Influence of the polymorphism of the DUSP14 gene on the expression of immune-related genes and development of pulmonary tuberculosis.

Recently, a genome-wide screening identified a functional single-nucleotide polymorphism in dual-specificity phosphatase 14 gene (DUSP14), which was associated with pulmonary tuberculosis (TB) in a West African study. DUSP14 regulates T-cell proliferation and cytokine production in a negative way via dephosphorylation and inactivation of key signaling molecules. The aim of this study is to further explore the possible significance of the DUSP14 polymorphism. Total RNA was extracted from the whole blood of 109 healthcare workers (HCWs) in Vietnam and subjected to quantitative reverse-transcription PCR for DUSP14 and 20 immune-related genes. DUSP14 rs1051838 was genotyped in 502 new pulmonary TB patients and 506 healthy controls. Among disease-free individuals (HCWs), T-helper type-1 (Th1)-related genes, interferon-gamma receptor 2 (IFNGR2) and signal transducer and activator of transcription-1 (STAT1) mRNA levels significantly increased as the number of A alleles of rs1051838 increased, whereas the DUSP14 mRNA level tended to decrease. The AA genotype was associated with protection against active TB in younger patients (⩽45 years old, OR=0.63, 95% CI 0.44-0.90). Our results suggest that a low-expression genotype of DUSP14 accompanied by high transcript levels of Th1 immune-related genes may confer protection against early TB development.

Association of TLR polymorphisms with development of tuberculosis in Indonesian females.

Tuberculosis (TB) is caused by Mycobacterium tuberculosis and is a major cause of morbidity and mortality worldwide. Many candidate genes have been investigated for a possible association with TB. Toll-like receptors (TLRs) are known to play important roles in human innate immune systems. Polymorphisms in and functions of TLRs have been investigated to identify associations with specific infectious diseases, including TB. Here, we examined whether single-nucleotide polymorphisms (SNPs) in TLRs and genes in TLR signaling were associated with TB susceptibility in Indonesian and Vietnamese populations. A statistically significant association was observed between TB susceptibility in a classified Indonesian female group and rs352139, an SNP located in the intron of TLR9, using the genotype (P = 2.76E-04) and recessive (AA vs AG+GG, P = 2.48E-04, odds ratio = 1.827, 95% confidence interval = 1.321-2.526) models. Meta-analysis of the Indonesian and Vietnamese populations showed that rs352139 was significantly associated with TB in the recessive model. This finding indicated that a TLR9 polymorphism might have an important role in the susceptibility to M. tuberculosis in Asian populations.

Association analysis of susceptibility candidate region on chromosome 5q31 for tuberculosis.

Chromosome 5q31 spans the T helper (Th) 2-related cytokine gene cluster, which is potentially important in Th1/Th2 immune responses. The chromosome 5q23.2-31.3 has been recently identified as a region with suggestive evidence of linkage to tuberculosis in the Asian population. With the aim of fine-mapping a putative tuberculosis susceptibility locus, we investigated a family-based association test between the dense single nucleotide polymorphism (SNP) markers within chromosome 5q31 and tuberculosis in 205 Thai trio families. Of these, 75 SNPs located within candidate genes covering SLC22A4, SLC22A5, IRF1, IL5, RAD50, IL13, IL4, KIF3A and SEPT8 were genotyped using the DigiTag2 assay. Association analysis revealed the most significant association with tuberculosis in haplotypes comprising SNPs rs274559, rs274554 and rs274553 of SLC22A5 gene (P(Global)=2.02 x 10(-6)), which remained significant after multiple testing correction. In addition, two haplotypes within the SLC22A4 and KIF3A region were associated with tuberculosis. Haplotypes of SLC22A5 were significantly associated with the expression levels of RAD50 and IL13. The results show that the variants carried by the haplotypes of SLC22A4, SLC22A5 and KIF3A region potentially contribute to tuberculosis susceptibility among the Thai population.

Genome-wide SNP-based linkage analysis of tuberculosis in Thais.

