Crosstalk between CpG Methylation and Polymorphisms (CpG-SNPs) in the Promotor Region of DIO2 in Kashin-Beck Disease / 中国医学科学杂志(英文版)

Objective This study was designed to determine the methylation profile of four CpGs and the genotypes of two CpG-SNPs located in promoter region of DIO2 in patients with Kashin-Beck disease (KBD). We also analyzed the interaction between the CpGs methylations and CpG-SNPs. Methods Whole blood specimens were collected from 16 KBD patients and 16 healthy subjects. Four CpGs and two CpG-SNPs in the promoter regions of DIO2 were detected using matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). The CpGs methylation levels were compared between samples from KBD patients and healthy subjects. The methylation levels were also analyzed in KBD patients with different CpG-SNP genotypes. Results The mRNA expression of DIO2 in whole blood of KBD patients was significnatly lower than in healthy controls (P <0.05). The methylation levels of DIO2-1_CpG_3 in KBD patients were significantly higher than those in healthy controls (P <0.05). The methylation levels of four CpGs were not significantly different between KBD patients and healthy controls. The methylation level of DIO2-1_CpG_3 in the promoter region of DIO2 in KBD patients with GA/AA genotype was significantly higher than that of KBD patients with GG genotype (P <0.05). Conclusion The methylation level of DIO2 increases in KBD patients. Similar trends exist in KBD carriers of variant genotypes of CpG-SNPs DIO2 rs955849187.

Serum Metabolomic Indicates Potential Biomarkers and Metabolic Pathways of Pediatric Kashin-Beck Disease / 生物医学与环境科学（英文）

Objective@#To explore potential serum biomarkers of children with Kashin-Beck Disease (KBD) and the metabolic pathways to which the biomarkers belong.@*Methods@#A two-stage metabolomic study was employed. The discovery cohort included 56 patients, 51 internal controls, and 50 external controls. The metabolites were determined by HPLC-(Q-TOF)-MS and confirmed by Human Metabolome Databases (HMDB) and Metlin databases. MetaboAnalyst 3.0 and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database were used to analyze the metabolic pathways of the candidate metabolites. The use of HPLC-(Q-TRAP)-MS enabled quantitative detection of the target metabolites which were chosen using the discovery study and verified in another independent verification cohort of 31 patients, 41 internal controls, and 50 external controls.@*Results@#Eight candidate metabolites were identified out in the discovery study, namely kynurenic acid, N-α-acetylarginine, 6-hydroxymelatonin, sphinganine, ceramide, sphingosine-1P, spermidine, and glycine. These metabolites exist in sphingolipid, glutathione, and tryptophan metabolic pathways. In the second-stage study, five candidate metabolites were validated, including kynurenic acid, N-α-acetylarginine, sphinganine, spermidine, and sphingosine-1P. Except for spermidine, all substances exhibited low expression in the case group compared with the external control group, and the difference in levels of sphinganine, spermidine, and sphingosine-1P was statistically significant.@*Conclusion@#The direction of change of levels of sphinganine, spermidine, and sphingosine-1P in the two-stage study cohorts was completely consistent, and the differences were statistically significant. Therefore, these substances can be used as potential biomarkers of KBD. Furthermore, these results raise the possibility that sphingolipid metabolic pathways may be closely related to KBD.

Polymorphism in rs2229783 of the Alpha 1(XI) Collagen Gene Is Associated with Susceptibility to but not Severity of Kashin-Beck Disease in a Northwest Chinese Han Population / 生物医学与环境科学（英文）

