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Osteoporosis is a common disease with multiple environmental and genetic risk factors involved. Using a marker-by-marker approach, the role of different estrogen-related genes has been analyzed in different populations, but most of these studies ignore the complex multigenic nature of human osteoporosis. Looking for markers related to osteoporosis, we have analyzed five single nucleotide polymorphisms located in genes related to the estrogen pathway, Follicle Stimulating Hormone Receptor (FSHR) gene, the CYP19 aromatase (CYP19A1) gene, the Estrogen Receptor alpha (ESR1) gene, the Estrogen Receptor beta (ESR2) gene and the Nuclear Receptor Interacting Protein 1 (NRIP1) gene in 265 unrelated postmenopausal women. We have obtained nominal P values for the NRIP1 Gly75Gly and ESR2 *39AG markers (P=0.013 and P=0.02 respectively), but no gene seems to be associated after multiple test corrections. Reanalysis of this study using 437 postmenopausal women confirmed our results and only detect marginal effects for ESR2 marker (P=0.045). By contrast, multilocus analysis predicted epistatic interactions between ESR1, ESR2 and NRIP1 loci and its involvement in postmenopausal osteoporosis (P=0.003). We detected two digenic genotypes involving ESR2-NRIP1 and ESR2-ESR1 genes strongly associated with osteoporosis (P=0.007). Replication of multilocus studies using 437 patients confirmed the detected interactions (P0.01). We proposed a non-additive non-multiplicative oligogenic model including ESR2 AG genotype modulated by NRIP1 A+ or ESR1 TT genotypes involved in osteoporosis. Our results reaffirm the polygenic nature and the genetic complexity of osteoporosis trait adding a new candidate gene (NRIP1) for association studies of bone-related traits.

SummaryRecently, we reported that the polymorphism 1132T>C (Gene-Bank: AF519768.1) of the NOS3 gene was associated with susceptibility to metabolic syndrome (MS) in hypertensive patients. This suggests that other genes such as CAV1, whose product (CAV1) regulates eNOS activity, could also be related to this phenotype.In this work we investigated the following:i) whether CAV1 is a quantitative trait locus of clustering of atherothrombotic traits associated with MS; ii) whether CVA1 is associated with hypertension or MS in hypertensive patients; and iii) whether genetic interaction between NOS3 and CAV1 is involved in the susceptibility or protection to hypertension associated with MS.To carry out the study, we genotyped 285 randomly selected individuals and 175 hypertensive patients, all of them ≤ 60 years old, with two polymorphisms of the CAV1 gene: the 22285 C>T and the 22375–22375 del AC (GeneBank AF125348), and the 1132T>C polymorphism of the NOS3 gene. To perform sample genotyping, we used pyrosequencing and FRET techniques.The 22285 C-22375–22375 del (Cd) haplotype of CAV1 gene was associated with low levels of blood pressure in the general population. Moreover, it was a genetic protection factor against MS in hypertensive patients. In addition, we found no evidence of gene-gene interaction between NOS3 and CAV1 genes with regard to that phenotype.

Germline mutations in different genes are associated with premature ovarian failure (POF, OMIM 311360), defined as premature menopause with amenorrhea occurring before the age of 40 years along with increased gonadotropin concentrations [follicle-stimulating hormone (FSH) >40 IU/L]. A new candidate gene, bone morphogenetic protein 15 ( BMP15 ) has been investigated in POF. In a family affected by hypergonadotropic ovarian failure, a mutation in the pro-region of the BMP15 gene (Y235C) was found in 2 affected sisters (1), and 3 linked markers within the BMP15 gene (−673C>T, −9C>G and IVS1 + 905A>G) are associated with high follicle production in women undergoing recombinant FSH stimulation (2). Several heterozygous variations affecting the pro-region and mature peptide of the BMP15 gene have been identified in women with POF (3)(4)(5), but A180T was the only variant found in all reported studies, occurring with relatively high frequency in POF women (8 of 502, 1.6%) but not at all in control groups. To clarify the role of the A180T allele in early menopause and ovarian failure, we used a pyrosequencing protocol (Biotage) to evaluate the A180T …

Cardiovascular disease is the leading cause of morbidity and mortality in the industrialized world. Familial aggregation of cardiovascular risk factors is a frequent finding, but genetic factors affecting its presentation are still poorly understood. The calpain 10 gene (CAPN10) has been associated with type 2 diabetes (T2DM), a complex metabolic disorder with increased risk of cardiovascular disease. Moreover, the CAPN10 gene has been associated with the presence of metabolic syndrome (MS) in T2DM and in polycystic ovary syndrome (PCOS). In this work, we have analysed whether the polymorphisms UCSNP44, -43, -19 and -63 are related to several cardiovascular risk factors in the context of MS. Molecular analysis of CAPN10 gene was performed in 899 individuals randomly chosen from a cross-sectional population-based epidemiological survey. We have found that CAPN10 gene in our population is mainly associated with two indicators of the presence of insulin resistance: glucose levels two hours after a 75-g oral glucose tolerance test (OGTT) and HOMA values, although cholesterol levels and blood pressure values are also influenced by CAPN10 variants. In addition, the 1221/1121 haplogenotype is under-represented in individuals that fulfil the International Diabetes Federation (IDF) diagnostic criteria for MS. Our results suggest that CAPN10 gene is associated with insulin resistance phenotypes in the Spanish population.

