Tumour necrosis factor-alpha and heat-shock protein 70-2 gene polymorphisms in a family with rheumatoid arthritis

OBJECTIVES: The aim of this work was to investigate the prevalence of TNF-alpha-308 polymorphism among the 29 members of a family with RA and the association between the MHC-linked biallelic HSP70-2 gene and the TNF-alpha polymorphism. Five of the members with RA were diagnosed by using the revised 1987 ACR criteria, and 1 member suffered from SLE. METHODS: The variations in the TNF-alpha and the HSP70-2 genotypes were analyzed by PCR-RFLP, using NcoI and PstI restriction enzymes. RESULTS: Two of the 29 members were homozygotes for allele A, 18 were heterozygotes (TNF A/G) and 9 of them were homozygotes for allele G. Nineteen of the 29 were heterozygotes for HSP70-2 (A/G), 10 of them were homozygotes for the G allele, and none were homozygotes for allele A. Four of the 5 the RA patients carried the A allele for TNF-alpha all 5 were heterozygotes for HSP70-2 genotypes. CONCLUSION: The carriage of the A allele for TNF-alpha of -308 SNP in 4 of the 5 RA patients, and the high prevalence (68.0%) of TNF A allele carriers in this family confirms the important role of this candidate gene in the pathomechanism of RA, and might be of prognostic value for future clinical observations. Further, to test for association a much larger set of genetically independent patients and controls is needed.     

The CD14-260 C --> T promoter polymorphism co-segregates with the tumor necrosis factor-alpha (TNF-alpha)-308 G --> A polymorphism and is associated with the interleukin-1 beta (IL-1 beta) synthesis capacity of human leukocytes

Genetic variations contribute to the interindividual variance in the cytokine response to endotoxin. The gene of tumor necrosis factor-alpha (TNF-alpha) carries a polymorphism at position -308 of the promoter, consisting of a G/A exchange. To further elucidate the inherited mechanisms influencing cytokine levels, healthy human blood donors were studied. Genotyping for the TNF-alpha -308 and the CD14 -260 C/T promoter polymorphisms was carried out by real-time polymerase chain reaction assay using specific fluorescence-labelled hybridisation probes. A human whole blood assay was used to study the leukocyte TNF-alpha and IL-1 beta synthesis capacity upon endotoxin stimulation. We found a linkage disequilibrium between the TNF-alpha -308 G/A and the CD14 -260 C/T polymorphisms (p = 0.043). The CD14 -260 polymorphism was associated with IL-1 beta levels (p = 0.033) and higher values were found in C homozygotes. No association was found between the CD14 -260 genotypes or the TNF-alpha -308 - CD14 -260 genotypes and the TNF-alpha response.     

The -308 polymorphism in the promoter region of the tumor necrosis factor-alpha (TNF-alpha) gene and ex vivo lipopolysaccharide-induced TNF-alpha expression in patients with aggressive periodontitis and/or type 1 diabetes mellitus

Several single-nucleotide polymorphisms (SNPs) have been identified in the TNF-alpha gene promoter. The transition G-->A at position -308 generates the TNF-alpha1 (G/G) and TNF-alpha2 (G/A or A/A) alleles, where the polymorphic TNF-alpha2 allele is associated with a high, in vitro TNF-alpha expression and an increased susceptibility to diverse illnesses. Here we study the association of the -308 TNF-alpha SNP with the susceptibility for developing aggressive periodontitis (AP), AP combined with type 1 diabetes mellitus (DM) and DM. We also explore the TNF-alpha capability expression and the presence of the -308 polymorphism. For this purpose we recruited 27 individuals with AP (AP+ group), 27 individuals with AP combined with DM (AP+/DM+ group), and 27 individuals with DM without signs of periodontitis upon clinical examination (DM+ group). The control group was comprised of 30 subjects. Genotyping for TNF-alpha promoter was performed by PCR-RFLP analysis. For TNF-alpha expression we used a blood culture system.     

