An expressed retrogene of the master embryonic stem cell gene POU5F1 is associated with prostate cancer susceptibility.

Genetic association studies of prostate and other cancers have identified a major risk locus at chromosome 8q24. Several independent risk variants at this locus alter transcriptional regulatory elements, but an affected gene and mechanism for cancer predisposition have remained elusive. The retrogene POU5F1B within the locus has a preserved open reading frame encoding a homolog of the master embryonic stem cell transcription factor Oct4. We find that 8q24 risk alleles are expression quantitative trait loci correlated with reduced expression of POU5F1B in prostate tissue and that predicted deleterious POU5F1B missense variants are also associated with risk of transformation. POU5F1 is known to be self-regulated by the encoded Oct4 transcription factor. We further observe that POU5F1 expression is directly correlated with POU5F1B expression. Our results suggest that a pathway critical to self-renewal of embryonic stem cells may also have a role in the origin of cancer.

Associations of prostate cancer risk variants with disease aggressiveness: results of the NCI-SPORE Genetics Working Group analysis of 18,343 cases.

Genetic studies have identified single nucleotide polymorphisms (SNPs) associated with the risk of prostate cancer (PC). It remains unclear whether such genetic variants are associated with disease aggressiveness. The NCI-SPORE Genetics Working Group retrospectively collected clinicopathologic information and genotype data for 36 SNPs which at the time had been validated to be associated with PC risk from 25,674 cases with PC. Cases were grouped according to race, Gleason score (Gleason ≤ 6, 7, ≥ 8) and aggressiveness (non-aggressive, intermediate, and aggressive disease). Statistical analyses were used to compare the frequency of the SNPs between different disease cohorts. After adjusting for multiple testing, only PC-risk SNP rs2735839 (G) was significantly and inversely associated with aggressive (OR = 0.77; 95 % CI 0.69-0.87) and high-grade disease (OR = 0.77; 95 % CI 0.68-0.86) in European men. Similar associations with aggressive (OR = 0.72; 95 % CI 0.58-0.89) and high-grade disease (OR = 0.69; 95 % CI 0.54-0.87) were documented in African-American subjects. The G allele of rs2735839 was associated with disease aggressiveness even at low PSA levels (<4.0 ng/mL) in both European and African-American men. Our results provide further support that a PC-risk SNP rs2735839 near the KLK3 gene on chromosome 19q13 may be associated with aggressive and high-grade PC. Future prospectively designed, case-case GWAS are needed to identify additional SNPs associated with PC aggressiveness.

Prostate cancer risk variants of the HOXB genetic locus.

The G84E germline mutation of HOXB13 predisposes to prostate cancer and is clinically tested for familial cancer care. We investigated the HOXB locus to define a potentially broader contribution to prostate cancer heritability. We sought HOXB locus germline variants altering prostate cancer risk in three European-ancestry case-control study populations (combined 7812 cases and 5047 controls): the International Consortium for Prostate Cancer Genetics Study; the Nashville Familial Prostate Cancer Study; and the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial. Multiple rare genetic variants had concordant and strong risk effects in these study populations and exceeded genome-wide significance. Independent risk signals were best detected by sentinel variants rs559612720 within SKAP1 (OR = 8.1, P = 2E-9) and rs138213197 (G84E) within HOXB13 (OR = 5.6, P = 2E-11), separated by 567 kb. Half of carriers inherited both risk alleles, while others inherited either alone. Under mutual adjustment, the variants separately carried 3.6- and 3.1-fold risk, respectively, while joint inheritance carried 11.3-fold risk. These risks were further accentuated among men meeting criteria for hereditary prostate cancer, and further still for those with early-onset or aggressive disease. Among hereditary prostate cancer cases diagnosed under age 60 and with aggressive disease, joint inheritance carried a risk of OR = 27.7 relative to controls, P = 2E-8. The HOXB sentinel variant pair more fully captured genetic risk for prostate cancer within the study populations than either variant alone.

Practical genotyping by single-nucleotide primer extension.

