fourteen markers especially DNMT3a, DCD, and MT1X
2021, Cancer Biomarkers
Molecular and computational methods for the detection of microsatellite instability in cancer
2018, Frontiers in Oncology

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^f1: Author to whom all correspondence should be addressed at: Division of Molecular Diagnostics and Therapy, University Hospital, Im Neuenheimer Feld 116, D-69120 Heidelberg, Germany. Tel: +49 6221 56 2876/77; Fax: +49 6221 56 5981; E-mail:[email protected]

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Regular ArticleMolecular screening of potential HNPCC patients using a multiplex microsatellite PCR system

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Regular Article
Molecular screening of potential HNPCC patients using a multiplex microsatellite PCR system