Comprehensive analysis of RET common and rare variant patientsin a series of Spanish Hirschsprung patients confirms a synergistic effect of both kinds of events

Background. RET is the major gene associated to Hirschsprung disease (HSCR) with differential contributions of its rare and common, coding and noncoding mutations to the multifactorial nature of this pathology. In the present study, we have performed a comprehensive study of our HSCR series evaluating the involvement of both RET rare variants (RVs) and common variants (CVs) in the context of the disease. Methods. RET mutational screening was performed by dHPLC and direct sequencing for the identification of RVs. In addition Taqman technology was applied for the genotyping of 3 RET CVs previously associated to HSCR, including a variant lying in an enhancer domain within RET intron 1 (rs2435357). Statistical analyses were performed using the SPSS v.17.0 to analyze the distribution of the variants. Results. Our results confirm the strongest association to HSCR for the "enhancer" variant, and demonstrate a significantly higher impact of it in male versus female patients. Integration of the RET RVs and CVs analysis showed that in 91.66% of cases with both kinds of mutational events, the enhancer allele is in trans with the allele bearing the RET RV. Conclusions. A gender effect exists on both the transmission and distribution of rare coding and common HSCR causing mutations. In addition, these RET CVs and RVs seem to act in a synergistic way leading to HSCR phenotype.

Mutation screening of multiple genes in Spanish patients with autosomal recessive retinitis pigmentosa by targeted resequencing.

Retinitis Pigmentosa (RP) is a heterogeneous group of inherited retinal dystrophies characterised ultimately by the loss of photoreceptor cells. RP is the leading cause of visual loss in individuals younger than 60 years, with a prevalence of about 1 in 4000. The molecular genetic diagnosis of autosomal recessive RP (arRP) is challenging due to the large genetic and clinical heterogeneity. Traditional methods for sequencing arRP genes are often laborious and not easily available and a screening technique that enables the rapid detection of the genetic cause would be very helpful in the clinical practice. The goal of this study was to develop and apply microarray-based resequencing technology capable of detecting both known and novel mutations on a single high-throughput platform. Hence, the coding regions and exon/intron boundaries of 16 arRP genes were resequenced using microarrays in 102 Spanish patients with clinical diagnosis of arRP. All the detected variations were confirmed by direct sequencing and potential pathogenicity was assessed by functional predictions and frequency in controls. For validation purposes 4 positive controls for variants consisting of previously identified changes were hybridized on the array. As a result of the screening, we detected 44 variants, of which 15 are very likely pathogenic detected in 14 arRP families (14%). Finally, the design of this array can easily be transformed in an equivalent diagnostic system based on targeted enrichment followed by next generation sequencing.

Association between IL-18 gene polymorphisms and biopsy-proven giant cell

INTRODUCTION: The objective was to investigate the potential implication of the IL18 gene promoter polymorphisms in the susceptibility to giant-cell arteritis GCA). METHODS: In total, 212 patients diagnosed with biopsy-proven GCA were included in this study. DNA from patients and matched controls was obtained from peripheral blood. Samples were genotyped for the IL18-137 G>C (rs187238), the IL18-607 C>A (rs1946518), and the IL18-1297 T>C (rs360719) gene polymorphisms with polymerase chain reaction, by using a predesigned TaqMan allele discrimination assay. RESULTS: No significant association between the IL18-137 G>C polymorphism and GCA was found. However, the IL18 -607 allele A was significantly increased in GCA patients compared with controls (47.8% versus 40.9% in patients and controls respectively; P = 0.02; OR, 1.32; 95% CI, 1.04 to 1.69). It was due to an increased frequency of homozygosity for the IL18 -607 A/A genotype in patients with GCA (20.4%) compared with controls (13.4%) (IL18 -607 A/A versus IL18 -607 A/C plus IL18 -607 C/C genotypes: P = 0.04; OR, 1.59; 95% CI, 1.02 to 2.46). Also, the IL18-1297 allele C was significantly increased in GCA patients (30.7%) compared with controls (23.0%) (P = 0.003; OR, 1.48; 95% CI, 1.13 to 1.95). In this regard, an increased susceptibility to GCA was observed in individuals carrying the IL18-1297 C/C or the IL18-1297 C/T genotypes compared with those carrying the IL18-1297 T/T genotype (IL18-1297 C/C plus IL18-1297 T/C versus IL18-1297 T/T genotype in GCA patients compared with controls: P = 0.005; OR, 1.61; 95% CI, 1.15 to 2.25). We also found an additive effect of the IL18 -1297 and -607 polymorphisms with TLR4 Asp299Gly polymorphism. The OR for GCA was 1.95 for combinations of genotypes with one or two risk alleles, whereas carriers of three or more risk alleles have an OR of 3.7. CONCLUSIONS: Our results show for the first time an implication of IL18 gene-promoter polymorphisms in the susceptibility to biopsy-proven GCA. In addition, an additive effect between the associated IL18 and TLR4 genetic variants was observed.