Genome-wide association studies bring into focus specific genetic variants of particular interest for which validation is often sought in large numbers of study subjects. Practical alternative methods are limiting for the application of genotyping few variants in many samples. A common scenario is the need to genotype a study population at a specific high-value single nucleotide polymorphism (SNP) or insertion-deletion (indel). Not all such variants, however, will be amenable to assay by a given approach. We have adapted a single-nucleotide primer extension (SNuPE) method that may be tailored to genotype a required variant, and implemented it as a useful general laboratory protocol. We demonstrate reliable application for production-scale genotyping, successfully converting 87% of SNPs and indels for assay with an estimated error rate of 0.003. Our implementation of the SNuPE genotyping assay is a viable addition to existing alternative methods; it is readily customizable, scalable, and uses standard reagents and a laboratory plate reader.

8q24 genetic variation and comprehensive haplotypes altering familial risk of prostate cancer.

The 8q24 genomic locus is tied to the origin of numerous cancers. We investigate its contribution to hereditary prostate cancer (HPC) in independent study populations of the Nashville Familial Prostate Cancer Study and International Consortium for Prostate Cancer Genetics (combined: 2,836 HPC cases, 2,206 controls of European ancestry). Here we report 433 variants concordantly associated with HPC in both study populations, accounting for 9% of heritability and modifying age of diagnosis as well as aggressiveness; 183 reach genome-wide significance. The variants comprehensively distinguish independent risk-altering haplotypes overlapping the 648 kb locus (three protective, and four risk (peak odds ratios: 1.5, 4, 5, and 22)). Sequence of the near-Mendelian haplotype reveals eleven causal mutation candidates. We introduce a linkage disequilibrium-based algorithm discerning eight independent sentinel variants, carrying considerable risk prediction ability (AUC = 0.625) for a single locus. These findings elucidate 8q24 locus structure and correlates for clinical prediction of prostate cancer risk.

Heritable variation of ERBB2 and breast cancer risk.

Amplification of the epithelial growth factor receptor gene ERBB2 (HER2, NEU) in breast cancer is associated with a poor clinical prognosis. In mammary gland development, this receptor plays a role in ductal and lobuloalveolar differentiation. We conducted a systematic investigation of the role of genetic variation of the ERBB2 gene in breast cancer risk in a study of 842 histologically confirmed invasive breast cancer cases and 1,108 controls from the Shanghai Breast Cancer Study. We observed that the ERBB2 gene resides within a locus of high linkage disequilibrium, composed of three major ancestral haplotypes in the study population. These haplotypes are marked by simple tandem repeat and single nucleotide polymorphisms, including the missense variants I655V and P1170A. We observed a risk-modifying effect of a highly polymorphic simple tandem repeat within an evolutionarily conserved region, 4.4 kb upstream from the ERBB2 transcription start site. Under a dominant genetic model, the age-adjusted odds ratio was 1.74 (95% confidence interval, 1.27-2.37). Its association with breast cancer, and with breast cancer stratified by histology, by histologic grade, and by stage, remained significant after correction for multiple comparisons. In contrast, we observed no association of ERBB2 single nucleotide polymorphism haplotypes with breast cancer predisposition.

Haplotype analysis of CYP11A1 identifies promoter variants associated with breast cancer risk.

The CYP11A1 gene encodes the cholesterol side chain cleavage enzyme that catalyzes the initial and rate-limiting step of steroidogenesis. A large number of epidemiologic studies have implicated the duration and degree of endogenous estrogen exposure in the development of breast cancer in women. Here, we conduct a systematic investigation of the role of genetic variation of the CYP11A1 gene in breast cancer risk in a study of 1193 breast cancer cases and 1310 matched controls from the Shanghai Breast Cancer Study. We characterize the genetic architecture of the CYP11A1 gene in a Chinese study population. We then genotype tagging polymorphisms to capture common variation at the locus for tests of association. Variants designating a haplotype encompassing the gene promoter are significantly associated with both increased expression (P = 1.6e-6) and increased breast cancer risk: heterozygote age-adjusted odds ratio (OR), 1.51 [95% confidence interval (95% CI), 1.19-1.91]; homozygote age-adjusted OR, 2.94 (95% CI, 1.22-7.12), test for trend, P = 5.0e-5. Among genes controlling endogenous estrogen metabolism, CYP11A1 harbors common variants that may influence expression to significantly modify risk of breast cancer.

