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author:("Han, xinxiang")
1.  Heterozygous mutation of c.3521C>T in COL1A1 may cause mild osteogenesis imperfecta/Ehlers-Danlos syndrome in a Chinese family 
Summary
Osteogenesis imperfecta (OI) is an inheritable connective tissue disorder with a broad clinical heterozygosis, which can be complicated by other connective tissue disorders like Ehlers-Danlos syndrome (EDS). OI/EDS are rarely documented. Most OI/EDS mutations are located in the N-anchor region of type I procollagen and predominated by glycine substitution. We identified a c.3521C>T (p.A1174V) heterozygous mutation in COL1A1 gene in a four-generation pedigree with proposed mild OI/EDS phenotype. The affected individuals had blue sclera and dentinogenesis imperfecta (DI) was uniformly absent. The OI phenotype varied from mild to moderate, with the absence of scoliosis and increased skin extensibility. Easy bruising, joint dislocations and high Beighton score were present in some affected individuals. EDS phenotype is either mild or unremarkable in some individuals. The mutation is poorly conserved and in silico prediction support the relatively mild phenotype. The molecular mechanisms of the mutation that leads to the possible OI/EDS phenotype should be further identified by biochemical analysis of N-propeptide processing and steady state collagen analysis.
doi:10.5582/irdr.2014.01039
PMCID: PMC4322595
Osteogenesis imperfecta; Ehlers-Danlos syndrome; type I collagen; mutations; in silico prediction
2.  Pseudoxanthoma elasticum: A review of 86 cases in China 
Summary
Pseudoxanthoma elasticum (PXE) is a type of rare hereditary disease that affects connective tissue. PXE is found around the world, and its epidemiology in China is still unclear. A database search revealed that 86 patients in total were reported in China from 1985 to 2013. The vast majority of these reports concern single, sporadic cases. This review summarizes the clinical characteristics of PXE and its treatment in China. The hope is to provide a reliable basis for studies on the incidence of PXE and for formulation of relevant policies in the future.
doi:10.5582/irdr.2014.01011
PMCID: PMC4214240  PMID: 25364647
Rare diseases; prevalence; clinical features; literature search
3.  Effects of targeted modulation of miR-762 on expression of the IFITM5 gene in Saos-2 cells 
Summary
Interferon-induced transmembrane protein 5 (IFITM5) is an osteoblast-specific membrane protein that plays an important role in the mineralization of the matrix in mature osteoblasts. However, understanding of the regulatory mechanism of IFITM5 expression is limited. Emerging evidence indicates that microRNAs (miRNAs) act as pivotal regulators in various biological processes including osteoblast proliferation and differentiation. This study aimed to investigate the impact of miRNAs on IFITM5 expression. Bioinformatic analyses predicted that miR-762 would be a potential regulator of IFITM5. A Dual-Luciferase Reporter Assay System indicated that miR-762 could bond with the 3′untranslated region (3′UTR) of IFITM5 via wild-type or mutant recombinant vectors and Western blotting verified that miR-762 negatively regulated IFITM5 expression. Collectively, these data indicate that miR-762 is a novel regulator of IFITM5 and that it suppresses the expression of IFITM5 in Saos-2 cells.
doi:10.5582/irdr.3.12
PMCID: PMC4204544  PMID: 25343121
IFITM5 gene; miR-762; mineralization; target gene
4.  China launched a pilot project to improve its rare disease healthcare levels 
China is facing the great challenge of serving the world’s largest rare disease population. It is necessary to develop a specific medical plan to increase the levels of optimal prevention, diagnosis and treatment of rare diseases under the existing clinical service structures in China. In 2013, China launched its first pilot project focused on 20 representative rare diseases. A national network including approximately 100 provincial or municipal medical centers has been established to enable collaboration on rare diseases across China. The main objectives for this project are to develop and apply medical guidelines and clinical pathways for rare diseases, to establish a rare disease patient registry and data repository system, and to promote molecular testing for rare genetic disorders. This project also emphasizes building close links among the collaborative network, clinicians on the frontlines in basic medical services institutions and rare disease patient organizations. Primarily, this project expects to develop an actionable medical services plan to increase the delivery of quality healthcare for individuals and families living with rare diseases in China within five years.
