Targeting neuronal PAS domain protein 2 and KN motif/ankyrin repeat domains 1:Advances in type 2 diabetes therapy

This editorial summarizes the latest literature on the roles of neuronal PAS domain protein 2 and KN motif/ankyrin repeat domain 1 in type 2 diabetes(T2D).We highlight their involvement inβ-cell dysfunction,explore their potential as therapeutic targets,and discuss the implications for new treatment strategies.We offer valuable insights into relevant gene regulation and cellular mechanisms relevant for the targeted management of T2D.

Comparative study of network-based prioritization of protein domains associated with human complex diseases

Domains are basic structural and functional unit of proteins,and,thus,exploring associations between protein domains and human inherited diseases will greatly improve our understanding of the pathogenesis of human complex diseases and further benefit the medical prevention,diagnosis and treatment of these diseases.Based on the assumption that deleterious nonsynonymous single nucleotide polymorphisms(nsSNPs)underlying human complex diseases may actually change structures of protein domains,affect functions of corresponding proteins,and finally result in these diseases,we compile a dataset that contains 1174 associations between 433 protein domains and 848 human disease phenotypes.With this dataset,we compare two approaches(guilt-by-association and correlation coefficient)that use a domain-domain interaction network and a phenotype similarity network to prioritize associations between candidate domains and human disease phenotypes.We implement these methods with three distance measures(direct neighbor,shortest path with Gaussian kernel,and diffusion kernel),demonstrate the effectiveness of these methods using three large-scale leave-one-out cross-validation experiments(random control,simulated linkage interval,and whole-genome scan),and evaluate the performance of these methods in terms of three criteria(mean rank ratio,precision,and AUC score).Results show that both methods can effectively prioritize domains that are associated with human diseases at the top of the candidate list,while the correlation coefficient approach can achieve slightly higher performance in most cases.Finally,taking the advantage that the correlation coefficient method does not require known disease-domain associations,we calculate a genome-wide landscape of associations between 4036 protein domains and 5080 human disease phenotypes using this method and offer a freely accessible web interface for this landscape.

Eukaryotic expression, purification and activity characterization of human soluble DSG2 extracellular domain protein

Objective:To construct a secretory eukaryotic expression vector of DSG2 fused with the Fc region of the human IgG,to validate its expression in 293T cells,and to purify the secretory protein with biological activity.Methods:The DSG2 extracellular domain fragment gene(DSG2ex),was amplified by PCR,and was inserted into the eukaryotic expression plasmid pCMV3-IgG1 to construct the recombinant eukaryotic expression plasmid-pCMV3-DSG2ex-IgG1.The successfully constructed eukaryotic expression plasmid was transfected into 293T cells to express and secrete DSG2 extracellular domain protein.The targeted protein was purified from the cell culture supernatant by Protein A affinity chromatography and confirmed by Western Blotting and ELISA.Results:The pCMV3-DSG2ex-IgG1 eukaryotic expression plasmid was successfully constructed.The highest protein expression level was obtained with 293T cells after 96 h of transfection.The relative molecular mass of the purified product was between 100 and 130 kDa was estimated by SDS-PAGE,which was consistent with the expectation.The yield of the purified protein reached 0.8 mg/ml with a purity over 90%.The purified DSG2 extracellular domain protein with IgG1 tag was recognized by IgG monoclonal antibodies by Western blotting.Moreover,the ELISA results showed that the prepared DSG2 extracellular domain protein had significant binding activity to human type 55 adenovirus Fiber Knob protein(HAdV-55).Conclusion:A simple and efficient method for eukaryotic expression and purification of human soluble DSG2 extracellular domain protein was successfully established,and biologically active DSG2 extracellular domain protein was purified,which laid the foundation for the later study of its protein function and anti-adenovirus drugs.

Characterization of functional domains of human p100 protein interacting with signal transducer and activator of transcription-6 (STAT-6)

In the present study, the interaction of human p100 protein with signal transducer and activator of transcription-6 (STAT-6) was investigated. It was proved that the staphylococcal nuclease (SN)-like and tudor (TD) domains containing in p100 protein acting as a adaptor to recruit STAT-6 to the basal transcription machinery, enhanced the STAT-6 mediated transcription activity. The interaction between STAT-6 and the p100 protein was mediated by the full-length of the SN-like domain, whereas individual fragments of SN-like domain showed no binding activity to STAT-6. In line with these results, the SN-like domain was directly engaged in the enhancement of STAT-6 mediated activation of gene transcription in vivo. Yet the TD domain had no ability to increase the transcription activation, but it was still required for the sufficient activation of transcription.

