Estrogen receptor alpha gene variants are associated with Alzheimer's disease.

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.

The membrane-spanning 4-domains, subfamily A (MS4A) gene cluster contains a common variant associated with Alzheimer's disease.

BACKGROUND: In order to identify novel loci associated with Alzheimer's disease (AD), we conducted a genome-wide association study (GWAS) in the Spanish population. METHODS: We genotyped 1,128 individuals using the Affymetrix Nsp I 250K chip. A sample of 327 sporadic AD patients and 801 controls with unknown cognitive status from the Spanish general population were included in our initial study. To increase the power of the study, we combined our results with those of four other public GWAS datasets by applying identical quality control filters and the same imputation methods, which were then analyzed with a global meta-GWAS. A replication sample with 2,200 sporadic AD patients and 2,301 controls was genotyped to confirm our GWAS findings. RESULTS: Meta-analysis of our data and independent replication datasets allowed us to confirm a novel genome-wide significant association of AD with the membrane-spanning 4-domains subfamily A (MS4A) gene cluster (rs1562990, P = 4.40E-11, odds ratio = 0.88, 95% confidence interval 0.85 to 0.91, n = 10,181 cases and 14,341 controls). CONCLUSIONS: Our results underscore the importance of international efforts combining GWAS datasets to isolate genetic loci for complex diseases.

ESR1 promoter polymorphism is not associated with nonsyndromic cryptorchidism.

The ESR1 promoter microsatellite (TA)n was reported as a potential functional polymorphism. In a case-control study, we were unable to demonstrate any association between (TA)n and nonsyndromic cryptorchidism in Italian and Spanish study populations.

CALHM1 P86L polymorphism is associated with late-onset Alzheimer's disease in a recessive model.

CALHM1 gene coding non-synonymous SNP P86L (rs2986017) was reported to increase the risk of Alzheimer's disease (AD) in a recent study. We have investigated this genetic variant in 2470 individuals from Spain to conduct an independent replication study of the proposed SNP marker. By applying a recessive model, we observed weak evidence of an association between P86L mutation and late-onset AD (LOAD) susceptibility in our case-control study (OR =1.38 C.I. = [1.01-1.89]). Meta-analysis of available studies also supports a recessive model for CALHM1 P86L variant and provides evidence of between study heterogeneity. Importantly, we found that adjusted mean age at AD onset in P86L homozygous LOAD patients was significantly earlier that in the rest of patients (77.01 +/- 6.1 for P86L homozygous carriers versus 79.0 +/- 6.0 for the rest of patients, p=0.002). We concluded that the CALMH1 gene may contribute to AD risk in our study population. The observed genetic model (recessive) and the estimated magnitude of the effect both imply that virtually all studies performed to date were markedly underpowered to detect this effect and underscore the importance of follow up, replication, and meta-analyses of promising genetic signals.

Pyrosequencing for SNP genotyping.

Pyrosequencing is a real-time DNA sequencing method. It is based on the transformation of pyrophosphates, released during DNA elongation by DNA polymerase, into measurable light. During DNA elongation, a single pyrophosphate molecule is released following incorporation of a single nucleotide. In the pyrosequencing reaction, released pyrophosphates are then rapidly converted by sulfurylase to adenosine triphosphate, which in turn is utilized by luciferase to produce light. Within standardized conditions, this reaction is accomplished in a few milliseconds and the light produced can be registered with a CCD camera. Therefore, it becomes possible to quantitatively measure the nucleotides incorporated. This approach has been automated in different platforms and can be used for a wide variety of applications, such as single-nucleotide polymorphism (SNP) genotyping, DNA sequencing, loss of heterozygosity analysis, and CpG methylation studies. Here we describe the entire process, focusing our attention on SNP genotyping, and giving examples of some other applications.

Genetic and genomic insights into age at natural menopause.

The age at natural menopause shows great variability. It has been proposed that early age at menopause is a risk factor for osteoporosis and cardiovascular disease, whereas later age at menopause is a risk factor for breast cancer. In addition, it is thought that the genetic factors accounting for the genetic variability in age at menopause could also play a role in those diseases, as well as infertility in women. In this minireview we comment on the latest genetics and genomics insights into age at natural menopause.

