Spoligotyping and whole-genome sequencing analysis of lineage 1 strains of Mycobacterium tuberculosis in Da Nang, Vietnam.

BACKGROUND: Spacer oligonucleotide typing (spoligotyping), a widely used, classical genotyping method for Mycobacterium tuberculosis complex (MTBC), is a PCR-based dot-blot hybridization technique to detect the genetic diversity of the direct repeat (DR) region. Of the seven major MTBC lineages in the world, lineage 1 (Indo-Oceanic) mostly corresponds to the East African-Indian (EAI) spoligotype family in East Africa and Southeast Asia. OBJECTIVES: We investigated the genomic features of Vietnamese lineage 1 strains, comparing spoligotype patterns using whole-genome sequencing (WGS) data. METHODS: M. tuberculosis strains isolated in Da Nang, Vietnam were subjected to conventional spoligotyping, followed by WGS analysis using a high-throughput sequencer. Vietnamese lineage 1 strains were further analyzed with other lineage 1 strains obtained from a public database. RESULTS: Indicating a major spoligotype in Da Nang, 86 (46.2%) of the 186 isolates belonged to the EAI family or lineage 1. Although typical EAI4-VNM strains are characterized by the deletion of spacers 26 and 27, 65 (75.6%) showed ambiguous signals on spacer 26. De novo assembly of the entire DR region and in silico spoligotyping analysis suggested the absence of spacer 26, and direct sequencing revealed that the 17th spacer sequence not used for conventional typing, was cross-hybridized to the spacer 26 probe. Vietnamese EAI4-VNM, other EAI-like strains, and those showing a non-EAI pattern lacking many spacers formed a monophyletic group separate from other EAI families in the world. CONCLUSION: Information about the alignment of spacers in the entire DR region obtained from WGS data provides a clue for the determination of experimentally ambiguous spoligo patterns. WGS data also helped to analyze the hidden relationships between apparently distinct spoligo patterns.

Circulating granulysin levels in healthcare workers and latent tuberculosis infection estimated using interferon-gamma release assays.

BACKGROUND: Granulysin (GNLY) is produced by human lymphocyte subpopulations and exhibits antimicrobial activity against Mycobacterium tuberculosis. We examined the association between GNLY levels in blood and latent tuberculosis (TB) infection. METHODS: Latency of TB infection among Vietnamese healthcare workers was estimated using interferon-gamma release assays (IGRA), and serum GNLY concentrations were measured using enzyme-linked immunosorbent assays. The levels of GNLY expression in whole blood and the presence of GNLY alleles with the exon-4 polymorphism rs11127 were also determined using PCR-based methods. RESULTS: Among 109 study participants, 41 (37.6 %) were IGRA positive and had significantly lower serum GNLY concentrations compared with IGRA-negative participants (adjusted mean, 95 % confidence interval; 2.03, 1.72-2.44 vs. 2.48, 2.10-2.92 ng/ml, P = 0.0127; analysis of covariance). Serum GNLY concentrations and TB antigen-stimulated interferon-gamma values were weakly inversely correlated (r = -0.20, P = 0.0333). Serum GNLY concentrations varied with GNLY genotypes even after adjustment for gender and age (adjusted P = 0.0015) and were moderately correlated with GNLY expression in blood cells (r = 0.40, P < 0.0001). In subsequent analyses, low serum GNLY concentrations were significantly associated with IGRA status (adjusted odds ratio and 95 % confidence interval, 0.55 and 0.31-0.98, respectively), although GNLY genotype and mRNA levels were not. CONCLUSIONS: Decreased GNLY, presumably at the protein level, is linked to the immunological condition of latent TB infection.

Dynamics of immune parameters during the treatment of active tuberculosis showing negative interferon gamma response at the time of diagnosis.