Multiple sclerosis risk variant HLA-DRB1*1501 associates with high expression of DRB1 gene in different human populations.

The human leukocyte antigen (HLA) DRB1*1501 has been consistently associated with multiple sclerosis (MS) in nearly all populations tested. This points to a specific antigen presentation as the pathogenic mechanism though this does not fully explain the disease association. The identification of expression quantitative trait loci (eQTL) for genes in the HLA locus poses the question of the role of gene expression in MS susceptibility. We analyzed the eQTLs in the HLA region with respect to MS-associated HLA-variants obtained from genome-wide association studies (GWAS). We found that the Tag of DRB1*1501, rs3135388 A allele, correlated with high expression of DRB1, DRB5 and DQB1 genes in a Caucasian population. In quantitative terms, the MS-risk AA genotype carriers of rs3135388 were associated with 15.7-, 5.2- and 8.3-fold higher expression of DQB1, DRB5 and DRB1, respectively, than the non-risk GG carriers. The haplotype analysis of expression-associated variants in a Spanish MS cohort revealed that high expression of DRB1 and DQB1 alone did not contribute to the disease. However, in Caucasian, Asian and African American populations, the DRB1*1501 allele was always highly expressed. In other immune related diseases such as type 1 diabetes, inflammatory bowel disease, ulcerative colitis, asthma and IgA deficiency, the best GWAS-associated HLA SNPs were also eQTLs for different HLA Class II genes. Our data suggest that the DR/DQ expression levels, together with specific structural properties of alleles, seem to be the causal effect in MS and in other immunopathologies rather than specific antigen presentation alone.

Genetic polymorphisms of RANTES, IL1-A, MCP-1 and TNF-A genes in patients with prostate cancer

BACKGROUND Inflammation has been implicated as an etiological factor in several human cancers, including prostate cancer. Allelic variants of the genes involved in inflammatory pathways are logical candidates as genetic determinants of prostate cancer risk. The purpose of this study was to investigate whether single nucleotide polymorphisms of genes that lead to increased levels of pro-inflammatory cytokines and chemokines are associated with an increased prostate cancer risk. METHODS A case-control study design was used to test the association between prostate cancer risk and the polymorphisms TNF-A-308 A/G (rs 1800629), RANTES-403 G/A (rs 2107538), IL1-A-889 C/T (rs 1800587) and MCP-1 2518 G/A (rs 1024611) in 296 patients diagnosed with prostate cancer and in 311 healthy controls from the same area. RESULTS Diagnosis of prostate cancer was significantly associated with TNF-A GA + AA genotype (OR, 1.61; 95% CI, 1.09-2.64) and RANTES GA + AA genotype (OR, 1.44; 95% CI, 1.09-2.38). A alleles in TNF-A and RANTES influenced prostate cancer susceptibility and acted independently of each other in these subjects. No epistatic effect was found for the combination of different polymorphisms studied. Finally, no overall association was found between prostate cancer risk and IL1-A or MCP-1 polymorphisms. CONCLUSION Our results and previously published findings on genes associated with innate immunity support the hypothesis that polymorphisms in proinflammatory genes may be important in prostate cancer development.

Systematic survey of clonal complexity in tuberculosis at a populational level and detailed characterization of the isolates involved.

