Multiplex SNaPshot Genotyping for Detecting Loss of Heterozygosity in the Mismatch-Repair Genes MLH1 and MSH2 in Microsatellite-Unstable Tumors

Bujalkova, Maria Gerykova; Zavodna, K; Krivulcik, Tomas; Ilenčíková, Denisa; Wolf, Brigitte; Kováč, Michal; Karner‐Hanusch, Judith; Heinimann, Karl; Marra, Giancarlo; Jiricny, Josef; Bartosova, Zdena

doi:10.1373/clinchem.2008.108902

Cited by 19 publications

(16 citation statements)

References 34 publications

(33 reference statements)

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“…The three ApoM gene SNPs were rs805297 (C-1065A), rs9404941 (T-855C), and (T-778C), representing at least 5% minor allele frequencies in the promoter region of the ApoM gene in the Han Chinese. Genotyping was performed with a SNaPshot Multiplex sequencing method (Bujalkova et al 2008). Ten microliters of 12-h fasting venous blood was collected from each subject and stored in a tube containing disodium EDTA as an anticoagulant.…”

Section: Polymorphism Genotypingmentioning

confidence: 99%

Association of Apolipoprotein M Gene Polymorphisms with Ischemic Stroke in a Han Chinese Population

Zhao

Qin

et al. 2010

J Mol Neurosci

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The apolipoprotein M (ApoM) gene is critical in the formation of pre-β-high-density lipoprotein (HDL) and cholesterol efflux to HDL. In this case and control study, 314 ischemic stroke patients and 389 healthy controls were analyzed for three ApoM gene single-nucleotide polymorphisms (SNPs), i.e., C-1065A, T-855C, and T-778C, using a SNaPshot Multiplex sequencing assay. The genotype and allele frequencies of the T-855C were similar in both ischemic stroke patients and the controls. But the frequency of the TC genotype, the C allele of T-778C, and the A allele of the C-1065A SNPs in ischemic stroke patients was significantly higher than that of the healthy controls. After adjusting for confounding risk factors (such as hypertension, diabetes, tobacco smoking, and alcohol consumption), the ApoM gene TC genotype, C allele of T-778C, and A allele of C-1065A were associated with a risk of ischemic stroke. Moreover, plasma levels of total cholesterol were significantly higher in patients with CC or CT genotypes of T-778C than those with TT genotype in the controls. The current data demonstrated that ApoM T-778 C and C-1065A SNPs were associated with increased risk of ischemic stroke in this Han Chinese population.

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Section: Polymorphism Genotypingmentioning

confidence: 99%

Association of Apolipoprotein M Gene Polymorphisms with Ischemic Stroke in a Han Chinese Population

Zhao

Qin

et al. 2010

J Mol Neurosci

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“…Germline DNA of the SK-20 patient was heterozygous at the c.1277-118G>A locus in intron 7 and showed LOH at this SNP. We had previously detected LOH by SNaPshot in the same case at two MSH2 -SNPs located in intron 1 (c.211+9C>G, c.211+98T>C), which are difficult to sequence due to formation of secondary structures in the respective regions [28]. Taken together, two distinct LOH events interrupted by heterozygous dup5-6 (tandem arrangement assumed) are apparently present in the tumor of the SK-20 patient.…”

Section: Resultsmentioning

confidence: 85%

“…These two genes carry a number of single nucleotide polymorphisms (SNPs) that can be potentially utilized to delineate the conversion tracts more precisely than is possible using the extragenic microsatellite markers. The SNP markers, though less heterozygous than the microsatellite markers, have been proven to be useful in detecting LOH at MMR loci [27,28]. In their recent LOH study on the heterogeneously defined microsatellite unstable carcinomas, van Puijenbroek et al [29] used SNP arrays to assess LOH on a genome-wide level.…”

Section: Introductionmentioning

confidence: 99%

Partial loss of heterozygosity events at the mutated gene in tumors from MLH1/MSH2 large genomic rearrangement carriers