Sexual dimorphism in blood pressure (BP) regulation has been observed both in humans and experimental animals, and estrogens have been shown to contribute to this epidemiological observation. A key enzyme in determining estrogen levels is aromatase cytochrome P450. The aim of this study was to evaluate the role of the gene encoding aromatase, CYP19A1 , as an independent risk factor for hypertension and its relationship with systolic and diastolic BP measures. We genotyped 2 polymorphisms within the CYP19A1 gene, IVS4 rs11575899 and 3′UTR rs10046, in 3448 individuals. In quantitative analysis, we observed significant associations between the 2 polymorphisms and BP values in women, being these associations dependent on BMI and independent of menopause status. The case–control analysis revealed that the most prominent associations were found for nonobese women in diastolic hypertension (DHT): the IVS4_22 and 3 ′ UTR_11 are risk genotypes (OR=1.61, P =0.027 and OR=1.59, P =0.012, respectively), whereas IVS4_11 and 3 ′ UTR_22 genotypes have a protective effect against DHT (OR=0.63, P =0.009, and OR=0.61, P =0.020, respectively). Haplotype analysis confirmed the above associations: among nonobese women the haplotype 21 is overrepresented in hypertensive women (OR=1.33, P =0.004, for DHT and OR=1.25, P =0.026, for systolic hypertension, SHT) and, conversely, the haplotype 12 protects against hypertension (OR=0.78, P =0.015 for DHT and OR=0.82, P =0.04 for SHT). Our study has shown that the CYP19A1 gene may be involved in the genetic regulation of BP in women. This effect is dependent on BMI and independent of menopause status, suggesting that this action is mainly driven by aromatase activity in fat tissue.

Objective Controlled ovarian stimulation (COS) using recombinant follicle-stimulating hormone (rFSH) is the main treatment in assisted reproduction. We performed a pharmacogenetic analysis of bone morphogenetic protein 15 (BMP15) gene using single nucleotide polymorphisms (SNPs) in COS. We also investigated the role of the BMP15 gene in ovarian hyperstimulation syndrome (OHSS). Methods We analysed different intragenic SNPs located within the BMP15 gene in 307 women treated with rFSH, evaluating its involvement in COS outcome. Results First, we analysed two polymorphisms, by applying different tests for genetic association, and we found a minimum P-value in patients producing ≥12 follicles in COS (high responders) in both polymorphisms of the BMP15 gene. Using bi-directional DNA sequencing, we identified two additional single nucleotide DNA variants. Second, we conducted association studies with all polymorphisms together, and noticed that none of them seemed to fully explain the association of the BMP15 gene with over-response to rFSH. However, N103S missense mutation is predicted to disrupt the secondary structure of human BMP15 protein and is weakly associated with OHSS. This coding mutation of the BMP15 gene may partially explain the results obtained during our research. Using Thesias software, we reconstructed haplotypes with the four intragenic variants and calculated their frequencies in normal and over-responders to rFSH. The haplotype TGGA was over-represented in high responders when compared with the rest of patients. Moreover, this association was higher in patients with OHSS, with a significant global haplotypic effect of the BMP15 gene. Conclusion Our results suggest a direct relationship between increased follicle production during COS and BMP15 alleles in response to rFSH in humans. The use of BMP15 markers to prevent OHSS is also a possibility that requires thorough evaluation.

The calpain family is a group of cysteine proteases unique in their dependency on calcium to attain functionally active forms. Calpains are involved in a wide range of cellular calcium-regulated functions, including signal transduction, cell proliferation and differentiation, and apoptosis. Moreover, altered calpain activity has been observed in several human diseases. Specific calpain inhibitors hold promise for the treatment of neuromuscular and neurodegenerative diseases in which calpains have been shown to be upregulated (e.g. Parkinson's disease and Duchenne muscular dystrophy). Conversely, calpain activators could be a useful approach for those diseases where reduced calpain activity has been observed, such as type 2 diabetes or metabolic syndrome.

