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.

Synthesis and Magnetic Studies of Two Neutral, Bis-Ligand Fe(II) Complexes Containing Carbazole-Bis(tetrazole) Ligands.

Previously reported carbazole-bis(tetrazole) (CzTR) ligands (where R = iPr and CH2-2,4,6-C6H2Me3) were used to synthesize air-stable, six-coordinate, octahedral bis-ligand Fe(II) complexes (CzTR)2Fe. The synthesis and characterization of these complexes using 1H nuclear magnetic resonance (NMR), X-ray crystallography, Mössbauer spectroscopy, and density functional theory (DFT) calculations are reported. Analysis of the magnetic properties revealed that the isopropyl derivative displays thermally induced spin crossover (SCO) over a temperature range of 150-350 K. This transition appears as an abrupt two-step transition in the solid state but simplifies to a smooth one-step transition in solution. The two-step transition in the solid state has been postulated to be due to lattice and solvation effects. In contrast, the slightly bulkier substituted CH2-2,4,6-C6H2Me3 (CH2Mes) Fe complex displays dramatically different magnetic behavior with no SCO and magnetic data suggesting low-spin Fe(II) with a possible TIP contribution. DFT calculations support the postulate that the change in magnetic behavior is primarily due to the nature of the ligand substituents.

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.

Gone with Gondwana: Amphipod diversification in freshwaters followed the breakup of the supercontinent.

The evolutionary origins of modern taxa are best understood as arising from the interplay of vicariance and dispersal. Vicariant events have long been considered responsible for Gondwanan distributions; such species are relics of the eponymic supercontinent on which they were thought to have originated. One such set of taxa are the freshwater members of the amphipod superfamily Hyaloidea, which due to their marine relatives and current distributions serve as an excellent model for testing vicariance and dispersal hypotheses. We investigated the evolutionary and biogeographic histories of the Hyaloidea using a molecular phylogenetic approach. Maximum likelihood analyses and Bayesian inference, using two nuclear genes and one mitochondrial gene, reveal the freshwater amphipods within the superfamily (Hyalellidae/Chiltoniidae) as a monophyletic group diverging from their extant marine ancestors during the Mesozoic. This is suggestive of the group entering freshwater relatively early, instead of geologically recent marine invasions as have been previously hypothesized. Despite the group's apparent monophyly, it is likely that marine hyaloids exploited shallow water marine/brackish habitats created following the breakup of Gondwana to invade continental freshwaters. Given the divergence times recorded and shallow cladogenetic events observed, it is possible that this occurred through multiple invasions by closely related taxa. Mesozoic invasions by the Hyaloidea suggest that freshwater members represent a much older lineage than previously considered, occupying continental freshwaters prior to the gammarids in the Cenozoic and contemporaneouslywith the crangonyctids in the Mesozoic. Our results highlight the Gondwanan origin of taxa with enigmatic distributions and the utility of amphipods for testing biogeographic hypotheses that infer the origin of freshwater taxa.

Past climate cooling promoted global dispersal of amphipods from Tian Shan montane lakes to circumboreal lakes.

Climate changes have substantial impacts on the geographic distribution of montane lakes and evolutionary dynamics of cold-adapted species. Past climate cooling is hypothesized to have promoted the dispersal of cold-adapted species via montane lakes, while future climate warming is thought to constrain their distributions. We test this hypothesis by using phylogeographic analysis and niche modeling of the Holarctic crustacean Gammarus lacustris with global sampling comprised of 567 sequenced individuals and 3180 occurrence records. We found that the species arose in Tian Shan in Central Asia and dispersed into montane lakes along the Alps, Himalayas, Tibet, East Asia, and the North American Rocky Mountain ranges, with accelerated diversification rates outside Tian Shan. Climatically suitable regions for geographic lineages of G. lacustris were larger during cooling periods (LGM), but smaller during warming periods (Mid-Holocene). In the future (2070) scenario, potential distributions in the Himalayas, North Tibet, South Tibet and North America are predicted to expand, whereas ranges in East Asia, Europe and Tian Shan will decline. Our results suggest that Mid-Miocene-to-Pleistocene continuous cooling promoted multiple independent dispersal events out of Tian Shan due to increased availability of montane lakes via "budding" of lineages. Montane lakes are conduits through which cold-adapted amphipods globally dispersed, dominating circumboreal lakes. However, future climate warming is likely to force organisms to shift upward in altitude and northward in latitude, leading to a future change in local populations. These findings highlight the importance of conservation of montane lakes, especially in the context of climate change.

