VTRNA2-1: Genetic Variation, Heritable Methylation and Disease Association.

VTRNA2-1 is a metastable epiallele with accumulating evidence that methylation at this region is heritable, modifiable and associated with disease including risk and progression of cancer. This study investigated the influence of genetic variation and other factors such as age and adult lifestyle on blood DNA methylation in this region. We first sequenced the VTRNA2-1 gene region in multiple-case breast cancer families in which VTRNA2-1 methylation was identified as heritable and associated with breast cancer risk. Methylation quantitative trait loci (mQTL) were investigated using a prospective cohort study (4500 participants with genotyping and methylation data). The cis-mQTL analysis (334 variants ± 50 kb of the most heritable CpG site) identified 43 variants associated with VTRNA2-1 methylation (p < 1.5 × 10-4); however, these explained little of the methylation variation (R2 < 0.5% for each of these variants). No genetic variants elsewhere in the genome were found to strongly influence VTRNA2-1 methylation. SNP-based heritability estimates were consistent with the mQTL findings (h2 = 0, 95%CI: -0.14 to 0.14). We found no evidence that age, sex, country of birth, smoking, body mass index, alcohol consumption or diet influenced blood DNA methylation at VTRNA2-1. Genetic factors and adult lifestyle play a minimal role in explaining methylation variability at the heritable VTRNA2-1 cluster.

Rare germline genetic variants and risk of aggressive prostate cancer.

Few genetic risk factors have been demonstrated to be specifically associated with aggressive prostate cancer (PrCa). Here, we report a case-case study of PrCa comparing the prevalence of germline pathogenic/likely pathogenic (P/LP) genetic variants in 787 men with aggressive disease and 769 with nonaggressive disease. Overall, we observed P/LP variants in 11.4% of men with aggressive PrCa and 9.8% of men with nonaggressive PrCa (two-tailed Fisher's exact tests, P = .28). The proportion of BRCA2 and ATM P/LP variant carriers in men with aggressive PrCa exceeded that observed in men with nonaggressive PrCa; 18/787 carriers (2.3%) and 4/769 carriers (0.5%), P = .004, and 14/787 carriers (0.02%) and 5/769 carriers (0.01%), P = .06, respectively. Our findings contribute to the extensive international effort to interpret the genetic variation identified in genes included on gene-panel tests, for which there is currently an insufficient evidence-base for clinical translation in the context of PrCa risk.

Genetic testing in Poland and Ukraine: should comprehensive germline testing of BRCA1 and BRCA2 be recommended for women with breast and ovarian cancer?

PURPOSE: To characterize the spectrum of BRCA1 and BRCA2 pathogenic germline variants in women from south-west Poland and west Ukraine affected with breast or ovarian cancer. Testing in women at high risk of breast and ovarian cancer in these regions is currently mainly limited to founder mutations. METHODS: Unrelated women affected with breast and/or ovarian cancer from Poland (n = 337) and Ukraine (n = 123) were screened by targeted sequencing. Excluded from targeted sequencing were 34 Polish women who had previously been identified as carrying a founder mutation in BRCA1. No prior testing had been conducted among the Ukrainian women. Thus, this study screened BRCA1 and BRCA2 in the germline DNA of 426 women in total. RESULTS: We identified 31 and 18 women as carriers of pathogenic/likely pathogenic (P/LP) genetic variants in BRCA1 and BRCA2, respectively. We observed five BRCA1 and eight BRCA2 P/LP variants (13/337, 3.9%) in the Polish women. Combined with the 34/337 (10.1%) founder variants identified prior to this study, the overall P/LP variant frequency in the Polish women was thus 14% (47/337). Among the Ukrainian women, 16/123 (13%) women were identified as carrying a founder mutation and 20/123 (16.3%) were found to carry non-founder P/LP variants (10 in BRCA1 and 10 in BRCA2). CONCLUSIONS: These results indicate that genetic testing in women at high risk of breast and ovarian cancer in Poland and Ukraine should not be limited to founder mutations. Extended testing will enhance risk stratification and management for these women and their families.

FANCM and RECQL genetic variants and breast cancer susceptibility: relevance to South Poland and West Ukraine.