OBJECTIVES: In the performance of interferon gamma release assays (IGRA) for the diagnosis of tuberculosis (TB) infection, false-negative results are a major obstacle. In active TB patients, treatment-dependent changes of the negative test results remain unknown. METHODS: The treatment course of 19 smear-positive/culture-confirmed TB patients who had IGRA-negative results by QuantiFERON-TB in-tube (QFT-IT) method at the time of diagnosis (month 0) in a previous study, were monitored in the present study. Blood was further collected at months 2 and 7, and the concentrations of 27 immune molecules were measured in the plasma supernatants remaining after performing the IGRA, using a suspension array system. RESULTS: After initiating treatment, eight of the 19 QFT-IT-negative patients showed positive conversion, whereas the remaining 11 (58%) did not; the interferon gamma (IFN-γ) response was restored to levels higher than 1 IU/ml in only three of the eight patients with positive conversion. Plasma concentrations of interleukin 1 receptor antagonist, interleukin 2, and interferon gamma-induced protein 10 remained low after Mycobacterium tuberculosis-specific antigen stimulation at months 2 and 7 in the continuously QFT-IT-negative group, whereas the parameters were elevated only in the transiently QFT-IT-negative group. CONCLUSIONS: It was demonstrated that a majority of active TB patients showing negative IGRA results did not regain sufficient levels of immune responsiveness despite successful treatment.

Association between tuberculosis recurrence and interferon-γ response during treatment.

OBJECTIVES: We investigated the relationship between tuberculosis recurrence and Mycobacterium tuberculosis antigen-stimulated interferon-gamma (IFN-γ) responses during treatment. METHODS: Plasma IFN-γ levels in active pulmonary tuberculosis patients (n = 407) were analyzed using QuantiFERON-TB Gold In-Tube™ (QFT-IT) at 0, 2, and 7 months of the 8-month treatment received from 2007 to 2009 and the patients were followed up for another 16 months after treatment. Risk factors for recurrence were assessed using the log-rank test and Cox proportional hazard models. Random coefficient models were used to compare longitudinal patterns of IFN-γ levels between groups. RESULTS: QFT-IT showed positive results in 95.6%, 86.2%, and 83.5% at 0, 2, and 7 months, respectively. The antigen-stimulated IFN-γ responses varied significantly during the treatment course (P < 0.0001). Unexpectedly, positive-to-negative conversion of QFT-IT results between 0 and 2 months was significantly associated with earlier recurrence (adjusted hazard ratio, 5.57; 95% confidence interval, 2.28-13.57). Time-dependent changes in IFN-γ levels were significantly different between the recurrence and nonrecurrence groups (P < 0.0001). CONCLUSIONS: Although the IGRA response varies individually, early response during the treatment course may provide an insight into host immune responses underlying tuberculosis recurrence.

Age-dependent association of mannose-binding lectin polymorphisms with the development of pulmonary tuberculosis in Viet Nam.

Mannose-binding lectin (MBL) binds to pathogens and induces complement-mediated opsonophagocytosis. Although the association between MBL2 polymorphisms and tuberculosis (TB) has been studied in various populations, the results are controversial. We explored the stages of TB associated with MBL2 polymorphisms. X/Y (rs7096206) and A/B (rs1800450) were genotyped in 765 new patients with active pulmonary TB without HIV infection and 556 controls in Hanoi, Viet Nam. The MBL2 nucleotide sequences were further analyzed, and plasma MBL levels were measured in 109 apparently healthy healthcare workers and 65 patients with TB. Latent TB infection (LTBI) was detected by interferon-gamma release assay (IGRA). The YA/YA diplotype, which exhibited high plasma MBL levels, was associated with protection against active TB in younger patients (mean age = 32)≦ 45 years old (odds ratio, 0.61; 95% confidence interval, 0.46-0.80). The resistant diplotype was less frequently found in the younger patients at diagnosis (P = 0.0021). MBL2 diplotype frequencies and plasma MBL levels were not significantly different between the IGRA-positive and -negative groups. MBL2 YA/YA exhibited a protective role against the development of TB in younger patients, whereas the MBL2 genotype and MBL levels were not associated with LTBI. High MBL levels may protect against the early development of pulmonary TB after infection.