Clonally complex infections by Mycobacterium tuberculosis are progressively more accepted. Studies of their dimension in epidemiological scenarios where the infective pressure is not high are scarce. Our study systematically searched for clonally complex infections (mixed infections by more than one strain and simultaneous presence of clonal variants) by applying mycobacterial interspersed repetitive-unit (MIRU)-variable-number tandem-repeat (VNTR) analysis to M. tuberculosis isolates from two population-based samples of respiratory (703 cases) and respiratory-extrapulmonary (R+E) tuberculosis (TB) cases (71 cases) in a context of moderate TB incidence. Clonally complex infections were found in 11 (1.6%) of the respiratory TB cases and in 10 (14.1%) of those with R+E TB. Among the 21 cases with clonally complex TB, 9 were infected by 2 independent strains and the remaining 12 showed the simultaneous presence of 2 to 3 clonal variants. For the 10 R+E TB cases with clonally complex infections, compartmentalization (different compositions of strains/clonal variants in independent infected sites) was found in 9 of them. All the strains/clonal variants were also genotyped by IS6110-based restriction fragment length polymorphism analysis, which split two MIRU-defined clonal variants, although in general, it showed a lower discriminatory power to identify the clonal heterogeneity revealed by MIRU-VNTR analysis. The comparative analysis of IS6110 insertion sites between coinfecting clonal variants showed differences in the genes coding for a cutinase, a PPE family protein, and two conserved hypothetical proteins. Diagnostic delay, existence of previous TB, risk for overexposure, and clustered/orphan status of the involved strains were analyzed to propose possible explanations for the cases with clonally complex infections. Our study characterizes in detail all the clonally complex infections by M. tuberculosis found in a systematic survey and contributes to the characterization that these phenomena can be found to an extent higher than expected, even in an unselected population-based sample lacking high infective pressure.

Characterization of microevolution events in Mycobacterium tuberculosis strains involved in recent transmission clusters.

Under certain circumstances, it is possible to identify clonal variants of Mycobacterium tuberculosis infecting a single patient, probably as a result of subtle genetic rearrangements in part of the bacillary population. We systematically searched for these microevolution events in a different context, namely, recent transmission chains. We studied the clustered cases identified using a population-based universal molecular epidemiology strategy over a 5-year period. Clonal variants of the reference strain defining the cluster were found in 9 (12%) of the 74 clusters identified after the genotyping of 612 M. tuberculosis isolates by IS6110 restriction fragment length polymorphism analysis and mycobacterial interspersed repetitive units-variable-number tandem repeat typing. Clusters with microevolution events were epidemiologically supported and involved 4 to 9 cases diagnosed over a 1- to 5-year period. The IS6110 insertion sites from 16 representative isolates of reference and microevolved variants were mapped by ligation-mediated PCR in order to characterize the genetic background involved in microevolution. Both intragenic and intergenic IS6110 locations resulted from these microevolution events. Among those cases of IS6110 locations in intergenic regions which could have an effect on the regulation of adjacent genes, we identified the overexpression of cytochrome P450 in one microevolved variant using quantitative real-time reverse transcription-PCR. Our results help to define the frequency with which microevolution can be expected in M. tuberculosis transmission chains. They provide a snapshot of the genetic background of these subtle rearrangements and identify an event in which IS6110-mediated microevolution in an isogenic background has functional consequences.

Additive effects of LPL, APOA5 and APOE variant combinations on triglyceride levels and hypertriglyceridemia: results of the ICARIA genetic sub-study.

