Interaction between Retinoid Acid Receptor-Related Orphan Receptor Alpha (RORA) and Neuropeptide S Receptor 1 (NPSR1) in Asthma

Retinoid acid receptor-related Orphan Receptor Alpha (RORA) was recently identified as a susceptibility gene for asthma in a genome-wide association study. To investigate the impact of RORA on asthma susceptibility, we performed a genetic association study between RORA single nucleotide polymorphisms (SNPs) in the vicinity of the asthma-associated SNP (rs11071559) and asthma-related traits. Because the regulatory region of a previously implicated asthma susceptibility gene, Neuropeptide S receptor 1 (NPSR1), has predicted elements for RORA binding, we hypothesized that RORA may interact biologically and genetically with NPSR1. 37 RORA SNPs and eight NPSR1 SNPs were genotyped in the Swedish birth cohort BAMSE (2033 children) and the European cross-sectional PARSIFAL study (1120 children). Seven RORA SNPs confined into a 49 kb region were significantly associated with physician-diagnosed childhood asthma. The most significant association with rs7164773 (T/C) was driven by the CC genotype in asthma cases (OR = 2.0, 95%CI 1.36–2.93, p = 0.0003 in BAMSE; and 1.61, 1.18–2.19, p = 0.002 in the combined BAMSE-PARSIFAL datasets, respectively), and strikingly, the risk effect was dependent on the Gln344Arg mutation in NPSR1. In cell models, stimulation of NPSR1 activated a pathway including RORA and other circadian clock genes. Over-expression of RORA decreased NPSR1 promoter activity further suggesting a regulatory loop between these genes. In addition, Rora mRNA expression was lower in the lung tissue of Npsr1 deficient mice compared to wildtype littermates during the early hours of the light period. We conclude that RORA SNPs are associated with childhood asthma and show epistasis with NPSR1, and the interaction between RORA and NPSR1 may be of biological relevance. Combinations of common susceptibility alleles and less common functional polymorphisms may modify the joint risk effects on asthma susceptibility.     

Association of SLC22A4 Gene Polymorphism with Rheumatoid Arthritis in the Chinese Population

Rheumatoid arthritis (RA) is a chronic inflammatory disease with complex genetic factors. Single‐nucleotide polymorphisms (SNPs) in the SLC22A4 gene have been previously reported to be associated with RA in Japanese but not European populations. This study further investigated the association of SLC22A4 polymorphisms, in particular slc2F1/slc2F2, with RA in the Chinese population, the largest Asian population. A total of 160 human subjects with 95 RA patients and 65 healthy controls were genotyped for slc2F1‐G/A and slc2F2‐C/T polymorphisms. The results showed that there was a significant difference in the genotype distribution of these two polymorphisms between the two groups. In addition, the presence of slc2F1 A allele and slc2F2 T allele carries a 1.93‐fold and 2.14‐fold increased risk for anticyclic citrullinated peptide (CCP) positivity, respectively. Overall, this study provided evidence that SLC22A4 gene polymorphisms played important roles in the etiology of RA in the largest Asian population, the Chinese population.     

红鳍东方鲀(Takifugu rubripes)MC4R基因的多态性分析

采用PCR-SSCP(single strand conformation polymorphism)技术和DNA测序方法分析红鳍东方鲀MC4R(Melanocortin-4receptor)基因编码区多态性。在MC4R基因编码区48 nt和264 nt均发生了碱基的转换突变(G→A),两个突变位点分别位于M1和M2引物扩增产物中。引物M1扩增产物SSCP分析得到两种基因型:AA基因型和AB基因型,并且AA基因型和A等位基因频率明显高于AB基因型和B等位基因。引物M2扩增产物也得到两种基因型:CC基因型和CD基因型,CC基因型和C等位基因频率明显高于CD基因型和D等位基因。遗传变异结果分析表明,两个突变位点均属于低度多态性,而且群体遗传杂合度较低,反映了该群体的遗传一致性较高。     

Increase of Hemoglobin Levels by Anti-IL-6 Receptor Antibody (Tocilizumab) in Rheumatoid Arthritis

Objective

To compare the effect of tocilizumab (TCZ) with other biologic therapies in improving anemia of rheumatoid arthritis (RA) patients.