BACKGROUND: FANCM and RECQL have recently been reported as breast cancer susceptibility genes and it has been suggested that they should be included on gene panel tests for breast cancer predisposition. However, the clinical value of testing for mutations in RECQL and FANCM remains to be determined. In this study, we have characterised the spectrum of FANCM and RECQL mutations in women affected with breast or ovarian cancer from South-West Poland and West Ukraine. METHODS: We applied Hi-Plex, an amplicon-based enrichment method for targeted massively parallel sequencing, to screen the coding exons and proximal intron-exon junctions of FANCM and RECQL in germline DNA from unrelated women affected with breast cancer (n = 338) and ovarian cancer (n = 89) from Poland (n = 304) and Ukraine (n = 123). These women were at high-risk of carrying a genetic predisposition to breast and/or ovarian cancer due to a family history and/or early-onset disease. RESULTS: Among 427 women screened, we identified one carrier of the FANCM:c.1972C > T nonsense mutation (0.23%), and two carriers of the frameshift insertion FANCM:c.1491dup (0.47%). None of the variants we observed in RECQL were predicted to be loss-of-function mutations by standard variant effect prediction tools. CONCLUSIONS: Our study of the Polish and Ukrainian populations has identified a carrier frequency of truncating mutations in FANCM consistent with previous reports. Although initial reports suggesting that mutations in RECQL could be associated with increased breast cancer risk included women from Poland and identified the RECQL:c.1667_1667 + 3delAGTA mutation in 0.23-0.35% of breast cancer cases, we did not observe any carriers in our study cohort. Continued screening, both in research and diagnostic settings, will enable the accumulation of data that is needed to establish the clinical utility of including RECQL and FANCM on gene panel tests.

Is RNASEL:p.Glu265* a modifier of early-onset breast cancer risk for carriers of high-risk mutations?

BACKGROUND: Breast cancer risk for BRCA1 and BRCA2 pathogenic mutation carriers is modified by risk factors that cluster in families, including genetic modifiers of risk. We considered genetic modifiers of risk for carriers of high-risk mutations in other breast cancer susceptibility genes. METHODS: In a family known to carry the high-risk mutation PALB2:c.3113G>A (p.Trp1038*), whole-exome sequencing was performed on germline DNA from four affected women, three of whom were mutation carriers. RESULTS: RNASEL:p.Glu265* was identified in one of the PALB2 carriers who had two primary invasive breast cancer diagnoses before 50 years. Gene-panel testing of BRCA1, BRCA2, PALB2 and RNASEL in the Australian Breast Cancer Family Registry identified five carriers of RNASEL:p.Glu265* in 591 early onset breast cancer cases. Three of the five women (60%) carrying RNASEL:p.Glu265* also carried a pathogenic mutation in a breast cancer susceptibility gene compared with 30 carriers of pathogenic mutations in the 586 non-carriers of RNASEL:p.Glu265* (5%) (p < 0.002). Taqman genotyping demonstrated that the allele frequency of RNASEL:p.Glu265* was similar in affected and unaffected Australian women, consistent with other populations. CONCLUSION: Our study suggests that RNASEL:p.Glu265* may be a genetic modifier of risk for early-onset breast cancer predisposition in carriers of high-risk mutations. Much larger case-case and case-control studies are warranted to test the association observed in this report.

Mutation screening of PALB2 in clinically ascertained families from the Breast Cancer Family Registry.

Loss-of-function mutations in PALB2 are associated with an increased risk of breast cancer, with recent data showing that female breast cancer risks for PALB2 mutation carriers are comparable in magnitude to those for BRCA2 mutation carriers. This study applied targeted massively parallel sequencing to characterize the mutation spectrum of PALB2 in probands attending breast cancer genetics clinics in the USA. The coding regions and proximal intron-exon junctions of PALB2 were screened in probands not known to carry a mutation in BRCA1 or BCRA2 from 1,250 families enrolled through familial cancer clinics by the Breast Cancer Family Registry. Mutation screening was performed using Hi-Plex, an amplicon-based targeted massively parallel sequencing platform. Screening of PALB2 was successful in 1,240/1,250 probands and identified nine women with protein-truncating mutations (three nonsense mutations and five frameshift mutations). Four of the 33 missense variants were predicted to be deleterious to protein function by in silico analysis using two different programs. Analysis of tumors from carriers of truncating mutations revealed that the majority were high histological grade, invasive ductal carcinomas. Young onset was apparent in most families, with 19 breast cancers under 50 years of age, including eight under the age of 40 years. Our data demonstrate the utility of Hi-Plex in the context of high-throughput testing for rare genetic mutations and provide additional timely information about the nature and prevalence of PALB2 mutations, to enhance risk assessment and risk management of women at high risk of cancer attending clinical genetic services.

