Multi-ancestry epigenome-wide analyses identify methylated sites associated with aortic augmentation index in TOPMed MESA.

Despite the prognostic value of arterial stiffness (AS) and pulsatile hemodynamics (PH) for cardiovascular morbidity and mortality, epigenetic modifications that contribute to AS/PH remain unknown. To gain a better understanding of the link between epigenetics (DNA methylation) and AS/PH, we examined the relationship of eight measures of AS/PH with CpG sites and co-methylated regions using multi-ancestry participants from Trans-Omics for Precision Medicine (TOPMed) Multi-Ethnic Study of Atherosclerosis (MESA) with sample sizes ranging from 438 to 874. Epigenome-wide association analysis identified one genome-wide significant CpG (cg20711926-CYP1B1) associated with aortic augmentation index (AIx). Follow-up analyses, including gene set enrichment analysis, expression quantitative trait methylation analysis, and functional enrichment analysis on differentially methylated positions and regions, further prioritized three CpGs and their annotated genes (cg23800023-ETS1, cg08426368-TGFB3, and cg17350632-HLA-DPB1) for AIx. Among these, ETS1 and TGFB3 have been previously prioritized as candidate genes. Furthermore, both ETS1 and HLA-DPB1 have significant tissue correlations between Whole Blood and Aorta in GTEx, which suggests ETS1 and HLA-DPB1 could be potential biomarkers in understanding pathophysiology of AS/PH. Overall, our findings support the possible role of epigenetic regulation via DNA methylation of specific genes associated with AIx as well as identifying potential targets for regulation of AS/PH.

Pulmonary Function and Blood DNA Methylation: A Multiancestry Epigenome-Wide Association Meta-analysis.

Rationale: Methylation integrates factors present at birth and modifiable across the lifespan that can influence pulmonary function. Studies are limited in scope and replication. Objectives: To conduct large-scale epigenome-wide meta-analyses of blood DNA methylation and pulmonary function. Methods: Twelve cohorts analyzed associations of methylation at cytosine-phosphate-guanine probes (CpGs), using Illumina 450K or EPIC/850K arrays, with FEV1, FVC, and FEV1/FVC. We performed multiancestry epigenome-wide meta-analyses (total of 17,503 individuals; 14,761 European, 2,549 African, and 193 Hispanic/Latino ancestries) and interpreted results using integrative epigenomics. Measurements and Main Results: We identified 1,267 CpGs (1,042 genes) differentially methylated (false discovery rate, <0.025) in relation to FEV1, FVC, or FEV1/FVC, including 1,240 novel and 73 also related to chronic obstructive pulmonary disease (1,787 cases). We found 294 CpGs unique to European or African ancestry and 395 CpGs unique to never or ever smokers. The majority of significant CpGs correlated with nearby gene expression in blood. Findings were enriched in key regulatory elements for gene function, including accessible chromatin elements, in both blood and lung. Sixty-nine implicated genes are targets of investigational or approved drugs. One example novel gene highlighted by integrative epigenomic and druggable target analysis is TNFRSF4. Mendelian randomization and colocalization analyses suggest that epigenome-wide association study signals capture causal regulatory genomic loci. Conclusions: We identified numerous novel loci differentially methylated in relation to pulmonary function; few were detected in large genome-wide association studies. Integrative analyses highlight functional relevance and potential therapeutic targets. This comprehensive discovery of potentially modifiable, novel lung function loci expands knowledge gained from genetic studies, providing insights into lung pathogenesis.

Epigenome-wide association study of mitochondrial genome copy number.

