Denaturing High-Performance Liquid Chromatography Detects Reliably BRCA1 and BRCA2 Mutations

Wagner, Teresa; Stoppa‐Lyonnet, Dominique; Fleischmann, E.; Muhr, Daniela; Pagès, Sabine; Sandberg, Torsten; Caux, Virginie; Moeslinger, Regina; Langbauer, Gudrun; Borg, Åke; Oefner, Peter J.

doi:10.1006/geno.1999.6026

Cited by 204 publications

(164 citation statements)

References 19 publications

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“…21 In addition, gels need to be exposed to a sensitive film for a variable length of time, and the interpretation of band patterns can be cumbersome for the inexperienced eye. Denaturing HPLC has been successfully applied in the mutation detection of BRCA1 gene, [27][28][29] PTEN, 30,31 and the tuberous sclerosis genes TSC1 and TSC2. 24,32 It has been estimated that the sensitivity of the detection is greater than 95%; 30 similar or superior to that obtained by SSCP.…”

Section: Resultsmentioning

confidence: 99%

Validation of Denaturing High Performance Liquid Chromatography as a Rapid Detection Method for the Identification of Human INK4A Gene Mutations

Orlow

Roy

Barz

et al. 2001

The Journal of Molecular Diagnostics

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The incidence of melanoma is increasing rapidly in western countries. Genetic predisposition in familial and in some sporadic melanomas has been associated with the presence of INK4A gene mutations. To better define the risk for developing sporadic melanoma based on genetic and environmental interactions, large groups of cases need to be studied. Mutational analysis of genes lacking hot spots for sequence variations is time consuming and expensive. In this study we present the application of denaturing high performance liquid chromatography (DHPLC) for screening of mutations. Exons 1␣, 2, and 3 were amplified from 129 samples and 13 known mutants, yielding 347 products that were examined at different temperatures. Forty-two of these amplicons showed a distinct non-wild-type profile on the chromatogram. Independent sequencing analysis confirmed 16 different nucleotide variations in Leu 32 Pro; Ile49Thr; 88 del G; Gln50Arg; Arg24Pro; Met53Ile; Met53Thr; Arg58stop; Pro81Leu; Asp84Ala; Arg80stop; Gly101Trp; Val106Val; Ala148Thr; and in positions (؊2) in intron 1 (C 3 T); and in the 3 UTR, nucleotide 500 (C 3 G). No false negatives or false positives were obtained by DHPLC in samples with mutations or polymorphisms. We conclude that the DHPLC is a fast, sensitive, cost-efficient, and reliable method for the scanning of Cutaneous melanomas are being detected at an increasing rate worldwide. Even though many patients are diagnosed at an early stage, the death rate continues to rise due to the increasing incidence of more advanced lesions. 1,2 Genetic and environmental factors such as family history, skin type, previous tumors, and sun exposure have been identified as important risk factors. [3][4][5][6] In addition, germline mutations or variants of certain genes have been proposed as risk factors for the development of melanomas. One of these genes, the CDKN2A or INK4A, encodes for p16, an important cell cycle regulator capable of arresting cells in G1-phase by inhibiting the phosphorylation of the retinoblastoma protein by cyclinD1/Cdk4 complexes. 7 The INK4A gene has been found silenced by point mutation, deletion, and methylation of the promoter region in several sporadic tumor types. 8 -16 Analyses of INK4A in sporadic melanomas revealed a frequency of mutations and deletions that ranges from approximately 75% in cell lines 8 to 15% in primary multiple melanoma tumors. 17 In addition, INK4A germline mutations have been found in melanoma kindreds, ranging in prevalence from 10.3 to 72.2%, 18 -19 although in overall approximately 20% of the families that have been studied show mutations in this gene. 20 In an attempt to better define the gene-environment interactions in sporadic melanoma, our group expects to enroll 4000 newly diagnosed subjects to determine the relationship between germline INK4A mutations and environmental factors such as sun exposure. Typically, INK4A gene mutations have been analyzed by polymerase chain reaction-single stranded conformational polymorphism (PCR-SSCP) and sequencing. 16,18,21 D...

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Section: Resultsmentioning

confidence: 99%

Validation of Denaturing High Performance Liquid Chromatography as a Rapid Detection Method for the Identification of Human INK4A Gene Mutations

Orlow

Roy

Barz

et al. 2001

The Journal of Molecular Diagnostics

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“…The procedure used for molecular analysis, as well as the mode of information collection, such as family cancers, age at cancer diagnosis of relatives, and age at death or current age, have been reported previously. 23,24 Prophylactic SO was offered to these patients according to the recommendations of the French INSERM-FNCLCC collective expertise updated in 2004. 19 At Institut Curie, ultrasound surveillance of the pelvis and abdominal cavity before SO has been regularly performed once a year since 2002.…”

Section: Patientsmentioning

confidence: 99%

Prophylactic salpingo‐oophorectomy in a series of 89 women carrying a BRCA1 or a BRCA2 mutation