FAVR (Filtering and Annotation of Variants that are Rare): methods to facilitate the analysis of rare germline genetic variants from massively parallel sequencing datasets.

BACKGROUND: Characterising genetic diversity through the analysis of massively parallel sequencing (MPS) data offers enormous potential to significantly improve our understanding of the genetic basis for observed phenotypes, including predisposition to and progression of complex human disease. Great challenges remain in resolving genetic variants that are genuine from the millions of artefactual signals. RESULTS: FAVR is a suite of new methods designed to work with commonly used MPS analysis pipelines to assist in the resolution of some of the issues related to the analysis of the vast amount of resulting data, with a focus on relatively rare genetic variants. To the best of our knowledge, no equivalent method has previously been described. The most important and novel aspect of FAVR is the use of signatures in comparator sequence alignment files during variant filtering, and annotation of variants potentially shared between individuals. The FAVR methods use these signatures to facilitate filtering of (i) platform and/or mapping-specific artefacts, (ii) common genetic variants, and, where relevant, (iii) artefacts derived from imbalanced paired-end sequencing, as well as annotation of genetic variants based on evidence of co-occurrence in individuals. We applied conventional variant calling applied to whole-exome sequencing datasets, produced using both SOLiD and TruSeq chemistries, with or without downstream processing by FAVR methods. We demonstrate a 3-fold smaller rare single nucleotide variant shortlist with no detected reduction in sensitivity. This analysis included Sanger sequencing of rare variant signals not evident in dbSNP131, assessment of known variant signal preservation, and comparison of observed and expected rare variant numbers across a range of first cousin pairs. The principles described herein were applied in our recent publication identifying XRCC2 as a new breast cancer risk gene and have been made publically available as a suite of software tools. CONCLUSIONS: FAVR is a platform-agnostic suite of methods that significantly enhances the analysis of large volumes of sequencing data for the study of rare genetic variants and their influence on phenotypes.

Low penetrance breast cancer susceptibility loci are associated with specific breast tumor subtypes: findings from the Breast Cancer Association Consortium.

Breast cancers demonstrate substantial biological, clinical and etiological heterogeneity. We investigated breast cancer risk associations of eight susceptibility loci identified in GWAS and two putative susceptibility loci in candidate genes in relation to specific breast tumor subtypes. Subtypes were defined by five markers (ER, PR, HER2, CK5/6, EGFR) and other pathological and clinical features. Analyses included up to 30 040 invasive breast cancer cases and 53 692 controls from 31 studies within the Breast Cancer Association Consortium. We confirmed previous reports of stronger associations with ER+ than ER- tumors for six of the eight loci identified in GWAS: rs2981582 (10q26) (P-heterogeneity = 6.1 × 10(-18)), rs3803662 (16q12) (P = 3.7 × 10(-5)), rs13281615 (8q24) (P = 0.002), rs13387042 (2q35) (P = 0.006), rs4973768 (3p24) (P = 0.003) and rs6504950 (17q23) (P = 0.002). The two candidate loci, CASP8 (rs1045485, rs17468277) and TGFB1 (rs1982073), were most strongly related with the risk of PR negative tumors (P = 5.1 × 10(-6) and P = 4.1 × 10(-4), respectively), as previously suggested. Four of the eight loci identified in GWAS were associated with triple negative tumors (P ≤ 0.016): rs3803662 (16q12), rs889312 (5q11), rs3817198 (11p15) and rs13387042 (2q35); however, only two of them (16q12 and 2q35) were associated with tumors with the core basal phenotype (P ≤ 0.002). These analyses are consistent with different biological origins of breast cancers, and indicate that tumor stratification might help in the identification and characterization of novel risk factors for breast cancer subtypes. This may eventually result in further improvements in prevention, early detection and treatment.

