Liming Bao

Regions of homozygosity identified by SNP microarray analysis aid in the diagnosis of autosomal recessive disease and incidentally detect parental blood relationships

Purpose:The purpose of this study was to document the ability of single-nucleotide polymorphism microarray to identify copy-neutral regions of homozygosity, demonstrate clinical utility of regions of homozygosity, and discuss ethical/legal implications when regions of homozygosity are associated with a parental blood relationship.Methods:Study data were compiled from consecutive samples sent to our clinical laboratory over a 3-year period. A cytogenetics database identified patients with at least two regions of homozygosity >10 Mb on two separate chromosomes. A chart review was conducted on patients who met the criteria.Results:Of 3,217 single-nucleotide polymorphism microarrays, 59 (1.8%) patients met inclusion criteria. The percentage of homozygosity ranged from 0.9 to 30.1%, indicating parental relationships from distant to first-degree relatives. First-degree kinship was suspected in the parents of at least 11 patients with regions of homozygosity covering >21.3% of their autosome. In four patients from two families, homozygosity mapping discovered a candidate gene that was sequenced to identify a clinically significant mutation.Conclusion:This study demonstrates clinical utility in the identification of regions of homozygosity, as these regions may aid in diagnosis of the patient. This study establishes the need for careful reporting, thorough pretest counseling, and careful electronic documentation, as microarray has the capability of detecting previously unknown/unreported relationships.Genet Med 2013:15(1):70–78

The E3 Deubiquitinase USP17 Is a Positive Regulator of Retinoic Acid-related Orphan Nuclear Receptor γt (RORγt) in Th17 Cells

Background: RORγt is the master transcription factor in Th17 cells. Results: USP17 stabilizes RORγt via deubiquitination, and USP17 levels are up-regulated in systemic lupus erythematosus. Conclusion: USP17 is a positive regulator of RORγt. Significance: USP17 could be a potential drug target to modulate RORγt-mediated autoimmune diseases such as systemic lupus erythematosus. Stable retinoic acid-related orphan nuclear receptor γt (RORγt) expression is pivotal for the development and function of Th17 cells. Here we demonstrate that expression of the transcription factor RORγt can be regulated through deubiquitination, which prevents proteasome-mediated degradation. We establish that USP17 stabilizes RORγt protein expression by reducing RORγt polyubiquitination at its Lys-360 residue. In contrast, knockdown of endogenous USP17 in Th17 cells resulted in decreased RORγt protein levels and down-regulation of Th17-related genes. Furthermore, USP17 expression was up-regulated in CD4+ T cells from systemic lupus erythematosus patients. Our data reveal a molecular mechanism in which RORγt expression in Th17 cells can be positively regulated by USP17, thereby modulating Th17 cell functions.

Case–control study of risk factors of myelodysplastic syndromes according to World Health Organization classification in a Chinese population†

Risk factors of mydelodysplastic syndromes (MDS) remain largely unknown. We conducted a hospital‐based case–control study consisting of 403 newly diagnosed MDS patients according to World Health Organization classification and 806 individually gender and age‐matched patient controls from 27 major hospitals in Shanghai, China, to examine relation of lifestyle, environmental, and occupational factors to risk of MDS. The study showed that all MDS (all subtypes combined) risk factors included anti tuberculosis drugs [odds ratio (OR)adj = 3.15; 95% confidence interval (CI) = 1.22–8.12] as an independent risk factor, benzene (ORadj = 3.73; 95% CI = 1.32–10.51), hair dye use (OR = 1.46; 95% CI = 1.03–2.07), new building and renovations (OR = 1.69; 95% CI = 1.11–2.00), pesticides (OR = 2.16; 95% CI = 1.22–3.82), and herbicides (OR = 5.33; 95% CI = 1.41–20.10) as relative risk factors. Risk factors of MDS subtype refractory cytopenia with multiple dysplasia (RCMD) were benzene (ORadj = 5.99; 95% CI = 1.19–30.16) and gasoline (ORadj = 11.44; 95% CI = 1.31–100.03) as independent risk factors, and traditional Chinese medicines (OR = 2.17; 95% CI = 1.15–4.07), pesticides (OR = 2.92; 95% CI = 1.37–6.25), and herbicides (OR = 12.00; 95% CI = 1.44–99.67) as relative risk factors. Smoking tobacco was significantly associated with refractory anemia with excess of blasts (RAEB) (ORadj = 2.43; 95% CI = 1.02–5.77). Education is shown as an independent protective factor against all MDS (ORadj = 0.90; 95% CI = 0.83–0.99) and RCMD (ORadj = 0.89; 95% CI = 0.79–0.99). These findings suggest that multiple modifiable behavioral, environmental, and occupational factors play a role in MDS etiology, and various MDS subtypes may have different susceptibility. Am. J. Hematol. 86:163–169, 2011.