doi:10.1186/1750-1172-9-14
PMCID: PMC3937133  PMID: 24468030
5.  Epidemiology of Human Respiratory Viruses in Children with Acute Respiratory Tract Infections in Jinan, China 
The viral etiologies of UTRIs and LTRIs in children in Jinan city were investigated between July 2009 and June 2010. Nasal and throat swabs were collected from 397 children with URTIs and bronchoalveolar lavage fluid specimens were collected from 323 children with LRTIs. RT-PCR/PCR was used to examine all samples for IFV, PIV, RSV, RV, hMPV, HBoV, CoV, ADV, RSV, and EV. Viral pathogens were detected in 47.10% of URTI samples and 66.57% samples, and the incidence of viral coinfection was 5.29% and 21.05%, respectively. IFV was the most common virus in URTIs, with a detection rate of 19.40%, followed by PIV (10.83%), RV (10.58%), and EV (6.30%). For LRTIs, PIV and RV were both detected in 27% of samples, followed by RSV (9.91%), HBoV (8.36%), IFV (5.57%), and hMPV (5.57%). RSV and HBoV were more prevalent in the youngest children of no more than six months. Meanwhile, RV, PIV, and RSV were the most frequent viruses combined with bacterial pathogens in LRTIs. In conclusion, the spectrum of respiratory virus infections in URTIs and LRTIs differed in terms of the most common pathogens, seasonal distribution, and coinfection rate.
doi:10.1155/2013/210490
PMCID: PMC3865640  PMID: 24363757
6.  The molecular and cellular basis of Apert syndrome 
Summary
Apert syndrome (AS) is a rare genetic and congenital disease characterized by craniosynostosis and syndactly of hands and feet. AS patients generally require lifelong management, however there are still no effective treatment methods except surgery. In recent years, research has made great progress in the pathogenesis of AS. FGFR2 mediates extracellular signals into cells and the mutations in the FGFR2 gene cause AS occurrence. Activated FGFs/FGFR2 signaling disrupt the balance of cell proliferation, differentiation and apoptosis via its downstream signal pathways. However, how the pathways transform the balance is not well understood and contradictions have occurred in different studies. In this review, we'll focus on these problems to get a better understanding of AS pathogenesis.
doi:10.5582/irdr.2013.v2.4.115
PMCID: PMC4204555  PMID: 25343114
Apert syndrome; FGFR2 gene; pathogenesis; signal pathways
7.  Development of Recombinant Antigen Array for Simultaneous Detection of Viral Antibodies 
PLoS ONE  2013;8(9):e73842.
Protein microarrays have been developed to study antibody reactivity against a large number of antigens, demonstrating extensive perspective for clinical application. We developed a viral antigen array by spotting four recombinant antigens and synthetic peptide, including glycoprotein G of herpes simplex virus (HSV) type 1 and 2, phosphoprotein 150 of cytomegalovirus (CMV), Rubella virus (RV) core plus glycoprotein E1 and E2 as well as a E1 peptide with the optimal concentrations on activated glass slides to simultaneously detect IgG and IgM against HSV1, HSV2, CMV and RV in clinical specimens of sera and cerebrospinal fluids (CSFs). The positive reference sera were initially used to measure the sensitivity and specificity of the array with the optimal conditions. Then clinical specimens of 144 sera and 93 CSFs were tested for IgG and IgM antibodies directed against HSV1, HSV2, CMV and RV by the antigen array. Specificity of the antigen array for viral antibodies detection was satisfying compared to commercial ELISA kits but sensitivity of the array varied relying on quality and antigenic epitopes of the spotting antigens. In short, the recombinant antigen array has potential to simultaneous detect multiple viral antibodies using minute amount (3 µl) of samples, which holds the particularly advantage to detect viral antibodies in clinical CSFs being suspicious of neonatal meningitis and encephalitis.