MxA transcripts with distinct first exons and modulation of gene expression levels by single-nucleotide polymorphisms in human bronchial epithelial cells.

Myxovirus resistance A (MxA) is a major interferon (IFN)-inducible antiviral protein. Promoter single-nucleotide polymorphisms (SNPs) of MxA near the IFN-stimulated response element (ISRE) have been frequently associated with various viral diseases, including emerging respiratory infections. We investigated the expression profile of MxA transcripts with distinct first exons in human bronchial epithelial cells. For primary culture, the bronchial epithelium was isolated from lung tissues with different genotypes, and total RNA was subjected to real-time reverse transcription polymerase chain reaction. The previously reported MxA transcript (T1) and a recently registered transcript with a distinct 5' first exon (T0) were identified. IFN-ß and polyinosinic-polycytidylic acid induced approximately 100-fold higher expression of the T1 transcript than that of the T0 transcript, which also had a potential ISRE motif near its transcription start site. Even without inducers, the T1 transcript accounted for approximately two thirds of the total expression of MxA, levels of which were significantly associated with its promoter and exon 1 SNPs (rs17000900, rs2071430, and rs464138). Our results suggest that MxA observed in respiratory viral infections is possibly dominated by the T1 transcript and partly influenced by relevant 5' SNPs.

Differential effects of a common splice site polymorphism on the generation of OAS1 variants in human bronchial epithelial cells.

The 2',5'-oligoadenylate synthetase 1 (OAS1) is one of the major interferon-inducible proteins and a critical component of the host defense system against viral infection. A single nucleotide polymorphism (SNP), rs10774671, presumably responsible for alternate splicing of this gene, has frequently been associated with a variety of viral diseases, including emerging respiratory infections. We investigated the SNP-dependent expression of OAS1 variants in primary cultured human bronchial epithelial cells. Total RNA was subjected to real-time RT-PCR with specific primer sets designed to amplify each transcript variant. We found that the p46 transcript was mainly expressed in cells with the GG genotype, whereas the p42 transcript was highly expressed, and the p44a (alternate exon in intron 5), p48, and p52 transcripts were expressed to a lesser extent, in cells with the AA genotype. Immunoblot analysis revealed that the p46 isoform and a smaller amount of the p42 isoform were present in cells with the GG genotype, whereas only the p42 isoform was clearly observed in cells with the AA genotype. Cellular DNA fragmentation induced by neutrophil elastase was more preferentially found in cells with the AA genotype. Thus, our findings provide insights into the potential role of OAS1 polymorphisms in respiratory infection.

Circulating levels of adiponectin, leptin, fetuin-A and retinol-binding protein in patients with tuberculosis: markers of metabolism and inflammation.

BACKGROUND: Wasting is known as a prominent feature of tuberculosis (TB). To monitor the disease state, markers of metabolism and inflammation are potentially useful. We thus analyzed two major adipokines, adiponectin and leptin, and two other metabolic markers, fetuin-A and retinol-binding protein 4 (RBP4). METHODS: The plasma levels of these markers were measured using enzyme-linked immunosorbent assays in 84 apparently healthy individuals (=no-symptom group) and 46 patients with active pulmonary TB around the time of treatment, including at the midpoint evaluation (=active-disease group) and compared them with body mass index (BMI), C-reactive protein (CRP), chest radiographs and TB-antigen specific response by interferon-γ release assay (IGRA). RESULTS: In the no-symptom group, adiponectin and leptin showed negative and positive correlation with BMI respectively. In the active-disease group, at the time of diagnosis, leptin, fetuin-A and RBP4 levels were lower than in the no-symptom group [adjusted means 2.01 versus 4.50 ng/ml, P<0.0001; 185.58 versus 252.27 µg/ml, P<0.0001; 23.88 versus 43.79 µg/ml, P<0.0001, respectively]. High adiponectin and low leptin levels were associated with large infiltrates on chest radiographs even after adjustment for BMI and other covariates (P=0.0033 and P=0.0020). During treatment, adiponectin levels increased further and then decreased. Leptin levels remained low. Initial low levels of fetuin-A and RBP4 almost returned to the normal reference range in concert with reduced CRP. CONCLUSIONS: Our data and recent literature suggest that low fat store and underlying inflammation may regulate these metabolic markers in TB in a different way. Decreased leptin, increased adiponectin, or this ratio may be a promising marker for severity of the disease independent of BMI. We should further investigate pathological roles of the balance between these adipokines.