We conducted cohort- and race-specific epigenome-wide association analyses of mitochondrial deoxyribonucleic acid (mtDNA) copy number (mtDNA CN) measured in whole blood from participants of African and European origins in five cohorts (n = 6182, mean age = 57-67 years, 65% women). In the meta-analysis of all the participants, we discovered 21 mtDNA CN-associated DNA methylation sites (CpG) (P < 1 × 10-7), with a 0.7-3.0 standard deviation increase (3 CpGs) or decrease (18 CpGs) in mtDNA CN corresponding to a 1% increase in DNA methylation. Several significant CpGs have been reported to be associated with at least two risk factors (e.g. chronological age or smoking) for cardiovascular disease (CVD). Five genes [PR/SET domain 16, nuclear receptor subfamily 1 group H member 3 (NR1H3), DNA repair protein, DNA polymerase kappa and decaprenyl-diphosphate synthase subunit 2], which harbor nine significant CpGs, are known to be involved in mitochondrial biosynthesis and functions. For example, NR1H3 encodes a transcription factor that is differentially expressed during an adipose tissue transition. The methylation level of cg09548275 in NR1H3 was negatively associated with mtDNA CN (effect size = -1.71, P = 4 × 10-8) and was positively associated with the NR1H3 expression level (effect size = 0.43, P = 0.0003), which indicates that the methylation level in NR1H3 may underlie the relationship between mtDNA CN, the NR1H3 transcription factor and energy expenditure. In summary, the study results suggest that mtDNA CN variation in whole blood is associated with DNA methylation levels in genes that are involved in a wide range of mitochondrial activities. These findings will help reveal molecular mechanisms between mtDNA CN and CVD.

Variability in Cytogenetic Testing for Multiple Myeloma: A Comprehensive Analysis From Across the United States.

PURPOSE: Multiple myeloma (MM) treatment has changed tremendously, with significant improvement in patient out-comes. One group with a suboptimal benefit is patients with high-risk cytogenetics, as tested by conventional karyotyping or fluorescence in situ hybridization (FISH). Methodology for these tests has been published, but not necessarily standardized. METHODS: We address variability in the testing and reporting methodology for MM cytogenetics in the United States using the ongoing African American Multiple Myeloma Study (AAMMS). We evaluated clinical and cytogenetic data from 1,221 patients (1,161 with conventional karyotyping and 976 with FISH) tested between 1998 and 2016 across 58 laboratories nationwide. RESULTS: Interlab and intralab variability was noted for the number of cells analyzed for karyotyping, with a significantly higher number of cells analyzed in patients in whom cytogenetics were normal (P 5.0025). For FISH testing, CD138-positive cell enrichment was used in 29.7% of patients and no enrichment in 50% of patients, whereas the remainder had unknown status. A significantly smaller number of cells was analyzed for patients in which CD138 cell enrichment was used compared with those without such enrichment (median, 50 v 200; P, .0001). A median of 7 loci probes (range, 1-16) were used for FISH testing across all laboratories, with variability in the loci probed even within a given laboratory. Chromosome 13-related abnormalities were the most frequently tested abnormality (n5956; 97.9%), and t(14;16) was the least frequently tested abnormality (n 5 119; 12.2%). CONCLUSIONS: We report significant variability in cytogenetic testing across the United States for MM, potentially leading to variability in risk stratification, with possible clinical implications and personalized treatment approaches.

A meta-analysis of genome-wide association studies of multiple myeloma among men and women of African ancestry.

Persons of African ancestry (AA) have a twofold higher risk for multiple myeloma (MM) compared with persons of European ancestry (EA). Genome-wide association studies (GWASs) support a genetic contribution to MM etiology in individuals of EA. Little is known about genetic risk factors for MM in individuals of AA. We performed a meta-analysis of 2 GWASs of MM in 1813 cases and 8871 controls and conducted an admixture mapping scan to identify risk alleles. We fine-mapped the 23 known susceptibility loci to find markers that could better capture MM risk in individuals of AA and constructed a polygenic risk score (PRS) to assess the aggregated effect of known MM risk alleles. In GWAS meta-analysis, we identified 2 suggestive novel loci located at 9p24.3 and 9p13.1 at P < 1 × 10-6; however, no genome-wide significant association was noted. In admixture mapping, we observed a genome-wide significant inverse association between local AA at 2p24.1-23.1 and MM risk in AA individuals. Of the 23 known EA risk variants, 20 showed directional consistency, and 9 replicated at P < .05 in AA individuals. In 8 regions, we identified markers that better capture MM risk in persons with AA. AA individuals with a PRS in the top 10% had a 1.82-fold (95% confidence interval, 1.56-2.11) increased MM risk compared with those with average risk (25%-75%). The strongest functional association was between the risk allele for variant rs56219066 at 5q15 and lower ELL2 expression (P = 5.1 × 10-12). Our study shows that common genetic variation contributes to MM risk in individuals with AA.