Crocus sativus L.produces anti-inflammatory effects and regulates the NLRP3–NF-κB pathway

Objective:This study aimed to evaluate the anti-inflammatory effects of petal and stamen extracts of saffron crocus(Crocus sativus)and explore the underlying mechanism.Methods:Local and systemic inflammation models were used to investigate the anti-inflammatory effects of C.sativus.A xyleneinduced inflammation model or lipopolysaccharide(LPS)-induced inflammation model was used in this study.C.sativus petal and stamen extracts were each administered to the mice in the xylene and LPS models by gavage for 14 d at 0.1 and 0.4 g/kg doses,respectively.Enzyme-linked immunosorbent assay(ELISA)was used to measure the concentrations of tumor necrosis factor(TNF)-αand interleukin(IL)-1βin mouse serum.Hematoxylin and eosin(H&E)staining was used to observe the pathological changes in the ear in the xylene-induced inflammation model and in the spleen in the LPS-induced inflammation model.NOD-like receptor thermal protein domain associated protein 3(NLRP3)protein levels within the nuclear factor-kappa B(NF-κB)pathway were assessed using western blotting.RAW264.7 cells were treated with LPS(5μg/mL)and LPS+C.sativus(0.05,0.1,and 0.2 mg/mL)for 24 h,and a Cell Counting Kit-8 was used to measure cell proliferation.Changes in NLRP3 and NF-κB levels were evaluated by western blotting.Results:Petal and stamen extracts of C.sativus attenuated the anti-inflammatory effects in local or systemic inflammatory models and repaired pathological changes in the ear in the xylene-induced inflammation model and spleen in the LPS-induced inflammation model.These extracts also decreased the concentrations of TNF-αand IL-1βin the mouse serum in the LPS-induced inflammation model.C.sativus downregulated NLRP3 protein level through the NF-κB pathway and downregulated LC-3 and BECLIN1 in vivo and in vitro.Carbonyl Cyanide3-ChloroPhenylhydrazone(CCCP)weakened the effects of C.sativus on the NLRP3–NF-κB pathway.Conclusion:C.sativus has anti-inflammatory effects and regulates the NLRP3-NF-κB pathway.

A novel histone methyltransferase gene Cg SDG40 positively regulates carotenoid biosynthesis during citrus fruit ripening

The flesh color of pummelo(Citrus maxima)fruits is highly diverse and largely depends on the level of carotenoids,which are beneficial to human health.It is vital to investigate the regulatory network of carotenoid biosynthesis to improve the carotenoid content in pummelo.However,the molecular mechanism underlying carotenoid accumulation in pummelo is not fully understood.In this study,we identified a novel histone methyltransferase gene,CgSDG40,involved in carotenoid regulation by analyzing the flesh transcriptome of typical white-fleshed pummelo,red-fleshed pummelo and extreme-colored F1 hybrids from a segregated pummelo population.Expression of CgSDG40 corresponded to flesh color change and was highly coexpressed with CgPSY1.Interestingly,CgSDG40 and CgPSY1 are located physically adjacent to each other on the chromosome in opposite directions,sharing a partially overlapping promoter region.Subcellular localization analysis indicated that CgSDG40 localizes to the nucleus.Overexpression of CgSDG40 significantly increased the total carotenoid content in citrus calli relative to that in wild type.In addition,expression of CgPSY1 was significantly activated in overexpression lines relative to wild type.Taken together,our findings reveal a novel histone methyltransferase regulator,CgSDG40,involved in the regulation of carotenoid biosynthesis in citrus and provide new strategies for molecular design breeding and genetic improvement of fruit color and nutritional quality.

Clinical and genetic characteristics of a child with Sotos syndrome and attention-deficit/hyperactivity disorder:A case report

BACKGROUND Sotos syndrome is an autosomal dominant disorder,whereas attention-deficit/hyperactivity disorder(ADHD)is a neurodevelopmental condition.This report aimed to summarize the clinical and genetic features of a pediatric case of Soros syndrome and ADHD in a child exhibiting precocious puberty.CASE SUMMARY The patient presented with accelerated growth and advanced skeletal maturation;however,she lacked any distinct facial characteristics related to specific genetic disorders.Genetic analyses revealed a paternally inherited heterozygous synonymous mutation[c.4605C>T(p.Arg1535Arg)].Functional analyses suggested that this mutation may disrupt splicing,and bioinformatics analyses predicted that this mutation was likely pathogenic.After an initial diagnosis of Sotos syndrome,the patient was diagnosed with ADHD during the follow-up period at the age of 8 years and 7 months.CONCLUSION The potential for comorbid ADHD in Sotos syndrome patients should be considered to avoid the risk of a missed diagnosis.

