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Diabetes impacts approximately 200 million people worldwide, of whom approximately 10% are affected by type 1 diabetes (T1D). The application of genome-wide association studies (GWAS) has robustly revealed dozens of genetic contributors to the pathogenesis of T1D, with the most recent meta-analysis identifying in excess of 40 loci. To identify additional genetic loci for T1D susceptibility, we examined associations in the largest meta-analysis to date between the disease and ∼2.54 million SNPs in a combined cohort of 9,934 cases and 16,956 controls. Targeted follow-up of 53 SNPs in 1,120 affected trios uncovered three new loci associated with T1D that reached genome-wide significance. The most significantly associated SNP (rs539514, P = 5.66×10⁻¹¹) resides in an intronic region of the LMO7 (LIM domain only 7) gene on 13q22. The second most significantly associated SNP (rs478222, P = 3.50×10⁻⁹ resides in an intronic region of the EFR3B (protein EFR3 homolog B) gene on 2p23; however, the region of linkage disequilibrium is approximately 800 kb and harbors additional multiple genes, including NCOA1, C2orf79, CENPO, ADCY3, DNAJC27, POMC, and DNMT3A. The third most significantly associated SNP (rs924043, P = 8.06×10⁻⁹ lies in an intergenic region on 6q27, where the region of association is approximately 900 kb and harbors multiple genes including WDR27, C6orf120, PHF10, TCTE3, C6orf208, LOC154449, DLL1, FAM120B, PSMB1, TBP, and PCD2. These latest associated regions add to the growing repertoire of gene networks predisposing to T1D. Show less

ChREBP is an essential transcription factor for lipogenesis. Its physiological role in adipose tissue has been studied only to a small extent and the control of its expression remains unknown in human adipocytes. We have studied ChREBP mRNA and protein expression levels in the liver and the omental (OM) and subcutaneous (SC) adipose tissues from obese and lean subjects, as well as in human differentiated preadipocytes. Liver and OM and SC adipose tissue biopsies were obtained from lean and obese patients. Human preadipocytes were isolated from the adipose tissues from obese patients and differentiated under adipogenic conditions. ChREBP expression levels were quantified by RT-PCR and Western blot analysis. We found opposing results in terms of ChREBP regulation in the liver and adipose samples. ChREBP increased in the liver from obese compared to lean subjects, whereas the expression decreased in both adipose tissues. The mRNAs of other adipogenic markers were checked in these tissues. The pattern of FASN was similar to the one for ChREBP, ADCY3 decreased in both adipose tissues from obese patients, AP2 decreased only in OM adipose tissue of obese patients and ATGL did not change. The levels of ChREBP mRNA and protein showed dramatic increases during the differentiation of human OM and SC preadipocytes. In conclusion, ChREBP expression has an opposite regulation in the liver and adipose tissue from obese subjects which is compatible with the increased hepatic lipogenesis and decreased adipocytic lipogenesis found in these patients. The dramatic increase of ChREBP mRNA and protein levels during preadipocyte differentiation suggests a role in adipogenesis. Show less

To investigate differential gene expression profiles in the bladder of spontaneously hypertensive rat (SHR), as the underlying mechanisms involved in hypertension-associated bladder dysfunction remain to be clarified. SHR and normotensive Wistar-Kyoto (WKY) rats were distributed initially in three groups: group 1 received doxazosin (30 mg/kg/day); group 2 received nifedipine (30 mg/kg/day); and group 3 received the vehicle orally for 4 weeks. The alterations in gene expression levels of candidate genes identified by microarray analysis with potential biological relevance were verified by real-time reverse transcription-polymerase chain reaction (RT-PCR). Voiding frequency was significantly higher, and mean voided volume was significantly lower in untreated SHRs than untreated WKY rats. Microarray analysis revealed that 25 of the differentially expressed genes in untreated SHRs compared to untreated WKY rats were related to G(s), G(i), G(q) and G(12/13) signalling, calcium handling, ion transport and smooth muscle-related genes. Furthermore, RT-PCR data, in accord with the microarray analysis, indicated that untreated SHRs had lower mRNA expression levels of Adcy2, Adcy3, Rgs2, Rgs3, Rgs4 and Arhgdia, and higher mRNA expression levels of Arhgef1, Arhgef11, Arhgef12, Geft, Rock1 and Rock2 than untreated WKY rats. The differential alterations in the micturition patterns and in the expression of several genes related to G-protein signalling pathway observed in SHRs were attenuated by treatment with doxazosin, but not nifedipine. Our data suggest that differential alterations in the expression of several genes related to G(s), G(q) and G(12/13) signalling pathways in the SHR bladder might be important in hypertension-associated bladder dysfunction. Show less