Data-adaptive multi-locus association testing in subjects with arbitrary genealogical relationships.

Genome-wide sequencing enables evaluation of associations between traits and combinations of variants in genes and pathways. But such evaluation requires multi-locus association tests with good power, regardless of the variant and trait characteristics. And since analyzing families may yield more power than analyzing unrelated individuals, we need multi-locus tests applicable to both related and unrelated individuals. Here we describe such tests, and we introduce SKAT-X, a new test statistic that uses genome-wide data obtained from related or unrelated subjects to optimize power for the specific data at hand. Simulations show that: a) SKAT-X performs well regardless of variant and trait characteristics; and b) for binary traits, analyzing affected relatives brings more power than analyzing unrelated individuals, consistent with previous findings for single-locus tests. We illustrate the methods by application to rare unclassified missense variants in the tumor suppressor gene BRCA2, as applied to combined data from prostate cancer families and unrelated prostate cancer cases and controls in the Multi-ethnic Cohort (MEC). The methods can be implemented using open-source code for public use as the R-package GATARS (Genetic Association Tests for Arbitrarily Related Subjects) .

Novel Common Genetic Susceptibility Loci for Colorectal Cancer.

BACKGROUND: Previous genome-wide association studies (GWAS) have identified 42 loci (P < 5 × 10-8) associated with risk of colorectal cancer (CRC). Expanded consortium efforts facilitating the discovery of additional susceptibility loci may capture unexplained familial risk. METHODS: We conducted a GWAS in European descent CRC cases and control subjects using a discovery-replication design, followed by examination of novel findings in a multiethnic sample (cumulative n = 163 315). In the discovery stage (36 948 case subjects/30 864 control subjects), we identified genetic variants with a minor allele frequency of 1% or greater associated with risk of CRC using logistic regression followed by a fixed-effects inverse variance weighted meta-analysis. All novel independent variants reaching genome-wide statistical significance (two-sided P < 5 × 10-8) were tested for replication in separate European ancestry samples (12 952 case subjects/48 383 control subjects). Next, we examined the generalizability of discovered variants in East Asians, African Americans, and Hispanics (12 085 case subjects/22 083 control subjects). Finally, we examined the contributions of novel risk variants to familial relative risk and examined the prediction capabilities of a polygenic risk score. All statistical tests were two-sided. RESULTS: The discovery GWAS identified 11 variants associated with CRC at P < 5 × 10-8, of which nine (at 4q22.2/5p15.33/5p13.1/6p21.31/6p12.1/10q11.23/12q24.21/16q24.1/20q13.13) independently replicated at a P value of less than .05. Multiethnic follow-up supported the generalizability of discovery findings. These results demonstrated a 14.7% increase in familial relative risk explained by common risk alleles from 10.3% (95% confidence interval [CI] = 7.9% to 13.7%; known variants) to 11.9% (95% CI = 9.2% to 15.5%; known and novel variants). A polygenic risk score identified 4.3% of the population at an odds ratio for developing CRC of at least 2.0. CONCLUSIONS: This study provides insight into the architecture of common genetic variation contributing to CRC etiology and improves risk prediction for individualized screening.

The OncoArray Consortium: A Network for Understanding the Genetic Architecture of Common Cancers.

