Eric Karlins

FGFR2 variants and breast cancer risk: fine-scale mapping using African American studies and analysis of chromatin conformation

Genome-wide association studies have identified FGFR2 as a breast cancer (BC) susceptibility gene in populations of European and Asian descent, but a causative variant has not yet been conclusively identified. We hypothesized that the weaker linkage disequilibrium across this associated region in populations of African ancestry might help refine the set of candidate-causal single nucleotide polymorphisms (SNPs) previously identified by our group. Eight candidate-causal SNPs were evaluated in 1253 African American invasive BC cases and 1245 controls. A significant association with BC risk was found with SNP rs2981578 (unadjusted per-allele odds ratio = 1.20, 95% confidence interval 1.03-1.41, P(trend) = 0.02), with the odds ratio estimate similar to that reported in European and Asian subjects. To extend the fine-mapping, genotype data from the African American studies were analyzed jointly with data from European (n = 7196 cases, 7275 controls) and Asian (n = 3901 cases, 3205 controls) studies. In the combined analysis, SNP rs2981578 was the most strongly associated. Five other SNPs were too strongly correlated to be excluded at a likelihood ratio of < 1/100 relative to rs2981578. Analysis of DNase I hypersensitive sites indicated that only two of these map to highly accessible chromatin, one of which, SNP rs2981578, has previously been implicated in up-regulating FGFR2 expression. Our results demonstrate that the association of SNPs in FGFR2 with BC risk extends to women of African American ethnicity, and illustrate the utility of combining association analysis in datasets of diverse ethnic groups with functional experiments to identify disease susceptibility variants.

Rare germline mutations in the BRCA2 gene are associated with early-onset prostate cancer

Studies of families who segregate BRCA2 mutations have found that men who carry disease-associated mutations have an increased risk of prostate cancer, particularly early-onset disease. A study of sporadic prostate cancer in the UK reported a prevalence of 2.3% for protein-truncating BRCA2 mutations among patients diagnosed at ages ⩽55 years, highlighting the potential importance of this gene in prostate cancer susceptibility. To examine the role of protein-truncating BRCA2 mutations in relation to early-onset prostate cancer in a US population, 290 population-based patients from King County, Washington, diagnosed at ages <55 years were screened for germline BRCA2 mutations. The coding regions, intron–exon boundaries, and potential regulatory elements of the BRCA2 gene were sequenced. Two distinct protein-truncating BRCA2 mutations were identified in exon 11 in two patients. Both cases were Caucasian, yielding a mutation prevalence of 0.78% (95% confidence interval (95%CI) 0.09–2.81%) and a relative risk (RR) of 7.8 (95%CI 1.8–9.4) for early-onset prostate cancer in white men carrying a protein-truncating BRCA2 mutation. Results suggest that protein-truncating BRCA2 mutations confer an elevated RR of early-onset prostate cancer. However, we estimate that <1% of early-onset prostate cancers in the general US Caucasian population can be attributed to these rare disease-associated BRCA2 mutations.

A copy number variant at the KITLG locus likely confers risk for canine squamous cell carcinoma of the digit.

The domestic dog is a robust model for studying the genetics of complex disease susceptibility. The strategies used to develop and propagate modern breeds have resulted in an elevated risk for specific diseases in particular breeds. One example is that of Standard Poodles (STPOs), who have increased risk for squamous cell carcinoma of the digit (SCCD), a locally aggressive cancer that causes lytic bone lesions, sometimes with multiple toe recurrence. However, only STPOs of dark coat color are at high risk; light colored STPOs are almost entirely unaffected, suggesting that interactions between multiple pathways are necessary for oncogenesis. We performed a genome-wide association study (GWAS) on STPOs, comparing 31 SCCD cases to 34 unrelated black STPO controls. The peak SNP on canine chromosome 15 was statistically significant at the genome-wide level (Praw = 1.60×10−7; Pgenome = 0.0066). Additional mapping resolved the region to the KIT Ligand (KITLG) locus. Comparison of STPO cases to other at-risk breeds narrowed the locus to a 144.9-Kb region. Haplotype mapping among 84 STPO cases identified a minimal region of 28.3 Kb. A copy number variant (CNV) containing predicted enhancer elements was found to be strongly associated with SCCD in STPOs (P = 1.72×10−8). Light colored STPOs carry the CNV risk alleles at the same frequency as black STPOs, but are not susceptible to SCCD. A GWAS comparing 24 black and 24 light colored STPOs highlighted only the MC1R locus as significantly different between the two datasets, suggesting that a compensatory mutation within the MC1R locus likely protects light colored STPOs from disease. Our findings highlight a role for KITLG in SCCD susceptibility, as well as demonstrate that interactions between the KITLG and MC1R loci are potentially required for SCCD oncogenesis. These findings highlight how studies of breed-limited diseases are useful for disentangling multigene disorders.

