Piotr Kozlowski

Whole-miRNome sequencing: a panel for the targeted sequencing of all human miRNA genes

Abstract Interest in the genetic variation of noncoding genomic elements, including microRNAs (miRNAs), is growing, and several mutations in miRNA genes implicated in human diseases, including cancer, have already been detected. However, the lack of dedicated analytical tools severely hampers progress in this area. In this study, we developed the first whole-miRNome sequencing (WMS) platform, which enables the targeted sequencing of all human miRNA genes (n ∼2000) and 28 miRNA biogenesis genes. By sequencing various types of DNA samples, including ∼300 tumor/normal pairs, from lung, colorectal, ovarian, renal, and basal cell carcinomas, we identified ∼2000 mutations, including 879 in miRNA genes. These mutations were located in all parts of the genes, including seed or cleavage sites essential for the functioning of miRNA genes. The high reliability of the mutations was confirmed through various approaches, including different sequencing methods. The analysis identified several miRNA genes with functional enrichment of cancer mutations, including MIR3928, which was specifically mutated in basal cell carcinoma, suggesting its potential role in this cancer. WMS also allowed the identification of multiple copy number alterations, which often encompassed miRNA genes. WMS provides highly effective, low-cost sequencing of all miRNA genes in different types of samples, including highly degraded ones.

Summary of BARD1 Mutations and Precise Estimation of Breast and Ovarian Cancer Risks Associated with the Mutations

Over the last two decades, numerous BARD1 mutations/pathogenic variants (PVs) have been found in patients with breast cancer (BC) and ovarian cancer (OC). However, their role in BC and OC susceptibility remains controversial, and strong evidence-based guidelines for carriers are not yet available. Herein, we present a comprehensive catalog of BARD1 PVs identified in large cumulative cohorts of ~48,700 BC and ~20,800 OC cases (retrieved from 123 studies examining the whole coding sequence of BARD1). Using these resources, we compared the frequency of BARD1 PVs in the cases and ~134,100 controls from the gnomAD database and estimated the effect of the BARD1 PVs on BC and OC risks. The analysis revealed that BARD1 is a BC moderate-risk gene (odds ratio (OR) = 2.90, 95% CIs:2.25–3.75, p < 0.0001) but not an OC risk gene (OR = 1.36, 95% CIs:0.87–2.11, p = 0.1733). In addition, the BARD1 mutational spectrum outlined in this study allowed us to determine recurrent PVs and evaluate the variant-specific risk for the most frequent PVs. In conclusion, these precise estimates improve the understanding of the role of BARD1 PVs in BC and OC predisposition and support the need for BARD1 diagnostic testing in BC patients.

BRIP1, RAD51C, and RAD51D mutations are associated with high susceptibility to ovarian cancer: mutation prevalence and precise risk estimates based on a pooled analysis of ~30,000 cases

AbstractBackgroundIt is estimated that more than 20% of ovarian cancer cases are associated with a genetic predisposition that is only partially explained by germline mutations in theBRCA1andBRCA2genes. Recently, several pieces of evidence showed that mutations in three genes involved in the homologous recombination DNA repair pathway, i.e.,BRIP1,RAD51C, andRAD51D, are associated with a high risk of ovarian cancer. To more precisely estimate the ovarian cancer risk attributed toBRIP1,RAD51C, andRAD51Dmutations, we performed a meta-analysis based on a comparison of a total of ~ 29,400 ovarian cancer patients from 63 studies and a total of ~ 116,000 controls from the gnomAD database.ResultsThe analysis allowed precise estimation of ovarian cancer risks attributed to mutations inBRIP1,RAD51C, andRAD51D, confirming that all three genes are ovarian cancer high-risk genes (odds ratio (OR) = 4.94, 95%CIs:4.07–6.00,p < 0.0001; OR = 5.59, 95%CIs:4.42–7.07,p < 0.0001; and OR = 6.94, 95%CIs:5.10–9.44,p < 0.0001, respectively). In the present report, we show, for the first time, a mutation-specific risk analysis associated with distinct, recurrent, mutations in the genes.ConclusionsThe meta-analysis provides evidence supporting the pathogenicity ofBRIP1,RAD51C, andRAD51Dmutations in relation to ovarian cancer. The level of ovarian cancer risk conferred by these mutations is relatively high, indicating that afterBRCA1andBRCA2, theBRIP1,RAD51C, andRAD51Dgenes are the most important ovarian cancer risk genes, cumulatively contributing to ~ 2% of ovarian cancer cases. The inclusion of the genes into routine diagnostic tests may influence both the prevention and the potential treatment of ovarian cancer.

Variant Identification in BARD1 , PRDM9 , RCC1 , and RECQL in Patients with Ovarian Cancer by Targeted Next-generation Sequencing of DNA Pools

Abstract Several ovarian cancer susceptibility genes have been discovered, but more are likely to exist. In this study, we aimed to analyze knowledge-based selected genes, that is, BARD1, PRDM9, RCC1, and RECQL, in which pathogenic germline variants have been reported in patients with breast and/or ovarian cancer. As deep sequencing of DNA samples remains costly, targeted next-generation sequencing of DNA pools was utilized to screen the exons of BARD1, PRDM9, RCC1, and RECQL in approximately 400 Polish ovarian cancer cases. A total of 25 pools of 16 samples (including several duplicated samples with known variants) were sequenced on the NovaSeq6000 and analyzed with SureCall (Agilent) application. The set of variants was filtrated to exclude spurious variants, and, subsequently, the identified rare genetic variants were validated using Sanger sequencing. No pathogenic mutation was found within the analyzed cohort of patients with ovarian cancer. Validation genotyping of filtered rare silent and missense variants revealed that the majority of them were true alterations, especially those with a higher mutation quality value. The high concordance (R2 = 0.95) of population allele frequency for 44 common SNPs in the European control population (gnomAD) and our experiment confirmed the reliability of pooled sequencing. Mutations in BARD1, PRDM9, RCC1, and RECQL do not contribute substantially to the risk of ovarian cancer. Pooled DNA sequencing is a cost-effective and reliable method for the initial screening of candidate genes; however, it still requires validation of identified rare variants. Prevention Relevance: BARD1, PRDM9, RCC1, and RECQL are not high/moderate-risk ovarian cancer susceptibility genes. Pooled sequencing is a reliable and cost-effective method to detect rare variants in candidate genes.