Investigation of Mycoplasma pneumoniae infection in pediatric population from 12,025 cases with respiratory infection☆

Although Mycoplasma pneumoniae (MP) is a major pathogen of primary atypical pneumonia in children, the clinical and laboratory characteristics of MP infection in large pediatric population are less reported. Here, we retrospectively analyzed 12,025 hospitalized children with respiratory infection by using serology and polymerase chain reaction (PCR) methods simultaneously. The results showed that 2433 (20.23%) children had MP infection, which mainly occurred in November to April. The presence of sore throat and pharyngitis was peculiar to MP infection. The positive percentage of MP-DNA was higher than that of MP-IgM in children aged <1 (P < 0.0001) and 1-3 years (P < 0.0001). Moreover, the positive rate of P1 gene, the key adhesion gene for MP infection, was higher in children with MP infection than in those with other pathogens (P < 0.0001). Our work provides the clinical information of children MP infection and highlights the superiority of PCR and potential usage of P1 as a diagnosis target for MP infection.

Depletion of OLFM4 gene inhibits cell growth and increases sensitization to hydrogen peroxide and tumor necrosis factor-alpha induced-apoptosis in gastric cancer cells.

BackgroundHuman olfactomedin 4 (OLFM4) gene is a secreted glycoprotein more commonly known as the anti-apoptotic molecule GW112. OLFM4 is found to be frequently up-regulated in many types of human tumors including gastric cancer and it was believed to play significant role in the progression of gastric cancer. Although the function of OLFM4 has been indicated in many studies, recent evidence strongly suggests a cell or tissue type-dependent role of OLFM4 in cell growth and apoptosis. The aim of this study is to examine the role of gastric cancer-specific expression of OLFM4 in cell growth and apoptosis resistance.MethodsOLFM4 expression was eliminated by RNA interference in SGC-7901 and MKN45 cells. Cell proliferation, anchorage-independent growth, cell cycle and apoptosis were characterized in vitro. Tumorigenicity was analyzed in vivo. The apoptosis and caspase-3 activation in response to hydrogen peroxide (H2O2) or tumor necrosis factor-alpha (TNF α) were assessed in the presence or absence of caspase inhibitor Z-VAD-fmk.ResultsThe elimination of OLFM4 protein by RNA interference in SGC-7901 and MKN45 cells significantly inhibits tumorigenicity both in vitro and in vivo by induction of cell G1 arrest (all P < 0.01). OLFM4 knockdown did not trigger obvious cell apoptosis but increased H2O2 or TNF α-induced apoptosis and caspase-3 activity (all P < 0.01). Treatment of Z-VAD-fmk attenuated caspase-3 activity and significantly reversed the H2O2 or TNF α-induced apoptosis in OLFM4 knockdown cells (all P < 0.01).ConclusionOur study suggests that depletion of OLFM4 significantly inhibits tumorigenicity of the gastric cancer SGC-7901 and MKN45 cells. Blocking OLFM4 expression can sensitize gastric cancer cells to H2O2 or TNF α treatment by increasing caspase-3 dependent apoptosis. A combination strategy based on OLFM4 inhibition and anticancer drugs treatment may provide therapeutic potential in gastric cancer intervention.

TRAF5-mediated Lys-63-linked Polyubiquitination Plays an Essential Role in Positive Regulation of RORγt in Promoting IL-17A Expression.

