Additional file 2: Table S2. of BRCA2 carriers with male breast cancer show elevated tumour methylation

Methylation specific high resolution melting condition and primers (XLSX 36 kb

Additional file 1: Table S1. of BRCA2 carriers with male breast cancer show elevated tumour methylation

REMARK patient flow through study (XLSX 34 kb

Additional file 3: Figure S1. of BRCA2 carriers with male breast cancer show elevated tumour methylation

a) BRCA2 subgroup cluster analysis, b) BRCAX subgroup cluster analysis, c) Numbers and sizes of clusters within BRCA2 and BRCAX subgroups using various correlation coefficient cut-offs (listed on the x-axis), d) age of diagnosis of patient within Cluster A, B and other BRCA2 tumours (DOCX 234 kb

Chocolate City -­‐ Vanilla Suburbs. Eine Reise in Vergangenheit und Zukunft des Städtischen

<p>PCR-based RNA splicing assays are commonly used in diagnostic and research settings to assess the potential effects of variants of uncertain clinical significance in BRCA1 and BRCA2. The Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium completed a multicentre investigation to evaluate differences in assay design and the integrity of published data, raising a number of methodological questions associated with cell culture conditions and PCR-based protocols. We utilized targeted RNA-seq to re-assess BRCA1 and BRCA2 mRNA isoform expression patterns in lymphoblastoid cell lines (LCLs) previously used in the multicentre ENIGMA study. Capture of the targeted cDNA sequences was carried out using 34 BRCA1 and 28 BRCA2 oligonucleotides from the Illumina Truseq Targeted RNA Expression platform. Our results show that targeted RNA-seq analysis of LCLs overcomes many of the methodology limitations associated with PCR-based assays leading us to make the following observations and recommendations: (1) technical replicates (n > 2) of variant carriers to capture methodology induced variability associated with RNA-seq assays, (2) LCLs can undergo multiple freeze/thaw cycles and can be cultured up to 2 weeks without noticeably influencing isoform expression levels, (3) nonsense-mediated decay inhibitors are essential prior to splicing assays for comprehensive mRNA isoform detection, (4) quantitative assessment of exon:exon junction levels across BRCA1 and BRCA2 can help distinguish between normal and aberrant isoform expression patterns. Experimentally derived recommendations from this study will facilitate the application of targeted RNA-seq platforms for the quantitation of BRCA1 and BRCA2 mRNA aberrations associated with sequence variants of uncertain clinical significance.</p

image_6_Investigation of Experimental Factors That Underlie BRCA1/2 mRNA Isoform Expression Variation: Recommendations for Utilizing Targeted RNA Sequencing to Evaluate Potential Spliceogenic Variants.tif

<p>PCR-based RNA splicing assays are commonly used in diagnostic and research settings to assess the potential effects of variants of uncertain clinical significance in BRCA1 and BRCA2. The Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium completed a multicentre investigation to evaluate differences in assay design and the integrity of published data, raising a number of methodological questions associated with cell culture conditions and PCR-based protocols. We utilized targeted RNA-seq to re-assess BRCA1 and BRCA2 mRNA isoform expression patterns in lymphoblastoid cell lines (LCLs) previously used in the multicentre ENIGMA study. Capture of the targeted cDNA sequences was carried out using 34 BRCA1 and 28 BRCA2 oligonucleotides from the Illumina Truseq Targeted RNA Expression platform. Our results show that targeted RNA-seq analysis of LCLs overcomes many of the methodology limitations associated with PCR-based assays leading us to make the following observations and recommendations: (1) technical replicates (n > 2) of variant carriers to capture methodology induced variability associated with RNA-seq assays, (2) LCLs can undergo multiple freeze/thaw cycles and can be cultured up to 2 weeks without noticeably influencing isoform expression levels, (3) nonsense-mediated decay inhibitors are essential prior to splicing assays for comprehensive mRNA isoform detection, (4) quantitative assessment of exon:exon junction levels across BRCA1 and BRCA2 can help distinguish between normal and aberrant isoform expression patterns. Experimentally derived recommendations from this study will facilitate the application of targeted RNA-seq platforms for the quantitation of BRCA1 and BRCA2 mRNA aberrations associated with sequence variants of uncertain clinical significance.</p

table_10_Investigation of Experimental Factors That Underlie BRCA1/2 mRNA Isoform Expression Variation: Recommendations for Utilizing Targeted RNA Sequencing to Evaluate Potential Spliceogenic Variants.xlsx

