Bruno Filipe Bettencourt

Role of human leukocyte antigen, killer-cell immunoglobulin-like receptors, and cytokine gene polymorphisms in leptospirosis

Leptospirosis is an emerging zoonotic disease caused by pathogenic species of the genus Leptospira. It has a broad range of clinical presentations in humans. Although progress has been made in the characterization of the host immune system factors that may affect disease progression and outcome, to date few reports have addressed the role of genetic polymorphisms in the susceptibility to leptospirosis. In this work a group of patients with a history of leptospiral infection and a control group were compared for polymorphisms in the human leukocyte antigen (HLA), in killer-cell immunoglobulin-like receptors (KIR), and in cytokine genes. Alleles in the HLA-A and -B loci were associated with susceptibility, as were the class I haplotype A*01-B*08-Cw*07 and the 8.1 ancestral haplotype (A*01-B*08-Cw*07-DRB1*03-DQB1*02). Single nucleotide polymorphisms in the interleukin (IL)-4 and IL-4Ralpha genes also had significantly higher frequencies in the patient group. No association was reported between KIR gene profile and leptospirosis. This work highlights the importance of using genetic polymorphisms to better understand the mechanisms involved in the immune response to leptospirosis.

Protective effect of an ERAP1 haplotype in ankylosing spondylitis: investigating non-MHC genes in HLA-B27-positive individuals

OBJECTIVE The association of non-MHC genes with AS has been recently suggested. We aimed to investigate the association of the ERAP1, IL23R and TNFSF15 regions and the susceptibility to and protection from AS in HLA-B27-positive individuals. METHODS A total of 200 unrelated AS patients and 559 healthy unrelated subjects, all HLA-B27 positive, were tested. Twenty single nucleotide polymorphisms (SNPs) were investigated in and near IL23R (nine SNPs), in ERAP1 (five SNPs) and in TNFSF15 (six SNPs). RESULTS ERAP1 rs30187 [odds ratio (OR) = 1.5, P = 4.7 × 10(-3)] had the strongest association with AS susceptibility. A protective effect was found in three of the ERAP1 SNPs: rs17482078 (OR = 0.7, P = 2.8 × 10(-2)), rs10050860 (OR = 0.7, P = 2.3 × 10(-2)), rs2287987 (OR = 0.6, P = 1.3 × 10(-2)). The ERAP1 haplotype rs17482078/rs10050860/rs30187/rs2287987-CCTT showed an association with AS susceptibility (P = 6.8 × 10(-3)) and a protective effect was identified in rs17482078/rs10050860/rs30187/rs2287987-TTCC (P = 3.1 × 10(-2)). Significant association with AS susceptibility was found in one IL23R marker (rs1004819, P = 4.3 × 10(-2), OR = 1.3). No associations were observed in the TNFSF15 region. CONCLUSION The identification of a new protection haplotype in ERAP1 and the lack of association of the TNFSF15 region can provide new insights into the understanding of the mechanisms underlying the susceptibility to and protection from AS.

The effects of vitrification on gene expression in mature mouse oocytes by nested quantitative PCR

PurposeThis study was conducted on the effects of vitrification cryotop method on gene expression of mature oocytes in Mus musculus.MethodsTranscript analyses of three mouse genes, namely Mater, Hook1 and Sod1, were performed upon non-vitrified and vitrified oocytes with different concentrations of dimethyl sulfoxide (DMSO) and ethylene glycol (EG),15%: 7.5% DMSO + 7.5% EG, and 30%: 15% DMSO + 15% EG, using cryotop following normalization of transcripts with Hprt1 by nested quantitative PCR.ResultsVitrification caused down-regulation of Mater and Hook1 and up-regulation of Sod1 when lower concentrations of cryoprotectants were used as opposed to the control group. The relative expression of Sod1 in vit2 (30% v/v) was significantly higher than vit1 (15% v/v). Quantitative transcript analysis of Mater and Hook1 for the vit2 condition failed to produce any data. Survival rates were the same for both vitrification treatments and significantly lower than control group.ConclusionsAlthough vit1 treatment had lower survival rate compared to control group, it demonstrated better stability comparing to vit2 based on the transcript analysis.