Methods

We compared the change of hemoglobin (Hb) levels in a cohort of 147 consecutive RA patients who were treated with biologics for more than 12 weeks. Twenty eight patients were treated with TCZ, and 119 patients were treated with biologics other than TCZ (87 with TNF inhibitors and 32 with abatacept). The change of Hb levels from baseline to week 12 was compared between the TCZ and the non-TCZ groups. We performed univariate and multivariate analyses with adjustment of potential confounders such as baseline characteristics, concomitant treatment, and the clinical response to treatment.

Results

Hb levels generally increased after biologic therapies both in the TCZ and the non-TCZ groups. The increase of Hb levels was greater in the TCZ group than in the non-TCZ groups (1.1 g/dL in the TCZ group vs 0.3 g/dL in the non-TCZ group, p = 0.009). Univariate analysis revealed that increase of Hb levels was also significantly associated with lower Hb, higher Low Hemoglobin Density, and higher CRP levels at baseline and greater reduction in the clinical disease activity index. TCZ therapy was significantly associated with the increase of Hb levels even after adjustment for these factors by multivariate analysis (p<0.001, effect size 0.08–0.12).

Conclusion

TCZ therapy is an independent factor associated with the increase of Hb level after biologic therapies in RA patients. It will help in selecting appropriate biologics for RA patients with anemia.     

Gene Polymorphisms and Pharmacogenetics in Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a systemic, chronic and inflammatory disease of unknown etiology with genetic predisposition. The advent of new biological agents, as well as the more traditional disease-modifying antirheumatic drugs, has resulted in highly efficient therapies for reducing the symptoms and signs of RA; however, not all patients show the same level of response in disease progression to these therapies. These variations suggest that RA patients may have different genetic regulatory mechanisms. The extensive polymorphisms revealed in non-coding gene-regulatory regions in the immune system, as well as genetic variations in drug-metabolizing enzymes, suggest that this type of variation is of functional and evolutionary importance and may provide clues for developing new therapeutic strategies. Pharmacogenetics is a rapidly advancing area of research that holds the promise that therapies will soon be tailored to an individual patient’s genetic profile.     

A Single-Nucleotide Polymorphism of Human Neuropeptide S Gene Originated from Europe Shows Decreased Bioactivity

Using accumulating SNP (Single-Nucleotide Polymorphism) data, we performed a genome-wide search for polypeptide hormone ligands showing changes in the mature regions to elucidate genotype/phenotype diversity among various human populations. Neuropeptide S (NPS), a brain peptide hormone highly conserved in vertebrates, has diverse physiological effects on anxiety, fear, hyperactivity, food intake, and sleeping time through its cognate receptor-NPSR. Here, we report a SNP rs4751440 (L⁶-NPS) causing non-synonymous substitution on the 6^th position (V to L) of the NPS mature peptide region. L⁶-NPS has a higher allele frequency in Europeans than other populations and probably originated from European ancestors ∼25,000 yrs ago based on haplotype analysis and Approximate Bayesian Computation. Functional analyses indicate that L⁶-NPS exhibits a significant lower bioactivity than the wild type NPS, with ∼20-fold higher EC₅₀ values in the stimulation of NPSR. Additional evolutionary and mutagenesis studies further demonstrate the importance of the valine residue in the 6^th position for NPS functions. Given the known physiological roles of NPS receptor in inflammatory bowel diseases, asthma pathogenesis, macrophage immune responses, and brain functions, our study provides the basis to elucidate NPS evolution and signaling diversity among human populations.     

Genetic Variation of the Fc Gamma Receptor 3B Gene and Association with Rheumatoid Arthritis

Background

Fc gamma receptors (FcγRs) play a crucial role in immunity by linking IgG antibody-mediated responses with cellular effector and regulatory functions. Genetic variants in these receptors have been previously identified as risk factors for several chronic inflammatory conditions. The present study aimed to investigate the presence of copy number variations (CNVs) in the FCGR3B gene and its potential association with the autoimmune disease rheumatoid arthritis (RA).