doi:10.1371/journal.pone.0073842
PMCID: PMC3772810  PMID: 24058498
8.  Advances in research on and diagnosis and treatment of achondroplasia in China 
Summary
Achondroplasia is a rare autosomal dominant genetic disease. Research on achondroplasia in China, however, has received little emphasis. Around 80–90% of cases of neonatal achondroplasia result from mutations in fibroblast growth factor receptor 3 (FGFR3) according to polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). Recently, genetic research on achondroplasia in China made a major breakthrough by revealing two novel mutations located on the FGFR3 gene, thus helping to complete the pathological molecular map of achondroplasia. There are still, however, unknown aspects of the diagnosis and treatment of achondroplasia. This review will summarize advances in research on and the clinical diagnosis and treatment of achondroplasia in China.
doi:10.5582/irdr.2013.v2.2.45
PMCID: PMC4204580  PMID: 25343101
Rare diseases; gene mutation; chondrodystrophia fetalis; chondrodystrophic dwarfism
9.  A comparative proteomics study on matrix vesicles of osteoblast-like Saos-2 and U2-OS cells 
Summary
Matrix vesicles (MVs) play an important role in the initial stage of the process of bone mineralization, and are involved in multiple rare skeletal diseases with pathological mineralization or calcification. The aim of the study was to compare the proteomic profiling of osteoblast-like cells with and without mineralization ability (Saos-2 and U2-OS), and to identify novel mineralization-associated MV proteins. MVs were extracted using ExoQuick solution from mineralization-induced Saos-2 and U2-OS cells, and then were validated by transmission electron microscopy. A label-free quantitative proteomic method was used to compare the protein profiling of MVs from Saos-2 and U2-OS cells. Western-blots were used to confirm the expression of MVs proteins identified in proteomic studies. In our proteomic studies, we identified that 89 mineralization-related proteins were significantly up-regulated in Saos-2 MVs compared with U2-OS MVs. We further validated that two MVs proteins, protein kinase C α and ras-related protein Ral-A, were up-regulated in MVs of Saos-2 cells compared to those of U2-OS cells under mineralization-induction. Our findings suggest that protein kinase C α and ras-related protein Ral-A might be involved in bone mineralization as MVs components.
doi:10.5582/irdr.2013.v2.2.59
PMCID: PMC4204581  PMID: 25343104
Matrix vesicle; osteoblasts; mineralization; proteomics
10.  Viral Aetiology in Adults with Acute Upper Respiratory Tract Infection in Jinan, Northern China 
Our study investigated the epidemiology of respiratory viruses in adult patients with upper respiratory tract infection (URTI) between August 2009 and September 2010 in Jinan, northern China. Nasal and throat swabs (n = 596) were collected from adult patients with URTIs. Nine respiratory-related viruses, including IFV, PIV, HRV, HMPV, HBoV, HCoV, ADV, RSV, and EV, were detected in all samples by conventional and reverse transcription polymerase chain reactions. Positive detection rate for respiratory virus was 38.76% and codetection rate was 4.70% in adults with acute respiratory tract infections. IFV (20.81%) was the dominant agent detected and IFVB had a higher incidence (12.58%) than IFVA (7.72%). Detection rates of 8.22%, 5.03%, 3.69%, and 2.52% were observed for HBoV, HRV, EV, and RSV, respectively. HCoV had the lowest detection rate of 0.50%. HBoV, HRV, EV, and ADV infection rates were higher in the 14–25-year-old group than in the 26–65-year-old group. Codetection rates were higher (7.52%) in the 14–25-year-old group than in the older age group (2.64%). The spectrum of respiratory virus infection in adult patients with URTIs was different in Jinan compared with other cities in China.