Associations of common variants at 1p11.2 and 14q24.1 (RAD51L1) with breast cancer risk and heterogeneity by tumor subtype: findings from the Breast Cancer Association Consortium.

A genome-wide association study (GWAS) identified single-nucleotide polymorphisms (SNPs) at 1p11.2 and 14q24.1 (RAD51L1) as breast cancer susceptibility loci. The initial GWAS suggested stronger effects for both loci for estrogen receptor (ER)-positive tumors. Using data from the Breast Cancer Association Consortium (BCAC), we sought to determine whether risks differ by ER, progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), grade, node status, tumor size, and ductal or lobular morphology. We genotyped rs11249433 at 1p.11.2, and two highly correlated SNPs rs999737 and rs10483813 (r(2)= 0.98) at 14q24.1 (RAD51L1), for up to 46 036 invasive breast cancer cases and 46 930 controls from 39 studies. Analyses by tumor characteristics focused on subjects reporting to be white women of European ancestry and were based on 25 458 cases, of which 87% had ER data. The SNP at 1p11.2 showed significantly stronger associations with ER-positive tumors [per-allele odds ratio (OR) for ER-positive tumors was 1.13, 95% CI = 1.10-1.16 and, for ER-negative tumors, OR was 1.03, 95% CI = 0.98-1.07, case-only P-heterogeneity = 7.6 × 10(-5)]. The association with ER-positive tumors was stronger for tumors of lower grade (case-only P= 6.7 × 10(-3)) and lobular histology (case-only P= 0.01). SNPs at 14q24.1 were associated with risk for most tumor subtypes evaluated, including triple-negative breast cancers, which has not been described previously. Our results underscore the need for large pooling efforts with tumor pathology data to help refine risk estimates for SNP associations with susceptibility to different subtypes of breast cancer.

Using SNP genotypes to improve the discrimination of a simple breast cancer risk prediction model.

It has been shown that, for women aged 50 years or older, the discriminatory accuracy of the Breast Cancer Risk Prediction Tool (BCRAT) can be modestly improved by the inclusion of information on common single nucleotide polymorphisms (SNPs) that are associated with increased breast cancer risk. We aimed to determine whether a similar improvement is seen for earlier onset disease. We used the Australian Breast Cancer Family Registry to study a population-based sample of 962 cases aged 35-59 years, and 463 controls frequency matched for age and for whom genotyping data was available. Overall, the inclusion of data on seven SNPs improved the area under the receiver operating characteristic curve (AUC) from 0.58 (95 % confidence interval [CI] 0.55-0.61) for BCRAT alone to 0.61 (95 % CI 0.58-0.64) for BCRAT and SNP data combined (p < 0.001). For women aged 35-39 years at interview, the corresponding improvement in AUC was from 0.61 (95 % CI 0.56-0.66) to 0.65 (95 % CI 0.60-0.70; p = 0.03), while for women aged 40-49 years at diagnosis, the AUC improved from 0.61 (95 % CI 0.55-0.66) to 0.63 (95 % CI 0.57-0.69; p = 0.04). Using previously used classifications of low, intermediate and high risk, 2.1 % of cases and none of the controls aged 35-39 years, and 10.9 % of cases and 4.0 % of controls aged 40-49 years were classified into a higher risk group. Including information on seven SNPs associated with breast cancer risk, improves the discriminatory accuracy of BCRAT for women aged 35-39 years and 40-49 years. Given, the low absolute risk for women in these age groups, only a small proportion are reclassified into a higher category for predicted 5-year risk of breast cancer.

Cross-platform compatibility of Hi-Plex, a streamlined approach for targeted massively parallel sequencing.

Although per-base sequencing costs have decreased during recent years, library preparation for targeted massively parallel sequencing remains constrained by high reagent cost, limited design flexibility, and protocol complexity. To address these limitations, we previously developed Hi-Plex, a polymerase chain reaction (PCR) massively parallel sequencing strategy for screening panels of genomic target regions. Here, we demonstrate that Hi-Plex applied with hybrid adapters can generate a library suitable for sequencing with both the Ion Torrent and the TruSeq chemistries and that adjusting primer concentrations improves coverage uniformity. These results expand Hi-Plex capabilities as an accurate, affordable, flexible, and rapid approach for various genetic screening applications.

