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Background: Multiple breast cancer genome-wide association studies (GWAS) have been conducted, with most samples analyzed being of European or Asian origin. No GWAS has been published to date on US Latinas or Latin American women. Previous studies have demonstrated that among Latina women, those with the highest Indigenous American ancestry are protected from breast cancer. We performed the first genome-wide association study of breast cancer in Latinas in a total of 1523 cases and 3221 controls and validated the strongest associations in an additional 977 Latina cases and 1158 controls. Methods: For the discovery phase, samples were genotyped with either the 6.0 Affymetrix array or Illumina Infinitum 660W/Omni 2.5 arrays. Additional genotypes were imputed from 1000 genomes and the final analysis included ∼7 million SNPs. SNPs for replication were genotyped using Sequenom. Logistic regression analyses included the first 10 principal components as covariates. Results: We identified two new variants, located 5’ of the ESR1 gene (6q25 region), which are strongly associated with breast cancer in this population. The minor allele for these variants is strongly protective (rs140068132: OR 0.61, 95%CI 0.48-0.75, p=3x10-13 & rs147157845: OR 0.63 95% CI 0.49-0.76; p =2x10-12). These alleles are present only in Latinos, correlate with Indigenous American ancestry, and are uncorrelated with previously reported risk variants in the ESR1 region. We found a strong association for both ER-positive and ER-negative breast cancer, but significantly more protection for ER-negative disease (p=0.014). The rs140068132 variant is also associated with breast density. The average breast density among women who are homozygous for the protective allele is approximately half of that of women homozygous for the common allele [Mean (sd) density for Homozygous GG = 9 (8); Homozygous AA = 17 (14), p 0.0009]. rs140068132, is located within a putative binding region for multiple transcription factors. MAPPER predicts disruption of putative transcription factor binding sites for the rare allele at rs140068132 but not rs147157845. Electromotility shift assays showed differential binding of the two alleles at the rs140068132 locus, with the protective allele (G) showing substantially reduced binding affinity compared to the common A allele. Conclusions: The variants at this locus represent, to our knowledge, the strongest genome wide effect size on breast cancer for a common variant (frequency >0.05 in any major population). These results may help in understanding the previous observations of lower breast cancer risk among Latina women with more Indigenous American ancestry and highlight the importance of conducting GWAS in diverse populations. Citation Format: Laura Fejerman, Nasim Ahmadiyeh, Donglei Hu, Scott Huntsman, Kenneth Beckman, Jennifer Caswell, Esther M. John, Gabriela Torres-Mejia, Luis Carvajal-Carmona, Magdalena Echeverry, Anna Marie Tuazon, Carolina Ramirez, COLUMBUS Consortium, Christopher Gignoux, Celeste Eng, Esteban Gonzalez-Burchard, Brian Henderson, Loic Le Marchand, Eliseo J. Perez-Stable, Christopher A. Haiman, Elad Ziv. Genome wide association study of breast cancer in Latinas identifies protective variants of Indigenous American origin on 6q25. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-299. doi:10.1158/1538-7445.AM2014-LB-299

Background: The mitochondrial genome is highly specialized, encoding essential mitochondrial electron transport chain proteins required for aerobic metabolism, and involved in free radical production—two pathways that are involved in carcinogenesis. The role of the mitochondrial genome in colorectal cancer susceptibility has yet to be fully examined. We comprehensively characterized the genetic variation across the 16kb mitochondrial genome across five ancestral populations and examined the association between mitochondrial SNPs (mtSNPs) and colorectal cancer risk in a multiethnic population. Methods: We compiled mitochondrial sequencing data from the public domain for African, Asian, European, and Latino populations, and sequenced the mitochondrial genomes of 160 Native Hawaiians. A total of 863 mtSNPs (MAF>1%) were identified and genotyped in a multiethnic validation panel, identifying 621 mtSNPs with working assays and resulting in 446 mtSNPs detected in at least one sample. A total of 186 mtSNPs were prioritized based on MAF>∼2% for genotyping in a case-control study of colorectal cancer nested in the Multiethnic Cohort (cases/controls=1,923/10,555). To evaluate the association between mtSNPs and colorectal cancer risk, odds ratios (OR) and 95% confidence intervals (CI) were estimated by unconditional logistic regression, adjusting for age, sex, and maternal self-reported ancestry. Stratified analysis were conducted by maternal self-reported ancestry given the wide variation in allele frequencies across ancestral groups. A Bonferroni-corrected p-value of 8.77x10-5 was used to determine statistical significance of associations given the number of mtSNPs tested and stratified analyses. Results: At a nominal P1%) were associated with colorectal cancer for all groups combined, while 5 of 114 mtSNPs, 1 of 45 mtSNPs, 7 of 65 mtSNPs, 42 of 101 mtSNPs, and 12 of 70 mtSNPs were associated among African Americans, Native Hawaiians, Asian Americans, Latinos, and European Americans, respectively. Statistically significant associations with colorectal cancer were observed with seven mtSNPs (four largely independent signals) among Latinos (P Conclusion: Our study suggests that inherited variants of the mitochondrial genome may be associated with colorectal cancer risk in specific ancestral populations. Future work will further address population stratification of the mitochondrial genome, as well as investigate haplogroup associations and the cumulative effects of mitochondrial genes on colorectal cancer susceptibility. Citation Format: Iona C. Cheng, Christian Caberto, Kenneth Beckman, Annette Lum-Jones, Christopher Haiman, Loic Le Marchand, Daniel Stram, Richa Saxena. Genetic association study of the mitochondrial genome and colorectal cancer risk: The Multiethnic Cohort. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 945. doi:10.1158/1538-7445.AM2014-945