Tuberculosis, a potentially fatal infectious disease, affects millions of individuals annually worldwide. Human protective immunity that contains tuberculosis after infection has not been clearly defined. To gain insight into host genetic factors, nonparametric linkage analysis was performed using high-throughput microarray-based single nucleotide polymorphism (SNP) genotyping platform, a GeneChip array comprised 59 860 bi-allelic markers, in 93 Thai families with multiple siblings, 195 individuals affected with tuberculosis. Genotyping revealed a region on chromosome 5q showing suggestive evidence of linkage with tuberculosis (Z(lr) statistics=3.01, logarithm of odds (LOD) score=2.29, empirical P-value=0.0005), and two candidate regions on chromosomes 17p and 20p by an ordered subset analysis using minimum age at onset of tuberculosis as the covariate (maximum LOD score=2.57 and 3.33, permutation P-value=0.0187 and 0.0183, respectively). These results imply a new evidence of genetic risk factors for tuberculosis in the Asian population. The significance of these ordered subset results supports a clinicopathological concept that immunological impairment in the disease differs between young and old tuberculosis patients. The linkage information from a specific ethnicity may provide unique candidate regions for the identification of the susceptibility genes and further help elucidate the immunopathogenesis of tuberculosis.

HLA-A, -B, -C, -DRB1 and -DQB1 alleles and haplotypes in the Kinh population in Vietnam.

Allele and haplotype frequencies of the human leukocyte antigens (HLA) were studied in the Kinh Vietnamese population. We analyzed 170 unrelated healthy individuals. DNA-based HLA typing was performed using a microsphere-based array genotyping platform with sequence-specific oligonucleotide probes to distinguish HLA-A, -B, -C, -DRB1 and -DQB1 alleles. A total of 21 HLA-A, 37 HLA-B, 18 HLA-C, 25 HLA-DRB1, and 14 HLA-DQB1 alleles were identified. HLA-A*1101, A*2402, A*3303, B*1502, B*4601, Cw*0102, Cw*0702, Cw*0801, DRB1*1202, DQB1*0301, DQB1*0303, and DQB1*0501 were found with frequencies higher than 10%. Two representative haplotypes bearing two to five HLA loci were A*1101-B*1502 and A*3303-B*5801 for HLA-A-B; Cw*0801-B*1502 and Cw*0102-B*4601 for HLA-C-B; B*1502-DRB1*1202 and B*4601-DRB1*0901 for HLA-B-DRB1; DRB1*1202-DQB1*0301 and DRB1*0901-DQB1*0303 for HLA-DRB1-DQB1; A*1101-Cw*0801-B*1502 and A*3303-Cw*0302-B*5801 for HLA-A-C-B; A*1101-B*1502-DRB1*1202 and A*2901-B*0705-DRB1*1001 for HLA-A-B-DRB1, A*1101-Cw*0801-B*1502-DRB1*1202-DQB1*0301 and A*2901-Cw*1505-B*0705-DRB1*1001-DQB1*0501 for HLA-A-C-B-DRB1-DQB1. Allele distribution and haplotype analysis demonstrated that the Vietnamese population shares HLA patterns with southern Chinese, Thai, Javanese and Micronesians, while it also retains unique characteristics.

Pulmonary Mycobacterium avium complex infection associated with the IVS8-T5 allele of the CFTR gene.

BACKGROUND: The T5 allele in intron 8 (IVS8) on specific haplotype backgrounds (e.g., long TG repeats) causes abnormal splicing in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, and is also known to be associated with chronic airway diseases. OBJECTIVE: To investigate the role of CFTR variations for susceptibility to pulmonary Mycobacterium avium complex (MAC) infection. PARTICIPANTS: Three hundred patients with pulmonary MAC infection (72 males, 228 females; mean age at onset 61.6 + or - 12.4 years) took part in this study. Diagnosis of MAC infection was based on American Thoracic Society criteria. Clinical profiles were collected and blood samples were genotyped for TG repeats, poly-T and M470V polymorphisms. RESULTS: We found significantly higher T5 frequency in MAC patients than in healthy controls from our own study (0.035 and 0.005, respectively, P = 0.023) and other reports. Homozygote for the T5 allele was found in two MAC patients. All T5 alleles were associated with longer TG repeats, the TG12 or TG13 allele. Seventeen of the 21 T5 alleles appeared to be associated with the V470 allele. Other polymorphisms did not show any significant differences in frequency. CONCLUSIONS: These findings suggest that the IVS8 5T allele might be involved in susceptibility to pulmonary MAC infection.

A survey of tuberculosis prevalence in Hanoi, Vietnam.

