Adong Shen

Intrachromosomal Looping Is Required for Activation of Endogenous Pluripotency Genes during Reprogramming

Generation of induced pluripotent stem cells (iPSCs) by defined factors is an extremely inefficient process, because there is a strong epigenetic block preventing cells from achieving pluripotency. Here we report that virally expressed factors bound to the promoters of their target genes to the same extent in both iPSCs and unreprogrammed cells (URCs). However, expression of endogenous pluripotentcy genes was observed only in iPSCs. Comparison of local chromatin structure of the OCT4 locus revealed that there was a cohesin-complex-mediated intrachromosomal loop that juxtaposes a downstream enhancer to the genes promoter, enabling activation of endogenous stemness genes. None of these long-range interactions were observed in URCs. Knockdown of the cohesin-complex gene SMC1 by RNAi abolished the intrachromosomal interaction and affected pluripotency. These findings highlight the importance of the SMC1-orchestrated intrachromosomal loop as a critical epigenetic barrier to the induction of pluripotency.

Pediatric Tuberculosis at Beijing Children’s Hospital: 2002–2010

OBJECTIVE: Our aim was to describe the patient characteristics, clinical–epidemiological profile, and treatment outcome of childhood tuberculosis (TB). METHODS: A retrospective, descriptive study was undertaken of 1212 children aged 0 to 18 years admitted to Beijing Children’s Hospital for the treatment of TB from January 2002 to December 2010. Statistical significance of category variables was evaluated by using Fisher’s exact test. RESULTS: Fifty-four percent of patients had extrapulmonary tuberculosis (EPTB), 38.8% had tuberculous meningitis, and 31.3% had disseminated TB. The last 2 types were defined as severe TB. Most patients with TB (81.6%) were cured or completed treatment. There were more patients aged <5 years and from rural areas with EPTB than with pulmonary tuberculosis. More severe cases of TB were found in patients aged <1 year than other less severe types of TB. Patients with no bacille Calmette-Guérin vaccination and a contact history at home had a significantly risk of contracting severe TB. Children aged <1 year and those with severe TB were more likely to have poor treatment outcomes (failed to improve or died). Among those with EPTB, only 61.3% and 61.1% had positive results on the purified protein derivative tuberculin skin test and chest radiograph, respectively. CONCLUSIONS: In this referral hospital setting, more pediatric EPTB and severe TB patients were found among children aged <1 year. Age <1 year and having severe TB were risk factors for treatment failure. Thus, prevention and health care in pediatric TB should focus on both EPTB and severe TB.

Interferon gamma release assay in diagnosis of pediatric tuberculosis: a meta-analysis

Although interferon gamma release assays (IGRAs) have been widely used for the diagnosis of latent and active tuberculosis in adults, a relative lack of validation studies in children has led to caution in their clinical interpretation. This meta-analysis systematically evaluated two IGRAs (ELISA and ELISPOT) and the tuberculin skin test (TST). We searched databases (PubMed, MEDLINE, Ovid) between January 2000 and January 2011 using search terms of latent tuberculosis infection or tuberculosis and interferon gamma release assay, or T-SPOT.TB test, or QuantiFERON-TB Gold, or ESAT-6, or CFP-10, and child, or childhood, or pediatrics. We also collected data by performing a manual search of references from relevant articles and communicating with selected authors. The meta-analysis was conducted with random effects models to account for heterogeneity between selected studies. The sensitivities of all three tests in active tuberculosis were similar. The pooled sensitivity was 70% for ELISA studies, 62% for ELISPOT studies and 71% for TST. Calculated sensitivities for IGRAs and the TST differ in culture-confirmed tuberculosis [ELISA (85%) vs. ELISPOT (76%) vs. TST (85%)] and clinical diagnosed cases [ELISA (64%) vs. ELISPOT (58%) vs. TST (66%)]. The pooled specificity was 100% for ELISA and 90% for ELISPOT, but was much lower for TST [56% in all included studies and 49% in children with bacillus Calmette-Guerin (BCG) vaccination]. The agreement between the TST and IGRAs in non-BCG-vaccinated children is higher than that in BCG-vaccinated children. In the diagnosis of active tuberculosis in children, the TST and IGRAs have similar sensitivity. By contrast, the specificity of IGRAs is far greater than the TST, particularly in children with previous BCG vaccination.

Lack of association between polymorphisms in the P2X7 gene and tuberculosis in a Chinese Han population.