A haplotype at chromosome Xq27.2 confers susceptibility to prostate cancer.

We conducted an association study to identify risk variants for familial prostate cancer within the HPCX locus at Xq27 among Americans of Northern European descent. We investigated a total of 507 familial prostate cancer probands and 507 age-matched controls without a personal or family history of prostate cancer. The study population was subdivided into a set of training subjects to explore genetic variation of the locus potentially impacting risk of prostate cancer, and an independent set of test subjects to confirm the association and to assign significance, addressing multiple comparisons. We identified a 22.9 kb haplotype nominally associated with prostate cancer among training subjects (292 cases, 292 controls; chi(2) = 5.08, P = 0.020), that was confirmed among test subjects (215 cases, 215 controls; chi(2) = 3.73, P = 0.040). The haplotype predisposed to prostate cancer with an odds ratio of 3.41 (95% CI 1.04-11.17, P = 0.034) among test subjects. The haplotype extending from rs5907859 to rs1493189 is concordant with a prior study of the region within the Finnish founder population, and warrants further independent investigation.

Association of mitochondrial DNA displacement loop (CA)n dinucleotide repeat polymorphism with breast cancer risk and survival among Chinese women.

Mitochondrial genome alternations may be involved in carcinogenesis. The noncoding region of the mitochondrial DNA (mtDNA) displacement loop (D-loop) has emerged as a mutational hotspot. Using data from a population-based case-control study conducted among Chinese women in Shanghai, we evaluated associations of breast cancer risk and survival with the mtDNA D-loop (CA)(n) dinucleotide repeat polymorphism. Included in the study were 1,058 cases and 1,129 age frequency-matched community controls that participated in the Shanghai Breast Cancer Study between 1996 and 1998. Breast cancer patients were followed to determine intervals of overall survival and disease-free survival. Overall, there was no association between the mtDNA D-loop (CA)(n) repeat polymorphism and breast cancer risk. Patients with multiple alleles of the mtDNA D-loop (CA)(n) polymorphism (heteroplasmy) had significantly poorer disease-free survival than those with one allele of the mtDNA D-loop (CA)(n) polymorphism (hazard ratio 1.62; 95% confidence interval, 1.16-2.26). These results suggest that the mtDNA D-loop (CA)(n) repeat polymorphism may be associated with breast cancer survival. Additional studies with a larger sample size are warranted.

Confirmation of the HOXB13 G84E germline mutation in familial prostate cancer.

BACKGROUND: A recent study of familial and early onset prostate cancer reported a recurrent rare germline mutation of HOXB13 among men of European descent. The gene resides within the 17q21 hereditary prostate cancer linkage interval. METHODS: We evaluated the G84E germline mutation (rs138213197) of HOXB13 in a case-control study of familial prostate cancer at Vanderbilt University (Nashville, TN) to independently evaluate the association of the mutation with familial prostate cancer. We genotyped 928 familial prostate cancer probands and 930 control probands without a personal or family history of prostate cancer. RESULTS: Our study confirmed the association between the G84E mutation of HOXB13 and risk of prostate cancer among subjects of European descent. We observed the mutation in 16 familial cases and in two controls, each as heterozygotes. The odds ratio (OR) for prostate cancer was 7.9 [95% confidence interval, (CI) 1.8-34.5, P = 0.0062] among carriers of the mutation. The carrier rate was 1.9% among all familial case probands and 2.7% among probands of pedigrees with ≥3 affected. In a separate case series of 268 probands of European descent with no additional family history of prostate cancer, the carrier rate was 1.5%. CONCLUSIONS: The germline mutation G84E of HOXB13 is a rare but recurrent mutation associated with elevated risk of prostate cancer in men of European descent, with an effect size that is greater than observed for previously validated risk variants of genome wide association studies. IMPACT: This study independently confirms the association of a germline HOXB13 mutation with familial prostate cancer.