Interest in the role of mitochondria in aging has intensified in recent years. This focus on mitochondria originated in part from the free radical theory of aging, which argues that oxidative damage plays a key role in degenerative senescence. Among the numerous mechanisms known to generate oxidants, leakage of the superoxide anion and hydrogen peroxide from the mitochondrial electron transport chain are of particular interest, due to the correlation between species-specific metabolic rate ("rate of living") and life span. Phenomenological studies of mitochondrial function long ago noted a decline in mitochondrial function with age, and on-going research continues to add to this body of knowledge. The extranuclear somatic mutation theory of aging proposes that the accumulation of mutations in the mitochondrial genome may be responsible in part for the mitochondrial phenomenology of aging. Recent studies of mitochondrial DNA (mtDNA) deletions have shown that they increase with age in humans and other mammals. Currently, there exist numerous important and fundamental questions surrounding mitochondria and aging. Among these are (1) How important are mitochondrial oxidants in determining overall cellular oxidative stress? (2) What are the mechanisms of mitochondrial oxidant generation? (3) How are lesions and mutations in mtDNA formed? (4) How important are mtDNA lesions and mutations in causing mitochondrial dysfunction? (5) How are mitochondria regulated, and how does this regulation change during aging? (6) What are the dynamics of mitochondrial turnover? (7) What is the relationship between mitochondrial damage and lipofuscinogenesis? (8) What are the relationships among mitochondria, apopotosis, and aging? and (9) How can mitochondrial function (ATP generation and the establishment of a membrane potential) and dysfunction (oxidant generation) be modulated and degenerative senescence thereby treated?

Publisher Summary Modifications and alternatives to the original HPLC-EC (high-performance liquid chromatography- electrochemical) methods have produced a burgeoning literature on oxidative DNA damage. At the same time, they have resulted in discordant results and disagreements about the most appropriate techniques. Because of the apparent importance of DNA damage, and the interest in 8-Oxo-2'-deoxyguanosine (oxo 8 dG) and oxo 8 Gua as biomarkers for this damage, it is important to identify the best available assay methods. This chapter reviews the HPLC-EC methods that find to be the most reliable, indicate the common pitfalls associated with these techniques, and uncertainties of interpretation. Methods for DNA isolation continue to improve, with minor modification of the chaotropic NaI technique producing the lowest oxo 8 dG values reported to date using HPLC-EC. Although the use of urinary oxo 8 Gua adducts to monitor DNA oxidation in vivo remains attractive, the results can be difficult to interpret.

Beckman, Kenneth B., and Bruce N. Ames. The Free Radical Theory of Aging Matures. Physiol. Rev. 78: 547–581, 1998. — The free radical theory of aging, conceived in 1956, has turned 40 and is rapidly attracting the interest of the mainstream of biological research. From its origins in radiation biology, through a decade or so of dormancy and two decades of steady phenomenological research, it has attracted an increasing number of scientists from an expanding circle of fields. During the past decade, several lines of evidence have convinced a number of scientists that oxidants play an important role in aging. (For the sake of simplicity, we use the term oxidant to refer to all “reactive oxygen species,” including O−2⋅, H2O2, and ⋅OH, even though the former often acts as a reductant and produces oxidants indirectly.) The pace and scope of research in the last few years have been particularly impressive and diverse. The only disadvantage of the current intellectual ferment is the difficulty in digesting the literature. Therefore, we have systematically reviewed the status of the free radical theory, by categorizing the literature in terms of the various types of experiments that have been performed. These include phenomenological measurements of age-associated oxidative stress, interspecies comparisons, dietary restriction, the manipulation of metabolic activity and oxygen tension, treatment with dietary and pharmacological antioxidants, in vitro senescence, classical and population genetics, molecular genetics, transgenic organisms, the study of human diseases of aging, epidemiological studies, and the ongoing elucidation of the role of active oxygen in biology.

