Heparin Increases HLA-G Levels in Primary Antiphospholipid Syndrome

Objectives. The aim of this study was to investigate the HLA-G serum levels in Primary Antiphospholipid Syndrome (PAPS) patients, its impact on clinical and laboratory findings, and heparin treatment. Methods. Forty-four PAPS patients were age and gender matched with 43 controls. HLA-G serum levels were measured using an enzyme-linked immunosorbent assay (ELISA). Results. An increase in soluble HLA-G levels was found in patients compared to controls (3.35 (0 22.9) versus 1.1 (0 14), P = 0.017). There were no significant differences in HLA-G levels between patients with and without obstetric events, arterial thrombosis, venous thrombosis, or stroke. Sixty-six percent of patients were being treated with heparin. Interestingly, patients treated with heparin had higher HLA-G levels than ones who were not treated with this medication (5 (0-22.9) versus 1.8 (0-16) ng/mL, P = 0.038). Furthermore, patients on heparin who experienced obstetric events had a trend to increased HLA-G levels compared to patients who were not on heparin and did not have obstetric events (5.8 (0-22.9) versus 2 (0-15.2) ng/mL, P = 0.05). Conclusion. This is the first study to demonstrate that serum HLA-G levels are increased in APS patients. We also demonstrated that heparin increases HLA-G levels and may increase tolerance towards autoantigens.

Human leukocyte antigen-G 3 ' untranslated region polymorphisms are associated with better kidney allograft acceptance

Human leukocyte antigen-G (FILA-G) plays a well-recognized role in the modulation of the immune response, and HLA-G expression has been associated with increased graft survival and decreased rejection episodes. To investigate the role of the HLA-G 3' untranslated region (3'UTR) in renal transplantation, we evaluated several polymorphic sites (14-bp Del/Ins +3003T/C, +3010C/G, +3027C/A, +3035C/T, +3142G/C, and +3187A/G) in patients exhibiting or not exhibiting rejection episodes. A total of 104 patients (15 with acute and 48 with chronic rejection, and 41 with no rejection) and 142 healthy individuals were studied. HLA-G 3'UTR was typed by direct sequencing. The +3035C-C genotype was more frequent in patients exhibiting chronic rejection compared with healthy controls, and the +3035C-T genotype was less frequent in chronic rejection compared with patients without rejection (acute plus chronic) or compared with healthy controls. The +3187G-A genotype, in which the A allele is associated with increased mRNA degradation, showed increased frequency in the rejection group (acute plus chronic) when compared with healthy controls. The 14 base pair Deletion/Insertion genotype was marginally increased in patients with acute rejection. This is the first study to show associations among numerous polymorphic sites in the HLA-G 3'UTR in kidney allotransplantation, which may contribute to the understanding of HLA-G post-transcriptional mechanisms. (C) 2012 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

MTP -493G/T gene polymorphism is associated with steatosis in hepatitis C-infected patients

The reduction of hepatic microsomal transfer protein (MTP) activity results in fatty liver, worsening hepatic steatosis and fibrosis in chronic hepatitis C (CHC). The G allele of the MTP gene promoter, -493G/T, has been associated with lower transcriptional activity than the T allele. We investigated this association with metabolic and histological variables in patients with CHC. A total of 174 untreated patients with CHC were genotyped for MTP -493G/T by direct sequencing using PCR. All patients were negative for markers of Wilson’s disease, hemochromatosis and autoimmune diseases and had current and past daily alcohol intake lower than 100 g/week. The sample distribution was in Hardy-Weinberg equilibrium. Among subjects with genotype 1, 56.8% of the patients with fibrosis grade 3+4 presented at least one G allele versus 34.3% of the patients with fibrosis grade 1+2 (OR = 1.8; 95%CI = 1.3-2.3). Logistic regression analysis with steatosis as the dependent variable identified genotypes GG+GT as independent protective factors against steatosis (OR = 0.4, 95%CI = 0.2-0.8; P = 0.01). The results suggest that the presence of the G allele of MTP -493G/T associated with lower hepatic MTP expression protects against steatosis in our CHC patients.