A method for detecting epistasis in genome-wide studies using case-control multi-locus association analysis.

BACKGROUND: The difficulty in elucidating the genetic basis of complex diseases roots in the many factors that can affect the development of a disease. Some of these genetic effects may interact in complex ways, proving undetectable by current single-locus methodology. RESULTS: We have developed an analysis tool called Hypothesis Free Clinical Cloning (HFCC) to search for genome-wide epistasis in a case-control design. HFCC combines a relatively fast computing algorithm for genome-wide epistasis detection, with the flexibility to test a variety of different epistatic models in multi-locus combinations. HFCC has good power to detect multi-locus interactions simulated under a variety of genetic models and noise conditions. Most importantly, HFCC can accomplish exhaustive genome-wide epistasis search with large datasets as demonstrated with a 400,000 SNP set typed on a cohort of Parkinson's disease patients and controls. CONCLUSION: With the current availability of genetic studies with large numbers of individuals and genetic markers, HFCC can have a great impact in the identification of epistatic effects that escape the standard single-locus association analyses.

Methylation alterations are not a major cause of PTTG1 misregulation.

BACKGROUND: On its physiological cellular context, PTTG1 controls sister chromatid segregation during mitosis. Within its crosstalk to the cellular arrest machinery, relies a checkpoint of integrity for which gained the over name of securin. PTTG1 was found to promote malignant transformation in 3T3 fibroblasts, and further found to be overexpressed in different tumor types. More recently, PTTG1 has been also related to different processes such as DNA repair and found to trans-activate different cellular pathways involving c-myc, bax or p53, among others. PTTG1 over-expression has been correlated to a worse prognosis in thyroid, lung, colorectal cancer patients, and it can not be excluded that this effect may also occur in other tumor types. Despite the clinical relevance and the increasing molecular characterization of PTTG1, the reason for its up-regulation remains unclear. METHOD: We analysed PTTG1 differential expression in PC-3, DU-145 and LNCaP tumor cell lines, cultured in the presence of the methyl-transferase inhibitor 5-Aza-2'-deoxycytidine. We also tested whether the CpG island mapping PTTG1 proximal promoter evidenced a differential methylation pattern in differentiated thyroid cancer biopsies concordant to their PTTG1 immunohistochemistry status. Finally, we performed whole-genome LOH studies using Affymetix 50 K microarray technology and FRET analysis to search for allelic imbalances comprising the PTTG1 locus. CONCLUSION: Our data suggest that neither methylation alterations nor LOH are involved in PTTG1 over-expression. These data, together with those previously reported, point towards a post-transcriptional level of misregulation associated to PTTG1 over-expression.

Analysis of the ERalpha germline PvuII marker in breast cancer risk.

BACKGROUND: Prolonged exposure to estrogens was found to be a risk factor for breast cancer. The molecular mechanism has been suggested to be the binding of estrogen receptors in mammary tissue, which promotes the proliferation of breast tissue. Different biomarkers mapping estrogen receptor alpha (ESR1) have been associated with breast cancer risk, although the size of the effect is not consistent among different reports. Variation in the estrogen receptor gene PvuII has been associated with an increased risk of developing breast cancer. However, some studies suggest that its effect might be constrained to a definite subgroup of patients. MATERIAL/METHODS: In this study the involvement of PvuII in breast cancer was analyzed in an independent sample of 444 unrelated breast cancer cases and 704 controls of Spanish origin. A case-control comparison was performed and the genotype distributions examined according to different tumor and population parameters. RESULTS: A trend towards association was observed in adjusted case-control association analysis (p=0.07). PvuII was associated with the familial forms of breast cancer (OR=3.81, p=0.02). T allele frequency was higher among patients with highly differentiated tumors (p=0.02), positive for steroid receptors (p=0.06), and negative for p53 (p=0.02). However, the PvuII genetic background did not affect disease-free survival time (p=0.65). CONCLUSIONS: The PvuII T allele may be a germline risk factor for familial forms of breast cancer and is associated with a specific subset of immunohistochemical tumor phenotype.

Controlled ovarian hyperstimulation pharmacogenetics: a simplified model to genetically dissect estrogen-related diseases.

