(GT)N dinucleotide repeat polymorphism of haem oxygenase-1 promotor region is not associated with inflammatory bowel disease risk or disease course

Haem oxygenase-1 (HO-1) up-regulation was suggested to reduce mucosal tissue damage in inflammatory bowel disease (IBD) and an up-regulation of HO-1 expression in patients with Crohn's disease (CD) and ulcerative colitis (UC) was demonstrated. A HO-1 gene promoter microsatellite (GT)(n) dinucleotide repeat polymorphism was associated with regulation of HO-1 in response to inflammatory stimuli. We therefore hypothesized that IBD patients might segregate into phenotypes with high or low HO-1 inducibility. Ethylenediamine tetraacetic acid blood samples were obtained from 179 CD patients, 110 UC patients and 56 control patients without inflammation. Genomic DNA was purified and the 5'-flanking region of the HO-1 gene containing the (GT)(n) dinucleotide repeat was amplified. Polymerase chain reaction (PCR) products were purified and the length of the PCR fragments was analysed. The number of (GT)(n) repeats in the population studied ranged from 13 to 42. The distribution of the allele frequencies was comparable in patients and controls for both the short and the long alleles. The frequencies of short-, middle- and long-sized alleles were not changed among the groups studied. No correlation was found between IBD and microsatellite instability detected in five individals. Our data indicate that (GT)(n) dinucleotide repeats of the HO-1 promotor region have no significance for the pathophysiology and disease course of IBD.     

Dinucleotide repeat polymorphism in intron II of human Toll-like receptor 2 gene and susceptibility to rheumatoid arthritis

Human Toll-like receptors (TLRs) participate in innate immune response and signal the activation of adaptive immunity. The presence of a functional intronic polymorphism consisting of guanine-thymine repeats in TLR2 gene was recently reported. Here, we investigated a dinucleotide repeat polymorphism in intron II of TLR2 in Korean patients with rheumatoid arthritis (RA). The numbers of guanine-thymine [(GT)(n)] repeats in intron II of the TLR 2 gene were counted in 183 patients with RA and in 148 healthy controls, using the gene scanning technique. We classified alleles into two subclasses for further analysis, 12-16 GT repeats (S allele) and 17-28 repeats (L allele). By subgroup analysis, we also examined whether the S allele is associated with the presence of shared epitope (SE), rheumatoid factor (RF), joint erosion and extra-articular complications. S-allele frequency was significantly increased in patients with RA than in healthy controls [30.3% vs. 23.0%, P = 0.03, or 1.46, 95% confidence interval (CI) 1.03-2.07], and genotypes containing S alleles were more frequent in patients with RA than in healthy controls (54.4% vs. 46.5%. P = 0.04, or 1.57, 95% CI 1.01-2.42). A skewed S-allele distribution was not found to be related to the presence of SE. Subgroup analysis showed no genotypic or allele frequency differences between patients with/without RF, joint erosion, or extra-articular complications. Genotype containing shorter GT repeats in intron II of the TLR2 gene may confer susceptibility to RA in Koreans.     

Association between late-onset Alzheimer's disease and microsatellite polymorphisms in intron II of the human toll-like receptor 2 gene

The amyloid beta protein (Aβ) deposits in the brains of patients with Alzheimer's disease (AD) are closely associated with innate immune responses that were assumed to play a pivotal role in the pathogenesis of AD. Toll-like receptor 2 (TLR2) is thought to contribute to Aβ clearance. Studies have reported the presence and functional implications of guanine-thymine (GT) repeat microsatellite polymorphisms in intron II of the human TLR2 gene. The present study evaluated the association of the microsatellite polymorphism and sporadic late-onset AD (LOAD) in the Han Chinese population. The numbers of (GT) repeats were counted in 137 AD patients and in 137 non-AD control subjects, using polymerase chain reaction and genescan analysis. The alleles were divided into three subclasses: (GT)16 or less as the S allele, (GT)17 to (GT)22 as the M allele, and (GT)23 or more as the L allele. Patients with AD had more S alleles (P < 0.001; odds ratio (OR) = 2.32; 95% confidence interval (CI) = 1.57–3.42) and fewer L alleles (P = 0.02; OR = 0.66; 95% CI = 0.46–0.93) than did healthy controls. Genotypes SS and SM were more common, whereas ML and SL were less common in patients with AD. In subgroup analyses, the genotypes including S alleles were associated with an increased risk of LOAD (OR = 2.05, 95% CI = 1.26–3.34), and this association was influenced by the presence of APOE ?4 alleles. This study demonstrates an association of microsatellite polymorphisms in intron II of the human TLR2 gene with risk for LOAD in Han Chinese.     