doi:10.1155/2013/869521
PMCID: PMC3649347  PMID: 23690828
11.  Low concentrations of zoledronic acid are better at regulating bone formation and repair 
Summary
The purpose of this study was to investigate optimal concentrations of zoledronic acid (ZA) in terms of their effect on the proliferation, differentiation, and mineralization of primary osteoblasts (OBs) and fibroblasts (FBs). Primary OBs and FBs isolated from patients with clinical osteogenesis imperfecta (OI) and developmental dysplasia of the hip (DDH) were treated in vitro with serial concentrations of ZA ranging from 10−3 M to 10−13 M. An MTT ((3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)) colorimetric assay, flow cytometry, alkaline phosphatase (ALP) determination activity, and alizarin red staining were used to measure the proliferation, differentiation, and mineralization of cells. The MTT assay indicated that high concentrations of ZA may be toxic to cultured cells. No obvious inhibition was observed with a ZA concentration of 10−7 M to 10−10 M. Proliferation was evident with a ZA concentration below 10−11 M (p < 0.05). Flow cytometry analysis revealed that cell cycle was arrested at G1/G0 stage with a ZA concentration ranging from 10−10 M to 10−8 M. ZA did not enhance ALP activity at a concentration of 10−8 M or 10−10 M. Alizarin red staining indicated the mineralization of primary OBs with a low concentration of ZA (10−12 M). In conclusion, this in vitro study indicated that ZA-mediated cell proliferation was dose-dependent and that ZA did not inhibit cell proliferation at concentrations below 10−8 M. These findings suggest low concentrations of ZA have more of an effect on cell differentiation and mineralization, so low concentrations are better at regulating bone formation and repair.
doi:10.5582/irdr.2013.v2.1.18
PMCID: PMC4204573  PMID: 25343096
Osteogenesis imperfecta; proliferation; differentiation; mineralization
12.  Subjective well-being of the elderly in Xi Cheng District, Beijing 
Shanghai Archives of Psychiatry  2012;24(6):335-346.
Background
In 2010 the Beijing Municipal Government promulgated a policy aimed at improving the quality of life and subjective well-being of elderly residents that included a component focused on mental health.
Aim
Identify factors associated with subjective well-being in a representative sample of elderly residents of Xi Cheng District in Beijing.
Methods
This cross-sectional study administered a self-completion survey to a stratified random sample of 2342 residents of Xi Cheng District who were 60 to 80 years of age. The level of well-being was assessed using a validated Chinese version of the Memorial University of Newfoundland Scale of Happiness (MUNSH). Detailed socioeconomic variables were obtained using a questionnaire developed by the authors. Social support, anxiety, and depression were assessed using validated Chinese versions of the Social Support Rating Scale (SSRS), Self-rating Anxiety Scale (SAS), and Self-rating Depression Scale (SDS).
Results
Among the 2342 respondents, 1616 (69.0%) had a total MUNSH score of 32 or above, indicating a high level of happiness; 423 (18.1%) has a total SSRS score 32 or below, indicating poor social support; 201 (8.6%) had a total SDS score of 53 or above, indicating significant depression; and 126 (5.3%) had a total SAS score of 50 or above, indicating significant anxiety. In the multivariate regression analysis the self-reported level of depression was the most important factor related to well-being. Anxiety, social support, income level, the quality of family relationships, the ability to self-regulate emotions, and regular exercise were also significantly related to well-being; but gender, marital status, age and educational level were not associated with well-being.
Conclusion
Among elderly urban residents in Beijing, self-reports of poor subjective well-being are closely associated with self-reports of depressive and anxiety symptoms and also associated with social factors such as social support, income level and family relationships. Prospective studies are needed to identify the causal relationships of these variables and, based on the findings, to develop targeted interventions aimed at improving the quality of life and well-being of elderly community members.