Several studies have suggested that genetic factors may affect the susceptibility of a population to tuberculosis, and it has been found that P2X7 is linked to an increased risk for tuberculosis in some West African, Southeast Asian, North American, and North European populations. To explore the potential role of P2X7 in the susceptibility to tuberculosis among members of the Chinese Han population, we evaluated the association of the 1513A-->C and -762T-->C polymorphisms in P2X7 with the risk for tuberculosis. PCR amplification of genomic DNA was followed by restriction fragment length polymorphism analysis, and allele-specific PCR was used. We found no significant differences in the genotypic and allelic frequencies of 1513A-->C polymorphisms in 96 patients with tuberculosis compared with 384 control subjects [P=0.856 and 0.316, respectively; odds ratio (OR) for the C allele=0.976; 95% confidence interval (CI)=0.755-1.262]. Similarly, no significant association was found between the -762T-->C polymorphism and tuberculosis (P=0.102 and 0.095 for the patients and controls, respectively; OR for the C allele=0.924; 95% CI=0.847-1.010). Thus, our analysis of P2X7 showed that the 1513A-->C and -762T-->C polymorphisms did not appear to be associated with the susceptibility of the Chinese Han population to tuberculosis.

Russian “Successful” Clone B0/W148 of Mycobacterium tuberculosis Beijing Genotype: a Multiplex PCR Assay for Rapid Detection and Global Screening

ABSTRACT We describe a multiplex PCR assay to detect the Mycobacterium tuberculosis Beijing genotype variant B0/W148, which is considered a “successful” clone of M. tuberculosis, widespread in Russia. Specificity and sensitivity of the assay were 100% based on the analysis of a collection of 516 M. tuberculosis isolates of different genotypes and origins. This assay may be used for accurate and simple detection and surveillance of this clinically and epidemiologically important variant of M. tuberculosis.

Tag SNP Polymorphism of CCL2 and Its Role in Clinical Tuberculosis in Han Chinese Pediatric Population

Background Chemokine (C-C motif) ligand 2 CCL2/MCP-1 is among the key signaling molecules of innate immunity; in particular, it is involved in recruitment of mononuclear and other cells in response to infection, including tuberculosis (TB) and is essential for granuloma formation. Methodology/Principal Findings We identified a tag SNP for the CCL2/MCP-1 gene (rs4586 C/T). In order to understand whether this SNP may serve to evaluate the contribution of the CCL2 gene to the expression of TB disease, we further analysed distribution of its alleles and genotypes in 301 TB cases versus 338 non-infected controls (all BCG vaccinated) representing a high-risk pediatric population of North China. In the male TB subgroup, the C allele was identified in a higher rate (P = 0.045), and, acting dominantly, was found to be a risk factor for clinical TB (P = 0.029). Homozygous TT genotype was significantly associated with lower CSF mononuclear leukocyte (ML) counts in patients with tuberculous meningitis (TBM) (P = 0.001). Conclusions/Significance The present study found an association of the CCL2 tag SNP rs4586 C allele and pediatric TB disease in males, suggesting that gender may affect the susceptibility to TB even in children. The association of homozygous TT genotype with decreased CSF mononuclear leukocyte (ML) count not only suggests a clinical significance of this SNP, but indicates its potential to assist in the clinical assessment of suspected TBM, where delay is critical and diagnosis is difficult.

Two novel mutations of the IRX4 gene in patients with congenital heart disease

IRX4 was the first identified cardiac transcription factor that is restricted to the ventricles at all stages of heart development. Irx4-deficient mice show ventricular dysfunction and develop cardiomyopathy. To study the potential impact of sequence variations in IRX4 on congenital heart disease (CHD) in humans, we examined the coding region of IRX4 in a cohort of 698 Chinese people with congenital heart disease and 250 healthy individuals as the controls. We found two potential disease-causing mutations, p. Asn85Tyr and p. Glu92Gly. A mammalian two-hybrid assay showed that both of the mutations significantly affected the interaction between IRX4 and RXRA. It demonstrated that IRX4 had a potential causative impact on the development of congenital heart disease, particularly ventricular septal defect.