Homologous Mutation to Human BRAF V600E Is Common in Naturally Occurring Canine Bladder Cancer—Evidence for a Relevant Model System and Urine-Based Diagnostic Test

Targeted cancer therapies offer great clinical promise, but treatment resistance is common, and basic research aimed at overcoming this challenge is limited by reduced genomic and biologic complexity in artificially induced rodent tumors compared with their human counterparts. Animal models that more faithfully recapitulate genotype-specific human pathology could improve the predictive value of these investigations. Here, a newly identified animal model for oncogenic BRAF-driven cancers is described. With 20,000 new cases in the United States each year, canine invasive transitional cell carcinoma of the bladder (InvTCC) is a common, naturally occurring malignancy that shares significant histologic, biologic, and clinical phenotypes with human muscle invasive bladder cancer. In order to identify somatic drivers of canine InvTCC, the complete transcriptome for multiple tumors was determined by RNAseq. All tumors harbored a somatic mutation that is homologous to the human BRAF(V600E) mutation, and an identical mutation was present in 87% of 62 additional canine InvTCC tumors. The mutation was also detectable in the urine sediments of all dogs tested with mutation-positive tumors. Functional experiments suggest that, like human tumors, canine activating BRAF mutations potently stimulate the MAPK pathway. Cell lines with the mutation have elevated levels of phosphorylated MEK, compared with a line with wild-type BRAF. This effect can be diminished through application of the BRAF(V600E) inhibitor vemurafenib. These findings set the stage for canine InvTCC as a powerful system to evaluate BRAF-targeted therapies, as well as therapies designed to overcome resistance, which could enhance treatment of both human and canine cancers Implications: This study demonstrates the activating BRAF mutation (V600E), which is found in multiple human cancers, is a driver of canine InvTCC, and highlights a urine-based test for quick diagnosis. Mol Cancer Res; 13(6); 993–1002. ©2015 AACR.

Comparison against 186 canid whole-genome sequences reveals survival strategies of an ancient clonally transmissible canine tumor.

Canine transmissible venereal tumor (CTVT) is a parasitic cancer clone that has propagated for thousands of years via sexual transfer of malignant cells. Little is understood about the mechanisms that converted an ancient tumor into the worlds oldest known continuously propagating somatic cell lineage. We created the largest existing catalog of canine genome-wide variation and compared it against two CTVT genome sequences, thereby separating alleles derived from the founders genome from somatic mutations that must drive clonal transmissibility. We show that CTVT has undergone continuous adaptation to its transmissible allograft niche, with overlapping mutations at every step of immunosurveillance, particularly self-antigen presentation and apoptosis. We also identified chronologically early somatic mutations in oncogenesis- and immune-related genes that may represent key initiators of clonal transmissibility. Thus, we provide the first insights into the specific genomic aberrations that underlie CTVTs dogged perseverance in canids around the world.

Association of FGFR4 genetic polymorphisms with prostate cancer risk and prognosis.

The fibroblast growth factor receptor 4 (FGFR4) is thought to be involved in many critical cellular processes and has been associated with prostate cancer risk. Four single nucleotide polymorphisms (SNPs) within or near FGFR4 were analyzed in a population-based study of 1458 prostate cancer patients and 1352 age-matched controls. We found no evidence to suggest that any of the FGFR4 SNP genotypes were associated with prostate cancer risk or with disease aggressiveness, Gleason score or stage. A weak association was seen between rs351855 and prostate cancer-specific mortality. Subset analysis of cases that had undergone radical prostatectomy revealed an association between rs351855 and prostate cancer risk. Although our results confirm an association between FGFR4 and prostate cancer risk in radical prostatectomy cases, they suggest that the role of FGFR4 in disease risk and outcomes at a population-based level appears to be minor.

Higher-than-expected population prevalence of potentially pathogenic germline TP53 variants in individuals unselected for cancer history

Li–Fraumeni syndrome (LFS) is an autosomal‐dominant cancer predisposition disorder associated with pathogenic germline variants in TP53, with a high penetrance over an individuals lifetime. The actual population prevalence of pathogenic germline TP53 mutations is still unclear, most likely due to biased selection of cancer affected families. The aim of this study was to estimate the population prevalence of potentially pathogenic TP53 exonic variants in three sequencing databases, totaling 63,983 unrelated individuals. Potential pathogenicity was defined using an original algorithm combining bioinformatic prediction tools, suggested clinical significance, and functional data. We identified 34 different potentially pathogenic TP53 variants in 131 out of 63,983 individuals (0.2%). Twenty‐eight (82%) of these variants fell within the DNA‐binding domain of TP53, with an enrichment for specific variants that were not previously identified as LFS mutation hotspots, such as the p.R290H and p.N235S variants. Our findings reveal that the population prevalence of potentially pathogenic TP53 variants may be up to 10 times higher than previously estimated from family‐based studies. These results point to the need for further studies aimed at evaluating cancer penetrance modifiers as well as the risk associated between cancer and rare TP53 variants.

