Programmed cell death 1 gene polymorphisms is associated with ankylosing spondylitis in Chinese Han population.

Programmed cell death 1 (PD-1) has been reported to have a genetic association in several autoimmune diseases. The aim of this study was to investigate the association of PD-1 polymorphisms and haplotypes with ankylosing spondylitis (AS) in Chinese Han population. In a case-control association study, three single-nucleotide polymorphisms (SNP), PD-1.3 G/A, PD-1.5 C/T and PD-1.9 T/C, were genotyped in 216 AS patients and 264 healthy controls using polymerase chain reaction-restriction fragment length polymorphism assay. All genotype distributions in the patients and in the controls were in Hardy-Weinberg equilibrium. The associations of genotypes and alleles with AS were analyzed. The genotype distributions of PD-1.9 were significantly different between the patients with AS and the controls (P = 0.025). The frequencies of TC genotype and T allele of PD-1.9 were higher in the patients than those in the controls (P = 0.026 and 0.004). No association for PD-1.5 in AS was found, and PD-1.3 was non-polymorphic in Chinese Han population. Moreover, the frequency of the CT haplotype (PD-1.5 C/T, PD-1.9 T/C) was significantly higher in AS patients than the controls (21.6 vs. 13.9%, P = 0.002). The CC haplotype was more common in the controls than in the patients (57.1 vs. 44.6%, P = 0.000). The results support a genetic association between the PD-1 polymorphism and susceptibility to AS in Chinese Han population.

High frequencies of HLA-B27 in Chinese patients with suspected of ankylosing spondylitis.

This study was performed to investigate the frequency of human leukocyte antigen (HLA)-B27 in Chinese patients with suspected of ankylosing spondylitis (AS) and to assess the clinical significance of HLA-B27 typing. A total of 1,016 patients suspected of AS were classified into six groups based on one major AS-related clinical manifestation. HLA-B27 was determined by polymerase chain reaction using sequence-specific primers. The frequency of B27 ranged between 24.3 and 46.7% among the patient groups, significantly higher than in healthy controls (2.4%). In the same group, the frequency of B27 in young (< or = 40 years) and in male patients was significantly higher than in the old and in female (P < 0.01). During a 1-year follow-up, 102 subjects were definitely diagnosed as AS, but only one B27(-) patient. Of the 102 definite patients, 69 (67.6%) definite patients were distributed in group 1 (low back pain and stiffness) with the higher incidence (28.5%) of AS. The incidence of AS in the same group was found with a similar pattern to the frequency of B27, in male and young patients significantly greater, except groups 4 and 6 (peripheral arthritis and alteration of skin). These findings confirm that HLA-B27 is one of sensitive diagnostic tools for early AS and suggest that there was a remarkable clinical significance of HLA-B27 typing in Chinese patients suspected of AS, particularly a young man who presents with low back pain and stiffness for > 3 months.

The association of HLA-B*27 subtypes with ankylosing spondylitis in Wuhan population of China.

The aim of this study was to investigate the association of the B27 subtypes with ankylosing spondylitis (AS) in the Wuhan population of China. We selected 317 HLA-B27-positive individuals (145 controls and 172 patients with ankylosing spondylitis). The B27 subtypes were characterized using a PCR-SSP method. Six B27 subtypes were determined: B*2702, 03, 04, 05, 06 and B*13. HLA-B*2704 and HLA-B*2705 were the two high frequency genotypes in controls and patients. Compared with the controls, the AS patients had high frequency of B*2704 (patients 69.2% vs. controls 53.8%) and low frequency of B*2705 (patients 23.8% vs. controls 33.1%). B*2703 was detected in 10 (5.8%) patients and in 13 (8.9%) controls. B*2702, 06 and B*2713 were relatively rare. Our results show that the allele conferring risk to AS in the Wuhan population of China was B*2704 and B*2705. B*2704 is strongly associated with AS.

A quantitative DNA methylation assay using mismatch hybridization and chemiluminescence.

OBJECTIVE: To develop a quantitative method for methylation analysis of the p16 gene based on mismatch hybridization and chemiluminescence. METHODS: Genomic DNA was modified by sodium bisulfite to convert all unmethylated but not methylated cytosines to uracil, and subsequently a pair of primer having no CpG sites was designed for amplification target DNA containing methylated or unmethylated CpG sites. The PCR product spanning CpG sites were hybridized with two oligonucleotide probes which perfectly matched the methylated and unmethylated CpG sequences respectively, and the hybrids were detected by chemiluminescent method. The percentage of methylated target sequences could be estimated by calculating the ratio of signals obtained with two probes. RESULTS: The percentage of methylation of artificial mixtures DNA showed a linear relation. There was a negative correlation between the methyaltion index with p16 transcriptional mRNA of p16 gene in tumor cell lines. CONCLUSION: Compared with existing methods, this assay is nonisotopic, rapid, simple, and can be widely applied to the study of DNA methylation.

Bioluminescent method for detecting telomerase activity.

BACKGROUND: Telomerase is a promising biomarker in cancer diagnosis and therapy. The elongation of telomeric repeats catalyzed by telomerase is accompanied by release of six PP(i) for each TTAGGG repeat (1 pmol PP(i)/310 pg telomeric repeats). We developed a novel method to measure telomerase activity by use of an enzymatic luminometric PP(i) assay (ELIPA). METHODS: Extracts of cell lines and tissues were incubated with primer at 30 degrees C for 30 min. Released PP(i) was converted to ATP by sulfurylase, and ATP was detected by a luciferase bioluminescence system. The ELIPA results were compared with results obtained with the conventional telomeric repeat amplification (TRAP)-ELISA in 42 lung carcinoma tissues and 27 control tissues without malignancy. RESULTS: The lower detection limits of ELIPA and TRAP-ELISA were 5 and 10 cells, respectively. The within-run imprecision (CV) of ELIPA was < or =12%. When compared with TRAP-ELISA, the correlation coefficient (r) was 0.79. When we used the cutoff value from ROC analysis to distinguish malignant and nonmalignant tissues, the sensitivity and specificity of ELIPA were 83% and 96%, respectively, whereas the sensitivity and specificity of TRAP-ELISA were 71% and 96%, respectively. CONCLUSION: ELIPA is a simple and sensitive homogeneous method to quantify telomerase activity.