Association of SLC11A1 (NRAMP1) polymorphisms with pulmonary Mycobacterium avium complex infection.

Although genetic variants in SLC11A1 (NRAMP1) have been associated with mycobacterial diseases, these findings have not been extensively validated in pulmonary Mycobacterium avium complex (MAC) infection. This study investigated the genomic structure of SLC11A1 and its association with MAC infection. Nineteen polymorphic loci were genotyped in European descendents and the Japanese population. Linkage disequilibrium (LD) structures and frequencies of major haplotypes differed between these 2 populations. Tag single nucleotide polymorphisms (SNPs) were chosen from the data set, and 6 polymorphic sites were genotyped in 122 pulmonary MAC cases and 211 controls from Japan. We observed that the T allele of rs2279014 in the 3' untranslated region was associated with protection from MAC disease when comparing allele frequencies with an odds ratio of 0.582 (95% confidence interval 0.379-0.894, p = 0.013). The frequencies of haplotypes constructed with the above 6 variants did not differ between cases and controls. Allele-specific expression imbalance of SLC11A1 mRNA was evaluated in peripheral blood cells from heterozygous individuals, but no difference was observed among haplotypes. Although the significance was modest, rs2279014 is in strong LD with nearby SNPs and further studies are required for conclusive validation.

Association of IFNGR2 gene polymorphisms with pulmonary tuberculosis among the Vietnamese.

Interferon-γ (IFN-γ) is a key molecule of T helper 1 (Th1)-immune response against tuberculosis (TB), and rare genetic defects of IFN-γ receptors cause disseminated mycobacterial infection. The aim of the present study was to investigate whether genetic polymorphisms found in the Th1-immune response genes play a role in TB. In our study, DNA samples were collected from two series of cases including 832 patients with new smear-positive TB and 506 unrelated individuals with no history of TB in the general population of Hanoi, Vietnam. Alleles of eight microsatellite markers located around Th1-immune response-related genes and single nucleotide polymorphisms near the promising microsatellites were genotyped. A set of polymorphisms within the interferon gamma receptor 2 gene (IFNGR2) showed a significant association with protection against TB (P = 0.00054). Resistant alleles tend to be less frequently found in younger age at diagnosis (P = 0.011). Luciferase assays revealed high transcriptional activity of the promoter segment in linkage disequilibrium with resistant alleles. We conclude that the polymorphisms of IFNGR2 may confer resistance to the TB development of newly infected individuals. Contribution of the genetic factors to TB appeared to be different depending on age at diagnosis.

Analysis of factors lowering sensitivity of interferon-γ release assay for tuberculosis.

BACKGROUND: Imperfect sensitivity of interferon-γ release assay (IGRA) is a potential problem to detect tuberculosis. We made a thorough investigation of the factors that can lead to false negativity of IGRA. METHODS: We recruited 543 patients with new smear-positive pulmonary tuberculosis in Hanoi, Viet Nam. At diagnosis, peripheral blood was collected and IGRA (QuantiFERON-TB Gold In-Tube) was performed. Clinical and epidemiological information of the host and pathogen was collected. The test sensitivity was calculated and factors negatively influencing IGRA results were evaluated using a logistic regression model in 504 patients with culture-confirmed pulmonary tuberculosis. RESULTS: The overall sensitivity of IGRA was 92.3% (95% CI, 89.6%-94.4%). The proportions of IGRA-negative and -indeterminate results were 4.8% (95% CI, 3.1%-7.0%) and 3.0% (95% CI, 1.7%-4.9%). Age increased by year, body mass index <16.0, HIV co-infection and the increased number of HLA-DRB1*0701 allele that patients bear showed significant associations with IGRA negativity (OR = 1.04 [95% CI, 1.01-1.07], 5.42 [1.48-19.79], 6.38 [1.78-22.92] and 5.09 [2.31-11.22], respectively). HIV co-infection and the same HLA allele were also associated with indeterminate results (OR = 99.59 [95% CI, 15.58-625.61] and 4.25 [1.27-14.16]). CONCLUSIONS: Aging, emaciation, HIV co-infection and HLA genotype affected IGRA results. Assessment of these factors might contribute to a better understanding of the assay.