Association of SLC11A1 promoter polymorphisms with the incidence of autoimmune and inflammatory diseases: a meta-analysis

Solute carrier family 11 member a1 (SLC11A1) exerts pleiotropic effects on macrophage function. Expression of SLC11A1 is regulated by a (GT)(n) microsatellite promoter repeat polymorphism of which nine alleles have been described. Enhanced activation of macrophages, associated with increased expression from allele 3, may be functionally linked to the development of autoimmune and inflammatory diseases. Conversely, low expression, driven by allele 2, may afford resistance. We have performed a meta-analysis to determine the association of SLC11A1 promoter alleles 2 and 3 with autoimmunity and inflammation. A random effects pooled odds ratio (OR) of 1.04 (95% confidence interval [CI]=0.20) for allele 3 suggested a weak association of this allele with an increased risk of disease. Calculation of the OR in the absence of asymmetry yielded a random effects pooled OR of 0.88 (95% CI=0.66), effectively reversing the above association. A fixed effects pooled OR of 0.90 (95% CI=0.24) was obtained for allele 2, suggesting a weak predominance of disease in the absence of this allele. Application of the trim-and-fill method resulted in a fixed effects OR of 0.80 (95% CI=0.22), thus strengthening this association. Associations of allele 3 with autoimmune and inflammatory diseases reported in several association studies may be attributable to some form of bias amongst published results.     

Evidence of a linkage disequilibrium between polymorphisms in the human estrogen receptor alpha gene and their relationship to bone mass variation in postmenopausal Italian women

Bone mineral density (BMD), the major determinant of osteoporotic fracture risk, has a strong genetic component. The discovery that inactivation of estrogen receptor alpha (ERalpha) gene is associated with low BMD indicated ERalpha as a candidate gene for osteoporosis. We have investigated the role of three ERalpha gene polymorphisms [intron 1 PVU:II and XBA:I RFLPs and TA dinucleotide repeat polymorphism 5' upstream of exon 1] in 610 postmenopausal women. There was a strong linkage disequilibrium between intron 1 polymorphic sites and also between these sites and the microsatellite (TA)(n) dinucleotide polymorphism, with a high degree of coincidence of the short TA alleles and the presence of PVU:II and XBA:I restriction sites. No significant relationship between intron 1 RFLPs and BMD was observed. A statistically significant correlation between (TA)(n) repeat allelic variants and lumbar BMD was observed (P = 0.04, ANCOVA), with subjects with a low number of repeats (TA < 15) showing the lowest BMD values. We observed a statistically significant difference in the mean +/- SD number of TA repeats between analyzed women with a vertebral fracture (n = 73) and the non-fracture group, equivalent to 2.9 (95% CI 1.56-5.72) increased fracture risk in women with a low number of repeats (TA < 15). We conclude that in this large population sample the (TA)(n) dinucleotide repeat polymorphism at the 5' end of the ERalpha gene accounts for part of the heritable component of BMD and might prove useful in the prediction of vertebral fracture risk in postmenopausal osteoporosis.     

Compound microsatellite repeats: practical and theoretical features.

Most linkage and population genetic studies that use microsatellites assume that the polymorphism observed at these loci is due simply to variation in the number of units of a single repeat. Variation is far more complex, however, for the numerous microsatellites that contain interruptions within the repeat or contain more than one type of repeat. We observed that for D18S58, a compound microsatellite containing (CG)(m), as well as (CA)(n) repeats, the apparent length of certain alleles varied between genotyping experiments. Similar results were obtained with other (CG)(m)-(CA)(n) repeats. Sequencing demonstrated that the D18S58 alleles demonstrating variable mobility contained longer (CG)(m) stretches than those alleles whose length did not appear to vary between experiments. These results suggest that (CG)(m) repeats, which are frequently present in compound human microsatellites, are prone to form an unusually stable secondary structure. We discuss the relative frequency of different classes of compound microsatellites identified through database searches, as well as their patterns of sequence and variation. Further characterization of such variation is important for elucidating the origin, mutational processes, and structure of these widely used, but incompletely understood, sequences.     