<p>PCR-based RNA splicing assays are commonly used in diagnostic and research settings to assess the potential effects of variants of uncertain clinical significance in BRCA1 and BRCA2. The Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium completed a multicentre investigation to evaluate differences in assay design and the integrity of published data, raising a number of methodological questions associated with cell culture conditions and PCR-based protocols. We utilized targeted RNA-seq to re-assess BRCA1 and BRCA2 mRNA isoform expression patterns in lymphoblastoid cell lines (LCLs) previously used in the multicentre ENIGMA study. Capture of the targeted cDNA sequences was carried out using 34 BRCA1 and 28 BRCA2 oligonucleotides from the Illumina Truseq Targeted RNA Expression platform. Our results show that targeted RNA-seq analysis of LCLs overcomes many of the methodology limitations associated with PCR-based assays leading us to make the following observations and recommendations: (1) technical replicates (n > 2) of variant carriers to capture methodology induced variability associated with RNA-seq assays, (2) LCLs can undergo multiple freeze/thaw cycles and can be cultured up to 2 weeks without noticeably influencing isoform expression levels, (3) nonsense-mediated decay inhibitors are essential prior to splicing assays for comprehensive mRNA isoform detection, (4) quantitative assessment of exon:exon junction levels across BRCA1 and BRCA2 can help distinguish between normal and aberrant isoform expression patterns. Experimentally derived recommendations from this study will facilitate the application of targeted RNA-seq platforms for the quantitation of BRCA1 and BRCA2 mRNA aberrations associated with sequence variants of uncertain clinical significance.</p

image_10_Investigation of Experimental Factors That Underlie BRCA1/2 mRNA Isoform Expression Variation: Recommendations for Utilizing Targeted RNA Sequencing to Evaluate Potential Spliceogenic Variants.tif

<p>PCR-based RNA splicing assays are commonly used in diagnostic and research settings to assess the potential effects of variants of uncertain clinical significance in BRCA1 and BRCA2. The Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium completed a multicentre investigation to evaluate differences in assay design and the integrity of published data, raising a number of methodological questions associated with cell culture conditions and PCR-based protocols. We utilized targeted RNA-seq to re-assess BRCA1 and BRCA2 mRNA isoform expression patterns in lymphoblastoid cell lines (LCLs) previously used in the multicentre ENIGMA study. Capture of the targeted cDNA sequences was carried out using 34 BRCA1 and 28 BRCA2 oligonucleotides from the Illumina Truseq Targeted RNA Expression platform. Our results show that targeted RNA-seq analysis of LCLs overcomes many of the methodology limitations associated with PCR-based assays leading us to make the following observations and recommendations: (1) technical replicates (n > 2) of variant carriers to capture methodology induced variability associated with RNA-seq assays, (2) LCLs can undergo multiple freeze/thaw cycles and can be cultured up to 2 weeks without noticeably influencing isoform expression levels, (3) nonsense-mediated decay inhibitors are essential prior to splicing assays for comprehensive mRNA isoform detection, (4) quantitative assessment of exon:exon junction levels across BRCA1 and BRCA2 can help distinguish between normal and aberrant isoform expression patterns. Experimentally derived recommendations from this study will facilitate the application of targeted RNA-seq platforms for the quantitation of BRCA1 and BRCA2 mRNA aberrations associated with sequence variants of uncertain clinical significance.</p

table_1_Investigation of Experimental Factors That Underlie BRCA1/2 mRNA Isoform Expression Variation: Recommendations for Utilizing Targeted RNA Sequencing to Evaluate Potential Spliceogenic Variants.PDF