Identification of a Biomarker in Cerebrospinal Fluid for Neuronopathic Forms of Gaucher Disease

Gaucher disease, a recessive inherited metabolic disorder caused by defects in the gene encoding glucosylceramidase (GlcCerase), can be divided into three subtypes according to the appearance of symptoms associated with central nervous system involvement. We now identify a protein, glycoprotein non-metastatic B (GPNMB), that acts as an authentic marker of brain pathology in neurological forms of Gaucher disease. Using three independent techniques, including quantitative global proteomic analysis of cerebrospinal fluid (CSF) in samples from Gaucher disease patients that display neurological symptoms, we demonstrate a correlation between the severity of symptoms and GPNMB levels. Moreover, GPNMB levels in the CSF correlate with disease severity in a mouse model of Gaucher disease. GPNMB was also elevated in brain samples from patients with type 2 and 3 Gaucher disease. Our data suggest that GPNMB can be used as a marker to quantify neuropathology in Gaucher disease patients and as a marker of treatment efficacy once suitable treatments towards the neurological symptoms of Gaucher disease become available.     

Effect of ScrF I polymorphism in the 2nd intron of the HMGCR gene on lipid-lowering response to simvastatin in Chinese diabetic patients

OBJECTIVE: To investigate whether ScrF I polymorphism in the 2nd intron of the HMG-COA reductase gene (HMGCR) influences serum lipid levels and whether this polymorphism affects the efficiency of the cholesterol lowering HMG-CoA reductase inhibitor, simvastatin. METHODS: One hundred sixty-eight patients with type 2 diabetes mellitus (T2DM) prospectively received simvastatin as a single-agent therapy (20mg day-1 p.o.) for 12 weeks. Serum lipid levels were determined before and after simvastatin treatment. Genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). RESULTS: Subjects with the AA homozygotes had significantly higher serum very low-density lipoprotein cholesterol (VLDL-C) levels than those with the aa homozygotes. In addition, in 168 patients with T2DM who took 20mg simvastatin, the VLDL-C lowering effect by simvastatin in subjects with the aa homozygotes was significantly lower than in those with the Aa heterozygotes and AA homozygotes. CONCLUSIONS: Simvastatin treatment significantly decreased plasma lipids in all patients (P<0.01). Importantly, we demonstrate that ScrF I polymorphism of the HMGCR gene in patients with T2DM groups is associated with significant elevation of serum VLDL-C levels. Subjects with the AA homozygotes had significantly higher serum high VLDL-C levels than those with the Aa heterozygotes and aa homozygotes (AA: 2.18+/-0.51; Aa: 2.04+/-0.59, aa: 1.86+/-0.43, P<0.05 for comparison among three genotypes and P<0.01 for difference between AA and aa). Furthermore, this polymorphism tends to show an enhanced response to an HMG-CoA reductase inhibitor in terms of the cholesterol-lowering effect. In 168 patients with T2DM who took 20mg simvastatin, the VLDL-C lowering effect by simvastatin in subjects with the AA homozygotes was significantly lower than in those with the Aa heterozygotes and aa homozygotes (the reduction in serum VLDL-C levels; 37.03+/-5.67 versus 28.97+/-4.96, P<0.01; 34.62+/-5.87 versus 28.97+/-4.96, P<0.05). These results suggest that the HMGCR gene may serve as a modifier gene for hypercholesterolemia in Chinese diabetic patients.     

在中国汉族人群中，以家系传递分析肿瘤坏死因子a-308A/G单核苷酸多 态性与抽动秽语综合征

目的:探讨在中国汉族人群中,肿瘤坏死因子a(TNF-alpha,TNF-a)基因启动子区-308A/G单核苷酸多态性与抽动秽语综合征(Tourette syndrome,TS)的遗传易感性。方法:91例TS患者及其父母组成的核心家系成员经聚合酶链式反应-限制性片段长度多态性(polymerase chain reaction-restriction fragment lengthpolymor phism,PCR-RFLP)方法进行基因分型,评估所有研究对象的TNF-a-308A/G位点的等位基因频率和基因型频率的分布,进行传递不平衡检验(transmission disequilibrium test,TDT),单体型相对风险(haplotype-based haplotyp erelativerisk,HHRR),单体型风险(haplotype relative riskHRR)的研究。结果:TS患者及其父母的等位基因分布经Hardy-Weinberg(H-W)平衡检验显示符合遗传平衡法则。(x2<3.84;P>0.05)TDT、HHRR和HRR研究结果显示该多态性位点的等位基因频率和基因型频率均不存在传递不平衡。结论:我们的数据表明肿瘤坏死因子a启动子区-308A/G单核苷酸多态性位点不是中国汉族人群TS的易感基因位点。     