Alcohol dependence is a complex disease, and although linkage and candidate gene studies have identified several genes associated with the risk for alcoholism, these explain only a portion of the risk. We carried out a genome-wide association study (GWAS) on a case-control sample drawn from the families in the Collaborative Study on the Genetics of Alcoholism. The cases all met diagnostic criteria for alcohol dependence according to the Diagnostic and Statistical Manual of Mental Disorders-Fourth Edition; controls all consumed alcohol but were not dependent on alcohol or illicit drugs. To prioritize among the strongest candidates, we genotyped most of the top 199 single nucleotide polymorphisms (SNPs) (p < or = 2.1 x 10(-4)) in a sample of alcohol-dependent families and performed pedigree-based association analysis. We also examined whether the genes harboring the top SNPs were expressed in human brain or were differentially expressed in the presence of ethanol in lymphoblastoid cells. Although no single SNP met genome-wide criteria for significance, there were several clusters of SNPs that provided mutual support. Combining evidence from the case-control study, the follow-up in families, and gene expression provided strongest support for the association of a cluster of genes on chromosome 11 (SLC22A18, PHLDA2, NAP1L4, SNORA54, CARS, and OSBPL5) with alcohol dependence. Several SNPs nominated as candidates in earlier GWAS studies replicated in ours, including CPE, DNASE2B, SLC10A2, ARL6IP5, ID4, GATA4, SYNE1, and ADCY3. We have identified several promising associations that warrant further examination in independent samples. Show less

Investigate the effect of hydrostatic pressure (HP) on 3', 5'-cyclic adenosine monophosphate (cAMP) levels and downstream signaling in cultures of normal optic nerve head (ONH) astrocytes from Caucasian American (CA) and African American (AA) donors. Intracellular cAMP levels were assayed after exposing ONH astrocytes to HP for varying times. Quantitative RT-PCR was used to determine the expression levels of selected cAMP pathway genes in human ONH astrocytes after HP treatment. Western blots were used to measure changes in the phosphorylation state of cAMP response element binding protein (CREB) in astrocytes subjected to HP, ATP, and phosphodiesterase or kinase inhibitors. The basal intracellular cAMP level is similar among AA and CA astrocytes. After exposure to HP for 15 min and 30 min in the presence of a phosphodiesterase inhibitor a further increase of intracellular cAMP was observed in AA astrocytes, but not in CA astrocytes. Consistent with activation of the cAMP-dependent protein kinase pathway, CREB phosphorylation (Ser-133) was increased to a greater extent in AA than in CA astrocytes after 3 h of HP. Exposure to elevated HP for 3-6 h differentially altered the expression levels of selected cAMP pathway genes (ADCY3, ADCY9, PTHLH, PDE7B) in AA compared to CA astrocytes. Treatment with ATP increased more CREB phosphorylation in CA than in AA astrocytes, suggesting differential Ca(2+) signaling in these populations. Activation of the cAMP-dependent signaling pathway by pressure may be an important contributor to increased susceptibility to elevated intraocular pressure and glaucoma in AA, a population at higher risk for the disease. Show less