et al. 2009

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BackgroundDepending on the population studied, large genomic rearrangements (LGRs) of the mismatch repair (MMR) genes constitute various proportions of the germline mutations that predispose to hereditary non-polyposis colorectal cancer (HNPCC). It has been reported that loss of heterozygosity (LOH) at the LGR region occurs through a gene conversion mechanism in tumors from MLH1/MSH2 deletion carriers; however, the converted tracts were delineated only by extragenic microsatellite markers. We sought to determine the frequency of LGRs in Slovak HNPCC patients and to study LOH in tumors from LGR carriers at the LGR region, as well as at other heterozygous markers within the gene to more precisely define conversion tracts.MethodsThe main MMR genes responsible for HNPCC, MLH1, MSH2, MSH6, and PMS2, were analyzed by MLPA (multiplex ligation-dependent probe amplification) in a total of 37 unrelated HNPCC-suspected patients whose MLH1/MSH2 genes gave negative results in previous sequencing experiments. An LOH study was performed on six tumors from LGR carriers by combining MLPA to assess LOH at LGR regions and sequencing to examine LOH at 28 SNP markers from the MLH1 and MSH2 genes.ResultsWe found six rearrangements in the MSH2 gene (five deletions and dup5-6), and one aberration in the MLH1 gene (del5-6). The MSH2 deletions were of three types (del1, del1-3, del1-7). We detected LOH at the LGR region in the single MLH1 case, which was determined in a previous study to be LOH-negative in the intragenic D3S1611 marker. Three tumors displayed LOH of at least one SNP marker, including two cases that were LOH-negative at the LGR region.ConclusionLGRs accounted for 25% of germline MMR mutations identified in 28 Slovakian HNPCC families. A high frequency of LGRs among the MSH2 mutations provides a rationale for a MLPA screening of the Slovakian HNPCC families prior scanning by DNA sequencing. LOH at part of the informative loci confined to the MLH1 or MSH2 gene (heterozygous LGR region, SNP, or microsatellite) is a novel finding and can be regarded as a partial LOH. The conversion begins within the gene, and the details of conversion tracts are discussed for each case.

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“…The aim of this study is to describe the possible approaches for mutational screening of KRAS gene in FFPE tissues and to introduce the mutation screening of the KRAS gene using the Real Time PCR analysis at our department and so to expand the molecular diagnoses already used in clinical practice in Slovak Republic [7][8][9][10][11]. We also suggested the possible effective screening strategies, with priority to the sensitivity and the economic costs.…”

Section: Introductionmentioning

confidence: 99%

Genetic analysis of KRAS mutation status in metastatic colorectal cancer patients

Zavodna

Konečný²,

Krivulcik³

et al. 2009

neo

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colorectal carcinoma (crc) represents a serious problem worldwide: in the slovak republic are diagnosed about 2600 new crc cases annually and its incidence is increasing. colorectal cancer patients may succumb to the disease because of local recurrence or local formation of metastasis. Therefore, it is necessary to modulate therapeutic algorithm with new methods, leading to early diagnostic of crc or changing the existing therapeutic procedures. recent progresses have been made in understanding of eGFr pathway involved in crc carcinogenesis, especially the role of ras protein. mutations in KRAS oncogene are frequently found in human cancers, particularly colorectal, pancreatic, billiary tract and lung tumors. The presence of the KRAS mutations in metastatic colorectal cancer patients correlates with lack of response to the certain epidemal growth factor receptor (eGFr) inhibitor therapies, such as panitumumab and cetuximab. consequently, screening for KRAS mutations status may be used as a prognostic marker, because the crc patients with KRAS positive tumors have a worse prognosis.The aim of our study was to establish the methods for rapid and sensitive detection of KRAS mutation status in formalin fixed paraffin embedded (FFpe) tissues dNa. We applied real Time pcr analysis (Therascreen Kras mutation Test Kit) and sequencing analysis (optimised for the analysis of FFpe tissues) to detect somatic mutations in codon 12 and 13 of KRAS gene. Both methods were used concurrently in the panel of dNa isolated from 25 colorectal FFpe tissues tumor. The positive or negative results from all 25 samples were identified by both methods independently. The KRAS mutations were presented in 8 of 25 patients (32%). our results demonstrate that the real Time pcr analysis can be used for detection of somatic KRAS mutations in FFpe clinical samples. however, we also recognize that the sequencing analysis of approximately 200bp amplicons may be used for KRAS mutations status screening, but with care of method sensitivity.

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Multiplex SNaPshot Genotyping for Detecting Loss of Heterozygosity in the Mismatch-Repair Genes MLH1 and MSH2 in Microsatellite-Unstable Tumors

Cited by 19 publications

References 34 publications

Association of Apolipoprotein M Gene Polymorphisms with Ischemic Stroke in a Han Chinese Population

Association of Apolipoprotein M Gene Polymorphisms with Ischemic Stroke in a Han Chinese Population

Partial loss of heterozygosity events at the mutated gene in tumors from MLH1/MSH2 large genomic rearrangement carriers

Genetic analysis of KRAS mutation status in metastatic colorectal cancer patients

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