TNF-alpha promoter, Nod2 and toll-like receptor-4 polymorphisms and the in vivo and ex vivo response to endotoxin

Humans exhibit substantial inter-individual differences in TNF-alpha production upon endotoxin stimulation. To determine to what extent the lipopolysaccharide-induced TNF-alpha production capacity in vivo and ex vivo is determined by polymorphisms in toll-like receptor-4 (TLR4), the TNF-alpha promoter region and Nod2, we screened for two TLR4 polymorphisms, a Nod2 polymorphism and the TNF-alpha promoter polymorphisms. We measured the perioperative endotoxemia and TNF-alpha production and the TNF-alpha production capacity of each patient in a whole-blood stimulation assay using blood drawn before anesthesia, using various LPS concentrations, in patients undergoing elective cardiac surgery. This operation represents a major surgical trauma associated with ischemia-reperfusion injury and triggers an endotoxemia and profound inflammatory response. In vivo TNF-alpha production was positively correlated with the level of endotoxemia after aortic declamping; thus TNF-alpha levels were higher in patients having endotoxemia compared to patients without endotoxemia. This correlation was observed in patients with any of the genotypes studied, and did not differ between the various genotypes. In vivo TNF-alpha levels correlated best with those ex vivo after stimulation with 1000 ng/mL LPS, and the estimated maximal TNF-alpha release capacity. Subjects with the wild-type TLR4 gene had similar levels of TNF-alpha upon LPS stimulation ex vivo as compared with patients carrying Asp299Gly and/or the Thr399Ile TLR4 polymorphism. Our results indicate that polymorphisms in the TLR4 receptor, Nod2 and TNF-alpha promoter region are not strongly associated with in vivo and ex vivo TNF-alpha production capacity upon endotoxin stimulation. This suggests that in this model of natural LPS release, the variation between individuals in TNF-alpha release can only modestly be determined by genetic background (TNF-alpha promoter, Nod2 and TLR4) of the individual.     

Genetic polymorphism of interleukin-8 (IL-8) is associated with Helicobacter pylori-induced duodenal ulcer

BACKGROUND AND AIMS: Helicobacter pylori infection almost invariably causes chronic gastritis, but only a proportion of the infected subjects develop peptic ulcers. The local inflammation associated with H. pylori infection is characterized by an increased production of the proinflammatory cytokines IL-1-B, IL-6, IL-8 and TNF-alpha. Since such cytokine production is often determined by the genetic polymorphism of regions regulating cytokine gene expression, we investigated the relationship between TNF-alpha and IL-8 polymorphisms and the development of duodenal ulcer disease. We also sought a correlation between the promoter polymorphism of the lipopolysaccharide (LPS) receptor CD14 and the formation of peptic ulcer, because CD14 plays a crucial role in the initiation of the cytokine cascade. METHODS: Genomic DNA extracted from the peripheral blood of 69 patients with H. pylori-positive duodenal ulcer disease and 47 H. pylori-positive healthy controls was analyzed for TNF-alpha -308 promoter polymorphism by RFLP, and for IL-8 -251 polymorphism by ARMS. Genetic polymorphism within the promoter of the CD14 gene was identified using the LightCycler instrument via melting point analysis. RESULTS: No significant correlation could be revealed between the TNF-alpha and CD14 promoter polymorphisms and the clinical outcome of H. pylori infection. The IL-8 A/T heterozygote mutant variant was detected with a significantly higher frequency (65.22%) among the ulcer patients than among the healthy, H. pylori-positive blood donors (36.17%), while the frequency of the normal allelic genotype (TT) was significantly higher in the control group (44.6% vs 15.9%). CONCLUSION: Analysis of the genetic predisposition to enhanced cytokine production revealed a significant association only for the IL-8 polymorphism. This observation draws attention to the possible importance of IL-8 polymorphism as a genetic predisposing factor in the pathomechanism of H. pylori-induced duodenal ulcer disease, and to the relative protection from duodenal ulcer disease that is associated with the TT genotype.     