The -308 tumour necrosis factor-alpha gene polymorphism predicts therapeutic response to TNFalpha-blockers in rheumatoid arthritis and spondyloarthritis patients

OBJECTIVE: To examine whether the G-to-A polymorphism at position -308 in the promoter of the tumour necrosis factor-alpha (TNFalpha) gene influences the therapeutic response to TNFalpha-blockers in patients with rheumatoid arthritis (RA), psoriatic arthritis (PsA) and ankylosing spondylitis (AS). METHODS: A total of 54 patients with RA, 10 with PsA and 22 with AS were genotyped by polymerase chain reaction for the -308 TNFalpha promoter polymorphism. They were treated with infliximab (n = 63), adalimumab (n = 10) or etanercept (n = 13). Clinical response was assessed after 24 weeks by the Disease Activity Score in 28 joints (DAS28) for RA and PsA, and the Bath Ankylosing Spondylitis Activity Index (BASDAI) for AS patients. RESULTS: All patients with the A/A genotype (n = 3, all RA) and two patients with the A/G genotype (AS) failed to respond to anti-TNF treatment. Irrespective of the underlying disease, moderate response (n = 44) was predominantly associated with the A/G genotype (A/G 18/22, G/G 4/22), whereas good response (n = 59) was exclusively seen in patients with the G/G genotype. The average improvement in the DAS28 score was 0.83 in the A/A, 1.50 in the A/G and 2.64 in the G/G group of RA and PsA patients (P < 0.0001). The BASDAI score in AS improved on average by 1.21 in the A/G and by 3.30 in the G/G group (P < 0.005). CONCLUSIONS: The data suggest that humans with a TNFalpha -308 G/G genotype are better responders to anti-TNFalpha treatment than those with A/A or A/G genotypes independent of the treated rheumatic disease (RA, PsA or AS).

Novel mitochondrial tRNA(Ile) m.4282A>G gene mutation leads to chronic progressive external ophthalmoplegia plus phenotype

BACKGROUND/AIM To investigate the underlying pathomechanism in a 33-year-old female Caucasian patient presenting with chronic progressive external ophthalmoplegia (CPEO) plus symptoms. METHODS Histochemical analysis of skeletal muscle and biochemical measurements of individual oxidative phosphorylation (OXPHOS) complexes. Genetic analysis of mitochondrial DNA in various tissues with subsequent investigation of single muscle fibres for correlation of mutational load. RESULTS The patient's skeletal muscle showed 20% of cytochrome c oxidase-negative fibres and 8% ragged-red fibres. Genetic analysis of the mitochondrial DNA revealed a novel point mutation in the mitochondrial tRNA(Ile) (MTTI) gene at position m.4282G>A. The heteroplasmy was determined in blood, buccal cells and muscle by restriction fragment length polymorphism (RFLP) combined with a last fluorescent cycle. The total mutational load was 38% in skeletal muscle, but was not detectable in blood or buccal cells of the patient. The phenotype segregated with the mutational load as determined by analysis of single cytochrome c oxidase-negative/positive fibres by laser capture microdissection and subsequent LFC-RFLP. CONCLUSIONS We describe a novel MTTI transition mutation at nucleotide position m.4282G>A associated with a CPEO plus phenotype. The novel variant at position m.4282G>A disrupts the middle bond of the D-stem of the tRNA(Ile) and is highly conserved. The conservation and phenotype-genotype segregation strongly suggest pathogenicity and is in good agreement with the MTTI gene being frequently associated with CPEO. This novel variant broadens the spectrum of MTTI mutations causing CPEO.