Identification of tuberculosis-associated proteins in whole blood supernatant.

BACKGROUND: Biological parameters are useful tools for understanding and monitoring complicated disease processes. In this study, we attempted to identify proteins associated with active pulmonary tuberculosis (TB) using a proteomic approach. METHODS: To assess TB-associated changes in the composition of human proteins, whole blood supernatants were collected from patients with active TB and healthy control subjects. Two-dimensional difference gel electrophoresis (2D-DIGE) was performed to analyze proteins with high molecular weights (approximately >20 kDa). Baseline protein levels were initially compared between patients with active TB and control subjects. Possible changes of protein patterns in active TB were also compared ex vivo between whole blood samples incubated with Mycobacterium tuberculosis (Mtb)-specific antigens (stimulated condition) and under unstimulated conditions. Immunoblot and enzyme-linked immunosorbent assays (ELISA) were performed to confirm differences in identified proteins. RESULTS: Under the baseline condition, we found that the levels of retinol-binding protein 4 (RBP4), fetuin-A (also called α-HS-glycoprotein), and vitamin D-binding protein differed between patients with active TB and control subjects on 2D gels. Immunoblotting results confirmed differential expression of RBP4 and fetuin-A. ELISA results further confirmed significantly lower levels of these two proteins in samples from patients with active TB than in control subjects (P < 0.0001). Mtb-specific antigen stimulation ex vivo altered clusterin expression in whole blood samples collected from patients with active TB. CONCLUSIONS: We identified TB-associated proteins in whole blood supernatants. The dynamics of protein expression during disease progression may improve our understanding of the pathogenesis of TB.

Molecular cloning of two novel mucin-like genes in the disease-susceptibility locus for diffuse panbronchiolitis.

Diffuse panbronchiolitis (DPB) is a rare complex genetic disease affecting East Asians and is strongly associated with the class I human leukocyte antigens (HLA)-B54 in Japanese and HLA-A11 in Koreans. We recently showed that an HLA-associated major susceptibility gene for DPB 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. We cloned two novel mucin-like genes designated panbronchiolitis related mucin-like 1 and 2 (PBMUCL1 and PBMUCL2) in the candidate region, which form a mucin-like gene cluster together with two adjacent genes, MUC21 and DPCR1. PBMUCL1 gene expression was remarkably upregulated by polyinosine-polycytidylic acid [poly(I:C)] stimulation in normal human bronchial epithelial cells redifferentiated at the air-liquid interface. We found genetic polymorphisms in PBMUCL1 gene which were associated with DPB: the A-allele of the PBMUCL1 intron 2 single nucleotide polymorphism (SNP) was positively associated and variable numbers of tandem repeats (VNTR) polymorphism in exon 3 (1,890-base pair deletion) was negatively associated. Despite a strong association with HLA-B in the Japanese, the mucin-like gene PBMUCL1 is also one of the candidate genes of DPB susceptibility.

Prevalence and risk factors for tuberculosis infection among hospital workers in Hanoi, Viet Nam.