A case-control study was conducted to investigate associations between organophosphate pesticide (OP) exposure, aggression, impulsivity, and attempted suicide. The purpose of this study was to explore whether genomic polymorphisms in the alpha 1(XI) collagen gene (COL11A1) were associated with the risk and severity of Kashin-Beck disease (KBD). Twenty-two single nucleotide polymorphisms (SNPs) in COL11A1 were genotyped in 274 KBD cases and 249 healthy controls using the Sequenom MassARRAY system. The expression of type XI collagen (COL11A) in the knee articular cartilage of 22 KBD patients and 21 controls was analyzed by immunohistochemistry. Our results showed that the frequency distribution of genotypes of the rs2229783 polymorphism in COL11A1 was significantly different between the KBD and control groups (P = 0.0003). Moreover, the expression level of COL11A in cartilage was significantly lower in the KBD group than in the controls (t = 2.637, P = 0.02). However, no association was found between the rs2229783 and the severity of KBD, suggesting a role of COL11A1 in the susceptibility to but not the severity of KBD.

Increased Chondrocyte Apoptosis in Kashin-Beck Disease and Rats Induced by T-2 Toxin and Selenium Deficiency / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To investigate chondrocyte apoptosis and the expression of biochemical markers associated with apoptosis in Kashin-Beck disease (KBD) and in an established T-2 toxin- and selenium (Se) deficiency-induced rat model.</p><p><b>METHODS</b>Cartilages were collected from the hand phalanges of five patients with KBD and five healthy children. Sprague-Dawley rats were administered a selenium-deficient diet for 4 weeks prior to T-2 toxin exposure. The apoptotic chondrocytes were observed by terminal deoxynucleotidyl transferase dUTP nick end labeling staining. Caspase-3, p53, Bcl-2, and Bax proteins in the cartilages were visualized by immunohistochemistry, their protein levels were determined by Western blotting, and mRNA levels were determined by real-time reverse transcription polymerase chain reaction.</p><p><b>RESULTS</b>Increased chondrocyte apoptosis was observed in the cartilages of children with KBD. Increased apoptotic and caspase-3-stained cells were observed in the cartilages of rats fed with normal and Se-deficient diets plus T-2 toxin exposure compared to those in rats fed with normal and Se-deficient diets. Caspase-3, p53, and Bax proteins and mRNA levels were higher, whereas Bcl-2 levels were lower in rats fed with normal or Se-deficiency diets supplemented with T-2 toxin than the corresponding levels in rats fed with normal diet.</p><p><b>CONCLUSION</b>T-2 toxin under a selenium-deficient nutritional status induces chondrocyte death, which emphasizes the role of chondrocyte apoptosis in cartilage damage and progression of KBD.</p>

Detection of the Urinary Biomarkers PYD, CTX-II, and DPD in Patients with Kashin-Beck Disease in the Qinghai Province of China / 生物医学与环境科学（英文）

Kashin-Beck disease (KBD) is an endemic degenerative osteoarthropathy of uncertain etiology. The aim of our study was to identify changes in C-telopeptide of type II collagen (CTX-II), pyridinoline (PYD), and deoxypyridinoline (DPD) among KBD patients. 54 KBD patients and 78 healthy controls were included this study. Urinary samples were collected and measured by ELISA. The median quantities of PYD, CTX-II, and DPD of KBD patients were 1107.73 ng/μmol.cre, 695.11 ng/μmol.cre, and 1342.34 pml/μmol.cre, while the median quantities of healthy controls were 805.59 ng/μmol.cre, 546.47 ng/μmol.cre, and 718.15 pml/μmol.cre, respectively. The differences between KBD patients and healthy controls were statistically significant (Z = 4.405, 3.653, and 3.724; P < 0.001). The higher levels of PYD, CTX-II, and DPD detected in KBD patients indicate that they could be used as biomarkers of KBD.