BACKGROUND: Common cancers develop through a multistep process often including inherited susceptibility. Collaboration among multiple institutions, and funding from multiple sources, has allowed the development of an inexpensive genotyping microarray, the OncoArray. The array includes a genome-wide backbone, comprising 230,000 SNPs tagging most common genetic variants, together with dense mapping of known susceptibility regions, rare variants from sequencing experiments, pharmacogenetic markers, and cancer-related traits. METHODS: The OncoArray can be genotyped using a novel technology developed by Illumina to facilitate efficient genotyping. The consortium developed standard approaches for selecting SNPs for study, for quality control of markers, and for ancestry analysis. The array was genotyped at selected sites and with prespecified replicate samples to permit evaluation of genotyping accuracy among centers and by ethnic background. RESULTS: The OncoArray consortium genotyped 447,705 samples. A total of 494,763 SNPs passed quality control steps with a sample success rate of 97% of the samples. Participating sites performed ancestry analysis using a common set of markers and a scoring algorithm based on principal components analysis. CONCLUSIONS: Results from these analyses will enable researchers to identify new susceptibility loci, perform fine-mapping of new or known loci associated with either single or multiple cancers, assess the degree of overlap in cancer causation and pleiotropic effects of loci that have been identified for disease-specific risk, and jointly model genetic, environmental, and lifestyle-related exposures. IMPACT: Ongoing analyses will shed light on etiology and risk assessment for many types of cancer. Cancer Epidemiol Biomarkers Prev; 26(1); 126-35. ©2016 AACR.

A Meta-analysis of Multiple Myeloma Risk Regions in African and European Ancestry Populations Identifies Putatively Functional Loci.

BACKGROUND: Genome-wide association studies (GWAS) in European populations have identified genetic risk variants associated with multiple myeloma. METHODS: We performed association testing of common variation in eight regions in 1,318 patients with multiple myeloma and 1,480 controls of European ancestry and 1,305 patients with multiple myeloma and 7,078 controls of African ancestry and conducted a meta-analysis to localize the signals, with epigenetic annotation used to predict functionality. RESULTS: We found that variants in 7p15.3, 17p11.2, 22q13.1 were statistically significantly (P < 0.05) associated with multiple myeloma risk in persons of African ancestry and persons of European ancestry, and the variant in 3p22.1 was associated in European ancestry only. In a combined African ancestry-European ancestry meta-analysis, variation in five regions (2p23.3, 3p22.1, 7p15.3, 17p11.2, 22q13.1) was statistically significantly associated with multiple myeloma risk. In 3p22.1, the correlated variants clustered within the gene body of ULK4 Correlated variants in 7p15.3 clustered around an enhancer at the 3' end of the CDCA7L transcription termination site. A missense variant at 17p11.2 (rs34562254, Pro251Leu, OR, 1.32; P = 2.93 × 10-7) in TNFRSF13B encodes a lymphocyte-specific protein in the TNF receptor family that interacts with the NF-κB pathway. SNPs correlated with the index signal in 22q13.1 cluster around the promoter and enhancer regions of CBX7 CONCLUSIONS: We found that reported multiple myeloma susceptibility regions contain risk variants important across populations, supporting the use of multiple racial/ethnic groups with different underlying genetic architecture to enhance the localization and identification of putatively functional alleles. IMPACT: A subset of reported risk loci for multiple myeloma has consistent effects across populations and is likely to be functional. Cancer Epidemiol Biomarkers Prev; 25(12); 1609-18. ©2016 AACR.

Assessment of Multifactor Gene-Environment Interactions and Ovarian Cancer Risk: Candidate Genes, Obesity, and Hormone-Related Risk Factors.