Evaluating the Causal Link Between Malaria Infection and Endemic Burkitt Lymphoma in Northern Uganda: A Mendelian Randomization Study

Background Plasmodium falciparum (Pf) malaria infection is suspected to cause endemic Burkitt Lymphoma (eBL), but the evidence remains unsettled. An inverse relationship between sickle cell trait (SCT) and eBL, which supports that between malaria and eBL, has been reported before, but in small studies with low power. We investigated this hypothesis in children in a population-based study in northern Uganda using Mendelian Randomization. Methods Malaria-related polymorphisms (SCT, IL10, IL1A, CD36, SEMA3C, and IFNAR1) were genotyped in 202 eBL cases and 624 controls enrolled during 2010–2015. We modeled associations between genotypes and eBL or malaria using logistic regression. Findings SCT was associated with decreased risk of eBL (adjusted odds ratio [OR] 0·37, 95% CI 0·21–0·66; p = 0·0003). Decreased risk of eBL was associated with IL10 rs1800896-CT (OR 0·73, 95% CI 0·50–1·07) and -CC genotypes (OR 0·53, 95% CI 0·29–0·95, ptrend = 0·019); IL1A rs2856838-AG (OR 0·56, 95% CI 0·39–0·81) and -AA genotype (OR 0·50, 95% CI 0·28–1·01, ptrend = 0·0016); and SEMA3C rs4461841-CT or -CC genotypes (OR 0·57, 95% CI 0·35–0·93, p = 0·0193). SCT and IL10 rs1800896, IL1A rs2856838, but not SEMA3C rs4461841, polymorphisms were associated with decreased risk of malaria in the controls. Interpretation Our results support a causal effect of malaria infection on eBL.

Genome-wide association study identifies the GLDC/IL33 locus associated with survival of osteosarcoma patients

Survival rates for osteosarcoma, the most common primary bone cancer, have changed little over the past three decades and are particularly low for patients with metastatic disease. We conducted a multi‐institutional genome‐wide association study (GWAS) to identify germline genetic variants associated with overall survival in 632 patients with osteosarcoma, including 523 patients of European ancestry and 109 from Brazil. We conducted a time‐to‐event analysis and estimated hazard ratios (HR) and 95% confidence intervals (CI) using Cox proportional hazards models, with and without adjustment for metastatic disease. The results were combined across the European and Brazilian case sets using a random‐effects meta‐analysis. The strongest association after meta‐analysis was for rs3765555 at 9p24.1, which was inversely associated with overall survival (HR = 1.76; 95% CI 1.41–2.18, p = 4.84 × 10−7). After imputation across this region, the combined analysis identified two SNPs that reached genome‐wide significance. The strongest single association was with rs55933544 (HR = 1.9; 95% CI 1.5–2.4; p = 1.3 × 10−8), which localizes to the GLDC gene, adjacent to the IL33 gene and was consistent across both the European and Brazilian case sets. Using publicly available data, the risk allele was associated with lower expression of IL33 and low expression of IL33 was associated with poor survival in an independent set of patients with osteosarcoma. In conclusion, we have identified the GLDC/IL33 locus on chromosome 9p24.1 as associated with overall survival in patients with osteosarcoma. Further studies are needed to confirm this association and shed light on the biological underpinnings of this susceptibility locus.

Whole exome sequencing in 75 high-risk families with validation and replication in independent case-control studies identifies TANGO2 , OR5H14 , and CHAD as new prostate cancer susceptibility genes

Prostate cancer (PCa) susceptibility is defined by a continuum from rare, high-penetrance to common, low-penetrance alleles. Research to date has concentrated on identification of variants at the ends of that continuum. Taking an alternate approach, we focused on the important but elusive class of low-frequency, moderately penetrant variants by performing disease model-based variant filtering of whole exome sequence data from 75 hereditary PCa families. Analysis of 341 candidate risk variants identified nine variants significantly associated with increased PCa risk in a population-based, case-control study of 2,495 men. In an independent nested case-control study of 7,121 men, there was risk association evidence for TANGO2 p.Ser17Ter and the established HOXB13 p.Gly84Glu variant. Meta-analysis combining the case-control studies identified two additional variants suggestively associated with risk, OR5H14 p.Met59Val and CHAD p.Ala342Asp. The TANGO2 and HOXB13 variants co-occurred in cases more often than expected by chance and never in controls. Finally, TANGO2 p.Ser17Ter was associated with aggressive disease in both case-control studies separately. Our analyses identified three new PCa susceptibility alleles in the TANGO2, OR5H14 and CHAD genes that not only segregate in multiple high-risk families but are also of importance in altering disease risk for men from the general population. This is the first successful study to utilize sequencing in high-risk families for the express purpose of identifying low-frequency, moderately penetrant PCa risk mutations.