Type 1 Gaucher Disease: Molecular,Biochemical, and Clinical Characterization of Patients from Northern Portugal

We report the study of 16 catholic type 1 Gaucher disease patients originating from a well-defined region in the north of Portugal where a relatively high incidence is observed. The patients were screened for mutations: 3060G → A, 5841A → G, 5976C → G, and 6433T → G, which enabled the identification of 27 of the 32 mutated alleles. Four different genotypes were identified, namely 5841G/6433C (n = 6). 5841G/5841G (n = 5), 5841G/? (n = 4). and 6433C/? (n = 1). All but one of the patients carried at least one 5841G mutated allele, making its frequency 62.5%, which is similar to that described for Ashkenazi Jewish patients. The 5841G homozygotes presented an overall milder clinical profile, whereas no clear genotype/phenotype correlation could he established for heterozygous patients. On the basis of residual glucocerebrosidase activity, no distinction could be made between 5841G homozygotes and 5841G/6433C compound heterozygotes. Patients that had at least one 5841G allele (encoding the Ser 370 mutated enzyme) all presented a cell-type-specific residual glucocerebrosidase activity as well as an increased molecular activity when measured in the presence of the physiological activators.     

Influence of the -308 TNF-alpha and -174 IL-6 polymorphisms on lipid profile in Mexican subjects

Polymorphisms in the promoter region of several cytokine genes have been associated with differential cytokine production. Several reports indicate that polymorphisms in the tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) genes are associated with lipid abnormalities. The aim of this study was to identify the genotype frequencies for -308G/ATNF-alpha and -174G/CIL-6 polymorphisms in Mexican subjects and to determine the influence of both polymorphisms on serum lipid levels. Serum lipid concentrations were measured in 100 healthy Mexican subjects. Screening of the -308G/ATNF-alpha and -174G/CIL-6 polymorphisms was performed in all participants using PCR-RFLPs. Genotype frequency for TNF-alpha polymorphism was: 87% GG and 13% GA, whereas IL-6 polymorphism was: 77% GG and 23% GC. The polymorphism frequencies obtained in this study were significantly different to Caucasian populations. High serum levels of triglycerides and total cholesterol were associated with GG genotype of the -308 TNF-alpha polymorphism, as well as low HDL-c levels, but no association was found between the -174 IL-6 polymorphism and serum lipid concentrations. We observed a significant association of the -308 TNF-alpha polymorphism with lipid profile in Mexican subjects. Furthermore, the genotype distribution of -308 TNF-alpha and -174 IL-6 polymorphisms in Mexican Mestizo population similar to populations in different continents may be due to our genetic background influenced by the mixture of Spaniards, Indian and black genes.     

G-308A TNF-alpha polymorphism is associated with an increased risk of invasive cervical cancer

Cervical cancer is initiated by high-risk human papillomaviruses (HPV-16 and HPV-18), but an effective immune response may control the progression of this disease. Tumor necrosis factor-alpha (TNF-alpha) is a pro-inflammatory cytokine, that has been implicated in several cancers. In a case-control study, we evaluated the association between the G-308A TNF-alpha promoter polymorphism and the risk for invasive cervical cancer (ICC). TNF-alpha polymorphism was analyzed by PCR-RFLP and confirmed by sequencing. DNA was obtained from blood samples of 439 individuals, including 195 patients with ICC and 244 normal healthy controls. According to our results, women carrying the A allele present a twofold increased risk of developing ICC (p=0.006; OR=1.88; 95% CI [1.20-2.94]). In conclusion, our study suggests that the presence of the high producer allele -308A in the TNF-alpha gene appears to be associated with an increased risk for the development of ICC.     

