OBJECTIVE

Receptor activator of nuclear factor-kappa B ligand (RANKL), its receptor activator of nuclear factor-kappa B (RANK), and decoy receptor osteoprotegerin (OPG) are three major proteins of the RANKL/RANK/OPG signaling pathway encoded by TNFSF11, TNFRSF11A, and TNFRSF11B, respectively. This pathway plays a critical role in bone remodeling and may have a role in the pathogenesis of type 2 diabetes mellitus (T2DM). The aim of this study was to investigate the relationship between gene polymorphisms in the RANKL/RANK/OPG pathway and T2DM in Southern Han Chinese women.

METHODS

A total of 1233 participants, including 514 T2DM patients and 719 healthy control subjects, were enrolled in this case-control study. Twenty-one single-nucleotide polymorphisms (SNPs) of TNFSF11, TNFRSF11A, and TNFRSF11B were genotyped using an improved multiplex ligation detection reaction technique.

RESULTS

Two SNPs of TNFRSF11B (rs11573819 and rs2073618) were significantly associated with T2DM (p = 0.04 and p = 0.009, respectively). Subjects with the GA genotype of rs11573819 had a lower risk of T2DM (odds ratio [OR] = 0.67, 95% confidence interval [CI] = 0.51-0.88, p = 0.005) compared with subjects with the GG genotype. The GG genotype of rs2073618 was associated with increased risk for T2DM (OR = 1.94, 95% CI = 1.14-3.30, p = 0.01) compared with the CC genotype.

CONCLUSIONS

This study suggests that TNFRSF11B but not TNFSF11 and TNFRSF11A genetic polymorphisms are associated with T2DM in Southern Han Chinese women. These findings provide preliminary support for the potential role of the RANKL/RANK/OPG pathway in T2DM.

We report a newly recognized bone disorder consisting of polyostotic expansile osteolysis affecting long bones and iliac bones; hyperostosis of the skull, thoracic cage, and medial portion of both clavicles; pectus carinatum; gigantiform synovial masses of the elbows and knees; atrial septal defect; cardiomegaly; unilateral cryptorchidism; and mental deficiency. Affected bones can be grouped into four general types of skeletal pathology: (1) expansile osteolysis, (2) osteolysis without expansion, (3) expansion without osteolysis, and (4) hyperostosis. Some bones remained unaffected. We have named the condition "polyosteolysis/hyperostosis syndrome." It is clearly at variance with any previously reported bone disorder, including familial expansile osteolysis, juvenile Paget disease, and McCune-Albright syndrome (and polyostotic fibrous dysplasia). Because our patient shared some features in common with juvenile Paget disease, we thought that mutational analysis of TNFRSF11B was indicated, even though our patient had some manifestations not found in juvenile Paget disease. Direct sequencing failed to identify a TNFRSF11B mutation. Because the parents of our propositus were first cousins suggests that polyosteolysis/hyperostosis syndrome may possibly have autosomal recessive inheritance.

The RANKL/OPG/RANK signalling pathway is a major regulatory system for osteoclast formation and activity. Mutations in TNFSF11, TNFRSF11B and TNFRSF11A cause defects in bone metabolism and development, thereby leading to skeletal disorders with changes in bone density and/or morphology. To date, nine kinds of monogenic skeletal diseases have been found to be causally associated with TNFSF11, TNFRSF11B and TNFRSF11A mutations. These diseases can be divided into two types according to the mutation effects and the resultant pathogenesis. One is caused by the mutations inducing constitutional RANK activation or OPG deficiency, which increase osteoclastogenesis and accelerate bone turnover, resulting in juvenile Paget's disease 2, Paget disease of bone 2, familial expansile osteolysis, expansile skeletal hyperphosphatasia, panostotic expansile bone disease, and Paget disease of bone 5. The other is caused by the de-activating mutations in TNFRSF11A or TNFSF11, which decrease osteoclastogenesis and elevate bone density, resulting in osteopetrosis, autosomal recessive 2 and 7, and dysosteosclerosis. Here we reviewed the current knowledge about these genetic disorders with paying particular attention to the updating genotype-phenotype association in the TNFRSF11A-caused diseases.

Keywords: Disease; OPG; Osteoclast; RANK; RANKL.

