Characterization of non-coding variants associated with transcription factor binding through ATAC-seq-defined footprint QTLs in liver.

Non-coding variants discovered by genome-wide association studies (GWAS) are enriched in regulatory elements harboring transcription factor (TF) binding motifs, strongly suggesting a connection between disease association and the disruption of cis-regulatory sequences. Occupancy of a TF inside a region of open chromatin can be detected in ATAC-seq where bound TFs block the transposase Tn5, leaving a pattern of relatively depleted Tn5 insertions known as a "footprint". Here, we sought to identify variants associated with TF-binding, or "footprint quantitative trait loci" (fpQTLs) in ATAC-seq data generated from 170 human liver samples. We used computational tools to scan the ATAC-seq reads to quantify TF binding likelihood as "footprint scores" at variants derived from whole genome sequencing generated in the same samples. We tested for association between genotype and footprint score and observed 693 fpQTLs associated with footprint-inferred TF binding (FDR < 5%). Given that Tn5 insertion sites are measured with base-pair resolution, we show that fpQTLs can aid GWAS and QTL fine-mapping by precisely pinpointing TF activity within broad trait-associated loci where the underlying causal variant is unknown. Liver fpQTLs were strongly enriched across ChIP-seq peaks, liver expression QTLs (eQTLs), and liver-related GWAS loci, and their inferred effect on TF binding was concordant with their effect on underlying sequence motifs in 80% of cases. We conclude that fpQTLs can reveal causal GWAS variants, define the role of TF binding site disruption in disease and provide functional insights into non-coding variants, ultimately informing novel treatments for common diseases.

Genotype inference from aggregated chromatin accessibility data reveals genetic regulatory mechanisms.

Background: Understanding the genetic causes for variability in chromatin accessibility can shed light on the molecular mechanisms through which genetic variants may affect complex traits. Thousands of ATAC-seq samples have been collected that hold information about chromatin accessibility across diverse cell types and contexts, but most of these are not paired with genetic information and come from diverse distinct projects and laboratories. Results: We report here joint genotyping, chromatin accessibility peak calling, and discovery of quantitative trait loci which influence chromatin accessibility (caQTLs), demonstrating the capability of performing caQTL analysis on a large scale in a diverse sample set without pre-existing genotype information. Using 10,293 profiling samples representing 1,454 unique donor individuals across 653 studies from public databases, we catalog 23,381 caQTLs in total. After joint discovery analysis, we cluster samples based on accessible chromatin profiles to identify context-specific caQTLs. We find that caQTLs are strongly enriched for annotations of gene regulatory elements across diverse cell types and tissues and are often strongly linked with genetic variation associated with changes in expression (eQTLs), indicating that caQTLs can mediate genetic effects on gene expression. We demonstrate sharing of causal variants for chromatin accessibility and diverse complex human traits, enabling a more complete picture of the genetic mechanisms underlying complex human phenotypes. Conclusions: Our work provides a proof of principle for caQTL calling from previously ungenotyped samples, and represents one of the largest, most diverse caQTL resources currently available, informing mechanisms of genetic regulation of gene expression and contribution to disease.

Evaluating the Contribution of Cell Type-Specific Alternative Splicing to Variation in Lipid Levels.

BACKGROUND: Genome-wide association studies have identified hundreds of loci associated with lipid levels. However, the genetic mechanisms underlying most of these loci are not well-understood. Recent work indicates that changes in the abundance of alternatively spliced transcripts contribute to complex trait variation. Consequently, identifying genetic loci that associate with alternative splicing in disease-relevant cell types and determining the degree to which these loci are informative for lipid biology is of broad interest. METHODS: We analyze gene splicing in 83 sample-matched induced pluripotent stem cell (iPSC) and hepatocyte-like cell lines (n=166), as well as in an independent collection of primary liver tissues (n=96) to perform discovery of splicing quantitative trait loci (sQTLs). RESULTS: We observe that transcript splicing is highly cell type specific, and the genes that are differentially spliced between iPSCs and hepatocyte-like cells are enriched for metabolism pathway annotations. We identify 1384 hepatocyte-like cell sQTLs and 1455 iPSC sQTLs at a false discovery rate of <5% and find that sQTLs are often shared across cell types. To evaluate the contribution of sQTLs to variation in lipid levels, we conduct colocalization analysis using lipid genome-wide association data. We identify 19 lipid-associated loci that colocalize either with an hepatocyte-like cell expression quantitative trait locus or sQTL. Only 2 loci colocalize with both a sQTL and expression quantitative trait locus, indicating that sQTLs contribute information about genome-wide association studies loci that cannot be obtained by analysis of steady-state gene expression alone. CONCLUSIONS: These results provide an important foundation for future efforts that use iPSC and iPSC-derived cells to evaluate genetic mechanisms influencing both cardiovascular disease risk and complex traits in general.