Methodology/Principal Findings

CNV of the FCGR3B gene was studied using Multiplex Ligation Dependent Probe Amplification (MLPA) in 518 Dutch RA patients and 304 healthy controls. Surprisingly, three independent MLPA probes targeting the FCGR3B promoter measured different CNV frequencies, with probe#1 and #2 measuring 0 to 5 gene copies and probe#3 showing little evidence of CNV. Quantitative-PCR correlated with the copy number results from MLPA probe#2, which detected low copy number (1 copy) in 6.7% and high copy number (≥3 copies) in 9.4% of the control population. No significant difference was observed between RA patients and the healthy controls, neither in the low copy nor the high copy number groups (p-values = 0.36 and 0.71, respectively). Sequencing of the FCGR3B promoter region revealed an insertion/deletion (indel) that explained the disparate CNV results of MLPA probe#1. Finally, a non-significant trend was found between the novel -256A>TG indel and RA (40.7% in healthy controls versus 35.9% in RA patients; P = 0.08).

Conclusions/Significance

The current study highlights the complexity and poor characterization of the FCGR3B gene sequence, indicating that the design and interpretation of genotyping assays based on specific probe sequences must be performed with caution. Nonetheless, we confirmed the presence of CNV and identified novel polymorphisms in the FCGR3B gene in the Dutch population. Although no association was found between RA and FCGR3B CNV, the possible protective effect of the -256A>TG indel polymorphism must be addressed in larger studies.     

猪Mu阿片受体基因单核苷酸多态性分析

Mu阿片受体（简称MOR）属于G蛋白藕联受体,分布在痛觉传导区,以及与情绪和行为有关的区域,影响动物的神经反应和行为表现。该研究以长白猪、大白猪和杜洛克猪为试验材料, 用8对引物对Mu阿片受体基因的5′ UTR区域、整个编码区和3′ UTR区域用PCR-SSCP方法进行了扫描,发现5处突变基因座（GenBank登录号：AF521309）。统计结果发现基因型频率分布与品种有关,大白猪突变基因型频率显著高于长白和杜洛克,本研究推测分布上的差异可能是由于长期的选择压力造成的。 Abstract:Mu opioid receptor (MOR) is a member of G protein-coupled receptor family,distributed in the pain transduction region in the brain and related to emotion and behaviour.This study was designed to investigate the Single Nucleotide Polymorphism (SNP) of Mu opioid receptor gene in various breeds,including duroc,landrace and Yorkshire.5′ UTR ( untranslate region),coding region and 3′ UTR of Mu opioid receptor gene were amplified by eight pairs of primers,and the Single Nucleotide Polymorphism (SNP) were detected by SSCP.Five polymorphisms were found (Genebank Accession number:AF521309).The results of χ2 test showed that the frequencies of genotypes in different breeds were significantly different (P＜0.01).The frequencies of mutation genotypes in Yorkshire were significantly higher than Duroc and Landrace.According to the above results,we can speculate the difference of the frequencies of genotypes may be the results of long term choice pressure.     

Structure and Polymorphism Analysis of Transforming Growth Factor Beta Receptor 1 (TGFBR1) in Pigs

Many quantitative trait loci (QTL) for growth and reproductive traits have been detected on the porcine chromosome region 1qter (SSC1qter), making it one of the most important genomic regions for pig breeding. SSC1q corresponds to human chromosome 9, on which lies transforming growth factor beta receptor 1 (TGFBR1). We cloned the porcine TGFBR1 cDNA and gene (as a candidate for QTL) and analyzed the gene structure and polymorphism. Porcine TGFBR1 consists of 9 exons and 8 introns. Intron 2 is alternatively spliced at the acceptor site, resulting in two kinds of mRNA, with putative open reading frames of 1500 and 1512 bp in length. The shorter one encodes 499 amino acid residues. The amino acid sequence has 96.2 and 97.2% sequence similarity to those of human and bovine TGFBR1, respectively. The sequence similarity between porcine and murine TGFBR1 is 95.6%. We detected three single-nucleotide substitutions in exons 1, 2, and 7. Those in exons 1 and 7 are nonsynonymous substitutions resulting in Pro8Ser and Ile413Val substitutions, respectively.     