A multistage genetic association study identifies breast cancer risk loci at 10q25 and 16q24.

BACKGROUND: Heritable risk for breast cancer includes an increasing number of common, low effect risk variants. We conducted a multistage genetic association study in a series of independent epidemiologic breast cancer study populations to identify novel breast cancer risk variants. METHODS: We tested 1,162 SNPs of greatest nominal significance from stage I of the Cancer Genetic Markers of Susceptibility breast cancer study (CGEMS; 1,145 cases, 1,142 controls) for evidence of replicated association with breast cancer in the Nashville Breast Cohort (NBC; 599 cases, 1,161 controls), the Collaborative Breast Cancer Study (CBCS; 1,552 cases, 1,185 controls), and BioVU Breast Cancer Study (BioVU; 1,172 cases, 1,172 controls). RESULTS: Among these SNPs, a series of validated breast cancer risk variants yielded expected associations in the study populations. In addition, we observed two previously unreported loci that were significantly associated with breast cancer risk in the CGEMS, NBC, and CBCS study populations and had a consistent, although not statistically significant, risk effect in the BioVU study population. These were rs1626678 at 10q25.3 near ENO4 and KIAA1598 (meta-analysis age-adjusted OR = 1.13 [1.07-1.20], P = 5.6 × 10(-5)), and rs8046508 at 16q23.1 in the eighth intron of WWOX (meta-analysis age-adjusted OR = 1.20 [1.10-1.31], P = 3.5 × 10(-5)). CONCLUSIONS: Our data supports the association of two novel loci, at 10q25.3 and 16q23.1, with risk of breast cancer. IMPACT: The expanding compendium of known breast cancer genetic risk variants holds increasing power for clinical risk prediction models of breast cancer, improving upon the Gail model.

An SNP-Based Linkage Map for Zebrafish Reveals Sex Determination Loci.

A surprising diversity of mechanisms controls sex determination of vertebrate organisms, even among closely related species. Both genetic and temperature-dependent systems of sex determination have been described in teleost fish. In the common zebrafish model organism, heteromorphic sex chromosomes are not observed, and the potential role of a genetic component of sex determination remains largely unknown. Here we report a genome-wide linkage study of sex determination in zebrafish using a novel SNP genetic map. We identified loci on zebrafish chromosomes 5 (LOD score 7.9) and 16 (LOD score 9.3) governing sex determination as a complex trait, rather than as an XY or ZW genetic system. Each of these loci contains a prominent candidate gene with a conserved role in sex determination across additional species that suggest potential mechanisms of sex determination in zebrafish. The chromosome 5 locus harbors dmrt1, a key gene in sex determination from fruit flies to humans; mutation of the human DMRT1 ortholog is a cause of complete sex reversal of XY individuals. The chromosome 16 locus harbors cyp21a2; mutation of the human CYP21A2 ortholog is one of the more common causes of pseudohermaphroditism. Mutation detection at each of these candidate genes within the zebrafish cross identified hypomorphic variants on the female-associated allele of each locus. The two loci together accounted for 16% of variance of the trait. Interacting environmental cues are likely to be an additional important component of sex determination in zebrafish.

A multistage association study identifies a breast cancer genetic locus at NCOA7.