Micro-endemic species of snails and amphipods show population genetic structure across very small geographic ranges.

Understanding variation in population genetic structure, even across small distances and for species with extremely limited ranges, is critical for conservation planning and the development of effective management strategies for imperiled species. Organisms that occupy the same geographic extent can maintain different population structures, ranging from highly diverged to panmictic. Such differences can result from differences in biological characteristics such as dispersal ability or demographic history. We used microsatellite loci to evaluate population genetic structure and variation of four desert spring invertebrates having high to low dispersal ability: the lung snail Physa acuta, two species of gilled snails (Juturnia kosteri and Pyrgulopsis roswellensis; family Hydrobiidae) and the amphipod Gammarus desperatus. The study location represents entire species ranges for the micro-endemic hydrobiids and G. desperatus, while P. acuta is ubiquitous throughout much of North America. We found little evidence of significant population genetic structure for P. acuta and J. kosteri, but much more for P. roswellensis and G. desperatus. Our results demonstrate differences in habitat preference and/or dispersal ability between the species. This information provides insight into how gene flow shapes varying population genetic structure between species across small spatial scales (<100 km2). Most importantly, our results suggest that conservation agencies should not consider these micro-endemic species to be composed of single populations, but rather, that management plans for such species should account for population genetic variation across the species' ranges.

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) .

Convolutional networks for fast, energy-efficient neuromorphic computing.

Deep networks are now able to achieve human-level performance on a broad spectrum of recognition tasks. Independently, neuromorphic computing has now demonstrated unprecedented energy-efficiency through a new chip architecture based on spiking neurons, low precision synapses, and a scalable communication network. Here, we demonstrate that neuromorphic computing, despite its novel architectural primitives, can implement deep convolution networks that (i) approach state-of-the-art classification accuracy across eight standard datasets encompassing vision and speech, (ii) perform inference while preserving the hardware's underlying energy-efficiency and high throughput, running on the aforementioned datasets at between 1,200 and 2,600 frames/s and using between 25 and 275 mW (effectively >6,000 frames/s per Watt), and (iii) can be specified and trained using backpropagation with the same ease-of-use as contemporary deep learning. This approach allows the algorithmic power of deep learning to be merged with the efficiency of neuromorphic processors, bringing the promise of embedded, intelligent, brain-inspired computing one step closer.

Isolation drives increased diversification rates in freshwater amphipods.

Vicariance and dispersal events affect current biodiversity patterns in desert springs. Whether major diversification events are due to environmental changes leading to radiation or due to isolation resulting in relict species is largely unknown. We seek to understand whether the Gammarus pecos species complex underwent major diversification events due to environmental changes in the area leading either to radiation into novel habitats, or formation of relicts due to isolation. Specifically, we tested the hypothesis that Gammarus in the northern Chihuahuan Desert of New Mexico and Texas, USA are descendants of an ancient marine lineage now containing multiple undescribed species. We sequenced a nuclear (28S) and two mitochondrial (16S, COI) genes from gammarid amphipods representing 16 desert springs in the northern Chihuahuan Desert. We estimated phylogenetic relationships, divergence times, and diversification rates of the Gammarus pecos complex. Our results revealed that the region contained two evolutionarily independent lineages: a younger Freshwater Lineage that shared a most-recent-common-ancestor with an older Saline Lineage ∼66.3  MYA (95.6-42.4  MYA). Each spring system generally formed a monophyletic clade based on the concatenated dataset. Freshwater Lineage diversification rates were 2.0-9.8 times higher than rates of the Saline Lineage. A series of post-Cretaceous colonizations by ancestral Gammarus taxa was likely followed by isolation. Paleo-geological, hydrological, and climatic events in the Neogene-to-Quaternary periods (23.03  MYA - present) in western North America promoted allopatric speciation of both lineages. We suggest that Saline Lineage populations include two undescribed Gammarus species, while the Freshwater Lineage shows repetition of fine-scale genetic structure in all major clades suggesting incipient speciation. Such ongoing speciation suggests that this region will continue to be a biodiversity hotspot for amphipods and other freshwater taxa.

A global database of nitrogen and phosphorus excretion rates of aquatic animals.