Mannose-binding lectin gene polymorphism predicts hospital admissions for COPD infections

Infection frequently causes exacerbations of chronic obstructive pulmonary disease (COPD). Mannose-binding lectin (MBL) is a pattern-recognition receptor that assists in clearing microorganisms. Polymorphisms in the MBL2 gene reduce serum MBL levels and are associated with risk of infection. We studied whether the MBL2 codon 54 B allele affected serum MBL levels, admissions for infective exacerbation in COPD and disease susceptibility. Polymorphism frequency was determined by PCR-RFLP in 200 COPD patients and 104 smokers with normal lung function. Serum MBL was measured as mannan-binding activity in a subgroup of 82 stable COPD patients. Frequency of COPD admissions for infective exacerbation was ascertained for a 2-year period. The MBL2 codon 54 B allele reduced serum MBL in COPD patients. In keeping, patients carrying the low MBL-producing B allele had increased risk of admission for infective exacerbation (OR 4.9, P-corrected = 0.011). No association of MBL2 genotype with susceptibility to COPD was detected. In COPD, serum MBL is regulated by polymorphism at codon 54 in its encoding gene. Low MBL-producing genotypes were associated with more frequent admissions to hospital with respiratory infection, suggesting that the MBL2 gene is disease-modifying in COPD. MBL2 genotype should be explored prospectively as a prognostic marker for infection risk in COPD.

The impact of the CACNA1C gene polymorphism on frontolimbic function in bipolar disorder

Genome-wide association studies in bipolar disorder (BD)1 have implicated a single-nucleotide polymorphism (rs1006737, G right arrow A) in the CACNA1C gene, which encodes for the alpha 1c (CAV1.2) subunit of the voltage-gated, L-type calcium channel. Neuroimaging studies of healthy individuals report that this risk allele modulates brain function within limbic (amygdala, anterior cingulate gyrus) and hippocampal regions during tasks of reward processing2, 3 and episodic memory. Moreover, animal studies suggest that the CaV1.2 L-type calcium channels influence emotional behaviour through enhanced neurotransmission via the lateral amygdala pathway. On the basis of this evidence, we tested the hypotheses that the CACNA1C rs1006737 risk allele will modulate neural responses within predefined prefrontal and subcortical regions of interest during emotional face processing and that this effect would be amplified in BD patients.

Effect of the kappa-casein gene polymorphism, breed and seasonality on physicochemical characteristics, composition and stability of bovine milk

The objective of this study was to evaluate the effect of genetic polymorphism of kappa-casein, breed and seasonality on the physicochemical characteristics, composition and stability of milk in commercial dairy herds. A total of 879 milk and blood samples were collected from 603 Holstein and 276 Girolando cows, obtained during rainy and dry seasons. Milk samples were analyzed to determine the physicochemical characteristics, composition and ethanol stability, while blood samples were subjected to polymerase chain reaction to identify the kappa-casein genotype. The frequencies of genotypes AA, AB and BB of k-casein were respectively, 66.83, 31.84 and 1.33% for Holstein, and 71.38, 27.90 and 0.72% for the Girolando cows, respectively. The A allele was more frequent than the B allele, both for Holstein (0.827 and 0.173) and Girolando cows (0.853 and 0.147), respectively. Cows of AB and BB genotypes showed a higher milk fat content compared to the AA genotype. There was an interaction between breed and seasonality on the concentration of milk urea with higher values for Holstein and Girolando cows in the rainy and dry season, respectively. The levels of lactose, total solids, crude protein, true protein, casein and the casein:true protein ratio were higher during the dry season, while during the rainy season, the somatic cell count and milk urea concentration were higher. There was no association between milk stability and k-casein genotypes, but Holstein cows showed higher milk stability than Girolando cows, and milk was more stable during the rainy season than during the dry season.