Controlled ovarian hyperstimulation (COH) is a routine treatment employed in most assisted reproductive techniques (ARTs). The existence of genetic factors involved in COH has been suspected. The main challenge for clinicians involved in ART is COH cycle cancellation, which usually occurs due to two opposing situations. On the one hand, there is the presence of a poor response during COH treatment, and on the other there is the presentation of a side effect related to gonadotropin hypersensitivity (ovarian hyperstimulation syndrome [OHSS]). Evidence for an association between single nucleotide polymorphisms and COH outcome has been obtained during the last decade. The genetic dissection of both extreme phenotypes of COH will be the main objective of this review. The development of predictive panels useful for the clinical management of COH is currently underway, and will improve the clinical management of patients undergoing ART.

This study aimed to evaluate the association between follicle-stimulating hormone (FSH) hormone efficacy and FSHR, CYP19, ESR1 and ESR2 genes using single nucleotide polymorphism analyses. One hundred and seventy women with conserved ovarian function undergoing controlled ovarian stimulation (COS) with daily exogenous recombinant FSH administration. Women were categorized as poor responders to FSH (three or less ovarian follicles observed at the end of cycle) or normal responders (more than three follicles). The outcome is the number of normal/poor responders as defined by the number of follicles obtained during COS. The DNA markers studied are located in genes related to the FSH mechanism of action (FSH receptor, CYP19 aromatase and oestrogen receptors alpha and beta genes). We conducted an association study between the COS outcome and selected DNA markers using two-point and multi-locus genetic association studies. Genotype pattern tracking in extreme phenotypes and multi-locus analysis using Sumstat and PM algorithms provided significant evidences of genetic interaction between FSHR, ESR1 and ESR2 markers in relation to COS outcome (P = 0.0015). Our results support the hypothesis that a discrete set of genes, related to the FSH hormone mechanism of action, controls the ovarian response to FSH in humans. An oligogenic model including specific FSHR, ESR1 and ESR2 genotype patterns may partially explain the poor response to FSH hormone during controlled ovarian stimulation treatments. The existence of genetic heterogeneity is also suspected.

The present research is aimed at assessing the role of 3 estrogen receptor alpha (ESR1) gene variants in late onset Alzheimer's disease (AD) susceptibility. One thousand one hundred thirteen unrelated late onset sporadic AD patients, 1109 healthy controls and 121 neurologically healthy elderly controls were used to carry out case-control genetic association studies with ESR1 rs3844508, rs2234693, and ESR1 noncoding deletion 1 (ESR1-NCD1) polymorphisms. Thirty-five healthy male samples were used for molecular analyses. The rs2234693 polymorphism is associated with AD in our population (odds ratio [OR], 1.29; p = 0.008). The rs3844508 marker confers protection against AD in males (OR, 0.57; p = 0.001) and the deletion ESR1-NCD1 is a risk factor for AD in women (OR, 1.67; p < 0.001). Molecular analyses on ESR1-NCD1 indicate that this deletion confers a higher response to estradiol activity on ESR1 receptor and it is also associated with differential expression of ESR1 isoforms. Our results support the involvement of ESR1 gene in AD and point to the existence of sexual dimorphism for ESR1 markers. In addition, carriers of ESR1-NCD1 deletion could overrespond to estradiol action.

1339 IL28B GENETIC VARIANTS AND GENDER ARE ASSOCIATED WITH SPONTANEOUS CLEARANCE OF HEPATITIS C VIRUS INFECTION H.Y. Rao, D.G. Sun, D. Jiang, R.F. Yang, F. Guo, J.H. Wang, F. Liu, H.Y. Zhang, H.H. Zhang, S.C. Du, A.S. Lok, L. Wei. Peking University People’s Hospital, Peking University Hepatology Institute, Beijing, Guan County Center for Disease Control and Prevention, Hebei Guan, China; Division of Gastroenterology, University of Michigan Medical Center, Ann Arbor, MI, USA E-mail: rao.huiying@163.com

Context Obesity is a multifactorial disorder, that is, a disease determined by the combined effect of genes and environment. In this context, polygenic approaches are needed. Objective To investigate the possibility of the existence of a crosstalk between the CALPAIN 10 homologue CALPAIN 5 and nuclear receptors of the peroxisome proliferator-activated receptors family. Design Cross-sectional, genetic association study and gene-gene interaction analysis. Subjects The study sample comprise 1953 individuals, 725 obese (defined as body mass index ≥ 30) and 1228 non obese subjects. Results In the monogenic analysis, only the peroxisome proliferator-activated receptor delta (PPARD) gene was associated with obesity (OR = 1.43 [1.04–1.97], p = 0.027). In addition, we have found a significant interaction between CAPN5 and PPARD genes (p = 0.038) that reduces the risk for obesity in a 55%. Conclusion Our results suggest that CAPN5 and PPARD gene products may also interact in vivo.