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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.

BACKGROUND: There is much evidence involving the KIT tyrosine kinase receptor and its ligand KITLG in the survival and proliferation of germ cells. Animal models and functional studies in humans suggest that this signalling pathway plays a role in male infertility. METHODS: We studied three and two single-nucleotide polymorphisms (SNPs) (rs3819392, rs3134885, rs2237012, rs10506957 and rs995030) located within the genomic region of the KIT and KITLG genes, respectively. A total of 167 idiopathic infertile men (sperm counts

BACKGROUND: Polycystic ovary syndrome (PCOS) is a common endocrine disorder in women of reproductive age. The aim of the present study was to investigate the role of CALPAIN-5 (CAPN5) gene in PCOS susceptibility. METHODS: We analysed four intronic polymorphisms of the CAPN5 gene in 148 well-characterized women with PCOS and 606 unrelated controls. We performed a case-control study and an intracohort analysis of clinical characteristics associated with PCOS. RESULTS: Analysis of haplotypes distribution between PCOS population compared to controls showed a strong deviation (P = 0.00029). The haplotypes GGCA and GGTG were overrepresented in PCOS patients (P = 0.009 and P = 0.001, respectively). In addition, we identified several CAPN5 haplotypes associated with phenotypic differences observed between PCOS patients, such as the presence of obesity (P = 0.02), cardiovascular complications (P = 0.02), familial antecedents of obesity (P = 0.003) and of hypertension (P = 0.007) and type 2 diabetes mellitus aggregation (P = 0.04). CONCLUSIONS: These results suggest a role of CAPN5 gene in PCOS susceptibility in humans. Moreover, novel candidate risk alleles have been identified, within CAPN5 gene, which could be associated with important phenotypic and prognosis differences observed in PCOS patients.

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.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) phenotypes appear in the same pedigrees [1] and can occur in the same individuals [2] (FTDALS, OMIM 105550). In fact about 2–3 % of ALS patients develop dementia [3]. Consequently, in some individuals, there appears to be a common genetic etiology for both diseases. Genetic variation identified in GRN, FUS, TDP43, and more recently, in C9ORF72 and UBQLN2 genes has been associated to familial ALS (FALS, OMIN 104105), familial FTD (OMIM 600274) and/or FTDALS consistent with the notion that both disorders may share genetic components and, probably, functional pathogenic mechanisms [4–8]. UBQLN2 gene was recently identified by Deng et al. [7] as a novel dominant but not fully penetrant X-linked FTDALS gene. Specifically, five segregating mutations in four different proline residues within the PXX repeat domain of UBQLN2 gene were identified in families afflicted with ALS/dementia. Importantly, the authors also found a correlation of hippocampal UBQLN2 pathology with dementia in ALS cases with or without UBQLN2 mutations, suggesting that UBQLN2 gene could be involved in ALS-related dementia, even without UBQLN2 mutations [7]. UBQLN2 gene encodes an ubiquitin-like protein (ubiquilin) that shares a high degree of similarity with related proteins in multiple eukaryotic organisms. Specifically, ubiquilins contain an N-terminal ubiquitin-like domain and a C-terminal ubiquitin-associated domain; therefore, UBQLN2 could be functionally linked to ubiquitination enzymatic processes. Functional analysis showed that mutations in UBQLN2 lead to an impairment of protein degradation [7]. Of note, UBQLN2 protein has also been shown to bind the Stch protein which is also involved in aggresome malfunction [9]. The role of ubiquitination in neurodegeneration is well established in different human neurodegenerative disorders such Parkinson’s disease or the Marinesco-Sjogren syndrome [10, 11]. Furthermore, it has been suggested that there is a link between Alzheimer’s disease and ubiquitins [12]. The purpose of the present study is to further explore the role of UBQLN2 mutations in familial FTD [13]. We examined 77 FTD index patients with a positive family history of dementia who had complete clinical evaluations. Importantly, we maintained FTD patients with observed male to male transmission (which makes a dominant X-linked transmission impossible) as negative controls of this study (n = 12). For the purpose of this study, we examined subjects with FTD and its subtypes (behavioral variant FTD (bvFTD), semantic dementia, progressive nonfluent aphasia), according to Neary et al. [14], as well as patients within the FTD spectrum, such as with those with combined phenotype of FTD and ALS, corticobasal syndrome or progressive supranuclear palsy (Table 1). All individuals agreed to participate in the study and blood samples were obtained after informed consent from Electronic supplementary material The online version of this article (doi:10.1007/s00415-012-6568-5) contains supplementary material, which is available to authorized users.

