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The sequence contexts of genomic variants play important roles in understanding biological significances of variants and potential sequencing related variant calling issues. However, methods for assessing the diverse sequence contexts of genomic variants such as tandem repeats and unambiguous annotations have been limited. Herein, we describe the Variant Sequence Context Annotation Tool (VarSCAT) for annotating the sequence contexts of genomic variants, including breakpoint ambiguities, flanking bases of variants, wildtype/mutated DNA sequences, variant nomenclatures, distances between adjacent variants, tandem repeat regions, and custom annotation with user customizable options. Our analyses demonstrate that VarSCAT is more versatile and customizable than the currently available methods or strategies for annotating variants in short tandem repeat (STR) regions or insertions and deletions (indels) with breakpoint ambiguity. Variant sequence context annotations of high-confidence human variant sets with VarSCAT revealed that more than 75% of all human individual germline and clinically relevant indels have breakpoint ambiguities. Moreover, we illustrate that more than 80% of human individual germline small variants in STR regions are indels and that the sizes of these indels correlated with STR motif sizes. VarSCAT is available from https://github.com/elolab/VarSCAT.

Insertions and deletions (indels) in human genomes are associated with a wide range of phenotypes, including various clinical disorders. High-throughput, next generation sequencing (NGS) technologies enable the detection of short genetic variants, such as single nucleotide variants (SNVs) and indels. However, the variant calling accuracy for indels remains considerably lower than for SNVs. Here we present a comparative study of the performance of variant calling tools for indel calling, evaluated with a wide repertoire of NGS datasets. While there is no single optimal tool to suit all circumstances, our results demonstrate that the choice of variant calling tool greatly impacts the precision and recall of indel calling. Furthermore, to reliably detect indels, it is essential to choose NGS technologies that offer a long read length and high coverage coupled with specific variant calling tools.

Population-based genome wide association studies have identified a locus at 9p22.2 associated with ovarian cancer risk, which also modifies ovarian cancer risk in BRCA1 and BRCA2 mutation carriers. We conducted fine-scale mapping at 9p22.2 to identify potential causal variants in BRCA1 and BRCA2 mutation carriers. Genotype data were available for 15,252 (2,462 ovarian cancer cases) BRCA1 and 8,211 (631 ovarian cancer cases) BRCA2 mutation carriers. Following genotype imputation, ovarian cancer associations were assessed for 4,873 and 5,020 SNPs in BRCA1 and BRCA 2 mutation carriers respectively, within a retrospective cohort analytical framework. In BRCA1 mutation carriers one set of eight correlated candidate causal variants for ovarian cancer risk modification was identified (top SNP rs10124837, HR: 0.73, 95%CI: 0.68 to 0.79, p-value 2× 10-16). These variants were located up to 20 kb upstream of BNC2. In BRCA2 mutation carriers one region, up to 45 kb upstream of BNC2, and containing 100 correlated SNPs was identified as candidate causal (top SNP rs62543585, HR: 0.69, 95%CI: 0.59 to 0.80, p-value 1.0 × 10-6). The candidate causal in BRCA1 mutation carriers did not include the strongest associated variant at this locus in the general population. In sum, we identified a set of candidate causal variants in a region that encompasses the BNC2 transcription start site. The ovarian cancer association at 9p22.2 may be mediated by different variants in BRCA1 mutation carriers and in the general population. Thus, potentially different mechanisms may underlie ovarian cancer risk for mutation carriers and the general population.