BACKGROUND Hypertriglyceridemia (HTG) is a well-established independent risk factor for cardiovascular disease and the influence of several genetic variants in genes related with triglyceride (TG) metabolism has been described, including LPL, APOA5 and APOE. The combined analysis of these polymorphisms could produce clinically meaningful complementary information. METHODS A subgroup of the ICARIA study comprising 1825 Spanish subjects (80% men, mean age 36 years) was genotyped for the LPL-HindIII (rs320), S447X (rs328), D9N (rs1801177) and N291S (rs268) polymorphisms, the APOA5-S19W (rs3135506) and -1131T/C (rs662799) variants, and the APOE polymorphism (rs429358; rs7412) using PCR and restriction analysis and TaqMan assays. We used regression analyses to examine their combined effects on TG levels (with the log-transformed variable) and the association of variant combinations with TG levels and hypertriglyceridemia (TG > or = 1.69 mmol/L), including the covariates: gender, age, waist circumference, blood glucose, blood pressure, smoking and alcohol consumption. RESULTS We found a significant lowering effect of the LPL-HindIII and S447X polymorphisms (p < 0.0001). In addition, the D9N, N291S, S19W and -1131T/C variants and the APOE-epsilon4 allele were significantly associated with an independent additive TG-raising effect (p < 0.05, p < 0.01, p < 0.001, p < 0.0001 and p < 0.001, respectively). Grouping individuals according to the presence of TG-lowering or TG-raising polymorphisms showed significant differences in TG levels (p < 0.0001), with the lowest levels exhibited by carriers of two lowering variants (10.2% reduction in TG geometric mean with respect to individuals who were homozygous for the frequent alleles of all the variants), and the highest levels in carriers of raising combinations (25.1% mean TG increase). Thus, carrying two lowering variants was protective against HTG (OR = 0.62; 95% CI, 0.39-0.98; p = 0.042) and having one single raising polymorphism (OR = 1.20; 95% CI, 1.39-2.87; p < 0.001) or more (2 or 3 raising variants; OR = 2.90; 95% CI, 1.56-5.41; p < 0.001) were associated with HTG. CONCLUSION Our results showed a significant independent additive effect on TG levels of the LPL polymorphisms HindIII, S447X, D9N and N291S; the S19W and -1131T/C variants of APOA5, and the epsilon4 allele of APOE in our study population. Moreover, some of the variant combinations studied were significantly associated with the absence or the presence of hypertriglyceridemia.

Human pregnane X receptor is expressed in breast carcinomas, potential heterodimers formation between hPXR and RXR-alpha.

BACKGROUND The human pregnane X receptor (hPXR) is an orphan nuclear receptor that induces transcription of response elements present in steroid-inducible cytochrome P-450 gene promoters. This activation requires the participation of retinoid X receptors (RXRs), needed partners of hPXR to form heterodimers. We have investigated the expression of hPXR and RXRs in normal, premalignant, and malignant breast tissues, in order to determine whether their expression profile in localized infiltrative breast cancer is associated with an increased risk of recurrent disease. METHODS Breast samples from 99 patients including benign breast diseases, in situ and infiltrative carcinomas were processed for immunohistochemistry and Western-blot analysis. RESULTS Cancer cells from patients that developed recurrent disease showed a high cytoplasmic location of both hPXR isoforms. Only the infiltrative carcinomas that relapsed before 48 months showed nuclear location of hPXR isoform 2. This location was associated with the nuclear immunoexpression of RXR-alpha. CONCLUSION Breast cancer cells can express both variants 1 and 2 of hPXR. Infiltrative carcinomas that recurred showed a nuclear location of both hPXR and RXR-alpha; therefore, the overexpression and the subcellular location changes of hPXR could be considered as a potential new prognostic indicator.

Dectin-1 and DC-SIGN polymorphisms associated with invasive pulmonary Aspergillosis infection.

The recognition of pathogen-derived structures by C-type lectins and the chemotactic activity mediated by the CCL2/CCR2 axis are critical steps in determining the host immune response to fungi. The present study was designed to investigate whether the presence of single nucleotide polymorphisms (SNPs) within DC-SIGN, Dectin-1, Dectin-2, CCL2 and CCR2 genes influence the risk of developing Invasive Pulmonary Aspergillosis (IPA). Twenty-seven SNPs were selected using a hybrid functional/tagging approach and genotyped in 182 haematological patients, fifty-seven of them diagnosed with proven or probable IPA according to the 2008 EORTC/MSG criteria. Association analysis revealed that carriers of the Dectin-1(rs3901533 T/T) and Dectin-1(rs7309123 G/G) genotypes and DC-SIGN(rs4804800 G), DC-SIGN(rs11465384 T), DC-SIGN(7248637 A) and DC-SIGN(7252229 C) alleles had a significantly increased risk of IPA infection (OR = 5.59 95%CI 1.37-22.77; OR = 4.91 95%CI 1.52-15.89; OR = 2.75 95%CI 1.27-5.95; OR = 2.70 95%CI 1.24-5.90; OR = 2.39 95%CI 1.09-5.22 and OR = 2.05 95%CI 1.00-4.22, respectively). There was also a significantly increased frequency of galactomannan positivity among patients carrying the Dectin-1(rs3901533_T) allele and Dectin-1(rs7309123_G/G) genotype. In addition, healthy individuals with this latter genotype showed a significantly decreased level of Dectin-1 mRNA expression compared to C-allele carriers, suggesting a role of the Dectin-1(rs7309123) polymorphism in determining the levels of Dectin-1 and, consequently, the level of susceptibility to IPA infection. SNP-SNP interaction (epistasis) analysis revealed significant interactions models including SNPs in Dectin-1, Dectin-2, CCL2 and CCR2 genes, with synergistic genetic effects. Although these results need to be further validated in larger cohorts, they suggest that Dectin-1, DC-SIGN, Dectin-2, CCL2 and CCR2 genetic variants influence the risk of IPA infection and might be useful in developing a risk-adapted prophylaxis.