A Single Nucleotide Polymorphism and Sequence Analysis of CSN1S1 Gene Promoter Region in Chinese BOS Grunniens (YAK)

The aim of this study was to investigate the polymorphism of the CSN1S1 gene promoter region in 4 Chinese yak breeds, and compare the yak CSN1S1 gene promoter region sequences with other ruminants. A Polymerase Chain Reaction-Single Strand Conformation Polymorphism protocol was developed for rapid genotyping of the yak CSN1S1 gene. One hundred fifty-eight animals from 4 Chinese yak breeds were genotyped at the CSN1S1 locus using the protocol developed. A single nucleotide polymorphism of the CSN1S1 gene promoter region has been identified in all yak breeds investigated. The polymorphism consists of a single nucleotide substitution G→A at position 386 of the CSN1S1 gene promoter region, resulting in two alleles named, respectively, G₃₈₆ and A₃₈₆, based on the nucleotide at position 386. The allele G₃₈₆ was found to be more common in the animals investigated. The corresponding nucleotide sequences in GenBank of yak (having the same nucleotides as allele G₃₈₆ in this study), bovine, water buffalo, sheep, and goat had similarity of 99.68%, 99.35%, 97.42%, 95.14%, and 94.19%, respectively, with the yak allele A_386.     

Tricyclic imidazole antagonists of the Neuropeptide S Receptor

A new structural class of potent antagonists of the Neuropeptide S Receptor (NPSR) is reported. High-throughput screening identified a tricyclic imidazole antagonist of NPSR, and medicinal chemistry optimization of this structure was undertaken to improve potency against the receptor as well as CNS penetration. Detailed herein are synthetic and medicinal chemistry studies that led to the identification of antagonists 15 and NPSR-PI1, which demonstrate potent in vitro NPSR antagonism and central exposure in vivo.     

10个牛品种线粒体12S rRNA基因多态性分析

以鲁西牛、渤海黑牛、大别山牛、南阳牛、晋南牛、秦川牛、中国西门塔尔牛、日本和牛、海福特牛和安格斯牛10个牛品种共计353头牛为研究对象,利用单链构象多态性（SSCP）分子标记方法对其线粒体DNA 12S rRNA基因进行了多态性分析,结果发现该基因出现分布频率不同的3种单倍型（Ⅰ、Ⅱ、Ⅲ）,其中单倍型Ⅰ出现的频率最低,仅在晋南牛1个个体中发现;单倍型Ⅱ在中国本地黄牛中都存在,在中国西门塔尔牛中的分布频率很低,但在3个国外品种海福特牛、安格斯牛和日本和牛中不存在;单倍型Ⅲ在这10个牛品种中均有分布,但以国外品种最丰富。计算平均多态信息含量,发现我国地方品种在0.232～0.423之间,基本属于中度多态,说明我国地方黄牛品种在12S rRNA上多态性比较丰富,国外品种较为贫乏;但是单倍型Ⅰ频率很低有丢失的趋势,需要对其进行保护。     

Protective Role of the Interleukin 33 rs3939286 Gene Polymorphism in the Development of Subclinical Atherosclerosis in Rheumatoid Arthritis Patients

Objectives

To determine whether the interleukin-33 (IL-33)-interleukin-1 receptor like 1 (IL-1RL1) signaling pathway is implicated in the risk of subclinical atherosclerosis in patients with rheumatoid arthritis (RA).

Methods

A total of 576 Spanish RA patients from Northern Spain were genotyped for 6 well-known IL33-IL1RL1 polymorphisms (IL33 rs3939286, IL33 rs7025417, IL33 rs7044343, IL1RL1 rs2058660, IL1RL1 rs2310173 and IL1RL1 rs13015714) by TaqMan genotyping assay. The presence of subclinical atherosclerosis was determined by the assessment of carotid intima-media thickness (cIMT) by carotid ultrasound (US).

Results

RA patients carrying the TT genotype of the IL33 rs3939286 polymorphism had lower cIMT values than those homozygous for the CC genotype (mean ± standard deviation (SD): 0.71 ± 0.14 mm versus 0.76 ± 0.16 mm, respectively) while patients carrying the CT genotype had intermediate cIMT values (mean ± SD: 0.73 ± 0.17 mm). Moreover, RA patients carrying the mutant allele T of the IL33 rs3939286 polymorphism exhibited significantly lower cIMT values than those carrying the wild allele C (mean ± SD: 0.72 ± 0.16 mm versus 0.75 ± 0.18 mm respectively; p = 0.04). The association of both genotype and allele frequencies of IL33 rs3939286 and cIMT levels remained statistically significant after adjustment for sex, age at the time of US study, follow-up and center (p = 0.006 and p = 0.0023, respectively), evidencing that the potential effect conferred by IL33 rs3939286 may be independent of confounder factors. No association with other IL33-IL1RL1 genetic variants was observed.