Discrimination of suballeles present at the TNFd microsatellite locus using induced heteroduplex analysis

Polymorphism at the TNFd locus has been implicated in a number of disease association studies. The TNFd locus consists of three regions of (GA)(n) repeats separated by an imperfect repeat of two guanine bases. TNFd alleles are genotyped by the number of repeats in the first (GA)(n) repeat region, and until now the second repeat region had been thought to be nonpolymorphic. We report the existence of suballeles present within the TNFd microsatellite locus, detected using induced heteroduplex generator (IHG) technology. These alleles cannot be detected using conventional typing strategies as they represent altered distribution of the (GA)(n) repeats or sequence variation within the repeat. The suballeles affect the frequencies of the conventional d3 and d4 alleles leading to significantly altered allele frequencies. Some studies have associated the d3 and d4 alleles with disease outcome. We re-analysed one such study cohort using IHG technology and demonstrated a high proportion of incorrectly assigned TNFd3 alleles.     

Association between microsatellite polymorphisms in intron II of the human Toll-like receptor 2 gene and nontuberculous mycobacterial lung disease in a Korean population

This study evaluated the association between the guanine-thymine (GT) repeat polymorphism in intron II of the Toll-like receptor 2 (TLR2) gene and lung disease caused by nontuberculous mycobacteria (NTM). Polymerase chain reaction and gene scans were used to determine the numbers of GT repeats for 193 patients with the nodular bronchiectatic form of NTM lung disease, including 110 patients with Mycobacterium avium-intracellulare (MAC) infection, 82 patients with Mycobacterium abscessus infection, and 1 patient with co-infection of both organisms. These values were compared with the results for 191 controls. Genotypes with shorter GT repeats were more common among patients with NTM lung disease (50.8 vs 37.7%, p = 0.01). In the subgroup analysis, genotypes that included S alleles were more common in the patients with MAC lung disease (53.6%, p = 0.01, OR, 1.91; 95% CI, 1.16, 3.16) than in healthy controls, whereas this difference was not statistically significant in patients with M. abscessus lung disease (47.6%, p = 0.13). In conclusion, these results suggest that the GT repeat microsatellite polymorphisms in intron II of the human TLR2 gene contribute to the development of NTM lung disease, especially MAC lung disease, in a Korean population.     

Genetic variation and evolutionary stability of the FMR1 CGG repeat in six closed human populations

In an attempt to understand the allelic diversity and mutability of the human FMR1 CGG repeat, we have analyzed the AGG substructure of this locus within six genetically-closed populations (Mbuti pygmy, Baka pygmy, R. surui, Karitiana, Mayan, and Hutterite). Most alleles (61/92 or 66%) possessed two AGG interspersions occurring with a periodicity of one AGG every nine or ten CGG repeats, indicating that this pattern is highly conserved in all human populations. Significant differences in allele distribution were observed among the populations for rare variants possessing fewer or more AGG interruptions than the canonical FMR1 CGG repeat sequence. Comparisons of expected heterozygosity of the FMR1 CGG repeat locus with 30 other microsatellite loci, demonstrated remarkably similar levels of polymorphism within each population, suggesting that most FMR1 CGG repeat alleles mutate at rates indistinguishable from other microsatellite loci. A single allele (1 out of 92) was identified with a large uninterrupted tract of pure repeats (42 pure CGG triplets). Retrospective pedigree analysis indicated that this allele had been transmitted unstably. Although such alleles mutate rapidly and likely represent evolving premutations, our analysis suggests that in spite of the estimated frequency of their occurrence, these unstable alleles do not significantly alter the expected heterozygosity of the FMR1 CGG repeat in the human population. © 1996 Wiley-Liss, Inc.     

IDDM2 and the polymorphism of the human tyrosine hydroxylase (hTH) gene in African Americans with type-1 diabetes

In this study, we investigate the polymorphic microsatellite repeat (TCAT)n, in the insulin gene region that has been associated with susceptibility to type-1 diabetes in some Caucasian populations. The microsatellite repeat polymorphism begins at base pair 1,170 in intron 1 of the hTH gene, which is located on the short arm of chromosome 11. This study is the first to investigate the association of this microsatellite repeat polymorphism in African-American type-1 diabetes patients and controls. The predicted amplified sequence was 254 bp. We found five alleles among African Americans in the Washington, DC area. The alleles were labeled K5 (244 bp), K4 (248 bp), K3 (252 bp), K2 (256 bp), and K1 (260 bp), and heterozygosity was greater than 0.75. The most frequent allele of the hTH microsatellite repeats was K5 (248 bp) with a frequency 0.62 in controls and 0.66 in type-1 diabetes patients, which did not differ significantly. Although the largest allele was more frequent in controls, the difference was not statistically significant. The five alleles of the hTH microsatellite generated 15 different genotypes. The most frequent genotype in controls and patients was K5/K4, whose frequencies were 0.19 and 0.17, respectively. No significant differences in genotype frequencies were found between type-1 diabetes patients and controls. This data shifts the focus from hTH to the VNTR at the insulin gene for IDDM2, the second major candidate gene for type-1 diabetes.     