doi:10.3969/j.issn.1002-0829.2012.06.005
PMCID: PMC4198900  PMID: 25324638
13.  Study and analysis of the state of rare disease research in Shandong Province, China 
Summary
As the world's most populous country, China has the world's largest number of rare disease groups in terms of prevalence. However, the country has no system of registering cases of most rare diseases, so there is very little documented information on the epidemiology of those diseases. The purpose of this study was to study the state of rare disease research and survey doctors in Shandong Province regarding their level of awareness of rare diseases. Types of rare diseases and numbers of cases were tallied and their geographical distribution over the decades was analyzed. Eight hundred and twenty-four doctors in tertiary hospitals and maternity and child care hospitals were surveyed by questionnaire. Data were descriptively analyzed and a map of disease distribution was created. Articles about rare diseases were retrieved from the Chinese Biomedical Literature Database to provide pertinent data. This study yielded 5,749 cases of 323 different types of rare diseases. The survey found that doctors lack awareness of research on rare diseases. An authoritative and information-rich platform for rare disease research is urgently needed. Key steps are to study epidemiological and statistical techniques and then obtain available data to provide a basis for the definition and regulation of rare diseases in China.
doi:10.5582/irdr.2012.v1.4.161
PMCID: PMC4204566  PMID: 25343091
Rare diseases; awareness survey; descriptive analysis
14.  A systematic review of genetic skeletal disorders reported in Chinese biomedical journals between 1978 and 2012 
Little information is available on the prevalence, geographic distribution and mutation spectrum of genetic skeletal disorders (GSDs) in China. This study systematically reviewed GSDs as defined in “Nosology and Classification of genetic skeletal disorders (2010 version)” using Chinese biomedical literature published over the past 34 years from 1978 to 2012. In total, 16,099 GSDs have been reported. The most frequently reported disorders were Marfan syndrome, osteogenesis imperfecta, fibrous dysplasia, mucopolysaccharidosis, multiple cartilaginous exostoses, neurofibromatosis type 1 (NF1), osteopetrosis, achondroplasia, enchondromatosis (Ollier), and osteopoikilosis, accounting for 76.5% (12,312 cases) of the total cases. Five groups (group 8, 12, 14, 18, 21) defined by “Nosology and Classification of genetic skeletal disorders” have not been reported in the Chinese biomedical literature. Gene mutation testing was performed in only a minor portion of the 16,099 cases of GSDs (187 cases, 1.16%). In total, 37 genes for 41 different GSDs were reported in Chinese biomedical literature, including 43 novel mutations. This review revealed a significant imbalance in rare disease identification in terms of geographic regions and hospital levels, suggesting the need to create a national multi-level network to meet the specific challenge of care for rare diseases in China.
doi:10.1186/1750-1172-7-55
PMCID: PMC3492206  PMID: 22913777
Rare diseases; Genetic skeletal diseases; China; Bibliographic study
15.  Peripheral blood microRNAs: A novel tool for diagnosing disease? 
Summary
Peripheral blood microRNAs (miRNAs) are endogenous, noncoding small RNAs present in blood. Because of their size, abundance, tissue specificity, and relative stability in peripheral circulation, they offer great promise of becoming a novel noninvasive biomarker. However, the mechanism by which they are secreted, their biological function, and the reason for the existence of extracellular miRNAs are largely unclear. This article describes advances in the study of the mechanism of origin and biological function of extracellular miRNAs along with approaches adopted by research and questions that remain. This work also discusses the potential for peripheral blood miRNAs to serve as a diagnostic tool.
doi:10.5582/irdr.2012.v1.3.98
PMCID: PMC4204598  PMID: 25343080
Peripheral blood miRNAs; diagnosis; biomarker; biological function
16.  Carbonic anhydrase I (CA1) is involved in the process of bone formation and is susceptible to ankylosing spondylitis 
Arthritis Research & Therapy  2012;14(4):R176.
Introduction
Ankylosing spondylitis (AS) is characterized by abnormal bone formation in the spine and the sacroiliac joints. In vitro assays demonstrate that carbonic anhydrase I (CA1) promotes calcium precipitation. This study investigated the function of CA1 for bio-mineralization and determined if common polymorphisms in the CA1 gene might contribute to AS risk.