Publisher Summary This chapter discusses the differences between the methods to identify protocols that minimize artifacts that lead to overestimates of oxidative damage. The chapter also discusses procedures that may lead to underestimates. It illustrates that different protocols have been used in virtually every step of the analysis: the isolation of mitochondria, the purification of mitochondrial DNA (mtDNA) from mitochondria, and the analysis of oxidative adducts in the mtDNA itself. Studies have used a number of different sources of mitochondria, including mammalian organs, tissue culture cells, and the mitochondria from entire arthropods. Although the absolute values obtained in studies of oxidative adducts may be in question, the relative differences seen between control values and those of treatment groups are often what is of greatest interest. In a number of the studies included in the chapter, such differences are noted and are statistically significant. This indicates that for comparative purposes, present methods are sufficient.

Abstract 2095 Celiac disease (CD) is an increasingly recognized disorder in Caucasian populations of European origin. Little is known about its prevalence in non-Caucasians. While CD is thought to be a cause of iron deficiency anemia, the extent to which CD contributes to iron deficiency in Caucasians and especially non-Caucasians is unknown. Iron deficiency is one of the most common nutritional deficiencies in both the developed world and in developing countries. Thus, it is important to know the prevalence of CD in different populations and in iron-deficient and iron-replete persons. To answer these questions, we tested serum collected from Caucasian and non-Caucasian men aged ≥ 25 y and women ≥ 50 y in the Hemochromatosis and Iron Overload Screening (HEIRS) Study. In the HEIRS Study, 101,168 participants were screened with serum biochemical tests of iron status, which not only detected participants with iron overload but also identified a unique multiethnic population of participants with iron deficiency. We hypothesized that CD is more common in those with iron deficiency than in iron-replete individuals. We also examined the difference in frequency of CD between Caucasians and non-Caucasians with iron deficiency. Cases with iron deficiency (defined as serum ferritin ≤ 12 μg/L [cases]) and iron-replete controls (serum ferritin > 100 μg/L in men, serum ferritin > 50 μg/L in women) were further evaluated by using a sequential serological testing scheme whereby all samples were tested with an ELISA for human recombinant tissue transglutaminase IgA antibodies, and positive values were confirmed by endomysial antibodies. Those positive for both tests were considered double positives and were presumed to have untreated CD. Those positive for tissue transglutaminase IgA antibodies but not for endomysial antibodies were termed single positives and were considered indeterminate and excluded from analysis. We further hypothesized that low serum ferritin and an appropriate human leukocyte antigen (HLA) variant, may be predictive of CD. We used SNP-based analysis to determine HLA genotype and DQ8, DQ2.2 or DQ4, and DQ2.5 risk variants. Fisher's exact test was used to examine the association between iron−deficient case and control status and the presence of celiac disease. Log-binomial regression was applied to estimate the odds of celiac disease in iron-deficient cases relative to that of controls. A total of 1713 subjects were tested for tissue transglutaminase IgA antibodies. Participants included 1100 Caucasians, 221 African Americans, 153 Asians, and 239 Hispanics. Fourteen of 571 iron-deficient cases were positive for the double serology compared to just one of 1142 controls. All of the seropositive tests were seen in Caucasians and none in the non-Caucasians. Excluding data from participants with indeterminate serological screen results (6 Caucasians, 4 non-Caucasians), we found that CD occurred in 14 of 567 cases (2.5%) and in only 1 of 1136 (0.1%) controls (Fisher's exact test, p=1.92 10−6). CD was more common in Caucasian cases (14 of 363, 4%) than in non-Caucasian cases (0 of 204, p=0.003). Only one Caucasian control and none of the non-Caucasian controls had CD. The odds of CD in individuals with iron deficiency was 28 (3.7, 212.8) times that in controls. Thirteen of 14 iron-deficient cases with CD carried the DQ2.5 variant of the HLA genotype. Presence of DQ2.5 was highly significantly associated with celiac disease status in iron-deficient cases compared to controls (Fisher's exact test, p = 7.183 × 10−8). The results indicate that CD is a small but significant contributor to iron deficiency in Caucasians. CD is very rare in other races, even among individuals with features frequently seen in CD, such as iron deficiency. CD is also rare in Caucasians who are iron replete. CD testing should be considered for adult Caucasian males or post-menopausal females having iron deficiency without evidence of other etiologies such as gastrointestinal blood loss. Patients identified with CD may benefit from appropriate treatment. Disclosures: No relevant conflicts of interest to declare.