Background: RORγt is required for Th17 cell function and differentiation. Results: TRAF5 stabilizes RORγt by ubiquitination and augments RORγt-mediated transcriptional expression of IL-17A. Conclusion: TRAF5 is a positive regulator for RORγt. Significance: TRAF5 could be a novel target for modulating RORγt-mediated inflammation and autoimmune diseases, including systemic lupus erythematosus. Retinoid-related orphan nuclear receptor γt (RORγt) is a key transcription factor for the development and function of Th17 cells. In this study, we show that tumor necrosis factor receptor-associated factor 5 (TRAF5), known as an E3 ubiquitin protein ligase and signal transducer, interacts with and ubiquitinates RORγt via Lys-63-linked polyubiquitination. TRAF5 stabilizes the RORγt protein level depending on its RING finger domain. Depletion of TRAF5 in Th17 cells destabilizes RORγt protein and down-regulates Th17-related genes, including IL-17A, an inflammatory cytokine involved in pathogenic mechanisms of several autoimmune diseases such as systemic lupus erythematosus. Moreover, up-regulation of the TRAF5 mRNA level was found in systemic lupus erythematosus patient CD4+ T cells. Our findings reveal a direct link between TRAF5-mediated ubiquitination and RORγt protein regulation, which may aggravate inflammatory progress and provide new therapeutic drug targets for autoimmune diseases.

Detecting 22q11.2 deletion in Chinese children with conotruncal heart defects and single nucleotide polymorphisms in the haploid TBX1 locus

BackgroundConotruncal heart defects (CTDs) are present in 75-85% of patients suffering from the 22q11.2 deletion syndrome. To date, no consistent phenotype has been consistently correlated with the 22q11.2 deletions. Genetic studies have implicated TBX1 as a critical gene in the pathogenesis of the syndrome. The aim of study was to determine the incidence of the 22q11.2 deletion in Chinese patients with CTDs and the possible mechanism for pathogenesis of CTDs.MethodsWe enrolled 212 patients with CTDs and 139 unrelated healthy controls. Both karyotypic analysis and multiplex ligation-dependent probe amplification were performed for all CTDs patients. Fluorescence in situ hybridization was performed for the patients with genetic deletions and their relatives. The TBX1 gene was sequenced for all patients and healthy controls. The χ2 and Fishers exact test were used in the statistical analysis.ResultsThirteen of the 212 patients with CTDs (6.13%) were found to have the 22q11.2 deletion syndrome. Of the 13 cases, 11 presented with a hemizygous interstitial microdeletion from CLTCL1 to LZTR1; one presented with a regional deletion from CLTCL1 to DRCR8; and one presented with a regional deletion from CDC45L to LZTR1. There were eight sequence variants in the haploid TBX1 genes of the del22q11 CTDs patients. The frequency of one single nucleotide polymorphism (SNP) in the del22q11 patients was different from that of the non-del patients (P < 0.05), and the frequencies of two other SNPs were different between the non-del CTDs patients and controls (P < 0.05).ConclusionsCTDs, especially pulmonary atresia with ventricular septal defect and tetralogy of Fallot, are the most common disorders associated with the 22q11.2 deletion syndrome. Those patients with both CTDs and 22q11.2 deletion generally have a typical or atypical deletion region within the TBX1 gene. Our results indicate that TBX1 genetic variants may be associated with CTDs.

WT1 mutations and single nucleotide polymorphism rs16754 analysis of patients with pediatric acute myeloid leukemia in a Chinese population

Abstract Acute myeloid leukemia (AML) is relatively rare in children. Somatic mutations including the single nucleotide polymorphism (SNP) rs16754 in Wilms tumor 1 gene (WT1) and their prognostic relevance in pediatric AML have not been studied in Chinese populations. We analyzed WT1 mutations and rs16754 genotypes in a cohort of 86 patients with de novo pediatric AML in a Chinese population. We detected WT1 mutations in approximately 20% of the patients. Most of the mutations identified were deletions and insertions clustered in exons 7 and 9. No differences were observed with respect to overall survival and relapse-free survival between patients with and without WT1 mutations. The analysis of rs16754 in WT1 exon 7 revealed G as the major allele. Patients with the rs16754GG genotype had improved overall survival (p =0.020) and relapse-free survival (p =0.025) compared with those with either rs16754GA or rs16754AA. Moreover, better overall survival (p =0.044) and relapse-free survival (p =0.068) were observed among patients with wild-type CEBPA with rs16754GG compared with those carrying rs16754GA/AA.