Association between SLC11A1 (formerly NRAMP1) and the risk of sarcoidosis in Poland

Sarcoidosis (SA) is a systemic granulomatous disorder of unknown etiology characterized by T helper 1-type inflammatory responses at sites of disease with signs of B cell hyperactivity. Like rheumatoid arthritis and diabetes, an infectious etiology has frequently been postulated but no single infectious trigger definitively identified. Polymorphic alleles at SLC11A1 have previously been associated with susceptibility to both the putative infectious agents and to these autoimmune disorders. We therefore investigated its candidacy as a genetic determinant of SA in Poland in an association-based study comparing 86 SA patients with 85 tuberculosis (TB) patients and 93 control subjects. The functional promoter (GT)(n) polymorphism and four of 10 other single nucleotide or insertion/deletion polymorphisms genotyped across SLC11A1 were informative in our sample. Consistent with previous autoimmune disease studies, allele 3 at the functional (GT)(n) promoter region repeat polymorphism was significantly associated with SA when compared with healthy controls (odds ratio 1.68; 95% CI: 1.01-2.81; P=0.04) or with TB patients (odds ratio 1.69; 95% CI: 1.042-0.78; P=0.03).     

Analysis of the polymorphic (GT)(n) repeat at the dopamine beta-hydroxylase gene in Spanish patients affected by schizophrenia

The presence of a polymorphic (GT)(n) repeat, a microsatellite repeat, at the human dopamine beta-hydroxylase (DBH) gene had been previously investigated in healthy people and in schizophrenic patients. The different DBH genotypes had been found to be associated to different DBH biochemical function, but no differences were found in the allelic and genotype frequencies between schizophrenic and control groups. To further clarify the potential involvement of the variation at the DBH gene in schizophrenia we have studied the DBH (GT)(n) repeat in a sample of 47 Spanish schizophrenic patients, in their healthy relatives (n = 72), and in a control population (n = 74). We have been able to identify five different variants of the DBH gene (A1, A2, A3, A4, A5) in the different groups. Subsequent statistical analysis revealed that the genotypes as well as the allele frequencies did not differ significantly among schizophrenic patients and the control population. Interestingly, the allelic variant A2 and the genotype A4/A2 were significantly more frequent in schizophrenic patients as compared with their healthy relatives. However, the association of the A2 allele with schizophrenia was not supported by the haplotype relative risk analysis of transmitted versus nontransmitted alleles. Therefore, although it will be important to extend the present analysis in a larger sample of schizophrenic patients and controls, our results suggest that the (GT)(n) does not seem to play a major role in the genetics of schizophrenia at least in this group of Spanish schizophrenic patients. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 96:88-92, 2000.     

Analysis of a GT microsatellite in the promoter of the foxp3/scurfin gene in autoimmune diseases

The aim of this study was to assess the possible association of the functional (GT)(n) microsatellite polymorphism in the FOXP3 gene with predisposition to several autoimmune diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), ulcerative colitis (UC), Crohn's disease, and celiac disease. We analyzed a case-control cohort composed of 231 SLE patients, 293 RA patients, 528 inflammatory bowel disease (354 Crohn's disease patients and 260 UC patients) patients, 103 celiac disease patients, and 274 healthy controls ethnically matched. Genotyping of (GT)(n) microsatellite was performed by polymerase chain reaction (PCR)-based method combined with fluorescent technology. We found no evidence for association of this polymorphism between controls and these autoimmune disease patients. Additionally, no differences in the genotype and allele distribution were found when patients were stratified according to clinical manifestation. The (GT)(n) microsatellite of the FOXP3 gene may not play a relevant role in the susceptibility to SLE, RA, inflammatory bowel disease, and celiac disease in our population.     

Association of interferon-γ and interferon regulatory factor 1 polymorphisms with asthma in a family-based association study in Taiwan

