Optimization of experimental conditions for RNA-based sequencing of MLH1 and MSH2 genes

Jakubowska, Anna; Górski, Bohdan; Kurzawski, Grzegorz; Dębniak, Tadeusz; Hadaczek, Piotr; Cybulski, Cezary; Kładny, Józef; Oszurek, Oleg; Scott, Rodney J.; Lubiński, Jan

doi:10.1002/1098-1004(2001)17:1<52::aid-humu6>3.0.co;2-e

Cited by 18 publications

(8 citation statements)

References 34 publications

(36 reference statements)

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“…However, cost and sensitivity limitations can partially be overcome by RNA-based analysis [60]. Jakubowska et al note that, "...primers specific for RT [reverse transcription] of MLH1 and MSH2 are crucial for increasing the sensitivity of cDNA analysis.…”

Section: Molecular Genetics Of Lynch Syndromementioning

confidence: 99%

HNPCC (Lynch Syndrome): Differential Diagnosis, Molecular Genetics and Management - a Review

Lynch

Lynch²,

Shaw³

et al. 2003

Hered Cancer Clin Pract

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HNPCC (Lynch syndrome) is the most common form of hereditary colorectal cancer (CRC), wherein it accounts for between 2-7 percent of the total CRC burden. When considering the large number of extracolonic cancers integral to the syndrome, namely carcinoma of the endometrium, ovary, stomach, hepatobiliary system, pancreas, small bowel, brain tumors, and upper uroepithelial tract, these estimates of its frequency are likely to be conservative. The diagnosis is based upon its natural history in concert with a comprehensive cancer family history inclusive of all anatomic sites. In order for surveillance and management to be effective and, indeed, lifesaving, among these high-risk patients, the linchpin to cancer control would be the physician, who must be knowledgeable about hereditary cancer syndromes, their molecular and medical genetics, genetic counseling, and, most importantly, the natural history of the disorders, so that the entirety of this knowledge can be melded to highly-targeted management.

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Section: Molecular Genetics Of Lynch Syndromementioning

confidence: 99%

HNPCC (Lynch Syndrome): Differential Diagnosis, Molecular Genetics and Management - a Review

Lynch

Lynch²,

Shaw³

et al. 2003

Hered Cancer Clin Pract

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“…RNA based sequencing resulted in the detection of an aberrant MLH1 transcript which was the result of a loss of exon 16, but no change could be identified in genomic DNA. 18 In addition to unequivocal mutations, sequence variants of uncertain pathological significance were detected in five families. One of these alterations (c.875T>C) occurred in two patients (table 1).…”

Section: Resultsmentioning

confidence: 99%

“…This is not, however, caused by the 3.5 kb genomic deletion observed frequently in Finland, since in experiments using long PCR with primers for exons 15 and 17 we observed only the product of normal length. 18 Two of the most frequent mutations identified in Poland were also found in Lithuanian families, suggesting a common history. Poland and the Baltic States may have more common mutations than reported here since the number of samples from Estonia, Latvia, and Lithuania were too small to make the appropriate comparisons.…”

Section: Discussionmentioning

confidence: 96%

Germline MSH2 and MLH1 mutational spectrum in HNPCC families from Poland and the Baltic States