Assessment of pathogenicity and functional characterization of APPL1 gene mutations in diabetic patients

BACKGROUND Adaptor protein,phosphotyrosine interacting with PH domain and leucine zipper 1(APPL1)plays a crucial role in regulating insulin signaling and glucose metabolism.Mutations in the APPL1 gene have been associated with the development of maturity-onset diabetes of the young type 14(MODY14).Currently,only two mutations[c.1655T>A(p.Leu552*)and c.281G>A p.(Asp94Asn)]have been identified in association with this disease.Given the limited understanding of MODY14,it is imperative to identify additional cases and carry out comprehensive research on MODY14 and APPL1 mutations.AIM To assess the pathogenicity of APPL1 gene mutations in diabetic patients and to characterize the functional role of the APPL1 domain.METHODS Patients exhibiting clinical signs and a medical history suggestive of MODY were screened for the study.Whole exome sequencing was performed on the patients as well as their family members.The pathogenicity of the identified APPL1 variants was predicted on the basis of bioinformatics analysis.In addition,the pathogenicity of the novel APPL1 variant was preliminarily evaluated through in vitro functional experiments.Finally,the impact of these variants on APPL1 protein expression and the insulin pathway were assessed,and the potential mechanism underlying the interaction between the APPL1 protein and the insulin receptor was further explored.RESULTS A total of five novel mutations were identified,including four missense mutations(Asp632Tyr,Arg633His,Arg532Gln,and Ile642Met)and one intronic mutation(1153-16A>T).Pathogenicity prediction analysis revealed that the Arg532Gln was pathogenic across all predictions.The Asp632Tyr and Arg633His variants also had pathogenicity based on MutationTaster.In addition,multiple alignment of amino acid sequences showed that the Arg532Gln,Asp632Tyr,and Arg633His variants were conserved across different species.Moreover,in in vitro functional experiments,both the c.1894G>T(at Asp632Tyr)and c.1595G>A(at Arg532Gln)mutations were found to downregulate the expression of APPL1 on both protein and mRNA levels,indicating their pathogenic nature.Therefore,based on the patient’s clinical and family history,combined with the results from bioinformatics analysis and functional experiment,the c.1894G>T(at Asp632Tyr)and c.1595G>A(at Arg532Gln)mutations were classified as pathogenic mutations.Importantly,all these mutations were located within the phosphotyrosinebinding domain of APPL1,which plays a critical role in the insulin sensitization effect.CONCLUSION This study provided new insights into the pathogenicity of APPL1 gene mutations in diabetes and revealed a potential target for the diagnosis and treatment of the disease.

Inhibition of Japanese Encephalitis Virus Infection by Flavivirus Recombinant E Protein Domain Ⅲ

Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus closely related to the human pathogens including yellow fever virus, dengue virus and West Nile virus. There are currently no effective antiviral therapies for all of the flavivirus and only a few highly effective vaccines are licensed for human use. In this paper, the E protein domain III (DIII) of six heterologous flaviviruses (DENV1-4, WNV and JEV) was expressed in Escherichia coli successfully. The proteins were purified after a solubilization and refolding procedure, characterized by SDS-PAGE and Western blotting. Competitive inhibition showed that all recombinant flavivirus DIII proteins blocked the entry of JEV into BHK-21 cells. Further studies indicated that antibodies induced by the soluble recombinant flavivirus DIII partially protected mice against lethal JEV challenge. These results demonstrated that recombinant flavivirus DIII proteins could inhibit JEV infection competitively, and immunization with proper folding flavivirus DIII induced cross-protection against JEV infection in mice, implying a possible role of DIII for the cross-protection among flavivirus as well as its use in antigens for immunization in animal models.

