A complete protein truncation test for BRCA1 and BRCA2

Garvin, Alex M.

doi:10.1038/sj.ejhg.5200172

Cited by 31 publications

(18 citation statements)

References 19 publications

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“…While nonsense-mediated mRNA decay may cause falsenegative results in PTT experiments, 9 false-positive results due to illegitimate splicing have not been previously described as a possible pitfall of PTT. In our series of PTT analyses, the BRCA2 splice variants were observed in both patient and control tissues.…”

Section: Discussionmentioning

confidence: 99%

Alternatively spliced, truncated human BRCA2 isoforms contain a novel coding exon

et al. 2003

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The protein truncation test (PTT) employs in vitro transcription and translation of amplified cDNA and exonic gDNA to reveal truncating germ-line mutations. In a series of PTT analyses, abnormal splicing in the region encompassing exons 20-23 of BRCA2 was discovered in leucocytes from high-risk breast cancer patients. Although sequencing of the genomic DNA in this region failed to reveal a detectable mutation in these patients, cDNA obtained from this region of BRCA2 uncovered numerous alternative splice isoforms. PTT analysis and nested RT-PCR using RNA from leucocytes from healthy individuals, normal tissue and breast and ovarian cancer tumours demonstrated the presence of these alternatively spliced transcripts in all cases. The splice forms appeared to be more prominent in RNA from aged blood, suggesting that isoform expression was conditional. It is therefore important to distinguish naturally occurring alternative splicing from true splice defects due to mutations when interpreting PTT results.

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Section: Discussionmentioning

confidence: 99%

Alternatively spliced, truncated human BRCA2 isoforms contain a novel coding exon

et al. 2003

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“…The protein truncation test (PTT) was performed for exon 11 of BRCA1 and exons 10, 11 and 27 of BRCA2. This test covers 65% of the coding region for each gene and identifies the most common types of pathogenic mutation in BRCA1/2 (Garvin, 1998). Sequence analysis or heteroduplex analysis of exons 2 and 20 of BRCA1 using either polyacrylamide gel electrophoresis or denaturing high-performance liquid chromatography were used to detect nonsense, frameshift and missense mutations within these exons.…”

Section: Mutation Screening For Brca1 and Brca2mentioning

confidence: 99%

Evaluation of models to predict BRCA germline mutations

et al. 2006

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The selection of candidates for BRCA germline mutation testing is an important clinical issue yet it remains a significant challenge. A number of risk prediction models have been developed to assist in pretest counselling. We have evaluated the performance and the inter-rater reliability of four of these models (BRCAPRO, Manchester, Penn and the Myriad-Frank). The four risk assessment models were applied to 380 pedigrees of families who had undergone BRCA1/2 mutation analysis. Sensitivity, specificity, positive and negative predictive values, likelihood ratios and area under the receiver operator characteristic (ROC) curve were calculated for each model. Using a greater than 10% probability threshold, the likelihood that a BRCA test result was positive in a mutation carrier compared to the likelihood that the same result would be expected in an individual without a BRCA mutation was 2.10 (95% confidence interval (CI) 1.66 -2.67) for Penn, 1.74 (95% CI 1.48 -2.04) for Myriad, 1.35 (95% CI 1.19 -1.53) for Manchester and 1.68 (95% CI 1.39 -2.03) for BRCAPRO. Application of these models, therefore, did not rule in BRCA mutation carrier status. Similar trends were observed for separate BRCA1/2 performance measures except BRCA2 assessment in the Penn model where the positive likelihood ratio was 5.93. The area under the ROC curve for each model was close to 0.75. In conclusion, the four models had very little impact on the pre-test probability of disease; there were significant clinical barriers to using some models and risk estimates varied between experts. Use of models for predicting BRCA mutation status is not currently justified for populations such as that evaluated in the current study.

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“…CHK2I157T genotyping was determined using PCR-RFLP as previously described [39]. The 155bp region prone to the Thymine-Cytosine mutation (amino acid 430) was amplified using specific primers Chk2157F: 5'-ACCCATGTATCTAGGAGAGCTG-3' and Chk2157R: 5'-CCACTGTGATCTTCTATGTCTGCA-3'.…”

Section: Chk2i157t Mutation Analysismentioning

confidence: 99%

CHK21100delC, I157T, IVS2 +IG > A, BRCA1 and BRCA2 Mutation Analysis in JF305: A Pancreatic Cancer Cell Line

Ndawula¹,

Yang²,

Gong³

et al. 2014

JBM

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JF305 is a highly prolific pancreatic cancer cell line that originated from a Chinese patient. The cell line bears a functional HR double strand DNA repair mechanism but very responsive to PARP treatment a phenomenon clearly suggesting presence of an anomaly in the mechanism. Brca1, Brca2 and CHK2 proteins are very important constituents of the HR mechanism whose respective gene coding mutations are strongly associated with several cancers and are widely exploited in anticancer chemotherapy. In this current study, the BRCA1, BRCA2 gene mutation status in JF305 was determined together with the presence of 3 widely reported cancer linked CHK2 founder mutations (1100delC, I157T, IVS2 +IG > A). CHK21100delC genotype was determined using allele specific PCR, while the PCR-RFLP assay was used for I157T, IVS2 +IG > A analysis. PCR and direct sequencing were used for assessing the BRCA1 and BRCA2 gene. Results revealed that JF305 is CHK21100delC heterozygous mutant, CHK2I157T and CHK2IVS2 +IG > A wild type. Furthermore, it was observed that JF305 lacked BRCA1 and BRCA2 gene mutations. The mutation status identification of CHK2 and BRCA1/2 in JF305 provides a major milestone towards elucidating the properties of the cell line which subsequently promises to be an excellent model for evaluating the role of parp inhibitors in pancreatic cancer chemotherapy most especially in the respective cancer cell lines without BRCA1 and BRCA2 gene mutations.

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A complete protein truncation test for BRCA1 and BRCA2

Cited by 31 publications

References 19 publications

Alternatively spliced, truncated human BRCA2 isoforms contain a novel coding exon

Alternatively spliced, truncated human BRCA2 isoforms contain a novel coding exon

Evaluation of models to predict BRCA germline mutations

CHK21100delC, I157T, IVS2 +IG > A, BRCA1 and BRCA2 Mutation Analysis in JF305: A Pancreatic Cancer Cell Line

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A complete protein truncation test for BRCA1 and BRCA2

Cited by 31 publications

References 19 publications

Alternatively spliced, truncated human BRCA2 isoforms contain a novel coding exon

Alternatively spliced, truncated human BRCA2 isoforms contain a novel coding exon

Evaluation of models to predict BRCA germline mutations

CHK21100delC, I157T, IVS2 +IG &gt; A, BRCA1 and BRCA2 Mutation Analysis in JF305: A Pancreatic Cancer Cell Line

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CHK21100delC, I157T, IVS2 +IG > A, BRCA1 and BRCA2 Mutation Analysis in JF305: A Pancreatic Cancer Cell Line