Fine Mapping of a Major Histocompatibility Complex in Ankylosing Spondylitis Association of the HLA-DPA1 and HLA-DPB1 Regions

OBJECTIVE To investigate the potential association of major histocompatibility complex (MHC) markers other than HLA-B27 with ankylosing spondylitis (AS). METHODS A total of 603 patients with AS and 542 healthy control subjects, all of whom were HLA-B27 positive, were selected for this study based on clinical criteria. First, high-density genotyping across the MHC region (2,360 single-nucleotide polymorphisms [SNPs]) was performed in a cohort of 191 patients and 241 control subjects. After a fine-mapping study, 5 SNPs from the HLA-DPA1/DPB1 region were validated in a second cohort of 412 patients with AS and 301 healthy control subjects. RESULTS Seventeen SNPs located within or near the HLA-DPA1 and HLA-DPB1 loci showed association with AS (P = 1.38 × 10⁻⁵ to 0.05). In addition, multimarker tests, both linkage disequilibrium and sliding windows, showed association of some groups of adjacent SNPs within the HLA-DPA1/DPB1 region with AS (P = 1.0 × 10⁻⁴ to 3.96 × 10⁻⁷). We validated the association by genotyping 5 SNPs from the DPA1/DPB1 region in an additional cohort and obtained P values from 6.42 × 10⁻⁵ to 0.01 in the analysis of the combined cohorts. Subtyping analysis of HLA-DPA1 and HLA-DPB1 showed that HLA-DPA1*01:03, A1*02:01, and B1*13:01 were the subtypes most susceptible to AS. CONCLUSION HLA markers and linkage disequilibrium blocks near HLA-DPA1 and HLA-DPB1 are statistically associated with AS. We identified a region located around the HLA-DPA1 and HLA-DPB1 loci associated with AS, another region within the MHC that is different from HLA-B27.

Evaluation of two methods for computational HLA haplotypes inference using a real dataset

BackgroundHLA haplotype analysis has been used in population genetics and in the investigation of disease-susceptibility locus, due to its high polymorphism. Several methods for inferring haplotype genotypic data have been proposed, but it is unclear how accurate each of the methods is or which method is superior. The accuracy of two of the leading methods of computational haplotype inference – Expectation-Maximization algorithm based (implemented in Arlequin V3.0) and Bayesian algorithm based (implemented in PHASE V2.1.1) – was compared using a set of 122 HLA haplotypes (A-B-Cw-DQB1-DRB1) determined through direct counting. The accuracy was measured with the Mean Squared Error (MSE), Similarity Index (IF) and Haplotype Identification Index (IH).ResultsNone of the methods inferred all of the known haplotypes and some differences were observed in the accuracy of the two methods in terms of both haplotype determination and haplotype frequencies estimation. Working with haplotypes composed by low polymorphic sites, present in more than one individual, increased the confidence in the assignment of haplotypes and in the estimation of the haplotype frequencies generated by both programs.ConclusionThe PHASE v2.1.1 implemented method had the best overall performance both in haplotype construction and frequency calculation, although the differences between the two methods were insubstantial. To our knowledge this was the first work aiming to test statistical methods using real haplotypic data from the HLA region.

Novel candidate blood-based transcriptional biomarkers of Machado-Joseph disease.

Machado‐Joseph disease (or spinocerebellar ataxia type 3) is a late‐onset polyglutamine neurodegenerative disorder caused by a mutation in the ATXN3 gene, which encodes for the ubiquitously expressed protein ataxin‐3. Previous studies on cell and animal models have suggested that mutated ataxin‐3 is involved in transcriptional dysregulation. Starting with a whole‐transcriptome profiling of peripheral blood samples from patients and controls, we aimed to confirm abnormal expression profiles in Machado‐Joseph disease and to identify promising up‐regulated genes as potential candidate biomarkers of disease status.