The application of pharmacogenetics and pharmacogenomics to assisted reproductive techniques will help clinicians to improve the efficacy of hormone treatments that are being routinely applied during assisted reproductive technique protocols. Genetic markers involving controlled ovarian hyperstimulation pharmacogenetics are being isolated within follicle-stimulating hormone and estrogen receptor signaling pathways using the candidate gene approach. Furthermore, the information obtained during controlled ovarian hyperstimulation pharmacogenetics studies could be applied to other estrogen-related diseases, such as osteoporosis, breast cancer, essential hypertension and many other diseases related to estrogen production or its mechanism of action. The theory that estrogen-related diseases may share some risk factors with controlled ovarian hyperstimulation efficacy, and side effects linked to genetic markers, is discussed.

Lack of association between NOS3 Glu298Asp and breast cancer risk: a case-control study.

Nitric oxide (NO) plays a central role in the physiololgy and pathology of diverse tissues. Different studies provide data suggesting that the endothelial cell nitric oxide synthase (NOS3) expression in peritumoral microvessels might be a prognostic indicator in breast cancer patients. However, the putative contribution of common NOS3 germline variants to breast cancer risk remained unknown. A recent work comprising 269 breast cancer patients and 244 controls suggested that NOS3 Glu298Asp polymorphism is associated to breast cancer risk (OR=1.9). We performed an independent analysis of these results in 440 unrelated patients and 321 controls from Spanish population. Although our study was 90% powered to detect ORs >/=1.55, did not find any significant difference in the Glu298Asp allele distribution between cases and controls (P > 0.42). These putative reasons for this result are discussed.

Preliminary molecular genetic analysis of the Receptor Interacting Protein 140 (RIP140) in women affected by endometriosis.

BACKGROUND: Endometriosis is a complex disease affecting 10-15% of women at reproductive age. Very few genes are known to be altered in this pathology. RIP140 protein is an important cofactor of oestrogen receptor and many other nuclear receptors. Targeting disruption experiments of nrip1 gene in mice have demonstrated that nuclear receptor interacting protein 1 gene (nrip1), the gene encoding for rip140 protein, is essential for female fertility. Specifically, mice null for nrip1 gene are viable, but females are infertile because of complete failure of mature follicles to release oocytes at ovulation stage. The ovarian phenotype observed in mice devoid of rip140 closely resembles the luteinized unruptured follicle (LUF) syndrome that is observed in a high proportion of women affected of endometriosis or idiopathic infertility. Here we present a preliminary work that analyses the role of NRIP1 gene in humans. METHODS: We have sequenced the complete coding region of NRIP1 gene in 20 unrelated patients affected by endometriosis. We have performed genetic association studies by using the DNA variants identified during the sequencing process. RESULTS: We identified six DNA variants within the coding sequence of NRIP1 gene, and five of them generated amino acid changes in the protein. We observed that three of twenty sequenced patients have specific combinations of amino-acid variants within the RIP140 protein that are poorly represented in the control population (p = 0.006). Moreover, we found that Arg448Gly, a common polymorphism located within NRIP1 gene, is associated with endometriosis in a case-control study (59 cases and 141 controls, Pallele positivity test = 0.027). CONCLUSION: Our results suggest that NRIP1 gene variants, separately or in combinations, might act as predisposing factors for human endometriosis.

Absence of de novo Y-chromosome microdeletions in male children conceived through intracytoplasmic sperm injection.

Molecular screening for Y-chromosome microdeletions in 96 Spanish male children conceived through intracytoplasmic sperm injection (ICSI) was conducted. No microdeletions were detected; these results support the notion that de novo Y-chromosome alterations are rare and unrelated to the ICSI technique itself.

Specific CAPN10 gene haplotypes influence the clinical profile of polycystic ovary patients.