Evaluation of the Sensitivity and Specificity of the New Clinical Diagnostic and Classification Criteria for Kashin-Beck Disease, an Endemic Osteoarthritis, in China / 生物医学与环境科学（英文）

This study aimed to evaluate the sensitivity and specificity of the new clinical diagnostic and classification criteria for Kashin-Beck disease (KBD) using six clinical markers: flexion of the distal part of fingers, deformed fingers, enlarged finger joints, shortened fingers, squat down, and dwarfism. One-third of the total population in Linyou County was sampled by stratified random sampling. The survey included baseline characteristics and clinical diagnoses, and the sensitivity and specificity of the new criteria was evaluated. We identified 3,459 KBD patients, of which 69 had early stage KBD, 1,952 had stage I, 1,132 had stage II, and 306 had stage III. A screening test classified enlarged finger joints as stage I KBD, with a sensitivity and specificity of 0.978 and 0.045, respectively. Shortened fingers were classified as stage II KBD, with a sensitivity and specificity of 0.969 and 0.844, respectively, and dwarfism was classified as stage III KBD with a sensitivity and specificity of 0.951 and 0.992, respectively. Serial screening test revealed that the new clinical classification of KBD classified stages I, II, and III KBD with sensitivities of 0.949, 0.945, and 0.925 and specificities of 0.967, 0.970, and 0.993, respectively. The screening tests revealed that enlarged finger joints, shortened fingers, and dwarfism were appropriate markers for the clinical diagnosis and classification of KBD with high sensitivity and specificity.

Exploring Genome-wide DNA Methylation Profiles Altered in Kashin-Beck Disease Using Infinium Human Methylation 450 Bead Chips / 生物医学与环境科学（英文）

To understand how differentially methylated genes (DMGs) might affect the pathogenesis of Kashin-Beck disease (KBD). Genome-wide methylation profiling of whole blood from 12 matched KBD and controls pairs was performed using a high-resolution Infinium 450 K methylation array. In total, 97 CpG sites were differentially methylated in KBD compared to the normal controls; of these sites, 36 sites were significantly hypermethylated (covering 22 genes) and 61 sites were significantly hypomethylated (covering 34 genes). Of these genes, 14 significant pathways were identified, the most significant P value pathway was type I diabetes mellitus pathway and pathways associated with autoimmune diseases and inflammatory diseases were included in this study. Subsequently, 4 CpG sites in HLA-DRB1 were validated using bisulfite sequencing polymerase chain reaction (BSP) in articular cartilage, and the results showed significant differences in the methylation status between KBD and controls, consistent with the results of the high-resolution array. These results suggested that differences in genome-wide DNA methylation exist between KBD and the controls, and the biological pathways support the autoimmune disease and inflammatory disease hypothesis of KBD.

Changing Grains for the Prevention and Treatment of Kashin-Beck Disease in Children: a Meta-analysis / 生物医学与环境科学（英文）

To evaluate the efficacy of changing grains on the prevention and treatment of Kashin-Beck Disease (KBD) in children, community-based trials were acquired from seven electronic databases (up to July 2014). As a result, the methodological quality of the six trials that have been included into our analysis was low. The pooled ORs favoring the prevention and treatment effects of changing grains were 0.15 (95% CI: 0.03-0.70) and 2.13 (95% CI: 1.44-3.16) respectively by meta-analysis. Subgroup analysis demonstrated the pooled OR favoring treatment effect of exchanging grains rather than drying grains both compared with endemic grains. The results showed that changing grains had obvious effects on the prevention and treatment of KBD in children. However, the evidences were limited by the potential biases and confounders. Large and well-designed trials are still needed.

Comparison of the expression profiles of cell death factors in articular cartilage between Kashin-Beck disease and osteoarthritis / 南方医科大学学报