Estrogen metabolism and growth factor signaling pathway genes play key roles in breast cancer development. We evaluated associations between breast cancer and tagging single-nucleotide polymorphisms (SNP) of 107 candidate genes of these pathways using single allele- and haplotype-based tests. We first sought concordance of associations between two study populations: the Nashville Breast Cohort (NBC; 510 cases, 988 controls), and the Cancer Genetic Markers of Susceptibility (CGEMS) breast cancer study (1,145 cases, 1,142 controls). Findings across the two study populations were concordant at tagging SNPs of six genes, and at previously published SNPs of FGFR2. We sought further replication of results for EGFR, NCOA7, and FGFR2 in the independent Collaborative Breast Cancer Study (CBCS; 1,552 cases, 1,185 controls). Associations at NCOA7 and FGFR2 replicated across all three studies. The association at NCOA7 on 6q22.32, detected by a haplotype spanning the initial protein-coding exon (5'-rs9375411, rs11967627, rs549438, rs529858, rs490361, rs17708107-3'), has not been previously reported. The haplotype had a significant inverse association with breast cancer in each study [OR(Het): 0.69 (NBC), 0.76 (CGEMS), 0.79 (CBCS)], and a meta-analysis OR(Het) of 0.75 (95% CI, 0.65-0.87, P = 1.4 × 10(-4)) in the combined study populations. The haplotype frequency was 0.07 among cases, and 0.09 among controls; homozygotes were infrequent and each OR(Hom) was not significant. NCOA7 encodes a nuclear receptor coactivator that interacts with estrogen receptor α to modulate its activity. These observations provide consistent evidence that genetic variants at the NCOA7 locus may confer a reduced risk of breast cancer.

Protein phosphatase 2A subunit gene haplotypes and proliferative breast disease modify breast cancer risk.

BACKGROUND: Protein phosphatase 2A (PP2A) is a major cellular phosphatase and plays key regulatory roles in growth, differentiation, and apoptosis. Women who are diagnosed with benign proliferative breast disease are at increased risk for the subsequent development of breast cancer. METHODS: The authors evaluated genetic variation of PP2A holoenzyme subunits for their potential contribution to breast cancer risk. A nested case-control investigation was performed on a cohort of women who had a history of benign breast disease. The women were followed for an average of 18 years, and DNA prepared from the original archival benign breast biopsy (1954-1995) was available for 450 women who were diagnosed with breast cancer on follow-up and for 890 of 900 women in a control group who were matched on race, age, and year of entry biopsy. RESULTS: Single allele-based and haplotype-based tests of association were conducted with assessment of significance by permutation testing. Significant risk and protective haplotypes of the PP2A structural/regulatory subunit A alpha isoform (PPP2R1A) were identified and had odds ratios of 1.63 (95% confidence interval [CI], 1.3-2.1) and 0.55 (95% CI, 0.41-0.76), respectively. These odds ratios remained significant after the analysis was adjusted for multiple comparisons. Women who had both the PPP2R1A risk haplotype and a history of proliferative breast disease had an odds ratio of 2.44 (95% CI, 1.7-3.5) for the subsequent development of breast cancer. The effects of haplotypes for 2 PP2A regulatory subunit genes, PP2 regulatory subunit B alpha isoform (PPP2R2A) and PP2A regulatory subunit B' epsilon isoform (PPP2R5E) on breast cancer risk were nominally significant but did not remain significant after the analysis was adjusted for multiple comparisons. CONCLUSIONS: The current findings supported the previously hypothesized role of PP2A as a tumor suppressor gene in breast cancer.

Genetic variants and prostate cancer risk: candidate replication and exploration of viral restriction genes.

The genetic variants underlying the strong heritable component of prostate cancer remain largely unknown. Genome-wide association studies of prostate cancer have yielded several variants that have significantly replicated across studies, predominantly in cases unselected for family history of prostate cancer. Additional candidate gene variants have also been proposed, many evaluated within familial prostate cancer study populations. Such variants hold great potential value for risk stratification, particularly for early-onset or aggressive prostate cancer, given the comorbidities associated with current therapies. Here, we investigate a Caucasian study population of 523 independent familial prostate cancer cases and 523 age-matched controls without a personal or family history of prostate cancer. We replicate identified associations at genome-wide association study loci 8q24, 11q13, and 2p15 (P = 2.9 x 10(-4) to P = 4.7 x 10(-5)), showing study population power. We also find evidence to support reported associations at candidate genes RNASEL, EZH2, and NKX3-1 (P = 0.031 to P = 0.0085). We further explore a set of candidate genes related to RNASEL and to its role in retroviral restriction, identifying nominal associations at XPR1 and RBM9. The effects at 8q24 seem more pronounced for those diagnosed at an early age, whereas at 2p15 and RNASEL the effects were more pronounced at a later age. However, these trends did not reach statistical significance. The effects at 2p15 were statistically significantly more pronounced for those diagnosed with aggressive disease.

