Dataset Information

Hi-Plex for high-throughput mutation screening: application to the breast cancer susceptibility gene PALB2.

ABSTRACT: Massively parallel sequencing (MPS) has revolutionised biomedical research and offers enormous capacity for clinical application. We previously reported Hi-Plex, a streamlined highly-multiplexed PCR-MPS approach, allowing a given library to be sequenced with both the Ion Torrent and TruSeq chemistries. Comparable sequencing efficiency was achieved using material derived from lymphoblastoid cell lines and formalin-fixed paraffin-embedded tumour.Here, we report high-throughput application of Hi-Plex by performing blinded mutation screening of the coding regions of the breast cancer susceptibility gene PALB2 on a set of 95 blood-derived DNA samples that had previously been screened using Sanger sequencing and high-resolution melting curve analysis (n?=?90), or genotyped by Taqman probe-based assays (n?=?5). Hi-Plex libraries were prepared simultaneously using relatively inexpensive, readily available reagents in a simple half-day protocol followed by MPS on a single MiSeq run.We observed that 99.93% of amplicons were represented at ?10X coverage. All 56 previously identified variant calls were detected and no false positive calls were assigned. Four additional variant calls were made and confirmed upon re-analysis of previous data or subsequent Sanger sequencing.These results support Hi-Plex as a powerful approach for rapid, cost-effective and accurate high-throughput mutation screening. They further demonstrate that Hi-Plex methods are suitable for and can meet the demands of high-throughput genetic testing in research and clinical settings.

SUBMITTER: Nguyen-Dumont T

PROVIDER: S-EPMC3829211 | biostudies-literature | 2013 Nov

REPOSITORIES: biostudies-literature

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Hi-Plex for high-throughput mutation screening: application to the breast cancer susceptibility gene PALB2.

Nguyen-Dumont Tú T Teo Zhi L ZL Pope Bernard J BJ Hammet Fleur F Mahmoodi Maryam M Tsimiklis Helen H Sabbaghian Nelly N Tischkowitz Marc M Foulkes William D WD Giles Graham G GG Hopper John L JL Southey Melissa C MC Park Daniel J DJ

BMC medical genomics 20131108

<h4>Background</h4>Massively parallel sequencing (MPS) has revolutionised biomedical research and offers enormous capacity for clinical application. We previously reported Hi-Plex, a streamlined highly-multiplexed PCR-MPS approach, allowing a given library to be sequenced with both the Ion Torrent and TruSeq chemistries. Comparable sequencing efficiency was achieved using material derived from lymphoblastoid cell lines and formalin-fixed paraffin-embedded tumour.<h4>Methods</h4>Here, we report h ...[more]

PMID: 24206657

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Project description:As a group, women who carry germline mutations in partner and localizer of breast cancer 2 susceptibility protein (PALB2) are at increased risk of breast cancer. Little is known about by how much or whether risk differs by mutation or family history, owing to the paucity of studies of cases unselected for family history.We screened 1,403 case probands for PALB2 mutations in a population-based study of Australian women with invasive breast cancer stratified by age at onset. The age-specific risk of breast cancer was estimated from the cancer histories of first- and second-degree relatives of mutation-carrying probands using a modified segregation analysis that included a polygenic modifier and was conditioned on the carrier case proband. Further screening for PALB2 c.3113G > A (W1038X) was conducted for 779 families with multiple cases of breast cancer ascertained through family cancer clinics in Australia and New Zealand and 764 population-based controls.We found five independent case probands in the population-based sample with the protein-truncating mutation PALB2 c.3113G > A (W1038X); 2 of 695 were diagnosed before age 40 years and 3 of 708 were diagnosed when between ages 40 and 59 years. Both of the two early-onset carrier case probands had very strong family histories of breast cancer. Further testing found that the mutation segregated with breast cancer in these families. No c.3113G > A (W1038X) carriers were found in 764 population-based unaffected controls. The hazard ratio was estimated to be 30.1 (95% confidence interval (CI), 7.5 to 120; P < 0.0001), and the corresponding cumulative risk estimates were 49% (95% CI, 15 to 93) to age 50 and 91% (95% CI, 44 to 100) to age 70. We found another eight families carrying this mutation in 779 families with multiple cases of breast cancer ascertained through family cancer clinics.The PALB2 c.3113G > A mutation appears to be associated with substantial risks of breast cancer that are of clinical relevance.

Project description:INTRODUCTION:Two major high-penetrance breast cancer genes, BRCA1 and BRCA2, are responsible for approximately 20% of hereditary breast cancer (HBC) cases in Finland. Additionally, rare mutations in several other genes that interact with BRCA1 and BRCA2 increase the risk of HBC. Still, a majority of HBC cases remain unexplained which is challenging for genetic counseling. We aimed to analyze additional mutations in HBC-associated genes and to define the sensitivity of our current BRCA1/2 mutation analysis protocol used in genetic counseling. METHODS:Eighty-two well-characterized, high-risk hereditary breast and/or ovarian cancer (HBOC) BRCA1/2-founder mutation-negative Finnish individuals, were screened for germline alterations in seven breast cancer susceptibility genes, BRCA1, BRCA2, CHEK2, PALB2, BRIP1, RAD50, and CDH1. BRCA1/2 were analyzed by multiplex ligation-dependent probe amplification (MLPA) and direct sequencing. CHEK2 was analyzed by the high resolution melt (HRM) method and PALB2, RAD50, BRIP1 and CDH1 were analyzed by direct sequencing. Carrier frequencies between 82 (HBOC) BRCA1/2-founder mutation-negative Finnish individuals and 384 healthy Finnish population controls were compared by using Fisher's exact test. In silico prediction for novel missense variants effects was carried out by using Pathogenic-Or-Not -Pipeline (PON-P). RESULTS:Three previously reported breast cancer-associated variants, BRCA1 c.5095C > T, CHEK2 c.470T > C, and CHEK2 c.1100delC, were observed in eleven (13.4%) individuals. Ten of these individuals (12.2%) had CHEK2 variants, c.470T > C and/or c.1100delC. Fourteen novel sequence alterations and nine individuals with more than one non-synonymous variant were identified. One of the novel variants, BRCA2 c.72A > T (Leu24Phe) was predicted to be likely pathogenic in silico. No large genomic rearrangements were detected in BRCA1/2 by multiplex ligation-dependent probe amplification (MLPA). CONCLUSIONS:In this study, mutations in previously known breast cancer susceptibility genes can explain 13.4% of the analyzed high-risk BRCA1/2-negative HBOC individuals. CHEK2 mutations, c.470T > C and c.1100delC, make a considerable contribution (12.2%) to these high-risk individuals but further segregation analysis is needed to evaluate the clinical significance of these mutations before applying them in clinical use. Additionally, we identified novel variants that warrant additional studies. Our current genetic testing protocol for 28 Finnish BRCA1/2-founder mutations and protein truncation test (PTT) of the largest exons is sensitive enough for clinical use as a primary screening tool.

Dataset Information

Hi-Plex for high-throughput mutation screening: application to the breast cancer susceptibility gene PALB2.

Publications

Hi-Plex for high-throughput mutation screening: application to the breast cancer susceptibility gene PALB2.

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