Methods
Calcification was induced in Saos-2 cells, a human osteosarcoma cell line, with ascorbic acid and β-glycerophosphate. Calcification was determined by Alizarin Red-S (AR-S) staining. Expressions of CA1, alkaline phosphatase (ALP), bone sialoprotein (BSP), osteocalcin (OCN), osterix (OSX) and runt-related transcription factor-2 (Runx2) were determined by real-time PCR and western blotting. The cells were also treated with acetazolamide, an anti-carbonic anhydrase drug. Genotyping was performed using Illumina VeraCode microarray in a case-control study including 51 AS patients, 267 rheumatoid arthritis (RA) patients and 160 healthy controls. The result was confirmed by Taqman assay, including 258 AS patients, 288 RA patients and 288 healthy controls.
Results
Following the induction of calcification, Saos-2 cells produced large amounts of calcium-rich deposits. Increased transcriptions of CA1, ALP, BSP, OCN, OSX and Runx2, essential genes for ossification, were detected in the cultured cells. Following treatmen with acetazolamide, the expression of CA1 obviously declined and mineralized nodule formation was also decreased. Illumina microarray indicates that SNP at rs7841425 also showed significant differences in allelic frequency (P = 0.01396) and genotypic frequency (P = 0.005902) between AS cases and controls. In addition, SNP at rs7827474 showed significant differences in allelic frequency (P = 5.83E-04) and genotypic frequency (P = 0.000186) between RA cases and controls (P values were adjusted to multiple comparisons). The Taqman assay revealed that rs725605 demonstrated statistically significant evidence of allele frequency (P = 0.022307) and gene frequency (P = 0.007731) for association with AS. This SNP did not show significant differences in allelic frequencies and gene frequencies between RA patients and controls.
Conclusions
CA1 may play an essential role in bio-mineralization and new bone formation. The gene encoding CA1 is susceptible to AS.
doi:10.1186/ar3929
PMCID: PMC3580570  PMID: 22838845
17.  Serum microRNA is a promising biomarker for osteogenesis imperfecta 
Summary
The purpose of our study was to screen preliminary differential expression bone-related microRNAs (miRNAs) in serum of patients with osteogenesis imperfacta and to clarify whether serum microRNA is a promising biomarker for osteogenesis imperfecta. geNorm and several other programes were performed to select suitable reference genes for quantitative detection of serum miRNAs from 6 candidate control genes. With geometric averaging of selected reference genes as a normalization factor, fluorescence-based quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) was used to detect expression levels of more than 100 bone-related miRNAs obtained by means of miRanda, Targetscan and Pictar software calculations and reading the literature. Through analysis of expression stability and pairwise variations, all 6 candidate reference genes had a stable expression level in serum of 8 healthy controls and 8 patients with different characrteristics, and the optimal number of reference genes for normalization was 4 (snRNAU6, miR-92a, miR-16, and Let-7a). For further validation, the expression stability of 4 reference genes remained steady in serum of another 8 healthy controls and 16 patients with osteogenesis imperfecta (M < 1.5). When normalized using multiple control genes, 11 bone-related miRNAs showed differential expression in serum of 8 osteogenesis imperfecta patients compared with 8 healthy controls. In conclusion, we identified snRNAU6, miR-92a, miR-16, and Let-7a as an internal reference gene group for qRT-PCR normalization and screening results revealed that there existed many differential expression bone-related miRNAs in serum of patients with osteogenesis imperfecta compared with healthy controls, and that these miRNAs had potential to be biomarkers for serologic tests and diagnosis of osteogenesis imperfecta with analysis of bioinformation.
doi:10.5582/irdr.2012.v1.2.81
PMCID: PMC4204563  PMID: 25343076
Osteogenesis imperfecta; serum miRNAs; reference gene; biomarker
18.  Rare diseases research in China: Opportunities, challenges, and solutions 
Summary
Rare diseases research in China can be traced back to the 1980s. Currently, control of rare diseases has become a national concern. This paper describes developments concerning rare diseases in China with regard to epidemiology, case registration, basic research, establishment of medical networks, and orphan drugs. A national program for rare disease research is being implemented in China to promote international cooperation in the future.