Conclusions

In conclusion, our results may suggest a potential protective effect of the IL33 rs3939286 allele T in the risk of subclinical atherosclerosis in patients with RA.     

Differential Gene Expression in Synovium of Rheumatoid Arthritis and Osteoarthritis

Rheumatoid arthritis (RA) and osteoarthritis (OA) are the major types of arthritis. Although both diseases are characterized by joint destruction, their etiologies are different. To get insights into pathophysiological pathways, we used the suppression subtractive hybridization (SSH) method to identify differentially expressed genes in RA. DNA sequencing identified 12 gene products including cytoskeletal γ-actin and extracellular matrix components such as fibronectin, collagen IIIα₁, and superficial zone protein. Interferon γ-inducible genes such as a novel thiol reductase, two genes of unknown function (HSIFNIN4, RING3), and annexin II were also found. Two genes encoded proteins involved in proliferation such as elongation factor 1α and the granulin precursor. Furthermore, the protease cathepsin B and synovial phospholipase A2 group IIA were detected by SSH. To confirm the differential expression of the genes, we performed RT-PCR analyses of RA and OA synovial tissues. Compared to OA patients, 9 of the 12 genes were overexpressed in RA, suggesting that SSH is a powerful tool for the detection of differential gene expression in synovial tissues. Further characterization of the gene products may help to identify pathophysiological mechanisms in arthritic diseases.     

寡核苷酸芯片技术用于检测过氧化物酶体活化物激活受体基因多态性

 通过寡核苷酸芯片技术检测PPARα基因Leu162Val、Val227Ala多态性和PPARγ Pro12Ala的基因多态性,建立一种快速、简便、准确的方法,为研究非酒精性脂肪性肝病的发病机制、临床诊断和治疗提供依据.收集人体外周血标本,提取DNA进行PCR扩增,设计相应的探针和引物,制备检测芯片,PCR产物与芯片杂交后,扫描芯片并分析结果.PCR产物进行测序验证.寡核苷酸芯片技术检测PPARα基因Leu162Val、Val227Ala多态性和PPARγ Pro12Ala基因多态性结果与测序结果一致.寡核苷酸芯片技术检测非酒精性脂肪性肝病(NAFLD)密切相关的PPAR基因多态性快速、准确,值得临床推广和应用.     

Association of Oxytocin Receptor Gene (OXTR) rs53576 Polymorphism with Sociality: A Meta-Analysis

A common variant in the oxytocin receptor gene (OXTR), rs53576, has been broadly linked to socially related personality traits and behaviors. However, the pattern of published results is inconsistent. Here, we performed a meta-analysis to comprehensively evaluate the association. The literature was searched for relevant studies and effect sizes between individuals homozygous for the G allele (GG) and individuals with A allele carriers (AA/AG). Specifically, two indices of sociality were evaluated independently: i) general sociality (24 samples, n = 4955), i.e., how an individual responds to other people in general; and ii) close relationships (15 samples, n = 5262), i.e., how an individual responds to individuals with closed connections (parent-child or romantic relationship). We found positive association between the rs53576 polymorphism and general sociality (Cohen’s d = 0.11, p = .02); G allele homozygotes had higher general sociality than the A allele carriers. However, the meta-analyses did not detect significant genetic association between rs53576 and close relationships (Cohen’s d = 0.01, p = .64). In conclusion, genetic variation in the rs53576 influences general sociality, which further implies that it is worthy to systematically examine whether the rs53576 is a valid genetic marker for socially related psychiatric disorders.     

IL-23 受体基因多态性和克罗恩病的相关性研究

目的:探讨白介素23受体(IL-23R)基因rs11465779位点多态性与克罗恩病(Crohn’s Disease,CD)的关系。方法:从98例上海本地汉族CD患者和90例正常对照者外周血标本中提取基因IBD组DNA,用聚合酶链式反应-限制性片段长度多态(PCR-RFLP)技术检测IL-23R基因rs11465779位点变异情况,分析其基因表型、基因频率与IBD的关系。结果:IL-23R基因rs11465779位点基因型和等位基因频率在CD组和对照组中分布差异无显著性(P>0.05),且该位点多态性和CD发病年龄及性别均无相关。结论:IL-23R基因rs11465779位点多态性变异,可能不是中国南方汉族人群CD发病的危险因素。