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.

addition to APOE exist. The objective of this study is to discover LOAD susceptibility genes other than the APOE. Methods: We conducted a three-stage genome-wide association study based on SNP in a total of 4,336 Japanese clinical subjects comprising 2,184 cases and 2,152 controls. In Stage 1, we genotyped 909,622 SNPs in 1,008 cases and 1,016 controls using Affymetrix GeneChip 6.0 microarrays. After stringent quality control (QC), 574,828 SNPs were analyzed in 951 cases and 894 controls. In Stage 2, we examined other sample sets consisting of 885 cases and 985 controls using Illumina Custom GoldenGate microarrays. After QC, 1,395 out of 1,418 SNPswere analyzed in 878 cases and 984 controls. In Stage 3, we genotyped 80 SNPs, comprising the high-ranked SNPs or SNPs of interest from Stage 2, in 1,977 cases and 2,128 controls by means of the TaqMan method. After QC, 80 SNPs were analyzed in 1,933 cases and 2,116 controls. Results: Multiple strong associations (Pallele < 1.7 3 10 ) for LOAD were observed in/around APOE region including BCL3, PVRL2, TOMM40,APOE,APOC1, and LRRC68 on chromosome 19. All association signals of these genes disappeared when subjects were stratified as to the APOE-e4 carrier status. In addition to these genes, we found several genes, albeit weak association, PAPOLOG (chr 2), ARHGEF3 (chr 3), EBF1 (chr 5), SORL1 (chr 11), FAM124A (chr 13) and GNAO1 (chr 16). To verify the association of these genes with LOAD in the Japanese population, we proceeded with replication studies in different ethnic cohorts, ADGC (11,840 cases and 10,931 controls) and Korean (333 cases and 1,128 controls).Conclusions: In addition to APOE, we found several candidate genes susceptibility to LOAD through a genome-wide association study.

BACE1 is a key enzyme involved in the production of amyloid ß-peptide (Aß) in Alzheimer's disease (AD) brains. Normally, its expression is constitutively inhibited due to the presence of the 5′untranslated region (5′UTR) in the BACE1 promoter. BACE1 expression is activated by phosphorylation of the eukaryotic initiation factor (eIF)2-alpha, which reverses the inhibitory effect exerted by BACE1 5′UTR. There are four kinases associated with different types of stress that could phosphorylate eIF2-alpha. Here we focus on the double-stranded (ds) RNA-activated protein kinase (PKR). PKR is activated during viral infection, including that of herpes simplex virus type 1 (HSV1), a virus suggested to be implicated in the development of AD, acting when present in brains of carriers of the type 4 allele of the apolipoprotein E gene. HSV1 is a dsDNA virus but it has genes on both strands of the genome, and from these genes complementary RNA molecules are transcribed. These could activate BACE1 expression by the PKR pathway. Here we demonstrate in HSV1-infected neuroblastoma cells, and in peripheral nervous tissue from HSV1-infected mice, that HSV1 activates PKR. Cloning BACE1 5′UTR upstream of a luciferase (luc) gene confirmed its inhibitory effect, which can be prevented by salubrinal, an inhibitor of the eIF2-alpha phosphatase PP1c. Treatment with the dsRNA analog poly (I:C) mimicked the stimulatory effect exerted by salubrinal over BACE1 translation in the 5′UTR-luc construct and increased Aß production in HEK-APPsw cells. Summarizing, our data suggest that PKR activated in brain by HSV1 could play an important role in the development of AD Neocodex is a private research organization (more than 70 articles in indexes of international research journals in the last 8 years). Neocodex’s work on this project was design, implementation, analysis and interpretation of genetic studies of the PKR gene that is included in this manuscript. Neocodex also coordinates the construction of the DNA bank employed in this draft. The work was carried out with funds from this entity and a project funded by the Alzheimur Foundation. The other funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. This w ork was also supported by the Spanish Ministerio de Ciencia y Tecnología (FIS: PI07/0593; Red HERACLES RD06/0009/002)

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.