Animals can be important in modulating ecosystem-level nutrient cycling, although their importance varies greatly among species and ecosystems. Nutrient cycling rates of individual animals represent valuable data for testing the predictions of important frameworks such as the Metabolic Theory of Ecology (MTE) and ecological stoichiometry (ES). They also represent an important set of functional traits that may reflect both environmental and phylogenetic influences. Over the past two decades, studies of animal-mediated nutrient cycling have increased dramatically, especially in aquatic ecosystems. Here we present a global compilation of aquatic animal nutrient excretion rates. The dataset includes 10,534 observations from freshwater and marine animals of N and/or P excretion rates. These observations represent 491 species, including most aquatic phyla. Coverage varies greatly among phyla and other taxonomic levels. The dataset includes information on animal body size, ambient temperature, taxonomic affiliations, and animal body N:P. This data set was used to test predictions of MTE and ES, as described in Vanni and McIntyre (2016; Ecology DOI: 10.1002/ecy.1582).

Predicting the effects of climate change on population connectivity and genetic diversity of an imperiled freshwater mussel, Cumberlandia monodonta (Bivalvia: Margaritiferidae), in riverine systems.

In the face of global climate change, organisms may respond to temperature increases by shifting their ranges poleward or to higher altitudes. However, the direction of range shifts in riverine systems is less clear. Because rivers are dendritic networks, there is only one dispersal route from any given location to another. Thus, range shifts are only possible if branches are connected by suitable habitat, and stream-dwelling organisms can disperse through these branches. We used Cumberlandia monodonta (Bivalvia: Unionoida: Margaritiferidae) as a model species to investigate the effects of climate change on population connectivity because a majority of contemporary populations are panmictic. We combined ecological niche models (ENMs) with population genetic simulations to investigate the effects of climate change on population connectivity and genetic diversity of C. monodonta. The ENMs were constructed using bioclimatic and landscape data to project shifts in suitable habitat under future climate scenarios. We then used forward-time simulations to project potential changes in genetic diversity and population connectivity based on these range shifts. ENM results under current conditions indicated long stretches of highly suitable habitat in rivers where C. monodonta persists; populations in the upper Mississippi River remain connected by suitable habitat that does not impede gene flow. Future climate scenarios projected northward and headwater-ward range contraction and drastic declines in habitat suitability for most extant populations throughout the Mississippi River Basin. Simulations indicated that climate change would greatly reduce genetic diversity and connectivity across populations. Results suggest that a single, large population of C. monodonta will become further fragmented into smaller populations, each of which will be isolated and begin to differentiate genetically. Because C. monodonta is a widely distributed species and purely aquatic, our results suggest that persistence and connectivity of stream-dwelling organisms will be significantly altered in response to future climate change.

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.

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.

Past climate change drives current genetic structure of an endangered freshwater mussel species.

Historical-to-recent climate change and anthropogenic disturbance affect species distributions and genetic structure. The Rio Grande watershed of the United States and Mexico encompasses ecosystems that are intensively exploited, resulting in substantial degradation of aquatic habitats. While significant anthropogenic disturbances in the Rio Grande are recent, inhospitable conditions for freshwater organisms likely existed prior to such disturbances. A combination of anthropogenic and past climate factors may contribute to current distributions of aquatic fauna in the Rio Grande basin. We used mitochondrial DNA and 18 microsatellite loci to infer evolutionary history and genetic structure of an endangered freshwater mussel, Popenaias popeii, throughout the Rio Grande drainage. We estimated spatial connectivity and gene flow across extant populations of P. popeii and used ecological niche models (ENMs) and approximate Bayesian computation (ABC) to infer its evolutionary history during the Pleistocene. structure results recovered regional and local population clusters in the Rio Grande. ENMs predicted drastic reductions in suitable habitat during the last glacial maximum. ABC analyses suggested that regional population structure likely arose in this species during the mid-to-late Pleistocene and was followed by a late Pleistocene population bottleneck in New Mexico populations. The local population structure arose relatively recently, perhaps due to anthropogenic factors. Popenaias popeii, one of the few freshwater mussel species native to the Rio Grande basin, is a case study for understanding how both geological and anthropogenic factors shape current population genetic structure. Conservation strategies for this species should account for the fragmented nature of contemporary populations.

A genome-wide meta-analysis of nodular sclerosing Hodgkin lymphoma identifies risk loci at 6p21.32.