Recently, several research groups have evaluated CAPN10 gene in polycystic ovarian syndrome (PCOS) patients and other phenotypes, including hirsutism or intermediate phenotypes of PCOS. Molecular genetic analysis of CAPN10 gene indicates that different alleles may play a role in PCOS susceptibility and could be associated with idiopathic hirsutism. However, these observations are not exempt from controversy, because independent studies cannot replicate these preliminary findings. We present a haplotype-phenotype correlation study of CAPN10 haplotypes in 148 women showing ecographically detected polycystic ovaries (PCO) combined with one or more of these clinical symptoms: amenorrhea or severe oligomenorrhea, hyperandrogenism, and anovulatory infertility, as well as 93 unrelated controls. We have reconstructed and analyzed 482 CAPN10 haplotypes in patients and controls. We detected the association of UCSNP-44 allele with PCO phenotype in the Spanish population (P = 0.02). In addition, we identified several CAPN10 alleles associated to phenotypic differences observed between PCO patients, such as the presence of hypercholesterolemia (haplotype 1121, P = 0.005), presence of hyperandrogenic features (P = 0.05), and familial cancer incidence (haplotype 1111, P = 0.0005). Our results confirm the association of UCSNP-44 allele with PCO phenotype in the Spanish population. Moreover, we have identified novel candidate risk alleles and genotypes, within CAPN10 gene, that could be associated with important phenotypic and prognosis differences observed in PCOS patients.

Scanning of Y-chromosome azoospermia factors loci using real-time polymerase chain reaction and melting curve analysis.

OBJECTIVE: To develop a novel method to scan AZF loci looking for microdeletions. DESIGN: Molecular method development. SETTING: Men undergoing reproductive techniques in a private fertility unit. Molecular methods were performed in a private center for biomedical research. PATIENT(S): : Fifty-eight men divided in two groups depending on seminal analyses. A group of 19 women were also included as positive controls (absence of amplification). INTERVENTION(S): Peripheral blood extraction and DNA purification. MAIN OUTCOME MEASURE(S): Our method is based on real-time polymerase chain reaction (PCR) and melting curve analysis. We performed the screening of 16 selected sequence tagged sites (STS) within AZF loci, and we also calculated the mean, range, and standard deviation for melting temperature patterns and the crossing points values for each STS tested. RESULT(S): We detected one azoospermic patient with several STS deleted within the AZFc region. No deletions were detected in a group of 13 healthy men, and no amplification for any of the STS tested were observed in the positive control group (19 healthy women). CONCLUSION(S): We have developed a novel method based on real-time PCR and melting curve analysis to scan AZF loci looking for microdeletions This method is fast and reliable and permits the scanning of DNA from one patient per hour, minimizing the risk of cross contamination, and false-positive and false-negative results.

Cloning, characterization and chromosome mapping of the human SMAP1 gene.

Stromal membrane associated protein (smap-1) is a new murine cell surface molecule on the stromal cells. The murine smap-1 protein is induced in stromal cells by the contact with erythroid cells, which suggests that this protein may be involved in the haematopoietic progenitor cells to stromal cells interactions. Here we report the structure, map location and expression analysis of the human SMAP1 gene, which cover approximately 100 kb on chromosome 6 between D6S455 and D6S1673 markers. This gene is composed of 11 exons and encodes a 468-amino-acid protein, which shows an 86% of homology with the murine smap-1 protein. The expression of smap-1 in erythropoietic organs as well as the correlation with the erythropoietic activity of the haematopoietic organs suggest that smap-1 is induced in stromal cells by the contact with erythroid cells, defining smap-1 as a key molecule that induced an erythropoietic microenvironment in haematopoietic organs. The high sequence conservation between murine and human SMAP1, as well as its expression in bone marrow, strongly suggest conserved functions of this protein in both organisms. Recently, a constitutional translocation t(6;10)(q13;q22) has been described in a patient with severe aplastic anaemia. SMAP1 gene localizes to 6q13 and is probably implicated in erythropoiesis, therefore it remains as an interesting candidate gene.

Cloning, characterization, and chromosome mapping of the human GlcAT-S gene.

We report on the structure, map location, and tissue expression of the human GlcAT-Sgene. The gene covers approximately 85 Kb on chromosome 6 (6q13) between the D6S455 and D6S1673 markers. GlcAT-S is composed of four exons and encodes a 324-amino-acid protein, which shows 89% homology with the rat glcat-s protein and is involved in the biosynthesis of the HNK-1 carbohydrate epitope on glycoproteins. Although GlcAT-Swas considered an interesting candidate gene for the RP25 locus, the absence of any pathogenic mutations in probands of RP25-linked families ruled out that candidacy.