Background The viable yellow allele of agouti (Avy) is remarkable for its unstable and partially heritable epigenetic state, which produces wide variation in phenotypes of isogenic mice. In the Avy allele an inserted intracisternal A particle (IAP) acts as a controlling element which deregulates expression of agouti by transcription from the LTR of the IAP; the phenotypic state has been linked to CpG methylation of the LTR. Phenotypic variation between Avy mice indicates that the epigenetic state of the IAP is unstable in the germline. Principal Findings We have made a detailed examination of somatic methylation of the IAP using bisulphite allelic sequencing, and find that the promoter is incompletely methylated even when it is transcriptionally silent. In utero exposure to supplementary methyl donors, which alters the spectrum of Avy phenotypes, does not increase the density of CpG methylation in the silent LTR. Conclusions Our findings suggest that, contrary to previous supposition, methyl donor supplementation acts through an indirect mechanism to silence Avy. The incomplete cytosine methylation we observe at the somatically silent Avy allele may reflect its unstable germline state, and the influence of epigenetic modifications underlying CpG methylation.

The genotyping of mother–father–child trios is a very useful tool in disease association studies, as trios eliminate population stratification effects and increase the accuracy of haplotype inference. Unfortunately, the use of trios for association studies may reduce power, since it requires the genotyping of three individuals where only four independent haplotypes are involved. We describe here a method for genotyping a trio using two DNA pools, thus reducing the cost of genotyping trios to that of genotyping two individuals. Furthermore, we present extensions to the method that exploit the linkage disequilibrium structure to compensate for missing data and genotyping errors. We evaluated our method on trios from CEPH pedigree 66 of the Coriell Institute. We demonstrate that the error rates in the genotype calls of the proposed protocol are comparable to those of standard genotyping techniques, although the cost is reduced considerably. The approach described is generic and it can be applied to any genotyping platform that achieves a reasonable precision of allele frequency estimates from pools of two individuals. Using this approach, future trio-based association studies may be able to increase the sample size by 50% for the same cost and thereby increase the power to detect associations.

The use of chemicals for high crop productivity and compensation for soil, water and biological resource degradation contribute to the high production costs and other problems of U.S. agriculture. This has prompted strong interest by U.S. farmers in low-input sustainable agriculture. The principles that underlie a low-input sustainable agricultural system are: (1) adapting the agricultural system to the environment of the region, including soil, water, climate and biota present at the site; (2) optimizing the use of biological and chemical/physical resources in the agroecosystem. In this investigation, it was demonstrated that high corn yields could be maintained and input costs reduced by adaptive management of soil, water, energy and biological resources. For example, soil erosion was reduced from 18 t ha −1 year to 1 t ha −1 year −1 and pest control accomplished without the use of pesticides. This reduced the costs of corn production 33% while reducing fossil energy inputs by about 50%.

Examination of ancestry-informative genetic markers shows that Puerto Rican and Mexican populations have shown strong assortative mating that continues to this day., Background While spouse correlations have been documented for numerous traits, no prior studies have assessed assortative mating for genetic ancestry in admixed populations. Results Using 104 ancestry informative markers, we examined spouse correlations in genetic ancestry for Mexican spouse pairs recruited from Mexico City and the San Francisco Bay Area, and Puerto Rican spouse pairs recruited from Puerto Rico and New York City. In the Mexican pairs, we found strong spouse correlations for European and Native American ancestry, but no correlation in African ancestry. In the Puerto Rican pairs, we found significant spouse correlations for African ancestry and European ancestry but not Native American ancestry. Correlations were not attributable to variation in socioeconomic status or geographic heterogeneity. Past evidence of spouse correlation was also seen in the strong evidence of linkage disequilibrium between unlinked markers, which was accounted for in regression analysis by ancestral allele frequency difference at the pair of markers (European versus Native American for Mexicans, European versus African for Puerto Ricans). We also observed an excess of homozygosity at individual markers within the spouses, but this provided weaker evidence, as expected, of spouse correlation. Ancestry variance is predicted to decline in each generation, but less so under assortative mating. We used the current observed variances of ancestry to infer even stronger patterns of spouse ancestry correlation in previous generations. Conclusions Assortative mating related to genetic ancestry persists in Latino populations to the current day, and has impacted on the genomic structure in these populations.