Common variation on 14q24.1, close to RAD51B, has been associated with breast cancer: rs999737 and rs2588809 with the risk of female breast cancer and rs1314913 with the risk of male breast cancer. The aim of this study was to investigate the role of RAD51B variants in breast cancer predisposition, particularly in the context of familial breast cancer in Finland. We sequenced the coding region of RAD51B in 168 Finnish breast cancer patients from the Helsinki region for identification of possible recurrent founder mutations. In addition, we studied the known rs999737, rs2588809, and rs1314913 SNPs and RAD51B haplotypes in 44,791 breast cancer cases and 43,583 controls from 40 studies participating in the Breast Cancer Association Consortium (BCAC) that were genotyped on a custom chip (iCOGS). We identified one putatively pathogenic missense mutation c.541C>T among the Finnish cancer patients and subsequently genotyped the mutation in additional breast cancer cases (n = 5259) and population controls (n = 3586) from Finland and Belarus. No significant association with breast cancer risk was seen in the meta-analysis of the Finnish datasets or in the large BCAC dataset. The association with previously identified risk variants rs999737, rs2588809, and rs1314913 was replicated among all breast cancer cases and also among familial cases in the BCAC dataset. The most significant association was observed for the haplotype carrying the risk-alleles of all the three SNPs both among all cases (odds ratio (OR): 1.15, 95% confidence interval (CI): 1.11-1.19, P = 8.88 x 10-16) and among familial cases (OR: 1.24, 95% CI: 1.16-1.32, P = 6.19 x 10-11), compared to the haplotype with the respective protective alleles. Our results suggest that loss-of-function mutations in RAD51B are rare, but common variation at the RAD51B region is significantly associated with familial breast cancer risk.

The Cancer Genetic Markers of Susceptibility genome-wide association study (GWAS) originally identified a single nucleotide polymorphism (SNP) rs11249433 at 1p11.2 associated with breast cancer risk. To fine-map this locus, we genotyped 92 SNPs in a 900kb region (120,505,799-121,481,132) flanking rs11249433 in 45,276 breast cancer cases and 48,998 controls of European, Asian and African ancestry from 50 studies in the Breast Cancer Association Consortium. Genotyping was done using iCOGS, a custom-built array. Due to the complicated nature of the region on chr1p11.2: 120,300,000-120,505,798, that lies near the centromere and contains seven duplicated genomic segments, we restricted analyses to 429 SNPs excluding the duplicated regions (42 genotyped and 387 imputed). Per-allelic associations with breast cancer risk were estimated using logistic regression models adjusting for study and ancestry-specific principal components. The strongest association observed was with the original identified index SNP rs11249433 (minor allele frequency (MAF) 0.402; per-allele odds ratio (OR) = 1.10, 95% confidence interval (CI) 1.08-1.13, P = 1.49 x 10-21). The association for rs11249433 was limited to ER-positive breast cancers (test for heterogeneity P≤8.41 x 10-5). Additional analyses by other tumor characteristics showed stronger associations with moderately/well differentiated tumors and tumors of lobular histology. Although no significant eQTL associations were observed, in silico analyses showed that rs11249433 was located in a region that is likely a weak enhancer/promoter. Fine-mapping analysis of the 1p11.2 breast cancer susceptibility locus confirms this region to be limited to risk to cancers that are ER-positive.

Genetic variations, such as single nucleotide polymorphisms (SNPs) in microRNAs (miRNA) or in the miRNA binding sites may affect the miRNA dependent gene expression regulation, which has been implicated in various cancers, including breast cancer, and may alter individual susceptibility to cancer. We investigated associations between miRNA related SNPs and breast cancer risk. First we evaluated 2,196 SNPs in a case-control study combining nine genome wide association studies (GWAS). Second, we further investigated 42 SNPs with suggestive evidence for association using 41,785 cases and 41,880 controls from 41 studies included in the Breast Cancer Association Consortium (BCAC). Combining the GWAS and BCAC data within a meta-analysis, we estimated main effects on breast cancer risk as well as risks for estrogen receptor (ER) and age defined subgroups. Five miRNA binding site SNPs associated significantly with breast cancer risk: rs1045494 (odds ratio (OR) 0.92; 95% confidence interval (CI): 0.88-0.96), rs1052532 (OR 0.97; 95% CI: 0.95-0.99), rs10719 (OR 0.97; 95% CI: 0.94-0.99), rs4687554 (OR 0.97; 95% CI: 0.95-0.99, and rs3134615 (OR 1.03; 95% CI: 1.01-1.05) located in the 3' UTR of CASP8, HDDC3, DROSHA, MUSTN1, and MYCL1, respectively. DROSHA belongs to miRNA machinery genes and has a central role in initial miRNA processing. The remaining genes are involved in different molecular functions, including apoptosis and gene expression regulation. Further studies are warranted to elucidate whether the miRNA binding site SNPs are the causative variants for the observed risk effects.