doi:10.5582/irdr.2012.v1.1.10
PMCID: PMC4204592  PMID: 25343065
Rare disease; China; development; epidemiology
19.  Phosphate/Pyrophosphate and MV-related Proteins in Mineralisation: Discoveries from Mouse Models 
During the process of matrix vesicle (MV)-mediated initiation of mineralisation, chondrocytes and osteoblasts mineralise the extracellular matrix by promoting the seeding of basic calcium phosphate crystals of hydroxyapatite (HA) along the collagen fibrils. This orchestrated process is carefully regulated by the balanced action of propagators and inhibitors of calcification. The primary antagonistic regulators of extracellular matrix mineralisation are phosphate (Pi) and inorganic pyrophosphate (PPi). Studies in mouse models and in humans have established critical roles for Pi/PPi homeostasis in biomineralisation. In this review, we present the regulators of Pi/PPi, as derived from animal models, and discuss their clinical relevance to physiological and pathological mineralisation.
doi:10.7150/ijbs.4538
PMCID: PMC3372882  PMID: 22719218
Mineralisation; Matrix vesicles; PPi; Pi; MV-related proteins; OPN.
20.  Investigating the Pathogenic Role of PADI4 in Oesophageal Cancer 
PADI4 post-translationally converts peptidylarginine to citrulline. PADI4 can disrupt the apoptotic process via the citrullination of histone H3 in the promoter of p53-target genes. The current study focused on PADI4 expression in various subtypes of oesophageal carcinoma (EC) by immunohistochemistry, western blotting and real time PCR. The study also investigated the effect of bile acid deoxycholate (DCA) on PADI4 expression in Eca-109 cells that originated from EC. Apoptosis and DCA-induced toxicity were analyzed by TUNEL, MTT assay and flow cytometry. Additionally, the present study investigated the correlation between single nucleotide polymorphism (SNP) in PADI4 gene and EC risk in Chinese population using Illumina GoldenGate assay. Compared with paraneoplastic tissues, the transcriptional and translational levels of PADI4 were significantly elevated in oesophageal squamous cell carcinoma (ESCC, n=9) and oesophageal adenocarcinoma (EAC, n=5) tissues. Immunolabeling detected expression of PADI4 in ESCC tissues (98.56%, n=139), EAC samples (87.5%, n=16) and oesophageal small cell undifferentiated carcinoma (91.7%, n=12) but not in normal tissues (0%, n=16). Furthermore, PADI4 levels is positively correlated with the pathological classification of ESCC (p=0.009). PADI4 expression levels were consistent with the number of apoptotic cells in the induced Eca-109 cells. rs10437048 [OR= 0.012831; 95% CI, 0.001746~0.094278; p=1.556×10-12] were significantly associated with decreased risk of EC, whereas rs41265997 [OR=12.7; 95% CI, 0.857077~33.207214; p=3.896×10-8] were significantly associated with increased risk of EC. rs41265997 in exon 3 of PADI4 gene is non-synonymous and converts ACG to ATG resulting in a threonine /methionine conversion at position 274 of the protein. Haplotypes GC that carries the variant alleles for rs2501796 and rs2477134 was significantly associated with increased risk of EC (frequency=0.085, p=0.0256, OR=2.7). The results suggest that PADI4 expression is related to the tumorigenic process of EC and the DCA-induced apoptosis. The PADI4 gene may be a valid EC susceptibility gene.
PMCID: PMC3119849  PMID: 21698003
Peptidyl arginine deaminase type 4 (PADI4/PAD4); oesophageal squamous cell carcinoma (ESCC); oesophageal adenocarcinoma (EAC); deoxycholate (DCA); apoptosis; genotyping; susceptibility; haplotype.
21.  Increased expression of carbonic anhydrase I in the synovium of patients with ankylosing spondylitis 
Background
One of the most distinctive features of ankylosing spondylitis (AS) is new bone formation and bone resorption at sites of chronic inflammation. Previous studies have indicated that the hyperplasia and inflammation of synovial tissues are significantly related to the pathogenic process of AS. The present study used a proteomic approach to identify novel AS-specific proteins by simultaneously comparing the expression profiles of synovial membranes from patients with AS, rheumatoid arthritis (RA) and osteoarthritis (OA).