Androgen regulated genes in human prostate xenografts in mice: relation to BPH and prostate cancer.

Benign prostatic hyperplasia (BPH) and prostate carcinoma (CaP) are linked to aging and the presence of androgens, suggesting that androgen regulated genes play a major role in these common diseases. Androgen regulation of prostate growth and development depends on the presence of intact epithelial-stromal interactions. Further, the prostatic stroma is implicated in BPH. This suggests that epithelial cell lines are inadequate to identify androgen regulated genes that could contribute to BPH and CaP and which could serve as potential clinical biomarkers. In this study, we used a human prostate xenograft model to define a profile of genes regulated in vivo by androgens, with an emphasis on identifying candidate biomarkers. Benign transition zone (TZ) human prostate tissue from radical prostatectomies was grafted to the sub-renal capsule site of intact or castrated male immunodeficient mice, followed by the removal or addition of androgens, respectively. Microarray analysis of RNA from these tissues was used to identify genes that were; 1) highly expressed in prostate, 2) had significant expression changes in response to androgens, and, 3) encode extracellular proteins. A total of 95 genes meeting these criteria were selected for analysis and validation of expression in patient prostate tissues using quantitative real-time PCR. Expression levels of these genes were measured in pooled RNAs from human prostate tissues with varying severity of BPH pathologic changes and CaP of varying Gleason score. A number of androgen regulated genes were identified. Additionally, a subset of these genes were over-expressed in RNA from clinical BPH tissues, and the levels of many were found to correlate with disease status. Our results demonstrate the feasibility, and some of the problems, of using a mouse xenograft model to characterize the androgen regulated expression profiles of intact human prostate tissues.

Genome-wide association and replication studies identify four variants associated with prostate cancer susceptibility.

We report a prostate cancer genome-wide association follow-on study. We discovered four variants associated with susceptibility to prostate cancer in several European populations: rs10934853[A] (OR = 1.12, P = 2.9 x 10(-10)) on 3q21.3; two moderately correlated (r2 = 0.07) variants, rs16902094[G] (OR = 1.21, P = 6.2 x 10(-15)) and rs445114[T] (OR = 1.14, P = 4.7 x 10(-10)), on 8q24.21; and rs8102476[C] (OR = 1.12, P = 1.6 x 10(-11)) on 19q13.2. We also refined a previous association signal on 11q13 with the SNP rs11228565[A] (OR = 1.23, P = 6.7 x 10(-12)). In a multivariate analysis using 22 prostate cancer risk variants typed in the Icelandic population, we estimated that carriers in the top 1.3% of the risk distribution are at a 2.5 times greater risk of developing the disease than members of the general population.

Common sequence variants on 2p15 and Xp11.22 confer susceptibility to prostate cancer.

We conducted a genome-wide SNP association study on prostate cancer on over 23,000 Icelanders, followed by a replication study including over 15,500 individuals from Europe and the United States. Two newly identified variants were shown to be associated with prostate cancer: rs5945572 on Xp11.22 and rs721048 on 2p15 (odds ratios (OR) = 1.23 and 1.15; P = 3.9 x 10(-13) and 7.7 x 10(-9), respectively). The 2p15 variant shows a significantly stronger association with more aggressive, rather than less aggressive, forms of the disease.

A major zebrafish polymorphism resource for genetic mapping.

We have identified 645,088 candidate polymorphisms in zebrafish and observe a single nucleotide polymorphism (SNP) validation rate of 71% to 86%, improving with polymorphism confidence score. Variant sites are non-random, with an excess of specific novel T- and A-rich motifs. We positioned half of the polymorphisms on zebrafish genetic and physical maps as a resource for positional cloning. We further demonstrate bulked segregant analysis using the anchored SNPs as a method for high-throughput genetic mapping in zebrafish.