Effect of Methyl-CpG Binding Domain Protein 2(MBD2) on AMD-like Lesions in ApoE-Deficient Mice

Summary： The role of methyl-CpG binding domain protein 2 （MBD2） in an ApoE-deficient mouse model of age-related macular degeneration （AMD） was investigated. Eight-week-old Mbd2/ApoE double deficient （Mbd2^-/- ApoE^-/-） mice （n=12, 24 eyes, experimental group） and MBD2 （wt） ApoE^-/- mice （n=12, 24 eyes, control group） were fed on Western-type diet for 4 months. The mice were sacrificed, and total serum cholesterol levels were analyzed and Bruch＇s membrane （BM） of the eyes was removed for ultrastructural observation by transmission electron microscopy. Moreover, intercellular adhesion molecule 1 （ICAM-1） immunoreactivities were evaluated by fluorescence microscopy in sections of the eyes in both groups for further understanding the function mechanism of MBD2. There was no significant difference in the total serum cholesterol levels between control group and experimental group （P〉0.05）. Transmission electron microscopy revealed that AMD-like lesions, various vacuoles accumulated on BM, notable outer collagenous layer deposits and dilated basal infoldings of retinal pigment epithelium （RPE） were seen in both groups, and the BM in control group was significantly thickened as compared with experimental group （P〈0.05）. Fluorescence micrographs exhibited the expression of ICAM-1 in choroid was higher in control group than in experimental group. We are led to conclude that MBD2 gene knockout may lead to accumulation of more deposits on the BM and influence the pathogenesis of AMD via triggering endothelial activation and inflammatory response in choroid, improving microcirculation, and reducing lipid deposition so as to inhibit the development of AMD-like lesions. Our study helps to provide a new therapeutic approach for the clinical treatment of AMD.

Mutual regulation between microRNA-373 and methyl-CpGbinding domain protein 2 in hilar cholangiocarcinoma

AIM:To investigate the reciprocal modulation between microRNA(miRNA) and DNA methylation via exploring the correlation between miR-373 and methyl-CpGbinding domain protein(MBD)2.METHODS:MiR-373 expression was examined using the TaqMan miRNA assay.Methylation of miR-373 was investigated using methylation-specific polymerase chain reaction,and recruitment of methyl binding proteins was studied using the chromatin immunoprecipitation assay.Mutation analysis was conducted using the QuikChange Site-Directed Mutagenesis kit.The activity of miR-373 gene promoter constructs and targeting at MBD2-three prime untranslated region(3'UTR) by miR-373 were evaluated by a dual-luciferase reporter gene assay.RESULTS:In hilar cholangiocarcinoma,miR-373 decreased and was closely associated with poor cell differentiation,advanced clinical stage,and shorter survival.The promoter-associated CpG island of miR-373 gene was hypermethylated and inhibited expression of miR-373.MBD2 was up-regulated and enriched at the promoter-associated CpG island of miR-373.Methylation-mediated suppression of miR-373 required MBD2 enrichment at the promoter-associated CpG island,and miR-373 negatively regulated MBD2 expression through targeting the 3'UTR.CONCLUSION:MiR-373 behaves as a direct transcriptional target and negative regulator of MBD2 activity through a feedback loop of CpG island methylation.

A Novel Method for Prediction of Protein Domain Using Distance-Based Maximal Entropy

Detecting the boundaries of protein domains is an important and challenging task in both experimental and computational structural biology. In this paper, a promising method for detecting the domain structure of a protein from sequence information alone is presented. The method is based on analyzing multiple sequence alignments derived from a database search. Multiple measures are defined to quantify the domain information content of each position along the sequence. Then they are combined into a single predictor using support vector machine. What is more important, the domain detection is first taken as an imbal- anced data learning problem. A novel undersampling method is proposed on distance-based maximal entropy in the feature space of Support Vector Machine （SVM）. The overall precision is about 80%. Simulation results demonstrate that the method can help not only in predicting the complete 3D structure of a protein but also in the machine learning system on general im- balanced datasets.

Evolution of a protein domain interaction network

In this paper, we attempt to understand complex network evolution from the underlying evolutionary relationship between biological organisms. Firstly, we construct a Pfam domain interaction network for each of the 470 completely sequenced organisms, and therefore each organism is correlated with a specific Pfam domain interaction network; secondly, we infer the evolutionary relationship of these organisms with the nearest neighbour joining method; thirdly, we use the evolutionary relationship between organisms constructed in the second step as the evolutionary course of the Pfam domain interaction network constructed in the first step. This analysis of the evolutionary course shows： （i） there is a conserved sub-network structure in network evolution; in this sub-network, nodes with lower degree prefer to maintain their connectivity invariant, and hubs tend to maintain their role as a hub is attached preferentially to new added nodes; （ii） few nodes are conserved as hubs; most of the other nodes are conserved as one with very low degree; （iii） in the course of network evolution, new nodes are added to the network either individually in most cases or as clusters with relative high clustering coefficients in a very few cases.

