Straightforward protocol for allele-specific chromatin conformation capture.

A key advance in our understanding of gene regulation came with the finding that the genome undergoes three-dimensional nuclear folding in a genetically determined process. This 3D conformation directly influences the association between enhancers and their target promoters. This complex interplay has been proven to be essential for gene regulation, and genetic variants affecting this process have been associated to human diseases. The development of new technologies that quantify these DNA interactions represented a revolution in the field. High throughput techniques like HiC provide a general picture of chromatin topology. However, they often lack resolution to evidence subtle effects that single nucleotide polymorphisms exert over the contacts between cis-regulatory regions and target promoters. Here we propose a cost-efficient approach to perform allele-specific chromatin conformation analysis. As a proof of concept, we analyzed the impact of a common deletion mapping between SIRPB1 promoter and one of its downstream enhancers.

De novo assembly of the Brown trout (Salmo trutta m. fario) brain and muscle transcriptome: transcript annotation, tissue differential expression profile and SNP discovery.

OBJECTIVES: The Brown trout is a salmonid species with a high commercial value in Europe. Life history and spawning behaviour include resident (Salmo trutta m. fario) and migratory (Salmo trutta m. trutta) ecotypes. The main objective is to apply RNA-seq technology in order to obtain a reference transcriptome of two key tissues, brain and muscle, of the riverine trout Salmo trutta m. fario. Having a reference transcriptome of the resident form will complement genomic resources of salmonid species. DATA DESCRIPTION: We generate two cDNA libraries from pooled RNA samples, isolated from muscle and brain tissues of adult individuals of Salmo trutta m. fario, which were sequenced by Illumina technology. Raw reads were subjected to de-novo transcriptome assembly using Trinity, and coding regions were predicted by TransDecoder. A final set of 35,049 non-redundant ORF unigenes were annotated. Tissue differential expression analysis was evaluated by Cuffdiff. A False Discovery Rate (FDR) ≤ 0.01 was considered for significant differential expression, allowing to identify key differentially expressed unigenes. Finally, we have identified SNP variants that will be useful tools for population genomic studies.

The rs2108622 polymorphism is related to the early risk of ischemic stroke in non-valvular atrial fibrillation subjects under oral anticoagulation.

Oral anticoagulant treatments, such as vitamin K antagonists (VKAs), are the main treatments administered to atrial fibrillation (AF) patients in order to prevent ischemic stroke (IS). However, the genes involved in the VKA metabolism can undergo variations in a single nucleotide (SNP). These SNPs may then affect the VKA target enzyme (VKORC1), VKA degradation enzyme (CYP2C9), and vitamin K bioavailability enzyme (CYP4F2). We genotyped these SNPs in a cohort of patients with non-valvular AF who were under VKA treatment after suffering an IS. Clinical variables, CHADS2-VASC score and data about the international normalized ratio (INR) within the therapeutic range were all recorded. DNA was extracted from blood and genotyping was carried out by DNA sequencing. The main endpoint was the time from VKA onset to IS. Of a total of 356 consecutive IS patients monitored, 33 were included in the study. The median time to the event was 2248.0 days (interquartile range [IQR] 896.3-3545.3). The median CHADS2-VASC score was 4.0 (IQR 3.0-6.0). When we considered the risk of IS within 2 years under VKA treatment, we found that only the rs2108622 AA genotype was significantly associated with this endpoint (early IS) (hazard ratio 6.81, 95% CI 1.37-33.92, p = 0.019). Kaplan-Meier curve analysis also showed a significant relationship between early IS and rs2108622 AA genotype (Log rank p = 0.022). The CYP4F2 gene rs2108622 polymorphism was associated with a risk of early IS in NV-AF patients under VKA treatment.

SIRPB1 copy-number polymorphism as candidate quantitative trait locus for impulsive-disinhibited personality.