Genetic variations in drug-induced liver injury (DILI): resolving the puzzle.

Despite stringent requirements for drug development imposed by regulatory agencies, drug-induced liver injury (DILI) is an increasing health problem and a significant cause for failure to approve drugs, market withdrawal of commercialized medications, and adoption of regulatory measures. The pathogenesis is yet undefined, though the rare occurrence of idiosyncratic DILI (1/100,000–1/10,000) and the fact that hepatotoxicity often recurs after re-exposure to the culprit drug under different environmental conditions strongly points toward a major role for genetic variations in the underlying mechanism and susceptibility. Pharmacogenetic studies in DILI have to a large extent focused on genes involved in drug metabolism, as polymorphisms in these genes may generate increased plasma drug concentrations as well as lower clearance rates when treated with standard medication doses. A range of studies have identified a number of genetic variants in drug metabolism Phase I, II, and III genes, including cytochrome P450 (CYP) 2E1, N-acetyltransferase 2, UDP-glucuronosyltransferase 2B7, glutathione S-transferase M1/T1, ABCB11, and ABCC2, that enhance DILI susceptibility (Andrade et al., 2009; Agundez et al., 2011). Several metabolic gene variants, such as CYP2E1c1 and NAT2 slow, have been associated with DILI induced by specific drugs based on individual drug metabolism information. Others, such as GSTM1 and T1 null alleles have been associated with enhanced risk of DILI development induced by a large range of drugs. Hence, these variants appear to have a more general role in DILI susceptibility due to their role in reducing the cell's antioxidative capacity (Lucena et al., 2008). Mitochondrial superoxide dismutase (SOD2) and glutathione peroxidase 1 (GPX1) are two additional enzymes involved in combating oxidative stress, with specific genetic variants shown to enhance the risk of developing DILI

A multicenter study confirms CD226 gene association with systemic sclerosis-related pulmonary fibrosis.

INTRODUCTION CD226 genetic variants have been associated with a number of autoimmune diseases and recently with systemic sclerosis (SSc). The aim of this study was to test the influence of CD226 loci in SSc susceptibility, clinical phenotypes and autoantibody status in a large multicenter European population. METHODS A total of seven European populations of Caucasian ancestry were included, comprising 2,131 patients with SSc and 3,966 healthy controls. Three CD226 single nucleotide polymorphisms (SNPs), rs763361, rs3479968 and rs727088, were genotyped using Taqman 5'allelic discrimination assays. RESULTS Pooled analyses showed no evidence of association of the three SNPs, neither with the global disease nor with the analyzed subphenotypes. However, haplotype block analysis revealed a significant association for the TCG haplotype (SNP order: rs763361, rs34794968, rs727088) with lung fibrosis positive patients (PBonf = 3.18E-02 OR 1.27 (1.05 to 1.54)). CONCLUSION Our data suggest that the tested genetic variants do not individually influence SSc susceptibility but a CD226 three-variant haplotype is related with genetic predisposition to SSc-related pulmonary fibrosis.

Analysis of the association between CD40 and CD40 ligand polymorphisms and systemic sclerosis.

