An ankylosing spondylitis risk variant alters osteoclast differentiation.

OBJECTIVE: To explore whether the variants in non MHC proteasome gene are associated with AS and explain the role of the variant in the disease. MATERIAL AND METHODS: Case-control analysis to identify AS predisposition genes; dual-luciferase reporter assay, immunoblot analysis and osteoclastogenesis assays to detect the function of the positive variant. Affected individuals were diagnosed according to the modified New York Criteria by at least two experienced rheumatologists, and rechecked by another rheumatologist. RESULTS: The study included 1037 AS patients and 1014 no rheumatic and arthritis disease controls. The main age of AS onset is between 16 and 35 years old. HLA-B27-positive subjects comprised 90.0% of patients. A nonsynonymous SNP rs12717 in proteasome gene PSMB1 significantly associated with AS. Individuals with CC genotype had a higher onset risk compared with those with GG/GC genotypes (OR = 1.89, P = 0.0047). We also discovered that PSMB1 regulates the receptor activator of nuclear factor-κB (RANK)/RANK ligand (RANKL) signalling pathway and the disease-associated variant PSMB1-Pro11 significantly inhibits RANKL-induced NF-κB pathway in osteoclast differentiation via the degradation of IKK-ß compared with PSMB1-Ala11. RANKL induced osteoclast differentiation was significantly lower in primary monocyte osteoclast precursor from individuals with genotype PSMB131C/31C compared with individuals with genotype PSMB131G/31G. CONCLUSIONS: These results reveal a novel understanding of the bone formation and reabsorbing imbalance in AS. The new bone formation phenotype can be attributed to the inhibition of osteoclast differentiation by a more functional PSMB1 gene.

A functional polymorphism affecting the APOA5 gene expression is causally associated with plasma triglyceride levels conferring coronary atherosclerosis risk in Han Chinese Population.

Apolipoprotein A5 (APOA5) gene plays a key role in plasma triglyceride (TG) metabolism, and shows the involvement in coronary artery disease (CAD). A set of single nucleotide polymorphisms around the APOA5 gene was identified to be associated with plasma TG levels. It is of biological and clinical importance to discern the genuine genetic determinants. A polymorphism in 3' untranslated region of the APOA5 gene, rs2266788, is deserving of investigation for suggestive clues from the association in multiple independent studies. In this study, rs2266788 was genotyped in 3222 unrelated subjects consisting of 2062 CAD cases and 1160 controls. The statistical analyses indicated that the minor C allele of rs2266788 was significantly associated with elevated plasma TG levels and higher CAD risk. In normal human liver tissues, comparison of global APOA5 mRNA levels among genotypes and allelic expression imbalance analysis showed the decreased gene expression for the C allele. Luciferase assays confirmed a concordant result that transcriptional activity was lowered for the C allele compared with the T allele in four cell lines. Multiple lines of evidence in our study supported that rs2266788 was causally associated with plasma TG levels conferring CAD risk in Han Chinese population owing to a cis-acting effect to the APOA5 gene expression.

Gene-wide characterization of common quantitative trait loci for ABCB1 mRNA expression in normal liver tissues in the Chinese population.

In order to comprehensively screen genetic variants leading to differential expression of the important human ABCB1 gene in the primary drug-metabolizing organ, ABCB1 mRNA expression levels were measured in 73 normal liver tissue samples from Chinese subjects. A set of Tag SNPs. were genotyped. In addition, imputation was performed within a 500 kb region around the ABCB1 gene using the reference panels of 1,000 Genome project and HapMap III. Bayesian regression was used to assess the strength of associations by compute Bayes Factors for imputed SNPs. Through imputation and linkage disequilibrium analysis, the imputed loci rs28373093, rs1002205, rs1029421, rs2285647, and rs10235835, may represent independent and strong association signals. rs28373093, a polymorphism 1.5 kb upstream from the ABCB1 transcription start site, has the strongest association. 2677 G>A/T and 3435C>T confer a clear gene-dosage effect on ABCB1 mRNA expression. The systematic characterization of gene-wide common quantitative trait loci associated with ABCB1 mRNA expression in normal liver tissues would provide the candidate markers to ABCB1-relevant clinical phenotypes in Chinese population.

Functional evaluation of genetic and environmental regulators of p450 mRNA levels.

