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Mutations in MLH1 gene and MSI status as molecular characteristics of sporadic colorectal cancer
Čaja, Fabián
Colorectal carcinoma (CRC) is one of the most prevalent malignancies in the Czech Republic. In general, there are two molecular pathways leading to CRC: one is characterized by chromosomal instability, the other by the deficiency in DNA mismatch repair (MMR) genes. MutL homologue 1 (MLH1) gene, a member of the MMR gene-family, represents a key component of the MMR system, responsible for recognition of nucleotide mismatches occurring during DNA replication, and for the recruitment of repair proteins to correct the replication errors. According to literature, somatic mutations in MMR genes, and MLH1 in particular, hallmark sporadic, MMR deficient, CRC cases. We aimed at analyzing somatic events in MLH1 gene and the determination of microsatellite instability (MSI) status in 99 DNA samples from 96 patients with sporadic CRC. Mutations were screened by high resolution melting (HRM) curve analysis. Positive cases in each run were subsequently verified by automated sequencing. Mainly gene variants were found in MLH1 gene: We discovered two new variants, one in exon 2 at position c. 204 C>G, p. Ile68Met (98 C/C, 1C/G) and the other in exon 11 at position c. 973 C>T, p. Arg325Trp (98 C/C, 1 C/T). Only the latter variant c. 973 C>T was identified as somatic mutation. All other variants found in MLH1 gene...
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Clinical and genetic aspects of familial breast cancer: Frequency of recurrent mutations in BRCA1 and BRCA2 genes in Czech republic and the role of NBN gene
Matějů, Martin ; Novotný, Jan (advisor) ; Konopásek, Bohuslav (referee) ; Vaňásek, Jaroslav (referee)
Summary: Background: An increased risk for development of hereditary breast cancer is associated with germline mutations in BRCA1/2 and the influence of NBN mutations is also supposed. The aim of this study is to specify the frequency of recurrent mutations in BRCA1/2 in unselected breast cancer patients and the frequency of most common pathogenic mutations in NBN in Czech republic, to assess current criteria for genetic testing and to consider the addition of NBN to the tested genes. Methods: Screening for recurrent mutations 5382insC and 300T>G in BRCA1 was performed by RFLP, screening for mutations in exon 11 of BRCA1 was performed by PTT, screening for mutations in a selected region of exon 11 of BRCA2 by DHPLC, and screening for mutations in exon 6 of NBN by HRMA. All the mutations were confirmed by direct sequencing. Results: In 679 unselected breast cancer patients 7 carriers of 5382insC, 3 of 300T>G, and 4 of other mutations in BRCA1 were identified. 2 locally prevalent mutations were found in BRCA2. In 730 controls only one 5382insC BRCA1 mutation was identified. Out of 5 NBN mutations found in 600 high-risk patients two were 657del5 and one R215W. A total of 8 NBN mutation carriers were identified among 703 breast cancer patients, 2 of them 657del5 carriers and three R215W carriers. In 915...
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Mutations in MLH1 gene and MSI status as molecular characteristics of sporadic colorectal cancer
Čaja, Fabián
Colorectal carcinoma (CRC) is one of the most prevalent malignancies in the Czech Republic. In general, there are two molecular pathways leading to CRC: one is characterized by chromosomal instability, the other by the deficiency in DNA mismatch repair (MMR) genes. MutL homologue 1 (MLH1) gene, a member of the MMR gene-family, represents a key component of the MMR system, responsible for recognition of nucleotide mismatches occurring during DNA replication, and for the recruitment of repair proteins to correct the replication errors. According to literature, somatic mutations in MMR genes, and MLH1 in particular, hallmark sporadic, MMR deficient, CRC cases. We aimed at analyzing somatic events in MLH1 gene and the determination of microsatellite instability (MSI) status in 99 DNA samples from 96 patients with sporadic CRC. Mutations were screened by high resolution melting (HRM) curve analysis. Positive cases in each run were subsequently verified by automated sequencing. Mainly gene variants were found in MLH1 gene: We discovered two new variants, one in exon 2 at position c. 204 C>G, p. Ile68Met (98 C/C, 1C/G) and the other in exon 11 at position c. 973 C>T, p. Arg325Trp (98 C/C, 1 C/T). Only the latter variant c. 973 C>T was identified as somatic mutation. All other variants found in MLH1 gene...
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Clinical and genetic aspects of familial breast cancerFrequency of recurrent mutations in BRCA1 and BRCA2 genes in Czech republic and the role of NBN gene
Matějů, Martin ; Novotný, Jan (advisor) ; Konopásek, Bohuslav (referee) ; Vaňásek, Jaroslav (referee)
Summary: Background: An increased risk for development of hereditary breast cancer is associated with germline mutations in BRCA1/2 and the influence of NBN mutations is also supposed. The aim of this study is to specify the frequency of recurrent mutations in BRCA1/2 in unselected breast cancer patients and the frequency of most common pathogenic mutations in NBN in Czech republic, to assess current criteria for genetic testing and to consider the addition of NBN to the tested genes. Methods: Screening for recurrent mutations 5382insC and 300T>G in BRCA1 was performed by RFLP, screening for mutations in exon 11 of BRCA1 was performed by PTT, screening for mutations in a selected region of exon 11 of BRCA2 by DHPLC, and screening for mutations in exon 6 of NBN by HRMA. All the mutations were confirmed by direct sequencing. Results: In 679 unselected breast cancer patients 7 carriers of 5382insC, 3 of 300T>G, and 4 of other mutations in BRCA1 were identified. 2 locally prevalent mutations were found in BRCA2. In 730 controls only one 5382insC BRCA1 mutation was identified. Out of 5 NBN mutations found in 600 high-risk patients two were 657del5 and one R215W. A total of 8 NBN mutation carriers were identified among 703 breast cancer patients, 2 of them 657del5 carriers and three R215W carriers. In 915...
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Mutations in MLH1 gene and MSI status as molecular characteristics of sporadic colorectal cancer
Čaja, Fabián ; Vodička, Pavel (advisor) ; Kadlecová, Jitka (referee)
Colorectal carcinoma (CRC) is one of the most prevalent malignancies in the Czech Republic. In general, there are two molecular pathways leading to CRC: one is characterized by chromosomal instability, the other by the deficiency in DNA mismatch repair (MMR) genes. MutL homologue 1 (MLH1) gene, a member of the MMR gene-family, represents a key component of the MMR system, responsible for recognition of nucleotide mismatches occurring during DNA replication, and for the recruitment of repair proteins to correct the replication errors. According to literature, somatic mutations in MMR genes, and MLH1 in particular, hallmark sporadic, MMR deficient, CRC cases. We aimed at analyzing somatic events in MLH1 gene and the determination of microsatellite instability (MSI) status in 99 DNA samples from 96 patients with sporadic CRC. Mutations were screened by high resolution melting (HRM) curve analysis. Positive cases in each run were subsequently verified by automated sequencing. Mainly gene variants were found in MLH1 gene: We discovered two new variants, one in exon 2 at position c. 204 C>G, p. Ile68Met (98 C/C, 1C/G) and the other in exon 11 at position c. 973 C>T, p. Arg325Trp (98 C/C, 1 C/T). Only the latter variant c. 973 C>T was identified as somatic mutation. All other variants found in MLH1 gene...
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