Background Genetic admixture is a common caveat for genetic association analysis. Therefore, it is important to characterize the genetic structure of the population under study to control for this kind of potential bias. Results In this study we have sampled over 800 unrelated individuals from the population of Spain, and have genotyped them with a genome-wide coverage. We have carried out linkage disequilibrium, haplotype, population structure and copy-number variation (CNV) analyses, and have compared these estimates of the Spanish population with existing data from similar efforts. Conclusions In general, the Spanish population is similar to the Western and Northern Europeans, but has a more diverse haplotypic structure. Moreover, the Spanish population is also largely homogeneous within itself, although patterns of micro-structure may be able to predict locations of origin from distant regions. Finally, we also present the first characterization of a CNV map of the Spanish population. These results and original data are made available to the scientific community. This work was supported in part by Agencia IDEA, Consejería de Innovación, Ciencia y Empresa (830882); Corporación Tecnológica de Andalucía (07/124); Ministerio de Educación y Ciencia (PCT-A41502790-2007 and PCT-010000-2007-18); Programa de Ayudas Torres Quevedo del Ministerio de Ciencia en Innovación (PTQ2002-0206, PTQ2003-0549, PTQ2003-0546, PTQ2003-0782, PTQ2003-0783, PTQ2004-0838, PTQ04-1-0006, PTQ04-3-0718, PTQ06-1-0002). CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM) is an ISCIII project.

[Background]: Hepatic encephalopathy is a major complication of cirrhosis and is associated with a poor prognosis. [Objective]: To identify mutations in the gene sequence for glutaminase in humans that could be responsible for the development of hepatic encephalopathy in patients with cirrhosis. [Design]: Cohort study. [Setting]: Outpatient clinics in 6 Spanish hospitals. [Patients]: 109 consecutive patients with cirrhosis in the estimation cohort, 177 patients in the validation cohort, and 107 healthy control participants. [Measurements]: Patients were followed every 3 or 6 months until the development of hepatic encephalopathy or liver transplantation, death, or the end of the study. [Results]: The genetic analyses showed that glutaminase TACC and CACC haplotypes were linked to the risk for overt hepatic encephalopathy. Mutation scanning of the glutaminase gene identified a section in the promoter region where base pairs were repeated (a microsatellite). Over a mean follow-up of 29.6 months, hepatic encephalopathy occurred in 28 patients (25.7%) in the estimation cohort. Multivariable Cox models were used to determine the following independent predictors: Child-Turcotte-Pugh stage (hazard ratio [HR], 1.6 [95% CI, 1.29 to 1.98]; P = 0.001), minimal hepatic encephalopathy (HR, 3.17 [CI, 1.42 to 7.09]; P = 0.006), and having 2 long alleles of the microsatellite (HR, 3.12 [CI, 1.39 to 7.02]; P = 0.006). The association between 2 long alleles of the microsatellite and overt hepatic encephalopathy was confirmed in a validation cohort (HR, 2.1 [CI, 1.17 to 3.79]; P = 0.012). Functional studies showed higher luciferase activity in cells transfected with the long form of the microsatellite, which suggests that the long microsatellite enhances glutaminase transcriptional activity. [Limitation]: Other genes and allelic variants might be involved in the clinical expression of hepatic encephalopathy. [Conclusion]: This study identifies a genetic factor that is associated with development of hepatic encephalopathy in patients with cirrhosis., Grant Support: By the Spanish Ministry of Health (Instituto de Salud Carlos III, grants PI040384 and PI070425).

GOLPH2 gene SNP variants Rs10868366 and Rs7019241 were reported to decrease the risk of Alzheimer's disease in a recent Whole Genome Association Study. We have investigated these genetic variants in 2470 individuals from Spain to conduct an independent replication study of the proposed SNP markers. We found no evidence of association between GOLPH2 markers and susceptibility to Alzheimer's disease in our series. We concluded that GOLPH2 gene does not contribute to risk of disease in this study sample.

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.

Whole-genome epistasis analysis may add a new layer of knowledge to whole-genome association studies, permitting the identification of new candidate genes which are completely transparent during conventional single-locus analysis. We present the first whole-genome conditional two-locus analysis in Parkinson’s disease (PD). We scanned the entire genome and selected markers that interacted with a set of well-known loci previously associated to PD (SNCA, Parkin, LRRK2, UCHL1, DJ-1, PINK and MAPT). Our work describes several loci potentially related to PD risk which interact with SNCA, PARK1 and LRRK2 markers. We propose conditional whole-genome two-locus association analysis as a valuable method that might be helpful in re-analysing and re-interpreting data from whole-genome association studies.

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 missregulation associated to PTTG1 over-expression.

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.