BACKGROUND: Many epithelial ovarian cancer (EOC) risk factors relate to hormone exposure and elevated estrogen levels are associated with obesity in postmenopausal women. Therefore, we hypothesized that gene-environment interactions related to hormone-related risk factors could differ between obese and non-obese women. METHODS: We considered interactions between 11,441 SNPs within 80 candidate genes related to hormone biosynthesis and metabolism and insulin-like growth factors with six hormone-related factors (oral contraceptive use, parity, endometriosis, tubal ligation, hormone replacement therapy, and estrogen use) and assessed whether these interactions differed between obese and non-obese women. Interactions were assessed using logistic regression models and data from 14 case-control studies (6,247 cases; 10,379 controls). Histotype-specific analyses were also completed. RESULTS: SNPs in the following candidate genes showed notable interaction: IGF1R (rs41497346, estrogen plus progesterone hormone therapy, histology = all, P = 4.9 × 10(-6)) and ESR1 (rs12661437, endometriosis, histology = all, P = 1.5 × 10(-5)). The most notable obesity-gene-hormone risk factor interaction was within INSR (rs113759408, parity, histology = endometrioid, P = 8.8 × 10(-6)). CONCLUSIONS: We have demonstrated the feasibility of assessing multifactor interactions in large genetic epidemiology studies. Follow-up studies are necessary to assess the robustness of our findings for ESR1, CYP11A1, IGF1R, CYP11B1, INSR, and IGFBP2 Future work is needed to develop powerful statistical methods able to detect these complex interactions. IMPACT: Assessment of multifactor interaction is feasible, and, here, suggests that the relationship between genetic variants within candidate genes and hormone-related risk factors may vary EOC susceptibility. Cancer Epidemiol Biomarkers Prev; 25(5); 780-90. ©2016 AACR.

Genome-wide association study of colorectal cancer identifies six new susceptibility loci.

Genetic susceptibility to colorectal cancer is caused by rare pathogenic mutations and common genetic variants that contribute to familial risk. Here we report the results of a two-stage association study with 18,299 cases of colorectal cancer and 19,656 controls, with follow-up of the most statistically significant genetic loci in 4,725 cases and 9,969 controls from two Asian consortia. We describe six new susceptibility loci reaching a genome-wide threshold of P<5.0E-08. These findings provide additional insight into the underlying biological mechanisms of colorectal cancer and demonstrate the scientific value of large consortia-based genetic epidemiology studies.

Breast cancer susceptibility variants and mammographic density phenotypes in norwegian postmenopausal women.

BACKGROUND: Mammographic density (MD) is one of the strongest known breast cancer risk factors. Twin studies have suggested that a large part of the variation in MD is genetically determined. We hypothesized that breast cancer susceptibility variants may affect MD, and that their effects may be modified by nongenetic factors. METHODS: We assessed MD, using a computer-assisted method, on 2,348 postmenopausal Caucasian women (50-69 years) who participated in the Norwegian Breast Cancer Screening Program (NBCSP) in 2004 or 2006-07. We used linear regression (additive models) to determine the association between each SNP and MD, adjusting for age, body mass index (BMI), and study. We evaluated MD associations with 17 established breast cancer SNPs, overall, and by strata defined by non-genetic factors. RESULTS: Two variants, 6q25.1-rs9383938 and TXNRD2-rs8141691, were statistically significantly associated with percent MD (P = 0.019 and 0.03, respectively), with the 6q25.1-rs9383938 association being consistent with the SNP effect on breast cancer risk. The effect of 6q25.1-rs3734805 on percent MD varied between parous and nulliparous women (Pinteraction = 0.02), whereas the effects of 9q31.2-rs865686 and MRPS30:FGF10-rs4415084 differed across strata of BMI (Pinteraction = 0.01 and 0.005, respectively). There was no evidence of effect modification by estrogen and progestin therapy use or alcohol consumption. CONCLUSION: This study provides novel evidence of shared genetic risk factors between MD and breast cancer and of possible MD genetic-environmental interactions. IMPACT: Although the results may be chance findings, they nevertheless highlight the need to investigate interactions with nongenetic factors in studies on the genetics of MD.

Genome-wide interaction studies reveal sex-specific asthma risk alleles.