在中国汉族人群中,以家系传递分析肿瘤坏死因子a-308A/G单核苷酸多态性与抽动秽语综合征

目的：探讨在中国汉族人群中,肿瘤坏死因子a（TNF-alpha,TNF-a）基因启动子区-308A/G单核苷酸多态性与抽动秽语综合征（Tourette syndrome,TS）的遗传易感性。方法：91例TS患者及其父母组成的核心家系成员经聚合酶链式反应-限制性片段长度多态性（polymerase chain reaction-restriction fragment lengthpolymor phism,PCR-RFLP）方法进行基因分型,评估所有研究对象的TNF-a-308A/G位点的等位基因频率和基因型频率的分布,进行传递不平衡检验（transmission disequilibrium test,TDT）,单体型相对风险（haplotype-based haplotyp erelativerisk,HHRR）,单体型风险（haplotype relative riskHRR）的研究。结果：TS患者及其父母的等位基因分布经Hardy-Weinberg（H-W）平衡检验显示符合遗传平衡法则。（x2〈3.84;P〉0.05）TDT、HHRR和HRR研究结果显示该多态性位点的等位基因频率和基因型频率均不存在传递不平衡。结论：我们的数据表明肿瘤坏死因子a启动子区-308A/G单核苷酸多态性位点不是中国汉族人群TS的易感基因位点。     

Frequencies of allelic polymorphism of endothelial NO-synthase gene in patients with acute coronary syndrome in Ukrainian population

Endothelial NO-synthase (eNOS) gene allelic polymorphism in 221 patients with acute coronary syndrome and in 83 practically healthy people was determined. It was shown that interrelations of normal homozygotes, heterozygotes and pathologic homozygotes in T/C promoter polymorphism analysis accout 48%, 36% and 16% correspondingly (in control--48%, 46%, 6%; P < 0.05 by chi2-test); in G894 --> T polymorphism ofexon 7 analysis--34%, 58%, 8% (in control--29%, 67%, 4%; P > 0.05), and in determination of 4a/4b polymorphism of intron 4--64.5%, 31% and 4.5% (in control--62.5%, 32.5%, 5%; P > 0.05). Obtained data show that eNOS C/C promoter variant is a risk factor of acute coronary syndrome in Ukrainian population.     

Polymorphism of the serotonin 2A receptor gene (5HTR2A) and personality traits

The interindividual variation of temperament features (such as anxiety, neuroticism, harm avoidance) is determined, in particular, by allele polymorphism of genes involved in serotonin metabolism and has earlier been associated with the insertion/deletion polymorphism of the serotonin transporter gene. Polymorphic alleles of the serotonin 2A receptor gene (5HTR2A) were tested for association with personality traits assessed with several tests. The T102C and A1438G polymorphisms were associated with a variation in emotionality, activity, and sociability, which are integral characteristics of temperament. With each polymorphism, differences were significant only between heterozygotes and homozygotes. Carriers of T102C genotype A1/A2 displayed a lower level of anxiety-related traits, a higher score on scale Hypomania, and a lower score on scale Social Introversion and were assumed to have higher activity and sociability. Carriers of A1428G genotype A/G differed from homozygotes G/G in having a lower level of social introversion and a lower score on scale No close friends, which testified to higher sociability of heterozygotes. Thus, the polymorphic alleles of SHTR2A proved to be associated with personality traits in mentally healthy people.     

Non-existence of a tight association between a 444leucine to proline mutation and phenotypes of Gaucher disease: high frequency of a NciI polymorphism in the non-neuronopathic form

Summary A ⁴⁴⁴leucine to proline mutation detected by a NciI polymorphism in the human glucocerebrosidase gene was studied to investigate the correlation of the three clinical phenotypes of Gaucher disease with this mutation in 11 Japanese patients with Gaucher disease (type I, 8 patients; type II, 1 patient; type III, 2 patients) and to determine the feasibility of the use of genomic probe DNA for carrier detection and prenatal diagnosis in 8 Japanese families with Gaucher disease and agreeable to family study (type I, 6 families; type III, 2 families). The homoallelic ⁴⁴⁴leucine to proline mutation was found only in patients with type I disease. Of the 8 type I patients, 5 had the homoallelic mutation and 2 had one mutant allele. One patient with type II disease did not have this mutant allele. Of the 2 type III patients, one had a single mutant allele whereas the other exhibited no mutation of this kind. These results suggest that the ⁴⁴⁴leucine to proline mutation is very common in the type I (non-neuronopathic form) disease and is not tightly associated only with neuronopathic types of Gaucher disease in Japanese patients. These findings seem to conflict with others showing that this mutation is partially responsible for the occurrence of neuronopathic Gaucher disease. Thus, the NciI polymorphism will not be useful for the diagnosis of subtypes of Gaucher disease. Carrier detection was feasible in three families with type I disease of the 8 families analyzed by the NciI polymorphism.     