Mutation Rates in Cancer Susceptibility Genes in Patients With Breast Cancer With Multiple Primary Cancers.

PURPOSE: Women with breast cancer have a 4%-16% lifetime risk of a second primary cancer. Whether mutations in genes other than BRCA1/2 are enriched in patients with breast and another primary cancer over those with a single breast cancer (S-BC) is unknown. PATIENTS AND METHODS: We identified pathogenic germline mutations in 17 cancer susceptibility genes in patients with BRCA1/2-negative breast cancer in 2 different cohorts: cohort 1, high-risk breast cancer program (multiple primary breast cancer [MP-BC], n = 551; S-BC, n = 449) and cohort 2, familial breast cancer research study (MP-BC, n = 340; S-BC, n = 1,464). Mutation rates in these 2 cohorts were compared with a control data set (Exome Aggregation Consortium [ExAC]). RESULTS: Overall, pathogenic mutation rates for autosomal, dominantly inherited genes were higher in patients with MP-BC versus S-BC in both cohorts (8.5% v 4.9% [P = .02] and 7.1% v 4.2% [P = .03]). There were differences in individual gene mutation rates between cohorts. In both cohorts, younger age at first breast cancer was associated with higher mutation rates; the age of non-breast cancers was unrelated to mutation rate. TP53 and MSH6 mutations were significantly enriched in patients with MP-BC but not S-BC, whereas ATM and PALB2 mutations were significantly enriched in both groups compared with ExAC. CONCLUSION: Mutation rates are at least 7% in all patients with BRCA1/2 mutation-negative MP-BC, regardless of age at diagnosis of breast cancer, with mutation rates up to 25% in patients with a first breast cancer diagnosed at age < 30 years. Our results suggest that all patients with breast cancer with a second primary cancer, regardless of age of onset, should undergo multigene panel testing.

Genomic Signatures Predict the Immunogenicity of BRCA-Deficient Breast Cancer.

PURPOSE: Breast cancers with BRCA1/2 alterations have a relatively high mutational load, suggesting that immune checkpoint blockade may be a potential treatment option. However, the degree of immune cell infiltration varies widely, and molecular features contributing to this variability remain unknown. EXPERIMENTAL DESIGN: We hypothesized that genomic signatures might predict immunogenicity in BRCA1/2 breast cancers. Using The Cancer Genome Atlas (TCGA) genomic data, we compared breast cancers with (89) and without (770) either germline or somatic BRCA1/2 alterations. We also studied 35 breast cancers with germline BRCA1/2 mutations from Penn using WES and IHC. RESULTS: We found that homologous recombination deficiency (HRD) scores were negatively associated with expression-based immune indices [cytolytic index (P = 0.04), immune ESTIMATE (P = 0.002), type II IFN signaling (P = 0.002)] despite being associated with a higher mutational/neoantigen burden, in BRCA1/2 mutant breast cancers. Further, absence of allele-specific loss of heterozygosity (LOH negative; P = 0.01) or subclonality (P = 0.003) of germline and somatic BRCA1/2 mutations, respectively, predicted for heightened cytolytic activity. Gene set analysis found that multiple innate and adaptive immune pathways that converge on NF-κB may contribute to this heightened immunogenicity. IHC of Penn breast cancers demonstrated increased CD45+ (P = 0.039) and CD8+ infiltrates (P = 0.037) and increased PDL1 expression (P = 0.012) in HRD-low or LOH-negative cancers. Triple-negative cancers with low HRD had far greater CD8+ T cells (P = 0.0011) and Perforin 1 expression (P = 0.014) compared with hormone receptor-positive HRD-high cancers. CONCLUSIONS: HRD scores and hormone receptor subtype are predictive of immunogenicity in BRCA1/2 breast cancers and may inform the design of optimal immune therapeutic strategies.

A single dose of neoadjuvant PD-1 blockade predicts clinical outcomes in resectable melanoma.