Genome wide association studies (GWAs) of breast cancer mortality have identified few potential associations. The concordance between these studies is unclear. In this study, we used a meta-analysis of two prognostic GWAs and a replication cohort to identify the strongest associations and to evaluate the loci suggested in previous studies. We attempt to identify those SNPs which could impact overall survival irrespective of the age of onset.To facilitate the meta-analysis and to refine the association signals, SNPs were imputed using data from the 1000 genomes project. Cox-proportional hazard models were used to estimate hazard ratios (HR) in 536 patients from the POSH cohort (Prospective study of Outcomes in Sporadic versus Hereditary breast cancer) and 805 patients from the HEBCS cohort (Helsinki Breast Cancer Study). These hazard ratios were combined using a Mantel-Haenszel fixed effects meta-analysis and a p-value threshold of 5×10(-8) was used to determine significance. Replication was performed in 1523 additional patients from the POSH study.Although no SNPs achieved genome wide significance, three SNPs have significant association in the replication cohort and combined p-values less than 5.6×10(-6). These SNPs are; rs421379 which is 556 kb upstream of ARRDC3 (HR = 1.49, 95% confidence interval (CI) = 1.27-1.75, P = 1.1×10(-6)), rs12358475 which is between ECHDC3 and PROSER2 (HR = 0.75, CI = 0.67-0.85, P = 1.8×10(-6)), and rs1728400 which is between LINC00917 and FOXF1.In a genome wide meta-analysis of two independent cohorts from UK and Finland, we identified potential associations at three distinct loci. Phenotypic heterogeneity and relatively small sample sizes may explain the lack of genome wide significant findings. However, the replication at three SNPs in the validation cohort shows promise for future studies in larger cohorts. We did not find strong evidence for concordance between the few associations highlighted by previous GWAs of breast cancer survival and this study.

1α,25-Dihydroxyvitamin D3 (1α,25(OH)2D3) had earlier been regarded as the only active hormone. The newly identified actions of 25-hydroxyvitamin D3 (25(OH)D3) and 24R,25-dihydroxyvitamin D3 (24R,25(OH)2D3) broadened the vitamin D3 endocrine system, however, the current data are fragmented and a systematic understanding is lacking. Here we performed the first systematic study of global gene expression to clarify their similarities and differences. Three metabolites at physiologically comparable levels were utilized to treat human and mouse fibroblasts prior to DNA microarray analyses. Human primary prostate stromal P29SN cells (hP29SN), which convert 25(OH)D3 into 1α,25(OH)2D3 by 1α-hydroxylase (encoded by the gene CYP27B1), displayed regulation of 164, 171, and 175 genes by treatment with 1α,25(OH)2D3, 25(OH)D3, and 24R,25(OH)2D3, respectively. Mouse primary Cyp27b1 knockout fibroblasts (mCyp27b1 (-/-)), which lack 1α-hydroxylation, displayed regulation of 619, 469, and 66 genes using the same respective treatments. The number of shared genes regulated by two metabolites is much lower in hP29SN than in mCyp27b1 (-/-). By using DAVID Functional Annotation Bioinformatics Microarray Analysis tools and Ingenuity Pathways Analysis, we identified the agonistic regulation of calcium homeostasis and bone remodeling between 1α,25(OH)2D3 and 25(OH)D3 and unique non-classical actions of each metabolite in physiological and pathological processes, including cell cycle, keratinocyte differentiation, amyotrophic lateral sclerosis signaling, gene transcription, immunomodulation, epigenetics, cell differentiation, and membrane protein expression. In conclusion, there are three distinct vitamin D3 hormones with clearly different biological activities. This study presents a new conceptual insight into the vitamin D3 endocrine system, which may guide the strategic use of vitamin D3 in disease prevention and treatment.