TRAF1/C5 but Not PTPRC Variants Are Potential Predictors of Rheumatoid Arthritis Response to Anti-Tumor Necrosis Factor Therapy

Background. The aim of our work was to replicate, in a Southern European population, the association reported in Northern populations between PTPRC locus and response to anti-tumor necrosis factor (anti-TNF) treatment in rheumatoid arthritis (RA). We also looked at associations between five RA risk alleles and treatment response. Methods. We evaluated associations between anti-TNF treatment responses assessed by DAS28 change and by EULAR response at six months in 383 Portuguese patients. Univariate and multivariate linear and logistic regression analyses were performed. In a second step to confirm our findings, we pooled our population with 265 Spanish patients. Results. No association was found between PTPRC rs10919563 allele and anti-TNF treatment response, neither in Portuguese modeling for several clinical variables nor in the overall population combining Portuguese and Spanish patients. The minor allele for RA susceptibility, rs3761847 SNP in TRAF1/C5 region, was associated with a poor response in linear and logistic univariate and multivariate regression analyses. No association was observed with the other allellic variants. Results were confirmed in the pooled analysis. Conclusion. This study did not replicate the association between PTPRC and the response to anti-TNF treatment in our Southern European population. We found that TRAF1/C5 risk RA variants potentially influence anti-TNF treatment response.

HLA-E, HLA-F and HLA-G — The Non-Classical Side of the MHC Cluster

Traditionally, the MHC is divided into the classes containing groups of genes with related functions; the MHC class I and II genes encode for human leukocyte antigens (HLA), proteins that are displayed on the cell surface. In humans, MHC class I molecules com‐ prise the classical (class I-a) HLA-A, -B, and -C, and the non-classical (class I-b) HLA-E, F, -G and – H (HFE) molecules (Pietra et al., 2009). Both categories are similar in their mechanisms of peptide binding and presentation and in the induced T-cell responses (Rodgers and Cook, 2005). The most remarkable feature of MHC class I-b molecules is their highly conserved nature (van Hall et al., 2010). In contrast with class Ia molecules they have been under a distinct selective pressure, exhibiting very low levels of allelic polymor‐ phism (Strong et al., 2003). Classical MHC class I gene transcription is mediated by several cis-acting regulatory elements, in the proximal promoter region (Gobin and van den Elsen, 2000). Those elements determine the constitutive and cytokine induced expression levels of the molecule (Gobin and van den Elsen, 2000).

Novel fast reverse transcriptase PCR assay for molecular detection of human influenza A (H1N1) virus.

A novel subtype of influenza A pandemic virus, A(H1N1)v, was recently reported by the Centers for Disease Control and Prevention (CDC) (Atlanta, GA) and WHO in April 2009 ([1][1]). Infection most commonly results in a mild respiratory tract infection, but there are reports of severe cases with

Genetic Predictors of Poor Prognosis in Portuguese Patients with Juvenile Idiopathic Arthritis: Data from Reuma.pt

Introduction. This study aimed to assess the genetic determinants of poor outcome in Portuguese patients with juvenile idiopathic arthritis (JIA). Methods. Our study was conducted in Reuma.pt, the Rheumatic Diseases Portuguese Register, which includes patients with JIA. We collected prospectively patient and disease characteristics and a blood sample for DNA analysis. Poor prognosis was defined as CHAQ/HAQ >0.75 at the last visit and/or the treatment with biological therapy. A selected panel of single nucleotide polymorphisms (SNPs) associated with susceptibility was studied to verify if there was association with poor prognosis. Results. Of the 812 patients with JIA registered in Reuma.pt, 267 had a blood sample and registered information used to define “poor prognosis.” In univariate analysis, we found significant associations with poor prognosis for allele A of TNFA1P3/20 rs6920220, allele G of TRAF1/C5 rs3761847, and allele G of PTPN2 rs7234029. In multivariate models, the associations with TRAF1/C5 (1.96 [1.17–3.3]) remained significant at the 5% level, while TNFA1P3/20 and PTPN2 were no longer significant. Nevertheless, none of associations found was significant after the Bonferroni correction was applied. Conclusion. Our study does not confirm the association between a panel of selected SNP and poor prognosis in Portuguese patients with JIA.