Association of β-fibrinogen gene -148C/T and -455G/A polymorphisms and coronary artery disease in Chinese population: A Meta analysis

The purpose of our study was to evaluate the correlation between the b-fibrinogen gene –148C/T and –455G/A polymorphisms and susceptibility to coronary artery disease in the Chinese population using a meta–analytic approach. Eligible studies about this correlation were identified by searching the PubMed, EMBASE, and CNKI databases. Of the 13 identified, 7 (with 1488 cases and 1234 controls) involved the –148C/T polymorphism and 9 (with 1023 cases and 1081 controls) involved the –455G/A polymorphism. No publication bias was detectable and heterogeneity testing found significant differ-ences between the ORs for both groups of studies. The combined OR for the 7 studies on susceptibility to coronary artery disease in –148T allele carriers compared to the –148C/C wild-type homozygotes was 1.31 (95%CI: 0.94－1.84, P=0.11). The combined OR for the 9 studies on susceptibility to coronary artery disease in –455A allele carriers compared to the –455G/G wild-type homozygotes was 1.75 (95%CI: 1.24－2.46, P=0.001). Our results suggest the absence of an association between the b-fibrinogen gene –148C/T polymorphism and susceptibility to coronary artery disease and the possibility that –455G/A polymorphism (in particular, allele A) increases susceptibility to this disease in the Chinese population.     

Complex alleles of the acid beta-glucosidase gene in Gaucher disease.

Gaucher disease is inherited in an autosomal recessive manner and is the most prevalent lysosomal storage disease. Gaucher disease has marked phenotypic variation and molecular heterogeneity, and seven point mutations in the acid beta-glucosidase (beta-Glc) gene have been identified. By means of sequence-specific oligonucleotides (SSO), mutation 6433C has been detected homozygously in neuronopathic type 2 (acute) and type 3 (subacute) patients, as well as in children with severe visceral involvement who are apparently free of neuronopathic disease. To investigate the molecular basis for this puzzling finding, amplified beta-Glc cDNAs from 6433C homozygous type 2 and type 3 Gaucher disease patients were cloned and sequenced. The Swedish type 3 Gaucher disease patient was truly homozygous for alleles only containing the 6433C mutation. In comparison, the type 2 patient contained a singly mutated 6433C allele and a "complex" allele with multiple discrete point mutations (6433C, 6468C, and 6482C). Each of the mutations in the complex allele also was present in the beta-Glc pseudogene. SSO hybridization of 6433C homozygotes revealed that both type 2 patients contained additional mutations in one allele, whereas the 6433C alone was detected in both type 3 and in young severe type 1 Gaucher disease patients. These results suggest that the presence of the complex allele influences the severity of neuronopathic disease in 6433C homozygotes and reveal the central role played by the pseudogene in the formation of mutant alleles of the beta-Glc gene. Analysis of additional cDNA clones also identified two new alleles in a type 3 patient, emphasizing the molecular heterogeneity of neuronopathic Gaucher disease.     

TNF-alpha and HLA-DR genotyping as potential prognostic markers in pulmonary sarcoidosis.

We have analyzed the HLA-DRB1 alleles and -308 TNF-alpha gene polymorphism in 78 sarcoidosis patients and 50 controls. The sarcoidosis group as a whole did not show any significant correlation with the TNF-A or the HLA-DR alleles compared to the control group. However, the patient subgroups of L?fgren and non-L?fgren sarcoidosis exhibited significant allele associations. In the L?fgren patient group, the TNF-A2 and the HLA-DR3 alleles were represented significantly higher, with a highly significant relative risk resulting from the presence of the TNF-A2 or the HLA-DR3 allele or both. In the non-L?fgren patient group, the phenotype expressing HLA-DR2 and lacking TNF-A2 was significantly higher than in the L?fgren patient group. Due to these significant genetic differences in the subgroups of L?fgren and non-L?fgren sarcoidosis patients, we conclude that the genotyping of these two loci (-308 TNF-alpha promoter polymorphism and HLA-DR) may be of prognostic value for the course of disease in sarcoidosis.