Two memory associated genes regulated by amyloid precursor protein intracellular domain Novel insights into the pathogenesis of learning and memory impairment in Alzheimer's disease

In this study, we employed chromatin immunoprecipitation, a useful method for studying the locations of transcription factors bound to specific DNA regions in specific cells, to investigate amyloid precursor protein intracellular domain binding sites in chromatin DNA from hippocampal neurons of rats, and to screen out five putative genes associated with the learning and memory functions. The promoter regions of the calcium/calmodulin-dependent protein kinase II alpha and glutamate receptor-2 genes were amplified by PCR from DNA products immunoprecipitated by amyloid precursor protein intracellular domain. An electrophoretic mobility shift assay and western blot analysis suggested that the promoter regions of these two genes associated with learning and memory were bound by amyloid precursor protein intracellular domain （in complex form）. Our experimental findings indicate that the amyloid precursor protein intracellular domain is involved in the transcriptional regulation of learning- and memory-associated genes in hippocampal neurons. These data may provide new insights into the molecular mechanism underlying the symptoms of progressive memory loss in Alzheimer＇s disease.

Coactivator p100 protein enhances histone acetyltransferase activity of CBP

Human p100 protein consists of four repeated domains of staphylococcal nuclease （SN）-like domain, as well as a tudor （TD） domain thereafter. We have previously shown that the SN-like domain of p100 interacted with STAT6 and the large subunit of RNA pol Ⅱ , resulting in the enhancement of STAT6-mediated gene transcriptional activation. Here, we show that SN-like domain also interacted with CREB binding protein （CBP） and directly enhanced the acetyl transferase activity of CBP on histone. On the other hand, overexpression of CBP alone had no ability to significantly increase STAT6- dependent transcriptional activation, however, together with p100 protein, sufficiently enhanced the activation of transcription which was in line with the previous result that p100 protein bridged STAT6 with CBP.

Decoding m^(6)A mRNA methylation by reader proteins in liver diseases

N6-methyladenosine(m^(6)A)is a dynamic and reversible epigenetic regulation.As the most prevalent internal post-transcriptional modification in eukaryotic RNA,it participates in the regulation of gene expression through various mechanisms,such as mRNA splicing,nuclear export,localization,translation efficiency,mRNA stability,and structural transformation.The involvement of m^(6)A in the regulation of gene expression depends on the specific recognition of m^(6)A-modified RNA by reader proteins.In the pathogenesis and treatment of liver disease,studies have found that the expression levels of key genes that promote or inhibit the development of liver disease are regulated by m^(6)A modification,in which abnormal expression of reader proteins determines the fate of these gene transcripts.In this review,we introduce m^(6)A readers,summarize the recognition and regulatory mechanisms of m^(6)A readers on mRNA,and focus on the biological functions and mechanisms of m^(6)A readers in liver cancer,viral hepatitis,non-alcoholic fatty liver disease(NAFLD),hepatic fibrosis(HF),acute liver injury(ALI),and other liver diseases.This information is expected to be of high value to researchers deciphering the links between m^(6)A readers and human liver diseases.

Up-regulated Expression of Tim-3/Gal-9 at Maternal-fetal Interface in Pregnant Woman with Recurrent Spontaneous Abortion

The relationship between T cell immunoglobulin domain and mucin domain protein 3（Tim-3）/Galectin（Gal）-9 pathway and recurrent spontaneous abortion（RSA） was studied. Thirty-one pregnant women with RSA and 27 normal early gravidas were investigated to detect the levels of Tim-3 and Gal-9 in villi and deciduas by Western blotting. Meanwhile, the concentration of interleukin（IL）-4 and IL-12 in peripheral blood plasma was determined by ELISA in 25 healthy fertile non-pregnant controls, the normal early gravidas and pregnant women with RSA mentioned above, respectively. It was found that the relative expression levels of Tim-3 and Gal-9 in villi and deciduas were significantly increased in pregnant women with RSA as compared with those in the normal early gravidas. The concentration of IL-4 in peripheral blood plasma of pregnant women with RSA was lower than that of the normal early gravidas（P〈0.05） and healthy fertile non-pregnant controls（P〈0.05）, but that of IL-2 in pregnant women with RSA was significantly higher than that of the normal early gravidas（P〈0.05） and healthy fertile non-pregnant controls（P〈0.05）. It was suggested that the overexpression of Tim-3/Gal-9 pathway may be related to the pathogenesis of RSA.