BACKGROUND: Transmission of tuberculosis (TB) to health care workers (HCWs) is a global issue. Although effective infection control measures are expected to reduce nosocomial TB, HCWs' infection has not been assessed enough in TB high burden countries. We conducted a cross-sectional study to determine the prevalence of TB infection and its risk factors among HCWs in Hanoi, Viet Nam. METHODOLOGY/PRINCIPAL FINDINGS: A total of 300 HCWs including all staff members in a municipal TB referral hospital received an interferon-gamma release assay (IGRA), QuantiFERON-TB Gold In-Tube(TM), followed by one- and two-step tuberculin skin test (TST) and a questionnaire-based interview. Agreement between the tests was evaluated by kappa statistics. Risk factors for TB infection were analyzed using a logistic regression model. Among the participants aged from 20 to 58 years (median = 40), prevalence of TB infection estimated by IGRA, one- and two-step TST was 47.3%, 61.1% and 66.3% respectively. Although the levels of overall agreement between IGRA and TST were moderate, the degree of agreement was low in the group with BCG history (kappa = 0.29). Working in TB hospital was associated with twofold increase in odds of TB infection estimated by IGRA. Increased age, low educational level and the high body mass index also demonstrated high odds ratios of IGRA positivity. CONCLUSIONS/SIGNIFICANCE: Prevalence of TB infection estimated by either IGRA or TST is high among HCWs in the hospital environment for TB care in Viet Nam and an infection control program should be reinforced. In communities with heterogeneous history of BCG vaccination, IGRA seems to estimate TB infection more accurately than any other criteria using TST.

Identification of MICA as a susceptibility gene for pulmonary Mycobacterium avium complex infection.

Host genetic susceptibility to adult pulmonary Mycobacterium avium complex disease remains unknown. To identify genetic loci for the disease, we prepared 3 sets of pooled DNA samples from 300 patients and 300 sex-matched control subjects and genotyped 19,651 microsatellite markers in a case-control manner. D6S0009i-located in the MICA (major histocompatibility complex class I chain-related A) gene, which encodes a ligand of the NKG2D receptor-had the lowest P value in pooled and individual DNA typing. The A6 allele of the microsatellite was significantly associated with female patients (P <. 001), whereas the classical HLA-B and HLA-DRB1 alleles did not show significant association. Functional analysis of allelic expression imbalance revealed that A6-derived messenger RNA was more highly expressed than non-A6-derived messenger RNA in human bronchial epithelial cells. MICA was expressed in bronchiolar epithelium, alveolar macrophages, and granulomatous lesions. These findings suggest that MICA might be one of the immune molecules affecting the pathogenesis of the disease.

Quality assessment of an interferon-gamma release assay for tuberculosis infection in a resource-limited setting.

BACKGROUND: When a test for diagnosis of infectious diseases is introduced in a resource-limited setting, monitoring quality is a major concern. An optimized design of experiment and statistical models are required for this assessment. METHODS: Interferon-gamma release assay to detect tuberculosis (TB) infection from whole blood was tested in Hanoi, Viet Nam. Balanced incomplete block design (BIBD) was planned and fixed-effect models with heterogeneous error variance were used for analysis. In the first trial, the whole blood from 12 donors was incubated with nil, TB-specific antigens or mitogen. In 72 measurements, two laboratory members exchanged their roles in harvesting plasma and testing for interferon-gamma release using enzyme linked immunosorbent assay (ELISA) technique. After intervention including checkup of all steps and standard operation procedures, the second trial was implemented in a similar manner. RESULTS: The lack of precision in the first trial was clearly demonstrated. Large within-individual error was significantly affected by both harvester and ELISA operator, indicating that both of the steps had problems. After the intervention, overall within-individual error was significantly reduced (P < 0.0001) and error variance was no longer affected by laboratory personnel in charge, indicating that a marked improvement could be objectively observed. CONCLUSION: BIBD and analysis of fixed-effect models with heterogeneous variance are suitable and useful for objective and individualized assessment of proficiency in a multistep diagnostic test for infectious diseases in a resource-constrained laboratory. The action plan based on our findings would be worth considering when monitoring for internal quality control is difficult on site.