<p>PCR-based RNA splicing assays are commonly used in diagnostic and research settings to assess the potential effects of variants of uncertain clinical significance in BRCA1 and BRCA2. The Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium completed a multicentre investigation to evaluate differences in assay design and the integrity of published data, raising a number of methodological questions associated with cell culture conditions and PCR-based protocols. We utilized targeted RNA-seq to re-assess BRCA1 and BRCA2 mRNA isoform expression patterns in lymphoblastoid cell lines (LCLs) previously used in the multicentre ENIGMA study. Capture of the targeted cDNA sequences was carried out using 34 BRCA1 and 28 BRCA2 oligonucleotides from the Illumina Truseq Targeted RNA Expression platform. Our results show that targeted RNA-seq analysis of LCLs overcomes many of the methodology limitations associated with PCR-based assays leading us to make the following observations and recommendations: (1) technical replicates (n > 2) of variant carriers to capture methodology induced variability associated with RNA-seq assays, (2) LCLs can undergo multiple freeze/thaw cycles and can be cultured up to 2 weeks without noticeably influencing isoform expression levels, (3) nonsense-mediated decay inhibitors are essential prior to splicing assays for comprehensive mRNA isoform detection, (4) quantitative assessment of exon:exon junction levels across BRCA1 and BRCA2 can help distinguish between normal and aberrant isoform expression patterns. Experimentally derived recommendations from this study will facilitate the application of targeted RNA-seq platforms for the quantitation of BRCA1 and BRCA2 mRNA aberrations associated with sequence variants of uncertain clinical significance.</p

table_4_Investigation of Experimental Factors That Underlie BRCA1/2 mRNA Isoform Expression Variation: Recommendations for Utilizing Targeted RNA Sequencing to Evaluate Potential Spliceogenic Variants.PDF

<p>PCR-based RNA splicing assays are commonly used in diagnostic and research settings to assess the potential effects of variants of uncertain clinical significance in BRCA1 and BRCA2. The Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium completed a multicentre investigation to evaluate differences in assay design and the integrity of published data, raising a number of methodological questions associated with cell culture conditions and PCR-based protocols. We utilized targeted RNA-seq to re-assess BRCA1 and BRCA2 mRNA isoform expression patterns in lymphoblastoid cell lines (LCLs) previously used in the multicentre ENIGMA study. Capture of the targeted cDNA sequences was carried out using 34 BRCA1 and 28 BRCA2 oligonucleotides from the Illumina Truseq Targeted RNA Expression platform. Our results show that targeted RNA-seq analysis of LCLs overcomes many of the methodology limitations associated with PCR-based assays leading us to make the following observations and recommendations: (1) technical replicates (n > 2) of variant carriers to capture methodology induced variability associated with RNA-seq assays, (2) LCLs can undergo multiple freeze/thaw cycles and can be cultured up to 2 weeks without noticeably influencing isoform expression levels, (3) nonsense-mediated decay inhibitors are essential prior to splicing assays for comprehensive mRNA isoform detection, (4) quantitative assessment of exon:exon junction levels across BRCA1 and BRCA2 can help distinguish between normal and aberrant isoform expression patterns. Experimentally derived recommendations from this study will facilitate the application of targeted RNA-seq platforms for the quantitation of BRCA1 and BRCA2 mRNA aberrations associated with sequence variants of uncertain clinical significance.</p

table_2_Investigation of Experimental Factors That Underlie BRCA1/2 mRNA Isoform Expression Variation: Recommendations for Utilizing Targeted RNA Sequencing to Evaluate Potential Spliceogenic Variants.xlsx

<p>PCR-based RNA splicing assays are commonly used in diagnostic and research settings to assess the potential effects of variants of uncertain clinical significance in BRCA1 and BRCA2. The Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium completed a multicentre investigation to evaluate differences in assay design and the integrity of published data, raising a number of methodological questions associated with cell culture conditions and PCR-based protocols. We utilized targeted RNA-seq to re-assess BRCA1 and BRCA2 mRNA isoform expression patterns in lymphoblastoid cell lines (LCLs) previously used in the multicentre ENIGMA study. Capture of the targeted cDNA sequences was carried out using 34 BRCA1 and 28 BRCA2 oligonucleotides from the Illumina Truseq Targeted RNA Expression platform. Our results show that targeted RNA-seq analysis of LCLs overcomes many of the methodology limitations associated with PCR-based assays leading us to make the following observations and recommendations: (1) technical replicates (n > 2) of variant carriers to capture methodology induced variability associated with RNA-seq assays, (2) LCLs can undergo multiple freeze/thaw cycles and can be cultured up to 2 weeks without noticeably influencing isoform expression levels, (3) nonsense-mediated decay inhibitors are essential prior to splicing assays for comprehensive mRNA isoform detection, (4) quantitative assessment of exon:exon junction levels across BRCA1 and BRCA2 can help distinguish between normal and aberrant isoform expression patterns. Experimentally derived recommendations from this study will facilitate the application of targeted RNA-seq platforms for the quantitation of BRCA1 and BRCA2 mRNA aberrations associated with sequence variants of uncertain clinical significance.</p