Association of mannose-binding lectin gene polymorphism but not of mannose-binding serine protease 2 with chronic severe aortic regurgitation of rheumatic etiology

N-Acetylglucosamine (GlcNAc) is the major immunoepitope of group A streptococcal cell wall carbohydrates. Antistreptococcal antibodies cross-reactive with anti-GlcNAc and laminin are present in sera of patients with rheumatic fever. The cross-reactivity of these antibodies with human heart valvular endothelium and the underlying basement membrane has been suggested to be a possible cause of immune-mediated valve lesion. Mannose-binding lectin (MBL) encoded by the MBL2 gene, a soluble pathogen recognition receptor, has high affinity for GlcNAc. We postulated that mutations in exon 1 of the MBL2 gene associated with a deficient serum level of MBL may contribute to chronic severe aortic regurgitation (AR) of rheumatic etiology. We studied 90 patients with severe chronic AR of rheumatic etiology and 281 healthy controls (HC) for the variants of the MBL2 gene at codons 52, 54, and 57 by using a PCR-restriction fragment length polymorphism-based method. We observed a significant difference in the prevalence of defective MBL2 alleles between patients with chronic severe AR and HC. Sixteen percent of patients with chronic severe AR were homozygotes or compound heterozygotes for defective MBL alleles in contrast to 5% for HC (P = 0.0022; odds ratio, 3.5 [ 95% confidence interval, 1.6 to 7.7]). No association was detected with the variant of the MASP2 gene. Our study suggests that MBL deficiency may contribute to the development of chronic severe AR of rheumatic etiology.

Mannan-binding lectin MBL2 gene polymorphism in chronic hepatitis C: association with the severity of liver fibrosis and response to interferon therapy

Hepatitis C virus (HCV) is a major cause of hepatic disease and of liver transplantation worldwide. Mannan-binding lectin (MBL), encoded by the MBL2 gene, can have an important role as an opsonin and complement activating molecule in HCV persistence and liver injury. We assessed the MBL2 polymorphism in 102 Euro-Brazilian patients with moderate and severe chronic hepatitis C, paired for gender and age with 102 HCV seronegative healthy individuals. Six common single nucleotide polymorphisms in the MBL2 gene, three in the promoter (H/L, X/Y and P/Q) and three in exon 1 (A, the wild-type, and B, C or D also known as O) were evaluated using real-time polymerase chain reaction with fluorescent hybridization probes. The concentration of MBL in plasma was measured by enzyme-linked immunosorbent assay. The frequency of the YA/YO genotype was significantly higher in the HCV patients compared with the controls (P = 0.022). On the other hand, the genotypes associated with low levels of MBL (XA/XA, XA/YO and YO/YO) were decreased significantly in the patients with severe fibrosis (stage F4), when compared with the patients with moderate fibrosis (stage F2) (P = 0.04) and to the control group (P = 0.011). Furthermore, MBL2 genotypes containing X or O mutations were found to be associated with non-responsiveness to pginterferon and ribavirin treatment (P = 0.023). MBL2 polymorphisms may therefore be associated not only with the development of chronic hepatitis C, but also with its clinical evolution and response to treatment.

Association of Interferon-gamma gene polymorphism (+874 T/A) with systemic sclerosis

Aperfeicoamento de Pessoal de Nivel Superior (CAPES)

Effect of a thrombin receptor (protease-activated receptor 1, PAR-1) gene polymorphism in chronic hepatitis C liver fibrosis

Background and Aim: Tissue injury leads to activation of coagulation and generation of thrombin. Inhibition of thrombin receptor protease-activated receptor 1 (PAR-1) has been shown to reduce liver fibrosis in animals. This study aimed to evaluate the effect of PAR-1 gene polymorphism on rate of liver fibrosis (RF) in chronic hepatitis C. Methods: Polymorphisms studied: C > T transition 1426 bp upstream of translation start site (-1426C/T), 13 bp repeat of preceding -506 5`-CGGCCGCGGGAAG-3` sequence (-506I/D), and A > T transversion in intervening sequence (IVS) 14 bp upstream of exon-2 start site (IVS-14A/T). A total of 287 European and 90 Brazilian patients were studied. Results: 1426C/T polymorphism: There was a trend to higher RF in patients with the TT genotype (P = 0.06) and an association between genotype CC and slow fibrosis (P = 0.03) in Europeans. In males, RF was significantly higher in those with the TT genotype compared to CT (P = 0.003) and CC (P = 0.007). There was a significant association between TT and fast fibrosis (P = 0.04). This was confirmed in an independent cohort of Brazilians where RF was higher in TT than in CC (P = 0.03). Analysis of -506I/D showed no difference in RF and distribution of slow/fast fibrosis among different genotypes in both populations. Analysis of IVS-14A/T showed no difference between genotypes. Conclusion: In conclusion, these findings suggest that PAR-1 receptor polymorphisms influence the progression of liver fibrosis.