[Progress in the genetics of chronic obstructive pulmonary disease].

Chronic obstructive pulmonary disease (COPD) is a multifactorial disease caused by the interaction of genetic susceptibility and environmental factors. Although cigarette smoking is the main environmental risk factor for developing COPD, only about 15 % of smokers develop clinically significant disease. Genetic researches in this field mainly focused on variants of genes involved in protease anti-protease systems, defense against oxidative stress and inflammation. In addition to the candidate gene studies, genome wide approach including genome wide association studies will be extensively done in the near future.

Identification of an alternative 5'-untranslated exon and new polymorphisms of angiotensin-converting enzyme 2 gene: lack of association with SARS in the Vietnamese population.

We analyzed genetic variations of angiotensin-converting enzyme 2 (ACE2), considering that it might influence patients' susceptibility to severe acute respiratory syndrome-associated coronavirus (SARS-CoV) or development of SARS as a functional receptor. By cloning of the full-length cDNA of the ACE2 gene in the lung, where replication occurs on SARS-CoV, it was shown that there are different splicing sites. All exons including the new alternative exon, exon-intron boundaries, and the corresponding 5'-flanking region of the gene were investigated and 19 single nucleotide polymorphisms (SNPs) were found. Out of these, 13 SNPs including one non-synonymous substitution and three 3'-UTR polymorphisms were newly identified. A case control study involving 44 SARS cases, 16 anti-SARS-CoV antibody-positive contacts, 87 antibody-negative contacts, and 50 non-contacts in Vietnam, failed to obtain any evidence that the ACE2 gene polymorphisms are involved in the disease process in the population. Nevertheless, identification of new 5'-untranslated exon and new SNPs is considered helpful in investigating regulation of ACE2 gene expression in the future.

Variations of the CFTR gene in the Hanoi-Vietnamese.

In order to investigate polymorphic backgrounds of the cystic fibrosis transmembrane conductance regulator gene (CFTR) in the Vietnamese, we analyzed 495 blood samples of randomly selected healthy individuals in Hanoi for the delta F508 mutation and TG-repeats, poly-T, and M470V polymorphisms. We compared their distributions with those of Caucasians and other Asian populations. No delta F508 mutation was found, being consistent with the extremely low incidence of cystic fibrosis (CF) in Vietnam. Allele frequency of the T5 allele promoting exon 9 skipping was 0.037. Greater number of TG-repeats, which is known to facilitate this aberrant splicing, was a predominant trend in the Vietnamese and other Asians. A "T5-TG12-V470" haplotype was most common (29/37) among T5-bearing haplotypes. Three major haplotypes, T7-TG12-M470, T7-TG11-V470, and T7-TG12-V470, estimated by PHASE program, related to 92% of the population. This is the first study of the CFTR gene among the Vietnamese.

Evaluation of microsatellite markers in association studies: a search for an immune-related susceptibility gene in sarcoidosis.

Association studies using linkage disequilibrium (LD) between candidate loci and nearby markers have been proposed to identify susceptibility genes for complex diseases. We analyzed polymorphisms of microsatellites (MSs) and LD patterns of the regions in which candidate genes related to the Th1 immune response have been annotated and attempted to identify a susceptibility gene for sarcoidosis in a marker-based association study. Nineteen MSs were identified in six Th1-related genes (IFNGR1, IFNGR2, IL12RB1, IL12RB2, STAT1 and STAT4) and then eight were further characterized as useful polymorphic markers. Most of these MSs showed LD with single nucleotide polymorphisms (SNPs) on both 5' and 3' ends of these candidate genes, in which r(2) values between at least one of the MS marker alleles and the SNPs were higher than 0.1. A significant association with one MS allele near STAT4 was shown and a cluster of SNPs in LD with the MS marker was associated with sarcoidosis. These results suggest that association studies using not only SNPs but also multi-allelic MS within or near candidate loci would be useful markers to search for a disease susceptibility gene, especially in populations with unknown LD structure.