Impulsive-disinhibited personality (IDP) is a behavioral trait mainly characterized by seeking immediate gratification at the expense of more enduring or long-term gains. This trait has a major role in the development of several disinhibitory behaviors and syndromes, including psychopathy, attention-deficit and hyperactivity disorder, cluster-B personality disorders, criminality and alcoholism. Available data consistently support a strong heritable component, accounting for 30-60% of the observed variance in personality traits. A genome-wide analysis of copy-number variants was designed to identify novel genetic pathways associated with the IDP trait, using a series of 261 male participants with maximized opposite IDP scores. Quantitative trait locus analysis of candidate copy-number variants (CNVs) was conducted across the entire IDP continuum. Functional effects of associated variants were evaluated in zebrafish embryos. A common CNV mapping to the immune-related gene SIRPB1 was significantly associated with IDP scores in a dose-dependent manner (ß=-0.172, P<0.017). Expression quantitative trait locus analysis of the critical region revealed higher SIRPB1 mRNA levels associated with the haplotype containing the deleted allele (P<0.0007). Epigenetic marks highlighted the presence of two potential insulators within the deleted region, confirmed by functional assays in zebrafish embryos, which suggests that SIRPB1 expression rates are affected by the presence/absence of the insulator regions. Upregulation of SIRPB1 has been described in prefrontal cortex of patients with schizophrenia, providing a link between SIRPB1 and diseases involving disinhibition and failure to control impulsivity. We propose SIRPB1 as a novel candidate gene to account for phenotypic differences observed in the IDP trait.

Absence of allelic imbalance involving EMSY, CAPN5, and PAK1 genes in papillary thyroid carcinoma.

Papillary thyroid cancer (PTC) accounts for 80% of all thyroid malignancies, and genetic alterations associated to its etiology remain largely unknown. Chromosomal band 11q13 seems to be one of the most frequently amplified regions in human cancer, providing several candidate genes that need detailed characterization. The aim of our study was to investigate the existence of allelic imbalance at EMSY, CAPN5, and PAK1, as candidate genes within 11q13.5-q14 region using a single nucleotide polymorphism-based analysis. We selected a panel of 9 polymorphisms that were analyzed in 41 thyroid carcinoma samples, their contralateral non-pathological tissue and 178 controls from the general population. We did not detect allelic imbalance at these loci in our series. However, we observed a difference in the EMSY-haplotype distribution among PTC patients when compared to controls (odds ratio=2.00; p=0.02). We conclude that 11q13.5-q14 is not imbalanced in PTC, but there is evidence suggesting that EMSY might be of relevance in PTC etiology.

Identification of a 2244 base pair interstitial deletion within the human ESR1 gene in the Spanish population.

BACKGROUND: The ESR1 AGATA haplotype is composed of five markers located within introns 5 and 6 of the human oestrogen receptor 1 (ESR1) gene. This haplotype has been studied in several male urogenital tract anomalies and male infertility. In one of these studies, a deviation from Hardy-Weinberg equilibrium (DHW) was found for the ESR1 AGATA marker rs3020375 in two groups of healthy controls. In the present study, we investigated whether the observed DHW is caused by structural variants present within the ESR1 gene. PARTICIPANTS: 229 family units achieving pregnancy through assisted reproductive technologies (n = 129) or by natural means (n = 100), 2465 general Spanish population controls and 162 men with idiopathic infertility. MAIN OUTCOME MEASUREMENTS: Segregation analyses of genetic markers in family units and case-control genetic association studies. RESULTS: We identified a new interstitial deletion of 2244 base pairs within intron 6 of the human ESR1 gene as the cause for the observed DHW. This new variant presents a 10% allelic frequency in the general Spanish population and it is associated with idiopathic male infertility (OR = 1.51; p = 0.03). The percentage of infertile couples in which both members carried the ESR1 deletion (10.08%) was also a higher than expected value of 6% (p = 0.03). CONCLUSIONS: We have characterised a novel structural variation in human ESR1 gene. The available data indicate a deleterious action of the ESR1 deletion in both male and couple fertility. The potential effects of this deletion on other oestrogen-related diseases need to be determined.

CAPN10 alleles modify laryngeal cancer risk in the Spanish population.

AIMS: Laryngeal carcinoma is a common upper respiratory tract cancer with different environmental and genetic factors involved in its development. To date, CAPN10 has been found to be extensively associated with hyperinsulinaemia and type 2 diabetes mellitus, by different groups. In addition, some reports have shown evidence that hyperinsulinaemia exerts a protective effect on laryngeal cancer risk. On the other hand, low circulating levels of IGF-1 have been recently found in patients with laryngeal carcinoma. Our objective was to examine the contribution of CAPN10 alleles to the development of laryngeal cancer. METHODS: Cases and controls were recruited from central and southern Spain. Genotypes were determined using pyrosequencing technology. We analysed CAPN10 UCSNP-44, -43, -19, and -63 allelic distribution in 218 unrelated laryngeal cancer patients and 606 controls from the general population. RESULTS: We found that the UCSNP-44 allele-C is significantly under-represented among patients with laryngeal cancer (OR=0.685, p=0.02). CONCLUSION: These results indicate that some CAPN10 alleles may be exerting a protective effect on laryngeal cancer risk in the Spanish population.