BACKGROUND: Asthma is a multi-factorial disorder caused by complex interactions between genetic and environmental factors. IFN-gamma and IFN regulatory factor 1 (IRF-1) affect Th1/Th2 cytokine balance, and influence the differentiation of Th2 cells, which influence the development of asthma. OBJECTIVE: This study investigated CA repeats polymorphism of the IFN-gamma gene and GT repeats polymorphism of the IRF-1 gene, which may predispose individuals to asthma pathogenesis. METHODS: In the present study, we used the transmission/disequilibrium test (TDT) to investigate the relationship between asthma and the IFN-gamma and IRF-1 polymorphisms by studying 348 subjects composed of 232 parents and 116 asthmatic children. RESULTS: For global TDT test, IFN-gamma CA repeats and IRF-1 GT repeat polymorphisms showed a significant association with asthma in children (P=0.009 and 0.017, respectively). We demonstrated that 13 CA repeats (138 bp) of IFN-gamma gene and 11 GT repeats (306 bp) of IRF-1 gene are significantly preferentially transmitted to asthmatic children (T/NT=89/61, chi2=8.43, P<0.005 and T/NT=75/49, chi2=8.18, P<0.005, respectively). The offspring will have an increased risk of asthma when their parents transmit IFN-gamma 13 CA repeats (OR=1.83, P=0.009) and IRF1 11 GT repeats (OR=1.88, P=0.007) to them. But we observed that the IFN-gamma and IRF-1 polymorphisms are not associated with IgE concentrations. CONCLUSION: These findings provide strong evidence of which IFN-gamma CA repeat and IRF-1 GT repeat polymorphisms influence the risk of asthma for children in Taiwan.     

Influence of Polymorphism within the Heme Oxygenase-I Promoter on Overall Survival and Transplantation-Related Mortality after Allogeneic Stem Cell Transplantation

Aside from major and minor histocompatibility antigens, genetic polymorphisms of various donor and host genes have been found to be risk factors for graft-versus-host disease and transplantation-related mortality (TRM). The heme oxygenase I (HO-I) protein has been implicated in regulating inflammatory response and has been described as a “protective gene” in solid organ transplantation. In humans, the promoter region displays length polymorphism due to a variable number of GT repeats. Individuals exhibiting 29 or fewer GT repeats express higher levels of HO-I on cellular stress compared with individuals with 30 or more GT repeats. We retrospectively analyzed length polymorphisms of 92 donor–host pairs undergoing allogeneic stem cell transplantation. Our findings demonstrate that mainly donor polymorphism leading to high expression of HO-1 (<30 GT repeats) on stress signals is associated with reduced overall survival, and that TRM is significantly increased in this group. This reduction in survival was most prominent when unrelated donors were used. Polymorphisms of the recipient HO-1 genes did not influence posttransplantation outcomes. We conclude that HO-1 polymorphism represents a new genetic risk factor for TRM and overall survival.     

New GABAA receptor alpha5 subunit gene polymorphism that may confound genotyping.

We report the discovery of a new GABAA receptor alpha5 subunit gene polymorphism close to the polymorphism described by Glatt et al. (GT)5GCGTGC(GT)21. This new polymorphism is of great importance, because it means that non-denaturing acrylamide gels used to separate the different alleles of the polymorphism described by Glatt et al. cannot distinguish an allele with the sequence: (GT)4GCGTGC(GT)n from another allele with the sequence: (GT)4(GCGT)4GC(GT)(n-6). These gel fragments are separated by size, which would be the same in these two cases. An alternative would be to use an analysis method that can detect base changes, for instance, single strand conformation polymorphism (SSCP) or denaturing gradient gel electrophoresis (DGGE).     

Polymorphisms of human CD19 gene: possible association with susceptibility to systemic lupus erythematosus in Japanese

CD19 regulates the signaling for B lymphocyte development, activation and proliferation. In mice, CD19 deficiency and overexpression were shown to result in hypogammaglobulinemia and autoantibody production, respectively. In the present study, we screened for the polymorphisms of CD19, and examined the detected polymorphisms for the association with rheumatoid arthritis (RA), Crohn's disease and systemic lupus erythematosus (SLE). Two SNPs, c.705G>T (P235P and IVS14-30C>T, were decreased (P = 0.0096 and P = 0.028, respectively), in SLE. A GT repeat polymorphism, c.*132(GT)(12-18), was detected within the 3'-untranslated region, and individuals with > or =15 times repeat was significantly increased in the independent two groups of Japanese SLE patients (P = 0.011 and P = 0.035, respectively); the overall difference between total SLE and controls was striking (P = 0.0061). No association was observed for RA and Crohn's disease. In addition, no variations other than the common polymorphisms were detected in four patients with common variable immunodeficiency, the phenotype of which resembles CD19 deficient mice. In Caucasian SLE families, this GT repeat polymorphism was rare. CD19 mRNA level in the isolated peripheral blood B lymphocytes was lower in individuals possessing (GT)(15-18) alleles compared with those without these alleles, both in controls and in SLE patients; however, the difference did not reach statistical significance. These results suggested that either the slight reduction in the CD19 mRNA level associated with the elongation of GT repeat, or an allele of another locus in linkage disequilibrium with CD19 (GT)(15-18), may be associated with susceptibility to SLE in Japanese.