Kurzawski

Suchy

Kładny

et al. 2002

Journal of Medical Genetics

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H ereditary non-polyposis colorectal cancer (HNPCC, Lynch syndrome) is an autosomal dominantly inherited syndrome predisposing to the early development of cancers of the colon, rectum, endometrium, small bowel, and urinary tract and accounts for ∼5% of all colon cancer cases.1 There are at least five genes involved in this cancer predisposition and they include MLH1, 2 MSH2, 3 MSH6, 4 PMS2, and PMS1.5 Currently, more than 300 different mutations have been described in these genes which account for approximately 500 HNPCC kindreds from different parts of the world.6 MLH1 and MSH2 genes show abnormalities in more than 90% of HNPCC families with identified germline mutations 7 (http://www.nfdht.nl). The majority of reported MLH1 and MSH2 mutations are dispersed throughout the 35 exons of these two genes. However, some changes are recurrent and are described as founder mutations in particular populations. [8][9][10][11] In order to develop efficient DNA testing, it is important to describe the nature and frequency of mutations that are characteristic of particular ethnic groups. The MSH2 and MLH1 mutation spectrum has not been investigated in the eastern European region and therefore there is no knowledge about any recurrent mutations which may significantly aid in the mutation screening procedures for this region. Here, we describe the results of DNA/RNA based mutation sequencing of both the MSH2 and MLH1 genes in a series of HNPCC families from Poland (89 cases) and the Baltic States (12 cases). MATERIAL AND METHODS PatientsA total of 101 unrelated patients affected by colorectal cancer or an HNPCC associated cancer (endometrium, small bowel, urinary tract) were from 17 families which fulfilled the Amsterdam II criteria 12 and from 84 families matching our modified criteria of suspected HNPCC, one colorectal cancer patient with a first degree relative affected by an HNPCC associated cancer, one of whom was diagnosed under the age of 50 years. 13 The clinical diagnosis of HNPCC was established or verified at the Hereditary Cancer Centre, Pomeranian Academy of Medicine, Szczecin, Poland. Patients used for this study were ascertained from the following regions: Bydgoszcz (3), Gdańsk (3), Kielce (14), Kraków (3), Legnica (1), Lublin (2), Lódz (1), Olsztyn (13), Poznań (7), Riga (3), Szczecin (33), Tartu (3), Wrocław (5), Vilnius (6), Zielona Góra (4). DNA isolationPeripheral blood samples were collected from the patients after obtaining informed consent. DNA was extracted directly from leucocytes by the classical phenol purification method or as described previously.14 DNA sequencing All exons and exon-intron junctions of MLH1 and MSH2 were amplified using the same protocols as described previously 15 with the same primer sequences as described by Wijnen et al 16 17 for DGGE but without the M13 and GC clamp sequences at the 5′ end. Dye terminator cycle sequencing reactions were performed using the ABI PRISM Dye-terminator Cycle Sequencing Ready Reaction kit (Perkin Elmer) according to the manufacturer's recommended p...

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“…The MLH1 p.Arg687Trp missense mutation has been identified in several CRC families from different populations, e.g., Spain, Japan, Poland and Sweden [34][35][36][37]. The missense mutation was detected in a Spanish proband with HNPCC and in 3 affected siblings and therefore considered pathogenic [37].…”

Section: Discussionmentioning

confidence: 99%

Functional characterization of rare missense mutations in MLH1 and MSH2 identified in Danish colorectal cancer patients

et al. 2009

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Recently, we have performed a population based study to analyse the frequency of colorectal cancer related MLH1 and MSH2 missense mutations in the Danish population. Half of the analyzed mutations were rare and most likely only present in the families where they were identified originally. Some of the missense mutations were located in conserved regions in the MLH1 and MSH2 proteins indicating a relation to disease development. In the present study, we functionally characterized 10 rare missense mutations in MLH1 and MSH2 identified in 13 Danish CRC families. To elucidate the pathogenicity of the missense mutations, we carried out in vitro functional analyses. The missense mutations were analyzed for their effect on protein expression and repair efficiency. The results of the functional analysis were correlated with clinical data on the families carrying these mutations. Eight missense mutations resulted in proteins with expression and repair efficiency similar to the wild type. One missense mutation (MSH2 p.Met688Val) caused reduced protein expression and one (MSH2 p.Leu187Arg) caused both reduced protein expression and repair deficiency. The MSH2 p.Leu187Arg mutation was found in an Amsterdam II family presenting with high microsatellite instability and loss of MSH2 and MSH6 proteins in tumours. In conclusion, only 1/10 missense mutations displayed repair deficiency and could be classified as pathogenic. No final conclusion can be drawn on the MSH2 p.Met688Val mutation, which caused reduced protein expression. Although, no deficiencies have been identified in the proteins harbouring the other missense mutations, pathogenicity of these variants cannot be unambiguously excluded.

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Optimization of experimental conditions for RNA-based sequencing of MLH1 and MSH2 genes

Cited by 18 publications

References 34 publications

HNPCC (Lynch Syndrome): Differential Diagnosis, Molecular Genetics and Management - a Review

HNPCC (Lynch Syndrome): Differential Diagnosis, Molecular Genetics and Management - a Review

Germline MSH2 and MLH1 mutational spectrum in HNPCC families from Poland and the Baltic States

Functional characterization of rare missense mutations in MLH1 and MSH2 identified in Danish colorectal cancer patients

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