Analysis of CXCL12 3'UTR G>A polymorphism in colorectal cancer.

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.

Association of genetic markers within the KIT and KITLG genes with human male infertility.

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 <5 million spz/ml) and 465 unrelated healthy controls from the same geographical region were genotyped for these SNPs. RESULTS: We found a statistically significant association of the rs3819392 polymorphism, which is located within the KIT gene, with idiopathic male infertility. In addition, a deviation from the Hardy-Weinberg equilibrium (HWE) law was observed for rs10506957 polymorphism within the KITLG gene only in the infertile group. CONCLUSIONS: Our data indicate that the KIT/KITLG system may be involved in a low sperm count trait in humans.

Improvement of recombinant protein yield by a combination of transcriptional amplification and stabilization of gene expression.

We explored the use of a cascade circuit for heterologous gene expression that consists of a regulatory module with a salicylate-inducible system that controls the expression of a second regulator, xylS2, whose product is activated by common inducers. Activation and increasing the concentration of the second regulator synergistically induced heterologous genes downstream of the Pm promoter in the expression module. This module can be placed in multicopy vectors or in the chromosome of a host strain by means of minitransposons. Using reporter genes, we evaluated gene regulation capacity and gross production of the system with different configurations. The highest yield was obtained when the expression module was in a multicopy plasmid after a 6-h induction. However, expression modules in plasmids showed low stability after induction even with selective pressure. The chromosomal configuration had the lowest basal levels and induced levels comparable to those of plasmid configurations, resulting in accumulation of more than 10% of the total protein. Unlike the configurations in plasmids, the yield was maintained for at least 3 days even without selective pressure. In conclusion, the cascade system in the chromosome configuration is more efficient for long-term fermentation because of the great stability of the overexpressing phenotype in spite of the high levels of expression.

Fluorescence resonance energy transfer analysis of CCR-V64I and SDF1-3'a polymorphisms: prevalence in southern Spain hiv type 1+ cohort and noninfected population.

The relationship between host genotype and AIDS, as well as the different genotype frequencies observed in different populations, have become important topics in HIV research. Therefore, the development of methods that provide faster and reliable results may contribute to further development and knowledge of those topics. We present the results of genotyping SDF1-3'A and CCR2-V64I in 440 HIV-1-infected people and 100 noninfected controls from southern Spain, using a novel method based on real-time PCR with LightCycler technology and fluorescence resonance energy transfer. Frequencies obtained were 23.8% for SDF1-3'A and 9.5% for CCR2-V64I for both HIV+ cohort and general population. Both polymorphisms are in accordance with the Hardy-Weinberg equilibrium law and no differences between patients and controls have been observed.

[Predictive factors for hospital infections caused by Gram-positive and Gram-negative organisms]. / Factores predictivos de las infecciones nosocomiales producidas por microorganismos grampositivos y gramnegativos.

BACKGROUND: Knowing the bacterian map and clinical profile of nosocomial infections (NI) in Spain may aid the better planning of empiric antimicrobian treatment. METHODS: A prospective incidence study carried out over 9 months was performed. Data collection out with the use of an EPINE project file. The chi square test and comparison of independent sample percentages were used for statistical analysis. RESULTS: During the study period 156 cases of NI (rate (5.5%) were detected: 65 patients with gram-negative bacilli infection (GNB), 34 by gram-positive cocci (GPC), 20 with mixed infection and 13 by Candida. The most frequent localization was urinary infection (63%) followed by surgical wound infection, pressure ulcers and respiratory infection. Of the 203 isolations, 57% corresponded to GNB, with E. coli being the most frequent microorganism. Staphylococcus aureus was the GPC most often found (95% methycilline sensitive). CONCLUSIONS: The profile of a patient with nosocomial infection in a hospital such as that in which the autors work would be as follows: if the patient were admitted in the department of internal medicine, was dementia or coma, denutrition, urinary catheter or neurologic disease and has NI (overall urinary infection) the infection would most likely be a caused by a gram-negative microorganism. If the patient has an i.v. line or is in a surgical ward, or has deep surgical wound infection the microorganism isolated would most likely be gram-positive.

[Endogenous digitalis]. / Digitálicos endógenos.

During the past 10 years, several groups of researchers from different laboratories have carried out research on an endogenous substance or substances which had cross immunoreacted to digoxin antibodies in patients without previous cardiotonic treatment. The increase has been demonstrated in various clinical cases which involve an augmentation of plasmatic volume. A considerable amount is known about its molecular structure and anatomic origin; the competitive inhibition of the sodium pump being quite clear.