Variations in the activities of Cytochrome P450s are one of the major factors responsible for inter-individual differences in drug clearance rates, which may cause serious toxicity or inefficacy of therapeutic drugs. Various mRNA level is one of the key factors for different activity of the major P450 genes. Although both genetic and environmental regulators of P450 gene expression have been widely investigated, few studies have evaluated the functional importance of cis- and trans-regulatory factors and environmental factors in the modulation of inter-individual expression variations of the P450 genes. In this study, we measured the mRNA levels of seven major P450 genes (CYP1A1, CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4 and CYP3A5) in 96 liver biopsy samples from Chinese population. Both trans-acting (mRNA levels and non-synonymous SNPs of putative regulator genes) and cis-acting (gene copy number and functional SNPs) factors were investigated to identify the determinants of the expression variations of these seven P450 genes. We found that expression variations of most P450 genes, regulator genes and housekeeping genes were positively correlated at the mRNA level. After partial correlation analysis using ACTB and GAPDH expression to eliminate the effect of global regulators, a UPGMA (Unweighted Pair Group Method with Arithmetic Mean) tree was constructed to reveal the effects of specific regulation networks potentially masked by global regulators. Combined with the functional analysis of regulators, our results suggested that expression variation at the mRNA level was mediated by several factors in a gene-specific manner. Cis-acting genetic variants might play key roles in the expression variation of CYP2D6 and CYP3A5, environmental inducers might play key roles in CYP1A1 and CYP1A2 variation and global regulators might play key roles in CYP2C9 variation. In addition, the functions of regulators that play less important roles in controlling expression variation for each P450 gene were determined.

A haplotype of the catalase gene confers an increased risk of essential hypertension in Chinese Han.

Our previous study in an isolated population showed an association between a genetic variant in the catalase gene (CAT) and essential hypertension (EH). This study indicates that three variants in the promoter and 5'-UTR region of CAT are predominant in Chinese Han, and they form two major haplotypes. A case-control study showed that the CATH2 haplotype confers susceptibility to EH (Pgenotype=0.0017, and Pallilc=0.00078). Subjects bearing CATH1/CATH2 and CATH2/CATH2 genotypes demonstrated a higher susceptibility to EH than CATH1/CATH1 homozygotes, with odds ratios of 1.474 and 1.625, respectively. Also, CATH1/CATH1 individuals had a later-onset age (P=0.015). Expression analysis using luciferase reporter vectors indicated that the CATH1 haplotype showed a lower transcriptional activity than the haplotype CATH2 (P<0.05 in all four cell lines), and we observed similar results in the endogenous allelic expression ratios of CATH1/CATH2 in cell lines. In contrast, most CATH1 haplotypes showed a higher transcription level than CATH2 haplotypes (10 out of 11 or 90.9%) in blood from normal individuals (P<0.01). We therefore hypothesize that CATH1 and CATH2 may play alternating roles at different level of oxidative stress.

Analysis of RGS2 expression and prognostic significance in stage II and III colorectal cancer.

The role of RGS2 (regulator of G-protein signalling 2) has been studied in several tumours. The purpose of the present study is to investigate the correlations between clinicopathological factors and patients' survival time and RGS2 expression in stage II and III CRC (colorectal cancer) patients. Real-time quantitative PCR was performed in 36 CRC tissues with recurrence and 28 without recurrence, and in three CRC-metastasis-derived cell lines (SW620, LoVo and Colo205) and 3 primary-CRC-derived ones (SW480, Caco-2 and HCT116) to examine RGS2 mRNA expression. In addition, to provide visualized evidence for RGS2 mRNA expression, random CRC samples were also performed with RT-PCR (reverse transcription-PCR). RGS2 protein was detected by immunostaining in 118 paraffin-embedded specimens, and the correlations between clinicopathological factors and survival time and RGS2 expression were analysed. We found that RGS2 mRNA was down-regulated both in CRC tissues with recurrence and metastasis-derived cell lines, and the expression level of RGS2 was unrelated to gender, age, tumour grade, or lymphovascular or perineural invasion. However, it was positively related to disease-free survival time (P<0.05). Furthermore, low RGS2 expression indicated a poorer survival rate (P<0.05, log-rank test). Multivariate analysis also showed that weak RGS2 protein expression was an independent adverse prognosticator in CRC (P<0.05). Taken together, we suggested that down-regulation of RGS2 might play an important role in CRC metastasis and predict poor prognosis in stage II and III CRC patients.

Sox17, the canonical Wnt antagonist, is epigenetically inactivated by promoter methylation in human breast cancer.

SRY-box 17 (Sox17) is a transcription factor which involved in a variety of developmental processes and can act as an antagonist of canonical Wnt/beta-catenin signaling pathway. However, the relationship between Sox17 gene expression, methylation status, and beta-catenin in breast cancer has not been established. Here we report that the expression level of Sox17 mRNA was dramatically decreased in five different breast cancer cell lines and 23 of 31 primary breast tumor samples, which significantly correlated with its methylation status. After treated with 5-aza-2'-deoxycytidine (5-aza-dC, a demethylation agent), the expression levels of Sox17 mRNA and protein were obviously increased. Restored expression of Sox17 by 5-aza-dC treatment decreased the expression level of beta-catenin in breast cancer cell lines. Furthermore, small interfering RNA (siRNA)-mediated knockdown of Sox17 in SKBR-3 and Bacp-37 cells enhanced beta-catenin expression. In 31 paired tissue samples, a significant difference between the expression level of Sox17 and beta-catenin was also observed (P < 0.001). Clinically, Sox17 methylation was detected in 74.3% breast tumors (84/113) and 31.9% (36/113) paired normal tissues, respectively (P < 0.0001). Sox17 methylation was also associated with tumor stage (P = 0.028) and lymph node metastasis (P = 0.013). These findings indicate that silencing of Sox17 due to promoter hypermethylation is a frequent event and may contribute to aberrant activation of Wnt signaling in breast cancer. Sox17 may be a valuable biomarker for the study of breast cancer carcinogenesis and progression.