Maternal SARS-CoV-2 exposure alters infant DNA methylation.

Background: Infection during pregnancy can increase the risk of neurodevelopmental disorders in offspring. The impact of maternal SARS-CoV-2 infection on infant neurodevelopment is poorly understood. The maternal immune response to infection may be mimicked in rodent models of maternal immune activation which recapitulate altered neurodevelopment and behavioural disturbances in the offspring. In these models, epigenetic mechanisms, in particular DNA methylation, are one pathway through which this risk is conferred in utero to offspring. We hypothesised that in utero exposure to SARS-CoV-2 in humans may alter infant DNA methylation, particularly in genes associated with neurodevelopment. We aimed to test this hypothesis in a pilot sample of children in Victoria, Australia, who were exposed in utero to SARS-CoV-2. Methods: DNA was extracted from buccal swab specimens from (n = 4) SARS-CoV-2 in utero exposed and (n = 4) non-exposed infants and methylation status assessed across 850,000 methylation sites using an Illumina EPIC BeadChip. We also conducted an exploratory enrichment analysis using Gene Ontology annotations. Results: 1962 hypermethylated CpG sites were identified with an unadjusted p-value of 0.05, where 1133 CpGs mapped to 959 unique protein coding genes, and 716 hypomethylated CpG sites mapped to 559 unique protein coding genes in SARS-CoV-2 exposed infants compared to non-exposed. One differentially methylated position (cg06758191), located in the gene body of AFAP1 that was hypomethylated in the SARS-CoV-2 exposed cohort was significant after correction for multiple testing (FDR-adjusted p-value <0.00083). Two significant differentially methylated regions were identified; a hypomethylated intergenic region located in chromosome 6p proximal to the genes ZP57 and HLA-F (fwer <0.004), and a hypomethylated region in the promoter and body of the gene GAREM2 (fwer <0.036). Gene network enrichment analysis revealed differential methylation in genes corresponding to pathways relevant to neurodevelopment, including the ERBB pathway. Conclusion: These pilot data suggest that exposure to SARS-CoV-2 in utero differentially alters methylation of genes in pathways that play a role in human neurodevelopment.

FAN1 variants identified in multiple-case early-onset breast cancer families via exome sequencing: no evidence for association with risk for breast cancer.

We are interested in the characterisation of previously undescribed contributions to the heritable component of human cancers. To this end, we applied whole-exome capture, followed by massively parallel sequence analysis to the germline DNA of two greater than third-degree affected relatives from four multiple-case, early-onset breast cancer families. Prior testing for variants in known breast cancer susceptibility, genes in these families did not identify causal mutations. We detected and confirmed two different variants in the DNA damage repair gene FAN1 (R377W, chr15:31197995 C>T and R507H, chr15:31202961 G>A [hg19]) which were not present in dbSNP131. In one family, FAN1 R377W, predicted to be damaging by SIFT and PolyPhen2, was present in all six tested members with cancer (five with breast cancer, one with malignant melanoma). In another family, FAN1 R507H, predicted to be damaging by SIFT but benign by PolyPhen2, was observed in one of two tested members with breast cancer. We genotyped FAN1 R377W and R507H variants across 1417 population-based cases and 1490 unaffected population-based controls (frequency-matched for age). These variants were rare in the Australian population (minor allele frequencies of 0.0064 and 0.010, respectively) and were not associated with breast cancer risk (OR = 0.80, 95% CI[0.39-1.61], P = 0.50 and OR = 0.74, 95% CI[0.41-1.29], P = 0.26, respectively). Analysis of breast cancer risks for relatives of case and control carriers did not find evidence of an increased risk. Despite the biological role of FAN1, the plausibility of its role as a breast cancer predisposition gene, and the possible deleterious nature of the identified variants, these two variants do not appear to be causal for breast cancer. Future studies to extend the genetic analysis of FAN1 will further explore its possible role as a breast cancer susceptibility gene.

Repeatability of methylation measures using a QIAseq targeted methyl panel and comparison with the Illumina HumanMethylation450 assay.