Methods
Synovial tissues were collected from the hip joints of patients with AS and knee joints of patients with RA or OA (n = 10 for each disease) during joint replacement surgery. Proteins extracted from the synovial tissues were separated by 2-D electrophoresis (2-DE), and the proteins with significantly increased expression in the AS samples were subjected to MALDI-TOF/TOF-MS analysis. The results were verified using western blotting and immunohistochemistry. Levels of the candidate proteins in synovial fluids from knee joints (n = 40 for each disease) were measured using ELISA.
Results
The proteomic approach revealed significantly increased expression of carbonic anhydrase I (CA1) in the synovial membrane of patients with AS as compared with the RA and OA tissue samples. Immunohistochemistry and western blotting analysis confirmed the findings described above. The ELISA detected a higher level of CA1 in synovial fluids from patients with AS than those with OA. The mean value of the CA1 level was also higher in AS patients as compared with RA patients. This study also detected increased expression of alpha-1-antitrypsin in the synovial tissues from AS patients, which is in agreement with other reports.
Conclusion
In vitro experiments by other groups indicated that CA1 catalyzes the generation of HCO3- through the hydration of CO2, which then combines with Ca2+ to form a CaCO3 precipitate. Calcification is an essential step of bone formation. Substantial evidence indicates that carbonic anhydrase also stimulates bone resorption. Hence, overexpression of CA1 in the synovial tissues of AS patients may promote improper calcification and bone resorption in AS.
doi:10.1186/1471-2474-11-279
PMCID: PMC3012668  PMID: 21143847
22.  Increased PADI4 expression in blood and tissues of patients with malignant tumors 
BMC Cancer  2009;9:40.
Background
Peptidylarginine deiminase type 4 (PAD4/PADI4) post-translationally converts peptidylarginine to citrulline. Recent studies suggest that PADI4 represses expression of p53-regulated genes via citrullination of histones at gene promoters.
Methods
Expression of PADI4 was investigated in various tumors and non-tumor tissues (n = 1673) as well as in A549, SKOV3 and U937 tumor cell lines by immunohistochemistry, real-time PCR, and western blot. Levels of PADI4 and citrullinated antithrombin (cAT) were investigated in the blood of patients with various tumors by ELISA (n = 1121).
Results
Immunohistochemistry detected significant PADI4 expression in various malignancies including breast carcinomas, lung adenocarcinomas, hepatocellular carcinomas, esophageal squamous cancer cells, colorectal adenocarcinomas, renal cancer cells, ovarian adenocarcinomas, endometrial carcinomas, uterine adenocarcinomas, bladder carcinomas, chondromas, as well as other metastatic carcinomas. However, PADI4 expression was not observed in benign leiomyomas of stomach, uterine myomas, endometrial hyperplasias, cervical polyps, teratomas, hydatidiform moles, trophoblastic cell hyperplasias, hyroid adenomas, hemangiomas, lymph hyperplasias, schwannomas, neurofibromas, lipomas, and cavernous hemangiomas of the liver. Additionally, PADI4 expression was not detected in non-tumor tissues including cholecystitis, cervicitis and synovitis of osteoarthritis, except in certain acutely inflamed tissues such as in gastritis and appendicitis. Quantitative PCR and western blot analysis showed higher PADI4 expression in gastric adenocarcinomas, lung adenocarcinomas, hepatocellular carcinomas, esophageal squamous cell cancers and breast cancers (n = 5 for each disease) than in the surrounding healthy tissues. Furthermore, western blot analysis detected PADI4 expression in cultured tumor cell lines. ELISA detected increased PADI4 and cAT levels in the blood of patients with various malignant tumors compared to those in patients with chronic inflammation and benign tumors. This was consistent with immunohistochemical results. Additionally, PADI4 and cAT levels were significantly associated with higher levels of known tumor markers.
Conclusion
Our results suggest that PADI4 expression is increased in the blood and tissues of many malignant tumors, a finding useful for further understanding of tumorigenesis.
doi:10.1186/1471-2407-9-40
PMCID: PMC2637889  PMID: 19183436

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