OBJECTIVE: To assess the prevalence of tuberculosis (TB) in Hanoi, Vietnam, in 2003/2004. METHODS: A random selection was carried out involving 11624 subjects from 20 communes within the city. RESULTS: On chest X-ray examination, 317 subjects (2.73%) showed abnormal lung opacity, of which 17 were sputum smear-positive, two concentrated smear-positive and three culture-positive, all with active TB. The prevalence of sputum smear-positive pulmonary TB was 146 per 100000 in persons aged >or=15 years (95%CI 65-228). CONCLUSION: This is the first large-scale assessment of the prevalence of TB in Hanoi. The prevalence rate was higher than expected, suggesting that a significant number of patients with active TB, particularly females, remain undiagnosed, thus representing a continuing potential source of transmission in the community.

Pulmonary Mycobacterium avium complex infection: association with NRAMP1 polymorphisms.

The present study aimed to elucidate risk factors for nonimmunocompromised pulmonary Mycobacterium avium complex (MAC) infection. Epidemiological data and variations of candidate genes for mycobacterial diseases were analysed in 111 patients with pulmonary MAC infection. Four polymorphisms of the human natural resistance-associated macrophage protein (NRAMP)1 gene, the 5'(GT)n, 469+14 G/C, D543N and the 3'untranslated region (3'TGTG) insertion/deletion, were genotyped using PCR-based methods. Fok I and Taq I polymorphisms of the vitamin D receptor gene and -221 X/Y and codon 54 A/B polymorphisms of the mannose binding lectin gene were also evaluated. Females were more susceptible to MAC infection mainly affecting the right middle lobe or lingular segment of the lung. Patients' residence at the onset of the disease was distributed evenly irrespective of a waterfront or city water supply system. As compared with homozygotes for major alleles of the D543N and TGTG insertion/deletion polymorphism of the NRAMP1 gene, heterozygotes containing minor alleles were less often observed in MAC cases than in controls. This genetic effect was more significant in patients without comorbidity but not in patients with comorbidity. Other polymorphisms did not show any association with the MAC infection. The human natural resistance-associated macrophage protein 1 gene might be involved in susceptibility to pulmonary Mycobacterium avium complex infection.

Assessment of synthetic peptides of severe acute respiratory syndrome coronavirus recognized by long-lasting immunity.

In order to determine highly immunogenic severe acute respiratory syndrome coronavirus (SARS-CoV) epitope peptides capable of inducing long-lasting immunity, we first screened immunoglobulin-G (IgG) antibodies reactive to 197 different overlapping 15-mers from the SARS-CoV proteins in the sera of three infected patients. Forty-two peptides among them were reactive to the sera from all three patients. Consequently, we tested for the reactivity of these 42 peptides to patients' sera (n = 45) at 6-month post-infection. The significantly higher levels of IgG antibodies specific to three (S791, M207 and N161) of 42 peptides were detectable in the post-infection sera from 23 (51%), 27 (60%) and 19 (42%) of 45 patients, respectively. These three peptides, recognized by their long-lasting immunity, may provide a better understanding of the immunogenicity of SARS-CoV.

[Genetic variation of NADPH/NADH oxidase and susceptibility to diffuse panbronchiolitis (DPB) and chronic obstructive pulmonary disease (COPD)].

Diffuse panbronchiolitis (DPB) and chronic obstructive pulmonary disease (COPD) are both characterized by chronic airflow obstruction of unknown etiology. It is hypothesized that neutrophils play the major role in the pathogenesis of these diseases. Recent studies have suggested that genetic factors may be related to individual susceptibility to these diseases. We have investigated the association between the C 242 T polymorphism of p 22 phox, a critical subunit of superoxide-generating NADH/NADPH oxidase, and susceptibility to DPB and COPD. Blood samples obtained from both patients with DPB (n = 82), COPD (n = 53), and control subjects (n = 82) were used for this genotyping assay; and the polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) were performed to genotype the gene. The frequency of the C allele was 0.91, 0.92, 0.92 in the DPB, COPD, and control groups, respectively. There were no differences in the distribution of the genotype of p 22 phox among these groups, either. We concluded that there is no association between the C 242 T polymorphism of p 22 phox, and susceptibility to DPB and COPD.

Human alveolar macrophages and granulocyte-macrophage colony-stimulating factor-induced monocyte-derived macrophages are resistant to H2O2 via their high basal and inducible levels of catalase activity.