Nodular sclerosing Hodgkin lymphoma (NSHL) is a distinct, highly heritable Hodgkin lymphoma subtype. We undertook a genome-wide meta-analysis of 393 European-origin adolescent/young adult NSHL patients and 3315 controls using the Illumina Human610-Quad Beadchip and Affymetrix Genome-Wide Human SNP Array 6.0. We identified 3 single nucleotide polymorphisms (SNPs) on chromosome 6p21.32 that were significantly associated with NSHL risk: rs9268542 (P = 5.35 × 10(-10)), rs204999 (P = 1.44 × 10(-9)), and rs2858870 (P = 1.69 × 10(-8)). We also confirmed a previously reported association in the same region, rs6903608 (P = 3.52 × 10(-10)). rs204999 and rs2858870 were weakly correlated (r(2) = 0.257), and the remaining pairs of SNPs were not correlated (r(2) < 0.1). In an independent set of 113 NSHL cases and 214 controls, 2 SNPs were significantly associated with NSHL and a third showed a comparable odds ratio (OR). These SNPs are found on 2 haplotypes associated with NSHL risk (rs204999-rs9268528-rs9268542-rs6903608-rs2858870; AGGCT, OR = 1.7, P = 1.71 × 10(-6); GAATC, OR = 0.4, P = 1.16 × 10(-4)). All individuals with the GAATC haplotype also carried the HLA class II DRB1*0701 allele. In a separate analysis, the DRB1*0701 allele was associated with a decreased risk of NSHL (OR = 0.5, 95% confidence interval = 0.4, 0.7). These data support the importance of the HLA class II region in NSHL etiology.

Genetic variation in insulin-like growth factor 2 may play a role in ovarian cancer risk.

The insulin-like growth factor (IGF) signaling axis plays an important role in cancer biology. We hypothesized that genetic variation in this pathway may influence risk of ovarian cancer. A three-center study of non-Hispanic whites including 1880 control women, 1135 women with invasive epithelial ovarian cancer and 321 women with borderline epithelial ovarian tumors was carried out to test the association between tag single-nucleotide polymorphisms (tSNPs) (n=58) in this pathway and risk of ovarian cancer. We found no association between variation in IGF1, IGFBP1 or IGFBP3 and risk of invasive disease, whereas five tSNPs in IGF2 were associated with risk of invasive epithelial ovarian cancer at P<0.05 and followed-up one of the associated SNPs. We conducted genotyping in 3216 additional non-Hispanic white cases and 5382 additional controls and were able to independently replicate our initial findings. In the combined set of studies, rs4320932 was associated with a 13% decreased risk of ovarian cancer per copy of the minor allele carried (95% confidence interval 0.81-0.93, P-trend=7.4 × 10(-5)). No heterogeneity of effect across study centers was observed (p(het)=0.25). IGF2 is emerging as an important gene for ovarian cancer; additional genotyping is warranted to further confirm these associations with IGF2 and to narrow down the region harboring the causal SNP.

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.

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.

Experimental verification of the homoaromaticity of 1,3,5-cycloheptatriene and evaluation of the aromaticity of tropone and the tropylium cation by use of the dimethyldihydropyrene probe.

By use of a dimethyldihydropyrene experimental probe for aromaticity, 1,3,5-cycloheptatriene (16) is demonstrated to be a neutral homoaromatic hydrocarbon! On the basis of (1)H NMR results, 16 is judged to be ~30%, tropone 18 ~20%, and tropylium 22 ~50% as aromatic as benzene. The latter result may be an underestimation because of charge delocalization. The B3LYP/6-31G* calculated geometries and GIAO-HF/6-31G*//B3LYP/6-31G* calculated NMR chemical shifts and nucleus-independent chemical shifts (NICS) support these conclusions. These estimates were obtained by synthesis of the annelated dihydropyrenes 7 (tropone fused), 9 (1,3,5-cycloheptatriene fused), and 10 (tropylium fused). [4 + 3] Cycloaddition of the isofuran 5 with an oxyallyl cation (prepared from 2,4-dibromopentan-3-one) gave the C7 fused dihydropyrene 6 in 77% yield. Elimination of water gave tropone 7 in 61% yield, which, via LiAlH(4) reduction to alcohol 8 (48% yield) and treatment with HBF(4), gave quantitatively tropylium cation 10. When ketone 7 was reduced with AlH(3) (generated from AlCl(3)/LiAlH(4)) in ether/benzene at 25 °C, the isomeric cycloheptatrienes 11 (70% yield) and 9 (15% yield) were obtained.

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.