Colorectal cancer is one of the most prevalent cancers in developed countries. However, the genetic factors influencing its appearance remain far from being fully characterized. Recently, a G>A functional transition mapping the 3' untranslated region of the CXCL12 gene (rs1801157) has been found to be under-represented among rectal cancer patients when compared to colon cancer patients from a Swedish series. Here we present the results from an independent analysis of CXCL12 rs1801157 in a larger CRC series of Spanish origin in order to analyse the robustness of this association within a different European population. No significant difference was observed between controls and colon or rectal cancer patients. We were also unable to find a correlation between rs1801157 and different prognostic markers such as metastasis development or disease-free survival time. The epidemiologic data involving CXCL12 rs1801157 in colorectal cancer risk are discussed.

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.

BACKGROUND: A specific haplotype (AGATA) in the estrogen receptor alpha (ER1) gene was recently described as a new risk factor for cryptorchidism in the Japanese population. In this ethnic group, single-nucleotide polymorphism 12 (SNP12) was concluded to be the tag SNP for the AGATA haplotype. MATERIALS AND METHODS: A large group of patients (total number = 335) and controls (total number = 567) of two Caucasian populations were analysed for the AGATA haplotype and SNP12 to verify whether this genetic variant and its tag SNP were associated with cryptorchidism or with severe spermatogenic failure. RESULTS: We confirm that SNP12 is the tag SNP for the AGATA haplotype also in Caucasians. However, in contrast with the Japanese population we found a protective effect for ESR1 SNP12 on cryptorchidism in the Italian population. No association between SNP12 and severe spermatogenic disturbances was observed. CONCLUSIONS: The observed associations (although with opposite effect) with cryptorchidism encourage future studies on independent cases and controls from different ethnic and geographic origins. On the other hand, in contrast with other ESR1 polymorphisms, SNP12 polymorphism is not associated with severe male factor infertility in two independent European population.

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.

Objective To examine whether polymorphisms within the ESR1 , FSHR , ESR2 , CYP19A1 , and NRIP1 genes are susceptibility factors for human male idiopathic infertility and to test the joint effects of these genes on male reproductive function. Design Genetic association study of male infertility with polymorphisms, using both single-gene and multilocus approaches. Setting Private and public fertility units and a private center for biomedical research. Patient(s) One hundred four Spanish men with azoospermia or severe oligozoospermia and 95 unselected race-matched healthy controls from the same geographic region. Intervention(s) Peripheral blood extraction, DNA purification, and ESR1 g.938T>C, FSHR Ser680Asn, ESR2 *39A>G, CYP19A1 *19C>T, and NRIP1 Gly75Gly polymorphism analyses. Main Outcome Measure(s) Single-gene statistical analyses and multilocus statistical analyses with Sumstat, Permutation and Model-free analysis, and Estimating Haplotypes software. Result(s) We observed an excess of homozygous infertile men for the ESR1 g.938T>C marker. Multilocus analyses detected genetic interaction between the five candidate gene markers that are influential over male infertility. In addition, we detected a five-loci protector genetic pattern with a frequency of 9.4% in controls but absent in infertile men. Conclusion(s) Our results support a relevant role for the estrogenic pathway, notably the ESR1 gene, in human male reproductive function and advocate a complex trait model for male infertility.

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.

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.

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.

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.

Alzheimer’s disease (AD) is a neurodegenerative disease characterized by senile plaques and neurofibrillary tangles. Senile plaques are deposits of amyloid ß-peptide (Aß) produced by the cleavage of a transmembrane protein termed Amyloid Precursor Protein (APP). The amyloidogenic cleavage of APP is performed by γ-secretase complex and ß-site APP cleaving enzyme 1 (BACE1), a key enzyme in AD that can be activated by different noxious stimuli. Interestingly, some viruses could activate double-stranded RNA-activated protein kinase (PKR), which phosphorylates Eukaryotic Initiation Factor 2 alpha (eIF2α). This phosphorylation stops global translation to avoid any synthesis of viral infective proteins, but paradoxically up-regulates BACE1 translation. One of the viral mechanisms to circumvent eIF2α phosphorylation is the recruitment of protein phosphatase 1 (PP1), to fully dephosphorylate eIF2α and allow viral protein synthesis. Due to the functional relationship between BACE1, PKR, PP1 and AD we have performed a large (1122 cases and 1191 control individuals) case-control genetic analysis using two biallelic polymorphisms rs2254958 and rs7480390, located within the genes coding for PKR and the catalytic unit A of PP1, respectively. Although a trend to association of the rs2254958 TT genotype with AD risk was found, our results show that neither rs7480390 nor rs2254958 are associated with AD susceptibility. This work was supported by the Spanish Ministerio de Ciencia y Tecnología (FIS: PI07/0593 and PI10/00587; ISCIII-RETIC RED HERACLES RD06/0009/002- FEDER).