Human alveolar macrophages (A-MPhi) and macrophages (MPhi) generated from human monocytes under the influence of granulocyte-macrophage colony-stimulating factors (GM-MPhi) express high levels of catalase activity and are highly resistant to H(2)O(2). In contrast, MPhi generated from monocytes by macrophage colony-stimulating factors (M-MPhi) express low catalase activity and are about 50-fold more sensitive to H(2)O(2) than GM-MPhi or A-MPhi. Both A-MPhi and GM-MPhi but not M-MPhi can induce catalase expression in both protein and mRNA levels when stimulated with H(2)O(2) or zymosan. M-MPhi but not GM-MPhi produce a large amount of H(2)O(2) in response to zymosan or heat-killed Staphylococcus aureus. These findings indicate that GM-MPhi and A-MPhi but not M-MPhi are strong scavengers of H(2)O(2) via the high basal level of catalase activity and a marked ability of catalase induction and that catalase activity of MPhi is regulated by colony-stimulating factors during differentiation.

Overestimated frequency of a possible emphysema-susceptibility allele when microsomal epoxide hydrolase is genotyped by the conventional polymerase chain reaction-based method.

A recent association study suggested that the His113 variant of microsomal epoxide hydrolase (mEPHX) may confer a risk for development of emphysema, presumably by increasing susceptibility to smoking injury. Before considering a possible role of this enzyme in pulmonary disease, we attempted to characterize the genetic polymorphism further. The Tyr/His113 polymorphism within exon 3 of mEPHX was initially examined in 62 healthy individuals by conventional methods involving polymerase chain reaction (PCR)-based determination of a restriction fragment length polymorphism (RFLP). Genomic nucleotide sequences, including the polymorphic site and the downstream primer sequence, were further analyzed in 95 unrelated, healthy Japanese volunteers by single-stranded conformation polymorphism (SSCP) analysis and direct sequencing. Genotyping by the first method (PCR-RFLP) revealed that the allelic distribution in our test population apparently deviated from Hardy-Weinberg equilibrium. Sequence analysis showed that a synonymous nucleotide substitution, AAG to AAA (Lys119), was located just within the published primer site. The AAA at codon 119 was present only in alleles with Tyr113, and its frequency reached 0.31 in our panel of 190 Japanese alleles. This substitution potentially hampered PCR amplification because of the nucleotide mismatch, with the result that the frequency of the Tyr113 variation was underestimated. The frequency of His113, a possible emphysema susceptibility allele of the mEPHX gene, was thus overestimated when human DNA samples were genotyped in the conventional way. Depending on the population(s) tested, this anomaly could represent a pitfall for PCR-based association studies.

Polymorphism of human leukocyte antigen-E gene in the Japanese population with or without recurrent abortion.

PROBLEM: The aim of this study was to investigate the gene frequencies and shared alleles of human leukocyte antigen (HLA)-E gene in Japanese couples with or without recurrent abortion. METHOD OF STUDY: Polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP) analysis was carried out to detect polymorphism in exon 3 of the HLA-E gene in 30 Japanese couples with recurrent abortion and 38 normal Japanese couples with proven fertility. RESULTS: No point mutation was detected in exon 3 of HLA-E in both recurrent aborters and normal controls. HLA-EG and HLA-ER alleles were detected with frequencies of 66.7% and 33.3% in couples with recurrent abortion and 69.2% and 30.8 in normal couples, respectively. The gene frequency of HLA-EG was higher than that of HLA-ER, which is contrary to that found in Caucasian, African-American and Hispanic people but similar to Chinese people. The frequency of each allele was not significantly different between recurrent aborters and normal controls. The number of shared alleles between each couple with recurrent abortion is not significantly different from that with normal controls. CONCLUSION: Allele frequencies of HLA-E were suggested to be different in Asian people from those in other ethnic people. In light of no specific distribution pattern in recurrent aborters, HLA-E polymorphism does not seem to play a role in the pathogenesis of recurrent abortion.

Association of Gc-globulin variation with susceptibility to COPD and diffuse panbronchiolitis.