Zinc finger E-box-binding homeobox 1 mediates aerobic glycolysis via suppression of sirtuin 3 in pancreatic cancer

AIM TO uncover the roles of tumor-promoting gene ZEB1 in aerobic glycolysis regulation and shed light on the underlying molecular mechanism.METHODS Endogenous zinc finger E-box binding homeobox-1 （ZEB1） was silenced using a and the impact of ZEB1 and lentivirus-mediated method, methyI-CpG binding domain protein 1 （MBD1） on aerobic glycolysis was measured using seahorse cellular flux analyzers, reactive oxygen species quantification, and mitochondrial membrane potential measurement. The interaction between ZEB1 and MBD1 was assessed by co-immunoprecipitation and immunofluorescence assays. The impact of ZEB1 and MBD1 interaction on sirtuin 3 （SIRT3） expression was confirmed by quantitative polymerase chain reaction, western blotting, and dual-luciferase and chromatinimmunoprecipitation assays.RESULTS ZEB1 was a positive regulator of aerobic glycolysis in pancreatic cancer. ZEB1 transcriptionally silenced expression of SIRT3, a mitochondrial-localized tumor suppressor, through interaction with MBD1.CONCLUSION ZEB1 silenced SIRT3 expression via interaction with MBD1 to promote aerobic glycolysis in pancreatic cancer.

EPAS1 and VEGFA gene variants are related to the symptoms of acute mountain sickness in Chinese Han population: a cross-sectional study

Background: More people ascend to high altitude(HA) for various activities, and some individuals are susceptible to HA illness after rapidly ascending from plains. Acute mountain sickness(AMS) is a general complaint that affects activities of daily living at HA. Although genomic association analyses suggest that single nucleotide polymorphisms(SNPs) are involved in the genesis of AMS, no major gene variants associated with AMS-related symptoms have been identified.Methods: In this cross-sectional study, 604 young, healthy Chinese Han men were recruited in June and July of 2012 in Chengdu, and rapidly taken to above 3700 m by plane. Basic demographic parameters were collected at sea level, and heart rate, pulse oxygen saturation(Sp O2), systolic and diastolic blood pressure and AMS-related symptoms were determined within 18–24 h after arriving in Lhasa. AMS patients were identified according to the latest Lake Louise scoring system(LLSS). Potential associations between variant genotypes and AMS/AMS-related symptoms were identified by logistic regression after adjusting for potential confounders(age, body mass index and smoking status).Results: In total, 320 subjects(53.0%) were diagnosed with AMS, with no cases of high-altitude pulmonary edema or high-altitude cerebral edema. Sp O2 was significantly lower in the AMS group than that in the non-AMS group(P=0.003). Four SNPs in hypoxia-inducible factor-related genes were found to be associated with AMS before multiple hypothesis testing correction. The rs6756667(EPAS1) was associated with mild gastrointestinal symptoms(P=0.013), while rs3025039(VEGFA) was related to mild headache(P=0.0007). The combination of rs6756667 GG and rs3025039 CT/TT further increased the risk of developing AMS(OR=2.70, P<0.001).Conclusions: Under the latest LLSS, we find that EPAS1 and VEGFA gene variants are related to AMS susceptibility through different AMS-related symptoms in the Chinese Han population;this tool might be useful for screening susceptible populations and predicting clinical symptoms leading to AMS before an individual reaches HA.Trial registration: Chinese Clinical Trial Registration, Chi CTR-RCS-12002232. Registered 31 May 2012.

Distribution Characteristics and Linkage Disequilibrium of TIM4 Promoter Polymorphisms in Asthma Patients of Chinese Han Population

To investigate the distribution characteristics and linkage disequilibrium of T cell immunoglobulin domain and mucin domain protein 4 （TIM4） promoter polymorphisms in asthma patients of Chinese Han population, the promoter region of TIM4 was re-sequenced by PCR-sequencing, and linkage disequilibrium was analyzed by SHEsis software. Four single nucleotide polymor- phisms （SNPs） in the promoter region of TIM4 were detected, including two new SNPs （at positions -1609, -153） and two reported SNPs （rs6874202, rs6882076）. The frequency distribution of rs6882076 was different among different races （P〈0.05）. In addition, linkage disequilibrium among the SNPs of the promoter region of TIM4 was found and GGTG was the predominant haplotype. There were four SNPs in the promoter region of TIM4 in asthma patients of Chinese Han population, which were in linkage disequilibrium.