OBJECTIVE: In previous studies using Illumina Infinium methylation arrays, we have identified DNA methylation marks associated with cancer predisposition and progression. In the present study, we have sought to find appropriate technology to both technically validate our data and expand our understanding of DNA methylation in these genomic regions. Here, we aimed to assess the repeatability of methylation measures made using QIAseq targeted methyl panel and to compare them with those obtained from the Illumina HumanMethylation450 (HM450K) assay. We included in the analysis high molecular weight DNA extracted from whole blood (WB) and DNA extracted from formalin-fixed paraffin-embedded tissues (FFPE). RESULTS: The repeatability of QIAseq-methylation measures was assessed at 40 CpGs, using the Intraclass Correlation Coefficient (ICC). The mean ICCs and 95% confidence intervals (CI) were 0.72 (0.62-0.81), 0.59 (0.47-0.71) and 0.80 (0.73-0.88) for WB, FFPE and both sample types combined, respectively. For technical replicates measured using QIAseq and HM450K, the mean ICCs (95% CI) were 0.53 (0.39-0.68), 0.43 (0.31-0.56) and 0.70 (0.59-0.80), respectively. Bland-Altman plots indicated good agreement between QIAseq and HM450K measurements. These results demonstrate that the QIAseq targeted methyl panel produces reliable and reproducible methylation measurements across the 40 CpGs that were examined.

Population-Based Estimates of the Age-Specific Cumulative Risk of Breast Cancer for Pathogenic Variants in CHEK2: Findings from the Australian Breast Cancer Family Registry.

Case-control studies of breast cancer have consistently shown that pathogenic variants in CHEK2 are associated with about a 3-fold increased risk of breast cancer. Information about the recurrent protein-truncating variant CHEK2 c.1100delC dominates this estimate. There have been no formal estimates of age-specific cumulative risk of breast cancer for all CHEK2 pathogenic (including likely pathogenic) variants combined. We conducted a population-based case-control-family study of pathogenic CHEK2 variants (26 families, 1071 relatives) and estimated the age-specific cumulative risk of breast cancer using segregation analysis. The estimated hazard ratio for carriers of pathogenic CHEK2 variants (combined) was 4.9 (95% CI 2.5-9.5) relative to non-carriers. The HR for carriers of the CHEK2 c.1100delC variant was estimated to be 3.5 (95% CI 1.02-11.6) and the HR for carriers of all other CHEK2 variants combined was estimated to be 5.7 (95% CI 2.5-12.9). The age-specific cumulative risk of breast cancer was estimated to be 18% (95% CI 11-30%) and 33% (95% CI 21-48%) to age 60 and 80 years, respectively. These findings provide important information for the clinical management of breast cancer risk for women carrying pathogenic variants in CHEK2.

Rare Germline Pathogenic Variants Identified by Multigene Panel Testing and the Risk of Aggressive Prostate Cancer.

While gene panel sequencing is becoming widely used for cancer risk prediction, its clinical utility with respect to predicting aggressive prostate cancer (PrCa) is limited by our current understanding of the genetic risk factors associated with predisposition to this potentially lethal disease phenotype. This study included 837 men diagnosed with aggressive PrCa and 7261 controls (unaffected men and men who did not meet criteria for aggressive PrCa). Rare germline pathogenic variants (including likely pathogenic variants) were identified by targeted sequencing of 26 known or putative cancer predisposition genes. We found that 85 (10%) men with aggressive PrCa and 265 (4%) controls carried a pathogenic variant (p < 0.0001). Aggressive PrCa odds ratios (ORs) were estimated using unconditional logistic regression. Increased risk of aggressive PrCa (OR (95% confidence interval)) was identified for pathogenic variants in BRCA2 (5.8 (2.7-12.4)), BRCA1 (5.5 (1.8-16.6)), and ATM (3.8 (1.6-9.1)). Our study provides further evidence that rare germline pathogenic variants in these genes are associated with increased risk of this aggressive, clinically relevant subset of PrCa. These rare genetic variants could be incorporated into risk prediction models to improve their precision to identify men at highest risk of aggressive prostate cancer and be used to identify men with newly diagnosed prostate cancer who require urgent treatment.