Decreased cardiac contractility and beta-adrenergic responsiveness have been observed in cirrhotic cardiomyopathy, but their molecular mechanisms remain unclear. To study beta-adrenergic-stimulated contractility and beta-adrenergic gene expression patterns, 20 Wistar Kyoto rats were treated with carbon tetrachloride to induce cirrhosis and 20 rats were used as controls. Left ventricular contractility was recorded in electrically driven isolated hearts perfused at constant flow with isoproterenol (10(-10) to 10(-6) M). A cardiac gene expression profile was obtained using a microarray for the myocyte adrenergic pathway. The cardiac contractility maximal response to isoproterenol was significantly reduced in cirrhotic rats in comparison to control rats, whereas the half-maximal effective concentration was not different. In cirrhotic rats, cardiac gene expression analysis showed a significant overexpression of G protein alpha-inhibiting subunit 2 (Galpha(i2)), cyclic nucleotide phosphodiesterase (PDE2a), regulator of G-protein signaling 2 (RGS2), and down-expression of adenylate cyclase (Adcy3). These results indicate that overexpression of Galpha(i2), PDE2a, and RGS2 down-regulates the beta-adrenergic signaling pathway, thus contributing to the pathogenesis of cirrhotic cardiomyopathy. Show less

Infertility is an important condition in reproductive medicine. According to this work, there is only one identified physiogenomic relationship on chromosome 5 (CAMK4) for male but there are four identified physiogenomic relationships on chromosome 12 (CD9), chromosome 19 (BSG), chromosome 2 (ADCY3) and chromosome 4 (AFP). Although it has been determined for a long time, there is no clear cut genetic difference between male and female infertility. Systemic approach on the pathophysiology and genomics might provide useful information to better understand the pathogenesis of infertility. In this work, physiogenomics analysis for infertility in male and female was performed. Show less

Capacitation of mammalian sperm, including alterations in flagellar motility, is presumably modulated by chemical signals encountered in the female reproductive tract. This work investigates signaling pathways for adenosine and catecholamine agonists that stimulate sperm kinetic activity. We show that 2-chloro-2'-deoxyadenosine and isoproterenol robustly accelerate flagellar beat frequency with EC50s near 10 and 0.05 microM, respectively. The several-fold acceleration is maximal by 60 sec. Although extracellular Ca2+ is required for agonist action on the flagellar beat, agonist treatment does not elevate sperm cytosolic [Ca2+] but does increase cAMP content. Acceleration does not require the conventional transmembrane adenylyl cyclase ADCY3, since it persists in sperm of ADCY3 knockout mice and in wild-type sperm in the presence of the inhibitors of conventional adenylyl cyclases SQ-22536, MDL-12330A, or 2', 5'-dideoxyadenosine. In contrast, the acceleration by these agents is absent in sperm that lack the predominant atypical adenylyl cyclase, SACY. Responses to these agonists are also absent in sperm from mice lacking the sperm-specific Calpha2 catalytic subunit of protein kinase A (PRKACA). Agonist responses also are strongly suppressed in wild-type sperm by the protein kinase inhibitor H-89. These results show that adenosine and catecholamine analogs activate sperm motility by mechanisms that require extracellular Ca2+, the atypical sperm adenylyl cyclase, cAMP, and protein kinase A. Show less

The adenylyl cyclases (AC) act as second messengers in regulatory processes in the central nervous system. They might be involved in the pathophysiology of diseases, but their biological function is unknown, except for AC type I, which has been implicated in learning and memory. We previously mapped the gene encoding AC I to human Chromosome (Chr) 7p12. In this study we report the mapping of the adenylyl cyclase genes type I-VI to mouse chromosomes by fluorescence in situ hybridization (FISH): Adcy1 to Chr 11A2, Adcy2 to 13C1, Adcy3 to 12A-B, Adcy4 to 14D3, Adcy5 to 16B5, and Adcy6 to 15F. We also confirmed previously reported mapping results of the corresponding human loci ADCY2, ADCY3, ADCY5, and ADCY6 to human chromosomes and, in addition, determined the chromosomal location of ADCY4 to human Chr 14q11.2. The mapping data confirm known areas of conservation between mouse and human chromosomes. Show less