<p><b>OBJECTIVE</b>To compare the expressions of programmed cell death 5 (PDCD5) and early growth response protein-1 (EGR-1) in the articular cartilage between Kashin-Beck disease (KBD) and primary osteoarthritis and the roles of these factors in KBD cartilage.</p><p><b>METHODS</b>Cartilage specimens were collected from 10 confirmed KBD patients, 15 osteoarthritic patients and 6 healthy subjects. The expression levels of PDCD5 and EGR-1 in the cartilage were detected by immunohistochemistry staining, and the positive chondrocyte counts were recorded in the different layers of KBD and OA cartilages.</p><p><b>RESULTS</b>The KBD cartilages contained a significantly higher percentage of PDCD5-positive chondrocytes in the middle layer [(41.35 ± 2.97)%] than OA cartilages [(26.48 ± 2.04)%, P=0.001] and normal cartilages [(19.02 ± 1.88)%, P=0.000] with also obvious PDCD5 over-expression in the deeper layer compared to OA (P=0.000) and normal cartilages (P=0.029), but PDCD5 expression in the superficial layer of the cartilages showed no significant difference among the 3 groups(P>0.05). The average EGR-1 positivity rate in the superficial layer of the cartilage was significantly higher in KBD patients than in OA patients (P=0.000) and healthy controls (P=0.000), but in the middle layer, its positivity rate in KBD patients was higher than that in the normal control (P=0.017) but lower than that of OA cartilage (P=0.002); EGR-1 expression in the deeper layer was comparable in KBD and OA cartilages but both was higher than that in normal cartilages. PDCD5 and EGR-1 expressions were not correlated in either KBD or normal cartilages, but were positively correlated in the superficial layer of OA cartilages.</p><p><b>CONCLUSIONS</b>KBD cartilages show a significantly increased PDCD5 expression in the deeper layer and enhanced EGR-1 expression in both superficial and deeper layers, suggesting the involvement of PDCD5 and EGR-1 in the pathogenesis of KBD.</p>

X-ray image characteristics and related measurements in the ankles of 118 adult patients with Kashin-Beck disease / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Kashin-Beck disease (KBD) is a chronic, degenerative osteoarthropathy that causes severe skeletal deformation. Although many researchers have proven that almost all KBD patients who showed an increaseing proximo-distal gradient had radiographic abnormalities of the ankle, few detailed description of radiographic changes in the ankles of patients with KBD has been reported, especially for variable measurements of ankle changes. The purpose of this study was to demonstrate the radiographic characteristics of the ankles of adult KBD patients.</p><p><b>METHODS</b>One hundred and eighteen adult KBD patients from september to October 2010 in Rongtang county in China were examined with lateral radiographs of the right ankle. The morphological abnormalities in the talus, calcaneus, navicular bone, distal tibia, and joint space were analyzed, and the calcaneus length, height, length-height ratio, tuber angle, front angle, plantar angle, and distal tibia anteroposterior (AP) length were measured using Riepert's method.</p><p><b>RESULTS</b>Eighty-one patients (68.6%) had abnormal ankle radiographs; 72 (88.9%) patients had talus changes, 69 (85.2%) patients had calcaneus changes, 28 (34.6%) patients had navicular bone changes, and 48 (59.2%) patients had distal tibia changes. For 118 KBD patients, the average calcaneus length was 7.4 cm, height was 4.3 cm, and the length-height ratio was 1.7. The calcaneus tuber angle was 28.2°, front angle was 38.0° and the plantar angle was 74.2°. The distal tibia anteroposterior length was 4.05 cm. Compared with 50 normal adults (control group), significant differences were found for the calcaneus length, the calcaneus length-height ratio, and the distal tibia AP length.</p><p><b>CONCLUSIONS</b>Patients with KBD have characteristic abnormalities on ankle radiographs; talus depression and deformity, calcaneus shortening deformity, and distal tibia deformity with AP length widening were the most typical changes.</p>

Nano-Se-chondroitin sulfate inhibits T-2 toxin-induced apoptosis of cultured chondrocytes from patients with Kashin-Beck disease / 南方医科大学学报