Asthma is a complex disease with sex-specific differences in prevalence. Candidate gene studies have suggested that genotype-by-sex interaction effects on asthma risk exist, but this has not yet been explored at a genome-wide level. We aimed to identify sex-specific asthma risk alleles by performing a genome-wide scan for genotype-by-sex interactions in the ethnically diverse participants in the EVE Asthma Genetics Consortium. We performed male- and female-specific genome-wide association studies in 2653 male asthma cases, 2566 female asthma cases and 3830 non-asthma controls from European American, African American, African Caribbean and Latino populations. Association tests were conducted in each study sample, and the results were combined in ancestry-specific and cross-ancestry meta-analyses. Six sex-specific asthma risk loci had P-values < 1 × 10(-6), of which two were male specific and four were female specific; all were ancestry specific. The most significant sex-specific association in European Americans was at the interferon regulatory factor 1 (IRF1) locus on 5q31.1. We also identify a Latino female-specific association in RAP1GAP2. Both of these loci included single-nucleotide polymorphisms that are known expression quantitative trait loci and have been associated with asthma in independent studies. The IRF1 locus is a strong candidate region for male-specific asthma susceptibility due to the association and validation we demonstrate here, the known role of IRF1 in asthma-relevant immune pathways and prior reports of sex-specific differences in interferon responses.

Reproducibility and reliability of SNP analysis using human cellular DNA at or near nanogram levels.

BACKGROUND: Illumina SNP arrays have been routinely used for genome-wide association studies to identify potential biomarkers for various diseases. The recommended 200 ng of DNA for high-quality results is a roadblock to utilizing this assay when such quantities of DNA are not available. The goal of this study is to determine the reproducibility and reliability of the assay when reduced amounts of DNA are used for the SNP arrays. FINDINGS: A serial 3-fold reduction of DNA from 200 ng to 0.8 ng was used for an Illumina SNP array in duplicates (200 ng, 66.7 ng, 22,2 ng, and 7.4 ng) or triplicates (2.47 ng and 0.8 ng). The reproducibility of the assay was determined by comparing allele calls (genotypes) at each locus within the duplicates or triplicates. The reliability of samples of reduced quantity was determined by comparing allele calls from samples of different quantities. As expected, the reproducibility and reliability both decrease with decreasing amounts of DNA used for the arrays. However, results of comparable quality to the 200 ng DNA recommended by Illumina can be obtained with much reduced amounts of DNA. CONCLUSION: Reasonably reproducible and reliable results can be obtained with quantities of DNA, as low as 0.8 ng (equivalent to 133 human cells), well below the manufacturer's recommendation. Results of nearly equal quality to that of using 200 ng DNA can be obtained with 22.2 ng of DNA reliably, and clearly acceptable data can be obtained using 7.4 ng of DNA for Illumina SNP arrays.

Testicular germ cell tumor susceptibility associated with the UCK2 locus on chromosome 1q23.

Genome-wide association studies (GWASs) have identified multiple common genetic variants associated with an increased risk of testicular germ cell tumors (TGCTs). A previous GWAS reported a possible TGCT susceptibility locus on chromosome 1q23 in the UCK2 gene, but failed to reach genome-wide significance following replication. We interrogated this region by conducting a meta-analysis of two independent GWASs including a total of 940 TGCT cases and 1559 controls for 122 single-nucleotide polymorphisms (SNPs) on chromosome 1q23 and followed up the most significant SNPs in an additional 2202 TGCT cases and 2386 controls from four case-control studies. We observed genome-wide significant associations for several UCK2 markers, the most significant of which was for rs3790665 (PCombined = 6.0 × 10(-9)). Additional support is provided from an independent familial study of TGCT where a significant over-transmission for rs3790665 with TGCT risk was observed (PFBAT = 2.3 × 10(-3)). Here, we provide substantial evidence for the association between UCK2 genetic variation and TGCT risk.

A genome-wide scan for breast cancer risk haplotypes among African American women.