Population-based estimates of breast cancer risk for carriers of pathogenic variants identified by gene-panel testing.

Population-based estimates of breast cancer risk for carriers of pathogenic variants identified by gene-panel testing are urgently required. Most prior research has been based on women selected for high-risk features and more data is needed to make inference about breast cancer risk for women unselected for family history, an important consideration of population screening. We tested 1464 women diagnosed with breast cancer and 862 age-matched controls participating in the Australian Breast Cancer Family Study (ABCFS), and 6549 healthy, older Australian women enroled in the ASPirin in Reducing Events in the Elderly (ASPREE) study for rare germline variants using a 24-gene-panel. Odds ratios (ORs) were estimated using unconditional logistic regression adjusted for age and other potential confounders. We identified pathogenic variants in 11.1% of the ABCFS cases, 3.7% of the ABCFS controls and 2.2% of the ASPREE (control) participants. The estimated breast cancer OR [95% confidence interval] was 5.3 [2.1-16.2] for BRCA1, 4.0 [1.9-9.1] for BRCA2, 3.4 [1.4-8.4] for ATM and 4.3 [1.0-17.0] for PALB2. Our findings provide a population-based perspective to gene-panel testing for breast cancer predisposition and opportunities to improve predictors for identifying women who carry pathogenic variants in breast cancer predisposition genes.

Mismatch repair gene pathogenic germline variants in a population-based cohort of breast cancer.

The advent of gene panel testing is challenging the previous practice of using clinically defined cancer family syndromes to inform single-gene genetic screening. Individual and family cancer histories that would have previously indicated testing of a single gene or a small number of related genes are now, increasingly, leading to screening across gene panels that contain larger numbers of genes. We have applied a gene panel test that included four DNA mismatch repair (MMR) genes (MLH1, MSH2, MSH6 and PMS2) to an Australian population-based case-control-family study of breast cancer. Altogether, eight pathogenic variants in MMR genes were identified: six in 1421 case-families (0.4%, 4 MSH6 and 2 PMS2) and two in 833 control-families (0.2%, one each of MLH1 and MSH2). This testing highlights the current and future challenges for clinical genetics in the context of anticipated gene panel-based population-based screening that includes the MMR genes. This testing is likely to provide additional opportunities for cancer prevention via cascade testing for Lynch syndrome and precision medicine for breast cancer treatment.

Hi-Plex2: a simple and robust approach to targeted sequencing-based genetic screening.

We have previously reported Hi-Plex, a multiplex PCR methodology for building targeted DNA sequencing libraries that offers a low-cost protocol compatible with high-throughput processing. Here, we detail an improved protocol, Hi-Plex2, that more effectively enables the robust construction of small-to-medium panel-size libraries while maintaining low cost, simplicity and accuracy benefits of the Hi-Plex platform. Hi-Plex2 was applied to three panels, comprising 291, 740 and 1193 amplicons, targeting genes associated with risk for breast and/or colon cancer. We show substantial reduction of off-target amplification to enable library construction for small-to-medium-sized design panels not possible using the previous Hi-Plex chemistry.

Hi-Plex for Simple, Accurate, and Cost-Effective Amplicon-based Targeted DNA Sequencing.

Hi-Plex is a suite of methods to enable simple, accurate, and cost-effective highly multiplex PCR-based targeted sequencing (Nguyen-Dumont et al., Biotechniques 58:33-36, 2015). At its core is the principle of using gene-specific primers (GSPs) to "seed" (or target) the reaction and universal primers to "drive" the majority of the reaction. In this manner, effects on amplification efficiencies across the target amplicons can, to a large extent, be restricted to early seeding cycles. Product sizes are defined within a relatively narrow range to enable high-specificity size selection, replication uniformity across target sites (including in the context of fragmented input DNA such as that derived from fixed tumor specimens (Nguyen-Dumont et al., Biotechniques 55:69-74, 2013; Nguyen-Dumont et al., Anal Biochem 470:48-51, 2015), and application of high-specificity genetic variant calling algorithms (Pope et al., Source Code Biol Med 9:3, 2014; Park et al., BMC Bioinformatics 17:165, 2016). Hi-Plex offers a streamlined workflow that is suitable for testing large numbers of specimens without the need for automation.