Laki

Kirova

This

et al. 2007

Cancer

Self Cite

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BACKGROUND.Prophylactic salpingo‐oophorectomy (SO), which is recommended in BRCA1/2 mutation carriers, still needs to be reappraised.METHODS.In all, 89 BRCA1/BRCA2 mutation carriers underwent SO between 1994–2004. Past medical and familial history, SO, results and survival after SO were analyzed.RESULTS.The series consisted of 56 BRCA1 and 33 BRCA2 mutation carriers. All but 1 had a family history of breast (BC) and/or ovarian cancer; 42 BRCA1 and 31 BRCA2 had a personal history of BC. The median age at SO was 44 (BRCA1) and 49.5 (BRCA2) years for women without previous BC (not significant) and 48 (BRCA1) and 53 BRCA2) years (P = .03) for women with previous BC. Occult ovarian (n = 2) and/or fallopian (n = 3) carcinomas were found in 4 patients (4.5%): 1 experienced recurrence (4 years), 2 are disease‐free (26 and 38 months of follow‐up), and 1 died from BC (12 months). Among the other 69 patients with previous BC (median follow‐up, 42 months), 14 developed ipsilateral or contralateral BC and 8 developed metastatic disease. Among the 16 patients without previous BC (median follow‐up, 27 months), 3 developed BC. Of the 89 patients, 85 are still alive: 3 died from BC and 1 died from pancreatic cancer. No peritoneal malignancy was observed.CONCLUSIONS.This study shows that prophylactic SO remains an important option for BRCA1/2 mutation carriers as asymptomatic ovarian/fallopian cancers were found in 4.5% of patients. However, a longer follow‐up and larger series are required to more precisely evaluate the benefits of this procedure in terms of BC incidence, peritoneal malignancy, or recurrence. Cancer 2007. © 2007 American Cancer Society.

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“…It is a robust, semiautomatic screening method that has been reported to be more sensitive than PCR/SSCP in a number of comparative studies. 24,25 …”

mentioning

confidence: 99%

“…In a large survey of 180 BRCA1/2 mutations in hereditary breast cancer families, the DHPLC identified 179/180 of the known mutations. 24 Our study is the first in which this method is used in the mutation screening of CDH1. In our families, 8 sequence variants were detected in only 20 samples.…”

mentioning

confidence: 99%

Germline mutations in E‐cadherin do not explain association of hereditary prostate cancer, gastric cancer and breast cancer

Jonsson

Bergh

Stattin

et al. 2002

Intl Journal of Cancer

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Somatic mutations in the E-cadherin (CDH1) gene have frequently been reported in cases with diffuse gastric and lobular breast cancers. Recently, germline mutations have been identified in families with diffuse gastric cancers. In families with hereditary prostate cancer (HPC), a significant association of prostate cancer, gastric and/or breast cancer has been observed in epidemiological studies. The aim of this study was to investigate if germline mutations in CDH1 could explain the risk for cancer in HPC families with an excess of gastric and breast cancer. In total, 17 members from 13 HPC families and 3 members from 3 families with hereditary gastric cancer (HGC) were screened for germline CDH1 sequence alterations using PCR/Denaturing HPLC for initial screening of nucleotide variants followed by confirmatory direct sequencing analysis. The frequency of identified novel germline mutations were tested for in 136 cases with hereditary prostate cancer and 215 cases of sporadic prostate cancer with 422 age matched controls in an allelic discrimination assay. In total, 8 sequence variants were detected in 20 samples tested. In the HPC families, we found 2 missense mutations, A592T in exon 12 and a novel D777N in exon 15 and a mutation in intron 5, 687؉92T>A. A previously known polymorphism in exon 13 and 3 sequence variations in introns and untranslated regions were also found, of which the significance is unknown. In HGC-023 with early onset diffuse gastric cancer a truncating mutation, R335X, was identified in exon 7. None of the missense mutations or 687؉92T>A were found in the extended HPC material or in the sporadic prostate cancer cases with age-matched controls in the allelic discrimination assay. We found several germline mutations of unknown clinical significance in the CDH1 gene that probably do not explain the association of prostate, gastric and/or breast cancers in the HPC-families. Two missense mutations and a mutation in intron 5 were identified that do not influence the risk of hereditary or sporadic prostate cancer in general and are considered to be pedigree specific. In a family with hereditary gastric cancer of the diffuse type, we identified the first truncating germline mutation in a Scandinavian family.

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Denaturing High-Performance Liquid Chromatography Detects Reliably BRCA1 and BRCA2 Mutations

Cited by 204 publications

References 19 publications

Validation of Denaturing High Performance Liquid Chromatography as a Rapid Detection Method for the Identification of Human INK4A Gene Mutations

Validation of Denaturing High Performance Liquid Chromatography as a Rapid Detection Method for the Identification of Human INK4A Gene Mutations

Prophylactic salpingo‐oophorectomy in a series of 89 women carrying a BRCA1 or a BRCA2 mutation

Germline mutations in E‐cadherin do not explain association of hereditary prostate cancer, gastric cancer and breast cancer

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