Genome-wide association studies (GWAS) simultaneously investigating hundreds of thousands of single nucleotide polymorphisms (SNP) have become a powerful tool in the investigation of new disease susceptibility loci. Haplotypes are sometimes thought to be superior to SNPs and are promising in genetic association analyses. The application of genome-wide haplotype analysis, however, is hindered by the complexity of haplotypes themselves and sophistication in computation. We systematically analyzed the haplotype effects for breast cancer risk among 5,761 African American women (3,016 cases and 2,745 controls) using a sliding window approach on the genome-wide scale. Three regions on chromosomes 1, 4 and 18 exhibited moderate haplotype effects. Furthermore, among 21 breast cancer susceptibility loci previously established in European populations, 10p15 and 14q24 are likely to harbor novel haplotype effects. We also proposed a heuristic of determining the significance level and the effective number of independent tests by the permutation analysis on chromosome 22 data. It suggests that the effective number was approximately half of the total (7,794 out of 15,645), thus the half number could serve as a quick reference to evaluating genome-wide significance if a similar sliding window approach of haplotype analysis is adopted in similar populations using similar genotype density.

A genome-wide association study of breast cancer in women of African ancestry.

Genome-wide association studies (GWAS) in diverse populations are needed to reveal variants that are more common and/or limited to defined populations. We conducted a GWAS of breast cancer in women of African ancestry, with genotyping of >1,000,000 SNPs in 3,153 African American cases and 2,831 controls, and replication testing of the top 66 associations in an additional 3,607 breast cancer cases and 11,330 controls of African ancestry. Two of the 66 SNPs replicated (p < 0.05) in stage 2, which reached statistical significance levels of 10(-6) and 10(-5) in the stage 1 and 2 combined analysis (rs4322600 at chromosome 14q31: OR = 1.18, p = 4.3 × 10(-6); rs10510333 at chromosome 3p26: OR = 1.15, p = 1.5 × 10(-5)). These suggestive risk loci have not been identified in previous GWAS in other populations and will need to be examined in additional samples. Identification of novel risk variants for breast cancer in women of African ancestry will demand testing of a substantially larger set of markers from stage 1 in a larger replication sample.

Polymorphisms in hormone metabolism and growth factor genes and mammographic density in Norwegian postmenopausal hormone therapy users and non-users.

INTRODUCTION: Mammographic density (MD) is one of the strongest known breast cancer risk factors. Estrogen and progestin therapy (EPT) has been associated with increases in MD. Dense breast tissue is characterized by increased stromal tissue and (to a lesser degree) increased numbers of breast epithelial cells. It is possible that genetic factors modify the association between EPT and MD, and that certain genetic variants are particularly important in determining MD in hormone users. We evaluated the association between MD and 340 tagging single nucleotide polymorphisms (SNPs) from about 30 candidate genes in hormone metabolism/growth factor pathways among women who participated in the Norwegian Breast Cancer Screening Program (NBCSP) in 2004. METHODS: We assessed MD on 2,036 postmenopausal women aged 50 to 69 years using a computer-assisted method (Madena, University of Southern California) in a cross-sectional study. We used linear regression to determine the association between each SNP and MD, adjusting for potential confounders. The postmenopausal women were stratified into HT users (EPT and estrogen-only) and non-users (never HT). RESULTS: For current EPT users, there was an association between a variant in the prolactin gene (PRL; rs10946545) and MD (dominant model, Bonferroni-adjusted P (Pb) = 0.0144). This association remained statistically significant among current users of norethisterone acetate (NETA)-based EPT, a regimen common in Nordic countries. Among current estrogen-only users (ET), there was an association between rs4670813 in the cytochrome P450 gene (CYP1B1) and MD (dominant model, Pb = 0.0396). In never HT users, rs769177 in the tumor necrosis factor (TNF) gene and rs1968752 in the region of the sulfotransferase gene (SULT1A1/SULT1A2), were significantly associated with MD (Pb = 0.0202; Pb = 0.0349). CONCLUSIONS: We found some evidence that variants in the PRL gene were associated with MD in current EPT and NETA users. In never HT users, variants in the TNF and SULT1A1/SULT1A2 genes were significantly associated with MD. These findings may suggest that several genes in the hormone metabolism and growth factor pathways are implicated in determining MD.