<p><b>OBJECTIVE</b>To observe the effect of nano-Se-chondroitin sulfate on the growth and apoptosis of chondrocytes from patients with Kashin-Beck disease (KBD) exposed to T-2 toxin in vitro.</p><p><b>METHODS</b>Samples of the articular cartilage were obtained from 6 patients with grade II/III KBD diagnosed in line with the National Clinical Diagnostic Criteria of KBD (WS/T 207-2010) for chondrocyte separation and culture in vitro. The separated chondrocytes were treated with synthesized nano-Se-chondroitin sulfate particles and T-2 toxin, alone or in combination, and the cell growth and apoptosis were observed using MTT assay, HE staining and flow cytometry.</p><p><b>RESULTS</b>The synthesized nano-Se-chondroitin sulfate, with a selenium entrapment ratio of 10.1%, spontaneously formed nanoparticles in distilled water with sizes ranging from 30 to 200 nm. Fourier-transform infrared spectroscopy suggested a possible covalent bond that bound Nano-Se and chondroitin sulfate. Within the concentration range of 50-200 ng/ml, nano-Se-chondroitin sulfate significantly inhibited T-2 toxin-induced apoptosis of the cultured chondrocytes and reduced the early apoptosis rate to (8.64∓1.57)% (P<0.05).</p><p><b>CONCLUSION</b>Nano-Se-chondroitin sulfate can inhibit T-2 toxin-induced apoptosis of cultured chondrocytes from KBD patients in vitro, and serves as a promising candidate therapeutic agent for KBD.</p>

Changes of death receptor regulator expression in the articular cartilage of patients with Kashin-Beck disease / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the changes in the expressions of Fas-associated death domain protein (FADD) and cellular-FLICE inhibitory protein (c-FLIP) in the articular cartilage of patients with Kashin-Beck disease (KBD) and the role of these proteins in the pathogenesis of KBD.</p><p><b>METHODS</b>The cartilage samples were collected from patients with established diagnosis of KBD and osteoarthritis and from healthy control subjects undergoing amputation due to traffic accidents. The expressions of Fas-associated death domain protein (FADD) and cellular-FLICE inhibitory protein (c-FLIP) in the cartilage were detected by immunohistochemistry, and the positive chondrocytes were counted in different layers of the articular cartilage under microscope.</p><p><b>RESULTS</b>The positivity rates of FADD in the middle layer of articular cartilage from patients with KBD [(28.68∓2.19)%] and osteoarthritis [(35.40∓2.34)%] were significantly higher than that in normal cartilage [(10.51∓5.02)%, F=16.245, P=0.000], but the rates in the upper and deeper layers were comparable among the 3 groups (P=0.206-0.761). In KBD cartilage, FADD expression was the highest in the middle layer [(28.68∓5.38)%] followed by the deeper layer [(17.94∓8.38)%]. Compared with the healthy controls, KBD and osteoarthritis patients showed significantly higher FLIP expression in the upper layer of the cartilage (F=5.929, P=0.018) but similar expressions in middle and deeper layers.</p><p><b>CONCLUSIONS</b>KBD patients have significant increased FADD expression in the middle layer but decreased FLIP expression in the upper layer of the cartilage, suggesting that the death receptor pathway and its regulators play important roles in the pathogenesis of KBD.</p>

Expression of Caspase-8 and Bcl-2 in the cartilage loose bodies in patients with Kashin-Beck disease / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the role of Caspase-8 and Bcl-2 in the formation of loose bodies in Kashin-Beck disease (KBD).</p><p><b>METHODS</b>Specimens of cartilage loose bodies were collected from 50 adult patients with KBD, and the samples of articular cartilage were collected from 10 healthy adults to serve as control. Avidin-biotin alkaline phosphatase immunohistochemistry was employed to examine Bcl-2 and Caspase-8 positivities in the chondrocytes in the loose bodies.</p><p><b>RESULTS</b>In KBD loose bodies, the percentage of chondrocytes positive for Bcl-2 and Caspase-8 [(18.40∓8.78)% and (67.54∓12.29)%, respectively] were significantly higher than those of the control group [(12.25∓1.58)% and (24.70∓4.35)%, respectively]. Caspase-8 was found to promote chondrocyte apoptosis in the loose bodies, and this effect overrode the apoptosis-suppressing effect of Bcl-2. Bcl-2 and Caspase-8 positivities were found mainly in the deep hypertrophic chondrocytes in the cartilage or in cells adjacent to the bone tissues.</p><p><b>CONCLUSION</b>KBD loose bodies contain an increased percentage of apoptotic chondrocytes positive for Bcl-2 and Caspase-8. The apoptosis-inducing effect of Caspase-8 was a dominant feature in the cartilage pathology of KBD compared to the apoptosis-suppressing effect of Bcl-2.</p>