Chronic obstructive pulmonary disease (COPD) and diffuse panbronchiolitis (DPB) are both characterized by chronic airflow limitation. Although the aetiology of these diseases is under investigation, it is commonly hypothesized that neutrophils have a major role in the disease pathogenesis. The variation of the genes related to chemotaxis of neutrophils may confer a risk for the development of both COPD and DPB. In the present report, the authors investigated the association between genetic variation that codes for the 416th and 420th amino acid of Gc-globulin, reported to be associated with chemotaxis of neutrophils, and susceptibility to COPD and DPB. Blood samples obtained from patients with COPD (n=63), DPB (n=82), and-control subjects (n=82) were used for the genotyping assay. The proportion of GC*1F homozygotes was significantly higher in the COPD patients than the control subjects (COPD 36.5% versus control 20.7%), and the odds ratio for GC*IF homozygotes was 2.2 (95%, confidence interval 1.1-4.6) for the COPD group. There was no difference on the distribution of the other genotypes (GC*1F-1S heterozygotes, GC*1S homozvgotes, GC*2-1F heterozygotes, GC*2-1S heterozygotes and GC*2 homozygotes) or the allele frequencies among these groups. These findings suggest that the GC*IF gene polymorphism of Gc-globulin may be one of the risk factors for chronic obstructive pulmonary disease. However, no association between this polymorphism of Gc-globulin and susceptibility to diffuse panbronchiolitis was found.

[Tuberculosis in patients with acquired immune deficiency syndrome].

HIV-1 infection is a major cause of worldwide epidemic of tuberculosis. In Japan, the cumulative number of the patients reported is 131 by the end of 1999 with 10 to 20 annual new cases. Most of Japanese cases are advanced AIDS patients with low CD4 number less than 100/microliter. The peak age of Japanese patient is 40 to 60 years old, whereas that of foreigners is 20-30 years old, suggesting that most Japanese cases are recurrent tuberculosis. There is increasing clinical evidence that coinfection with M. tuberculosis accelerates progression of AIDS. We found that, in vivo, HIV-1 load and mutation increase in involved lung segments in patients with pulmonary tuberculosis. We also reported that Mycobacterium tuberculosis stimulates HIV-1 replication by enhancing transcription on the 5' LTR in a macrophage cell line, THP-1, in vitro. In contrast, HIV-1 replication is suppressed by M. tuberculosis infection of monocytes derived macrophages (MDM) or differentiated monocytic THP-1 cells. We observed that HIV-1 5' LTR function was repressed in PMA differentiated THP-1 cells after co-infection with M. tuberculosis. Point mutations in C/EBP-beta binding domains of the HIV-1 LTR negative regulatory element (NRE) abolished promoter repression. Monocyte-derived macrophages and differentiated THP-1 cells increased expression of the 16 kDa inhibitory from of C/EBP-beta after M. tuberculosis coinfection. Bronchoalveolar lavage cells obtained from normal controls and alveolar macrophages from uninflamed lung of tuberculosis patients also expressed the 16 kDa inhibitory form of C/EBP-beta. However, alveolar macrophages from lung segments involved with pulmonary tuberculosis had markedly reduced C/EBP-beta expression. These data suggest that 16 kDa isoform of C/EBP-beta plays an important role for the control of HIV-1 replication in macrophages. We propose derepression of HIV-1 LTR mediated transcription as one mechanism for enhanced HIV-1 replication observed in pulmonary tuberculosis.

Fine localization of a major disease-susceptibility locus for diffuse panbronchiolitis.

Diffuse panbronchiolitis affecting East Asians is strongly associated with the class I human leukocyte antigen (HLA) alleles. Recent observations suggest that a major disease-susceptibility gene may be located between the HLA-B and HLA-A loci in the class I region of the major histocompatibility complex on chromosome 6. To test this possibility, we analyzed 14 polymorphic markers in 92 Japanese patients and 93 healthy controls. Of these, seven marker alleles, including HLA-B54 and HLA-A11, were significantly associated with the disease. Maximum-likelihood haplotype analysis and subsequent direct determination of individual haplotypes identified a group of disease-associated haplotypes, one of which contained all seven disease-associated marker alleles. Another haplotype, containing HLA-B*5504, was also associated with the disease. All these haplotypes seem to have diverged from a common ancestral haplotype in East Asians and share a specific segment containing three consecutive markers between the S and TFIIH loci in the class I region. Furthermore, one of the markers within the candidate region showed the highest delta value, indicating the strongest association. Of 20 Korean patients with diffuse panbronchiolitis, 17 also shared the combination of the disease-associated marker alleles within the candidate region. These results indicate that an HLA-associated major susceptibility gene for diffuse panbronchiolitis is probably located within the 200 kb in the class I region 300 kb telomeric of the HLA-B locus on the chromosome 6p21.3.