Combined 677CC/1298AC genotypes of methylenetetrahydrofolate reductase (MTHFR ) reduce susceptibility to precursor B lymphoblastic leukemia in a Chinese population.

The methylenetetrahydrofolate reductase (MTHFR) encodes a major enzyme in folate metabolism. It has been suggested that two MTHFR polymorphisms, 677C>T and 1298A>C, influence risk of acute lymphoblastic leukemia (ALL). Most studies on relation of MTHFR polymorphisms to ALL susceptibility have been in pediatric populations because ALL is relatively rare in adults. Here, we report a case–control study of 127 Chinese patients with adult precursor B lymphoblastic leukemia (B‐ALL) to examine correlation between the MTHFR polymorphisms and B‐ALL susceptibility in adults. Our data show that although the prevalence of genotype 1298CC was significantly higher in the female patients than in the controls (P = 0.04), the differences in distributions of combined genotypes of 1298CC with either 677CC or 677CT between the cases and the controls were statistically insignificant. Haplotype analysis revealed no significant difference between the cases and the controls. The prevalence for joint MTHFR genotypes 677CC/1298AC was significantly lower in the female B‐ALL cases than in the controls [odds ratio (OR) = 0.06, 95% CI = 0.00–0.53, P = 0.0033] and no differences among the men [OR = 0.71, 95% CI = 0.20–2.53, P = 0.55], suggesting that protective effects of combined MTHFR 677CC/1298AC genotypes on susceptibility of adult B‐ALL are gender bias toward women with 677CC/1298AC women being at a 17‐fold reduced odds to develop B‐ALL.

HLA class II alleles may influence susceptibility to adult dermatomyositis and polymyositis in a Han Chinese population

BackgroundPolymyositis (PM) and dermatomyositis (DM) are idiopathic inflammatory myopathies. Genetic variability in human leukocyte antigen (HLA) genes plays an important role in the pathogenesis of PM and DM. However, few studies on the subject in Chinese populations have been reported thus far.MethodsWe studied the influence of HLA polymorphisms on DM and PM susceptibility by analyzing HLA-DRB1, HLA-DQA1, and HLA-DQB1 alleles in 71 adult DM patients, 20 adult PM patients, and 113 controls in a Han Chinese population.ResultsA positive association was found between HLA-DQA1*0104 and DM (p = 0.01; corrected p (pcorr) NS; odds ratio (OR) = 2.58; 95% confidence interval (CI): 1.18–5.64), while an inverse correlation was noted between HLA-DQB1*0303 and myositis patients with interstitial lung inflammation (p = 0.01; pcorr NS; OR = 0.25; 95% CI: 0.07–0.73). A positive relationship was also observed between HLA-DRB1*07 and DM (p = 0.01; pcorr NS; OR = 2.26; 95% CI: 1.12–4.59), while HLA-DRB1*03 seems to be protective against DM (p = 0.01; pcorr NS; OR = 0.26; 95% CI: 0.06–0.81). The lung complication was closely associated with HLA-DRB1*04 (p = 0.01; pcorr NS; OR = 2.82; 95% CI: 1.15–6.76) and HLA-DRB1*12 (p = 0.02; pcorr NS; OR = 2.52; 95% CI: 1.02–6.07). The frequency of HLA-DRB1*07 was significantly higher among myositis patients with dysphagia than among controls (p = 0.01; pcorr NS; OR = 4.78; 95% CI: 1.03–24.42). The putative haplotype DRB1*07-DQA1*01-DQB1*02 was positively correlated with DM (p = 0.03; pcorr NS; OR = 2.90; 95% CI: 1.02–8.93) and the lung complication (p = 0.02; pcorr NS; OR = 3.45; 95% CI: 1.04–11.58).ConclusionsOur results demonstrate that HLA alleles may be involved in susceptibility to adult DM and PM in the Han Chinese population.