INTRODUCTION The aim of the present study was to investigate the possible role of CD40 and CD40 ligand (CD40LG) genes in the susceptibility and phenotype expression of systemic sclerosis (SSc). METHODS In total, 2,670 SSc patients and 3,245 healthy individuals from four European populations (Spain, Germany, The Netherlands, and Italy) were included in the study. Five single-nucleotide polymorphisms (SNPs) of CD40 (rs1883832, rs4810485, rs1535045) and CD40LG (rs3092952, rs3092920) were genotyped by using a predesigned TaqMan allele-discrimination assay technology. Meta-analysis was assessed to determine whether an association exists between the genetic variants and SSc or its main clinical subtypes. RESULTS No evidence of association between CD40 and CD40LG genes variants and susceptibility to SSc was observed. Similarly, no significant statistical differences were observed when SSc patients were stratified by the clinical subtypes, the serologic features, and pulmonary fibrosis. CONCLUSIONS Our results do not suggest an important role of CD40 and CD40LG gene polymorphisms in the susceptibility to or clinical expression of SSc.

The TT genotype of the STAT4 rs7574865 polymorphism is associated with high disease activity and disability in patients with early arthritis

BACKGROUND The number of copies of the HLA-DRB1 shared epitope, and the minor alleles of the STAT4 rs7574865 and the PTPN22 rs2476601 polymorphisms have all been linked with an increased risk of developing rheumatoid arthritis. In the present study, we investigated the effects of these genetic variants on disease activity and disability in patients with early arthritis. METHODOLOGY AND RESULTS We studied 640 patients with early arthritis (76% women; median age, 52 years), recording disease-related variables every 6 months during a 2-year follow-up. HLA-DRB1 alleles were determined by PCR-SSO, while rs7574865 and rs2476601 were genotyped with the Taqman 5' allelic discrimination assay. Multivariate analysis was performed using generalized estimating equations for repeated measures. After adjusting for confounding variables such as gender, age and ACPA, the TT genotype of rs7574865 in STAT4 was associated with increased disease activity (DAS28) as compared with the GG genotype (β coefficient [95% confidence interval] = 0.42 [0.01-0.83], p = 0.044). Conversely, the presence of the T allele of rs2476601 in PTPN22 was associated with diminished disease activity during follow-up in a dose-dependent manner (CT genotype = -0.27 [-0.56- -0.01], p = 0.042; TT genotype = -0.68 [-1.64- -0.27], p = 0.162). After adjustment for gender, age and disease activity, homozygosity for the T allele of rs7574865 in STAT4 was associated with greater disability as compared with the GG genotype. CONCLUSIONS Our data suggest that patients with early arthritis who are homozygous for the T allele of rs7574865 in STAT4 may develop a more severe form of the disease with increased disease activity and disability.

Evidence of new risk genetic factor to systemic lupus erythematosus: the UBASH3A gene

The ubiquitin associated and Src-homology 3 (SH3) domain containing A (UBASH3a) is a suppressor of T-cell receptor signaling, underscoring antigen presentation to T-cells as a critical shared mechanism of diseases pathogenesis. The aim of the present study was to determine whether the UBASH3a gene influence the susceptibility to systemic lupus erythematosus (SLE) in Caucasian populations. We evaluated five UBASH3a polymorphisms (rs2277798, rs2277800, rs9976767, rs13048049 and rs17114930), using TaqMan® allelic discrimination assays, in a discovery cohort that included 906 SLE patients and 1165 healthy controls from Spain. The SNPs that exhibit statistical significance difference were evaluated in a German replication cohort of 360 SLE patients and 379 healthy controls. The case-control analysis in the Spanish population showed a significant association between the rs9976767 and SLE (Pc = 9.9E-03 OR = 1.21 95%CI = 1.07-1.37) and a trend of association for the rs2277798 analysis (P = 0.09 OR = 0.9 95%CI = 0.79-1.02). The replication in a German cohort and the meta-analysis confirmed that the rs9976767 (Pc = 0.02; Pc = 2.4E-04, for German cohort and meta-analysis, respectively) and rs2277798 (Pc = 0.013; Pc = 4.7E-03, for German cohort and meta-analysis, respectively) UBASH3a variants are susceptibility factors for SLE. Finally, a conditional regression analysis suggested that the most likely genetic variation responsible for the association was the rs9976767 polymorphism. Our results suggest that UBASH3a gene plays a role in the susceptibility to SLE. Moreover, our study indicates that UBASH3a can be considered as a common genetic factor in autoimmune diseases.