Immunologic responses to anti-PD-1 therapy in melanoma patients occur rapidly with pharmacodynamic T cell responses detectable in blood by 3 weeks. It is unclear, however, whether these early blood-based observations translate to the tumor microenvironment. We conducted a study of neoadjuvant/adjuvant anti-PD-1 therapy in stage III/IV melanoma. We hypothesized that immune reinvigoration in the tumor would be detectable at 3 weeks and that this response would correlate with disease-free survival. We identified a rapid and potent anti-tumor response, with 8 of 27 patients experiencing a complete or major pathological response after a single dose of anti-PD-1, all of whom remain disease free. These rapid pathologic and clinical responses were associated with accumulation of exhausted CD8 T cells in the tumor at 3 weeks, with reinvigoration in the blood observed as early as 1 week. Transcriptional analysis demonstrated a pretreatment immune signature (neoadjuvant response signature) that was associated with clinical benefit. In contrast, patients with disease recurrence displayed mechanisms of resistance including immune suppression, mutational escape, and/or tumor evolution. Neoadjuvant anti-PD-1 treatment is effective in high-risk resectable stage III/IV melanoma. Pathological response and immunological analyses after a single neoadjuvant dose can be used to predict clinical outcome and to dissect underlying mechanisms in checkpoint blockade.

BRCA locus-specific loss of heterozygosity in germline BRCA1 and BRCA2 carriers.

Complete loss of BRCA1 or BRCA2 function is associated with sensitivity to DNA damaging agents. However, not all BRCA1 and BRCA2 germline mutation-associated tumors respond. Herein we report analyses of 160 BRCA1 and BRCA2 germline mutation-associated breast and ovarian tumors. Retention of the normal BRCA1 or BRCA2 allele (absence of locus-specific loss of heterozygosity (LOH)) is observed in 7% of BRCA1 ovarian, 16% of BRCA2 ovarian, 10% of BRCA1 breast, and 46% of BRCA2 breast tumors. These tumors have equivalent homologous recombination deficiency scores to sporadic tumors, significantly lower than scores in tumors with locus-specific LOH (ovarian, P = 0.0004; breast P < 0.0001, two-tailed Student's t-test). Absence of locus-specific LOH is associated with decreased overall survival in ovarian cancer patients treated with platinum chemotherapy (P = 0.01, log-rank test). Locus-specific LOH may be a clinically useful biomarker to predict primary resistance to DNA damaging agents in patients with germline BRCA1 and BRCA2 mutations.Most tumours associated with germline BRCA1/BRCA2 loss of function mutations respond to DNA damaging agents, however, some do not. Herein, the authors identify that a subset of breast/ovarian tumors retain a normal allele, which is associated with decreased overall survival after DNA damage-inducing platinum chemotherapy.

The contribution of pathogenic variants in breast cancer susceptibility genes to familial breast cancer risk.

Understanding the gene-specific risks for development of breast cancer will lead to improved clinical care for those carrying germline mutations in cancer predisposition genes. We sought to detail the spectrum of mutations and refine risk estimates for known and proposed breast cancer susceptibility genes. Targeted massively-parallel sequencing was performed to identify mutations and copy number variants in 26 known or proposed breast cancer susceptibility genes in 2134 BRCA1/2-negative women with familial breast cancer (proband with breast cancer and a family history of breast or ovarian cancer) from a largely European-Caucasian multi-institutional cohort. Case-control analysis was performed comparing the frequency of internally classified mutations identified in familial breast cancer women to Exome Aggregation Consortium controls. Mutations were identified in 8.2% of familial breast cancer women, including mutations in high-risk (odds ratio > 5) (1.4%) and moderate-risk genes (2 < odds ratio < 5) (2.9%). The remaining familial breast cancer women had mutations in proposed breast cancer genes (1.7%), Lynch syndrome genes (0.5%), and six cases had two mutations (0.3%). Case-control analysis demonstrated associations with familial breast cancer for ATM, PALB2, and TP53 mutations (odds ratio > 3.0, p < 10-4), BARD1 mutations (odds ratio = 3.2, p = 0.012), and CHEK2 truncating mutations (odds ratio = 1.6, p = 0.041). Our results demonstrate that approximately 4.7% of BRCA1/2 negative familial breast cancer women have mutations in genes statistically associated with breast cancer. We classified PALB2 and TP53 as high-risk, ATM and BARD1 as moderate risk, and CHEK2 truncating mutations as low risk breast cancer predisposition genes. This study demonstrates that large case-control studies are needed to fully evaluate the breast cancer risks associated with mutations in moderate-risk and proposed susceptibility genes.

Erratum: Author Correction: The contribution of pathogenic variants in breast cancer susceptibility genes to familial breast cancer risk.

[This corrects the article DOI: 10.1038/s41523-017-0024-8.].