Liver HLA-G expression is associated with multiple clinical and histopathological forms of chronic hepatitis B virus infection

As the mechanisms leading to the persistence of hepatitis B virus (HBV) infection are poorly understood and as the histocompatibility leucocyte antigen (HLA)-G is well described as a tolerogenic molecule, we evaluated HLA-G expression in 74 specimens of HBV liver biopsies and in 10 specimens obtained from previously healthy cadaver liver donors. HBV specimens were reviewed and classified by the METAVIR score, and HLA-G expression was assessed by immunohistochemistry. No HLA-G expression was observed in control hepatocytes. In contrast, 57 (77%) of 74 HBV specimens showed soluble and membrane-bound HLA-G expression in hepatocytes, biliary epithelial cells or both. No associations between the intensity of HLA-G expression and patient age or gender, HBeAg status, severity of liver fibrosis, and grade of histological findings were observed. Although significance was not reached (P = 0.180), patients exhibiting HLA-G expression presented a higher median HBV DNA viral load (105 copies/mL) than those who did not express HLA-G (103.7 copies/mL). These results indicate that HLA-G is expressed in most cases of chronic HBV infection in all stages and may play a role in the persistency of HBV infection.

Correlation between beta-2-glycoprotein I gene polymorphism and anti-beta-2 glycoprotein I antibodies in patients with multibacillary leprosy

Antiphospholipid antibodies, such as anti-beta 2-glycoprotein I (beta 2GPI), are present in multibacillary leprosy (MB) patients; however, MB patients do not usually present with antiphospholipid antibody syndrome (APS), which is characterized by thromboembolic phenomena (TEP). Rare cases of TEP occur in leprosy patients, but the physiopathology of this condition remains unclear. In this case-control study, we examined whether single-nucleotide polymorphisms (SNPs) of the beta 2GPI gene contributed to the risk of leprosy and APS co-morbidity. SNPs Ser88Asn, Leu247Val, Cys306Gly and Trp316Ser were identified in 113 Brazilian leprosy patients. Additionally, anti-beta 2GPI antibodies and plasma concentrations of beta 2GPI were quantified. The Ser88Asn, Cys306Gly and Trp316Ser SNPs were not risk factors for APS in leprosy. A higher frequency of Val/Val homozygosity was observed in leprosy patients compared to controls (36 vs. 5%; P < 0.001). Forty-two percent of MB and 17% of paucibacillary leprosy patients were positive for anti-beta 2GPI IgM (P = 0.014). There was no correlation between SNP Ser88Asn or Cys306Gly and anti-beta 2GPI antibody levels. In MB patients with positive anti-beta 2GPI IgM, the frequency of Val/Val homozygosity was higher than in controls (32 vs. 15%; P = 0.042). The frequency of the mutant allele Ser316 was higher in MB patients with positive rather than negative anti-beta 2GPI IgM levels (6 vs. 0%; P = 0.040) and was greater than in the control group (6 vs. 1%; P = 0.034). The studied polymorphisms did not influence the plasma concentrations of beta 2GPI. These results suggest that Leu247Val and Trp316Ser SNPs may represent genetic risk factors for anti-beta 2GPI antibody production in MB patients.