A Functional Promoter Polymorphism of IFITM3 Is Associated with Susceptibility to Pediatric Tuberculosis in Han Chinese Population

A susceptibility locus for tuberculosis, a re-emerging infectious disease throughout the world, was previously discovered to exist on chromosome 11p15. IFITM3 gene encoding for interferon inducible transmembrane protein 3, is located at 11p15. It acts as an effector molecule for interferon-gamma, which is essential for anti-tuberculosis immune response. In order to investigate the association between susceptibility to TB and genetic polymorphisms of the IFITM3 core promoter, a case-control study including 368 TB patients and 794 healthy controls was performed in Han Chinese children in northern China. The rs3888188 polymorphism showed significant association with susceptibility to TB. The rs3888188 G allele, acting recessively, was more frequent in TB patients (95% confidence interval: 1.08–1.56, Bonferroni P-value: 0.039). We further assessed the effect of rs3888188 polymorphism on IFITM3 transcription in vitro. As based on luciferase promoter assays, the promoter activity of haplotypes with rs3888188 G allele was lower than that of haplotypes with rs3888188 T allele. Moreover, peripheral-blood mononuclear cells carrying rs3888188 GG genotype showed a reduced IFITM3 mRNA level compared to cells carrying TT or GT genotype. In conclusion, rs3888188, a functional promoter polymorphism of IFITM3, was identified to influence the risk for pediatric TB in Han Chinese population.

Compensatory Mutations of Rifampin Resistance Are Associated with Transmission of Multidrug-Resistant Mycobacterium tuberculosis Beijing Genotype Strains in China

ABSTRACT Mycobacterium tuberculosis can acquire resistance to rifampin (RIF) through mutations in the rpoB gene. This is usually accompanied by a fitness cost, which, however, can be mitigated by secondary mutations in the rpoA or rpoC gene. This study aimed to identify rpoA and rpoC mutations in clinical M. tuberculosis isolates in northern China in order to clarify their role in the transmission of drug-resistant tuberculosis (TB). The study collection included 332 RIF-resistant and 178 RIF-susceptible isolates. The majority of isolates belonged to the Beijing genotype (95.3%, 486/510 isolates), and no mutation was found in rpoA or rpoC of the non-Beijing genotype strains. Among the Beijing genotype strains, 27.8% (89/320) of RIF-resistant isolates harbored nonsynonymous mutations in the rpoA (n = 6) or rpoC (n = 83) gene. The proportion of rpoC mutations was significantly higher in new cases (P = 0.023) and in strains with the rpoB S531L mutation (P < 0.001). In addition, multidrug-resistant (MDR) strains with rpoC mutations were significantly associated with 24-locus mycobacterial interspersed repetitive-unit–variable-number tandem-repeat clustering (P = 0.016). In summary, we believe that these findings indirectly suggest an epistatic interaction of particular mutations related to RIF resistance and strain fitness and, consequently, the role of such mutations in the spread of MDR M. tuberculosis strains.

Genetic Contribution of CISH Promoter Polymorphisms to Susceptibility to Tuberculosis in Chinese Children

Tuberculosis (TB) is the leading cause of death due to an infectious disease worldwide, particularly in developing countries. A series of candidate genes have been suggested to be associated with development of TB disease. Among them, the human Cytokine-inducible Src homology 2(SH2) domain protein (CISH) gene has been very recently reported to be involved in T cell activation and differentiation in response to Mycobacterium tuberculosis infection. Here, we studied the association between CISH promoter polymorphisms and pediatric TB. A case-control study enrolled 352 TB patients and 527 healthy controls, who were of Han Chinese ethnicity and aged from 0.2 to 18 years. CISH gene promoter SNPs rs414171, rs622502 and rs809451 were genotyped in all subjects and transcriptional activity, mRNA level, and plasma cytokine level of subjects with different genotypes were further examined. Carriers with rs414171TT homozygotes and rs809451GC heterozygotes had a 1.78-fold (95% CI,1.16–2.74) and 1.86-fold (95% CI, 1.26–2.74) excess risk of developing TB compared to those with wild-type genotypes. A greater risk of TB disease was observed in population carrying C−809451-T−414171-C−622502 haplotype (OR 3.66, 95% CI:2.12–6.32). The G−809451-A−414171-C−622502-containing CISH promoter drove a 5.43-fold increased reporter expression compared to the C−809451-T−414171-C−622502-containing counterpart in Hela cell lines (P = 0.0009). PBMCs carrying rs414171TT homozygotes and rs809451GC heterozygotes showed a reduced CISH mRNA level compared to cells carrying wild type genotypes. Individuals with the rs414171TT genotype had significantly increased IL-12p40 and IL-10 production. In conclusion, CISH promoter rs414171 and rs809451 polymorphisms may play a vital role in mediating individual susceptibility to tuberculosis.