Transmission disequilibrium test for 15 short tandem repeat loci in Kashin-Beck disease and their interaction with low selenium / 南方医科大学学报

<p><b>OBJECTIVE</b>To identify the genetic susceptibility to Kashin-Beck disease (KBD) and explore the interaction between low selenium (Se) and the susceptibility gene loci in KBD.</p><p><b>METHODS</b>The DNA samples collected from 23 KBD nuclear families were analyzed using PCR and GeneScan Analysis 3.7 and Genotyper3.7 software. The haplotype relative risk (HRR) and transmission disequilibrium test (TDT) were used to test the data of the genotypes. The serum selenium (Se) concentration was measured by atomic fluorescence spectrometry, and the interaction between low Se and the susceptibility loci was calculated using a binary logistic regression.</p><p><b>RESULTS</b>In the 23 nuclear families, the alleles of D2S151 (248 bp), D2S305 (320 bp), and D11S4094 (194 bp) showed significant correlation to KBD (P<0.05). Serum Se concentrations in the studied individuals was 0.037 µg/ml. No significant statistical interaction was observed between low Se exposure and the susceptibility loci (P>0.05).</p><p><b>CONCLUSION</b>The polymorphisms in the STR loci D2S305, D2S151, and D11S4094 or the polymorphism loci near them might been related to KBD susceptibility. Low Se exposure shows no significant interaction with the susceptibility loci.</p>

Pattern of joints involvement in Kashin-Beck disease: a local osteochondropathy in China

Kashin-Beck Disease [KBD] is an endemic osteochondropathy. The disease starts in childhood and attacks the growth of joint cartilage, the joints become deformed and painful, and the worst forms result in dwarfism. The most frequent joints involved are the finger, wrist, ankles, knees and elbows. In this study the pattern of joints involved in KBD was studied. A total of 368 patients aged above 13 from 6 villages in endemic areas of the Shaanxi province, located in the northwest of China, were selected through multistage stratified sampling. The patients were diagnosed based on the clinical criteria for diagnosis of KBD. The patients were administered with a battery of questionnaires along with clinical examination for joint involvement. The patients presented both as oligoarticular as well as polyarticular pattern. Smaller joints were more frequently involved with a proximal-distal pattern. The number of joints involved increased with the severity of the disease. Proximal smaller joints are more frequently involved in the disease. The involvement of bigger joints takes place in later stages of disease, i.e., in the second and third degree. The pattern of joint involvement shows some correlation with Rheumatoid Arthritis which needs further investigation and comparative studies

TrxR2 gene polymorphisms may not be associated with the susceptibility to Kashin-Beck disease / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the association between single nucleotide polymorphisms of thioredoxin reductase-2 (TrxR2) gene and the susceptibility to Kashin-Beck disease (KBD).</p><p><b>METHODS</b>Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to analyze the genotype frequencies of rs5748469 in TrxR2 gene in 84 KBD patients and 109 healthy control subjects.</p><p><b>RESULTS</b>The genotype frequencies of A/A, A/C, and C/C in the KBD patients were 83.33%, 15.48% and 1.19%, as compared with the frequencies of 74.31%, 25.69%, and 0.00% in the healthy control, respectively, showing no significant difference in the single nucleotide polymorphisms of TrxR2 gene between the two groups (P=0.13).</p><p><b>CONCLUSION</b>No obvious correlation can be found between rs5748469 polymorphisms in TrxR2 gene and the susceptibility to KBD.</p>