Frequent epigenetic inactivation of the receptor tyrosine kinase EphA5 by promoter methylation in human breast cancer.

EphA5 is a member of the Eph receptor tyrosine kinase family, which plays a critical role in the regulation of carcinogenesis. Our previous DNA methylation microarray results suggested that the CpG islands in the EphA5 promoter exhibited higher methylation levels in breast cancer tissues. In this study, we further analyzed EphA5 gene expression profiles, methylation status, and clinical implications in breast cancer. We found that the level of EphA5 mRNA was dramatically decreased in 5 different breast cancer cell lines. After treating the cell lines with 5-aza-2'-deoxycytidine (5-aza-dC, a demethylation agent), the levels of EphA5 mRNA and protein were significantly increased. Bisulfite sequencing and methylation-specific polymerase chain reaction detection showed that decreased expression of EphA5 was associated with its methylation status. We also found a significant correlation (P = .017) between the reduction of EphA5 mRNA levels and aberrant methylation of EphA5 in 31 paired tissue samples. In clinical samples, EphA5 methylation was detected in 64.1% (75/117) of breast tumors and 28.2% (33/117) of paired normal tissues (P < .001), which was associated with higher tumor grade (P = .024), lymph node metastasis (P = .004), and progesterone receptor-negative status (P = .008). Our data indicate that EphA5 might be a potential target for epigenetic silencing in primary breast cancer and a valuable molecular marker for breast cancer carcinogenesis and progression.

Polymorphisms in the ADRB2 gene and Graves disease: a case-control study and a meta-analysis of available evidence.

BACKGROUND: The beta-2-Adrenergic receptor (ADRB2) gene on chromosome 5q33.1 is an important immunoregulatory factor. We and others have previously implicated chromosomal region 5q31-33 for contribution to the genetic susceptibility to Graves disease (GD) in East-Asian populations. Two recent studies showed associations between the single nucleotide polymorphism (SNP) rs1042714 in the ADRB2 gene and GD. In this study, we aimed to fully investigate whether the ADRB2 gene conferred susceptibility to GD in Chinese population, and to perform a meta-analysis of association between ADRB2 and GD. METHODS: Approximately 1 kb upstream the transcription start site and the entire coding regions of the ADRB2 gene were resequenced in 48 Han Chinese individuals to determine the linkage disequilibrium (LD) patterns. Tag SNPs were selected and genotyped in a case-control collection of 1,118 South Han Chinese subjects, which included 428 GD patients and 690 control subjects. A meta-analysis was performed with the data obtained in the present samples and those available from prior studies. RESULTS: Fifteen SNPs in the ADRB2 gene were identified by resequencing and one SNP was novel. Ten tag SNPs were investigated further to assess association of ADRB2 in the case-control collection. Neither individual tag SNP nor haplotypes showed association with GD in Han Chinese population (P > 0.05). Our meta-analysis of the ADRB2 SNP rs1042714 measured heterogeneity between the ethnic groups (I2 = 53.1%) and no association to GD was observed in the overall three studies with a random effects model (OR = 1.13, 95% CI, 0.95 to 1.36; P = 0.18). However, significant association was found from the combined data of Caucasian population with a fixed effects model (OR = 1.18, 95% CI, 1.06 to 1.32; P = 0.002; I2 = 5.9%). CONCLUSION: Our study indicated that the ADRB2 gene did not exert a substantial influence on GD susceptibility in Han Chinese population, but contributed to a detectable GD risk in Caucasian population. This inconsistency resulted largely from between-ethnicity heterogeneity.

Polymorphisms in interleukin-15 gene on chromosome 4q31.2 are associated with psoriasis vulgaris in Chinese population.

Through a series of linkage analyses in a large Chinese family cohort of psoriasis, we previously identified and confirmed a non-HLA psoriasis linkage locus PSORS9 within a small region at 4q31.2-32.1. Within the critical region of the PSORS9 locus, IL-15 has been long recognized as a strong candidate gene for psoriasis. In this study, we investigated the association between IL-15 genetic polymorphisms and psoriasis in a large Chinese sample. Highly significant evidence for association was identified at a single-nucleotide polymorphism (SNP) (g.96516A --> T) within the 3'-untranslated region (UTR) of the IL-15 gene (P=0.00006, after correction for multiple testing). Haplotype analysis using the SNPs within the 3'UTR region also provided strong supporting evidence for association (P=0.00005), where we identified a haplotype of the 3'UTR region of IL-15 associated with increased risk to psoriasis (odds ratio=1.65). This association was also supported by the results of our expression activity analyses, where we demonstrated that the identified risk haplotype is associated with an increased activity of IL-15. Therefore, we provided early evidence for the important role of IL-15 genetic variants in the pathogenesis of psoriasis, probably by increasing interleukin production and inflammation in the lesions of psoriasis.