Objective: To screen and identify the mutations in Kawasaki disease by targeted enrichment of genomic region sequencing technique and investigate susceptibility genes associated with coronary artery lesion. Method: This was a case-control study.A total of 114 patients diagnosed as Kawasaki disease treated in Shanghai Children's Hospital between December 2015 and November 2016 were studied and another 45 healthy children who were physically examined in outpatient department were enrolled as control group. Patients were divided into two groups based on the results of echocardiogram. Peripheral venous blood was obtained from patients and controls. Genomic DNA was extracted. SeqCap EZ Choice libraries were prepared by targeted enrichment of genomic region technology. Then the libraries were sequenced to identify susceptibility genes associated with coronary artery lesion in patients diagnosed as Kawasaki disease.Susceptible genes were identified by Burden test, Pearson chi-square test or Fisher's exact probability test. Result: There was statistically significant difference in TNFRSF11B(rs2073618)G>C(p.N3K)mutation and GG/GC/CC genotype between Kawasaki disease group and control group(χ(2)=15.52, P=0.00). There was statistically significant difference in TNFRSF13B(rs34562254)C>T(p.P251L)mutation(χ(2)=10.40, P=0.01)and LEFTY1(rs360057)T>G(p.D322A)mutation(χ(2)=8.505, P=0.01)between patients with coronary artery lesions and those without. Conclusion: Targeted enrichment of genomic region sequencing technology can be used to do primary screening for the susceptible genes associated with coronary artery lesions in Chinese Kawasaki patients and may provide theoretical basis for larger sample investigation of risk prediction score standard in Kawasaki disease.

Background: Immune checkpoint inhibitors (ICIs) have been used as a novel treatment for diffuse gliomas, but the efficacy varies with patients, which may be associated with the tumor mutational burden (TMB) and immune infiltration. We aimed to explore the relationship between the two and their impacts on the prognosis.

Methods: The data of the training set were downloaded from The Cancer Genome Atlas (TCGA). "DESeq2" R package was used for differential analysis and identification of differentially expressed genes (DEGs). A gene risk score model was constructed based on DEGs, and a nomogram was developed combined with clinical features. With the CIBERSORT algorithm, the relationship between TMB and immune infiltration was analyzed, and an immune risk score model was constructed. Two models were verification in the validation set downloaded from the Chinese Glioma Genome Atlas (CGGA).

Results: Higher TMB was related to worse prognosis, older age, higher grade, and higher immune checkpoint expression. The gene risk score model was constructed based on BIRC5, SAA1, and TNFRSF11B, and their expressions were all negatively correlated with prognosis. The nomogram was developed combined with age and grade. The immune risk score model was constructed based on M0 macrophages, neutrophils, naïve CD4⁺ T cells, and activated mast cells. The proportions of the first two were higher in the high-TMB group and correlated with worse prognosis, while the latter two were precisely opposite.

Conclusions: In diffuse gliomas, TMB was negatively correlated with prognosis. The association of immune infiltration with TMB and prognosis varied with the type of immune cells. The nomogram and risk score models can accurately predict prognosis. The results can help identify patients suitable for ICIs and potential therapeutic targets, thus improve the treatment of diffuse gliomas.

Keywords: Diffuse glioma; Immune checkpoint inhibitor; Immune infiltration; Prognosis; Tumor mutational burden.

Background: Rheumatoid arthritis (RA) is a chronic autoimmune disease accompanied with joint destruction that often leads to disability. Wang-Bi capsule (WB), a traditional Chinese medicine-based herbs formula, has exhibited inhibition effect on joint destruction of collagen-induced arthritis (CIA) animal model in our previous study. But its molecular mechanisms are still obscure.

Methods: CIA rats were treated intragastrical with WB for eight weeks, and the effect of joints protection were evaluated by hematoxylin and eosin (H&E) staining, safranin O fast green staining, tartrate-resistant acid phosphatase (TRAP) staining and micro‑CT scanning analysis. The transcriptomic of tarsal joints were used to investigate how WB alleviated joint destruction.

Results: The histological examination of ankle joints showed WB alleviated both cartilage damage and bone destruction of CIA rats. This protective effect on joints were further evidenced by micro-CT analysis. The transcriptomic analysis showed that WB prominently changed 12 KEGG signaling pathways ("calcium signaling pathway", "cAMP signaling pathway", "cell adhesion molecules", "chemokine signaling pathway", "complement and coagulation cascades", "MAPK signaling pathway", "NF-kappa B signaling pathway", "osteoclast differentiation", "PI3K-Akt signaling pathway", "focal adhesion", "Gap junction" and "Rap1 signaling pathway") associated with bone or cartilage. Several genes (including Il6, Tnfsf11, Ffar2, Plg, Tnfrsf11b, Fgf4, Fpr1, Siglec1, Vegfd, Cldn1, Cxcl13, Chad, Arrb2, Fgf9, Egfr) regulating bone resorption, bone formation and cartilage development were identified by further analysis. Meanwhile, these differentially expressed genes were validated by real-time quantitative PCR.

Conclusions: Overall, the protective effect of WB treatment on joint were confirmed in CIA rats, and its basic molecular mechanisms may be associated with regulating some genes (including Il6, Tnfsf11, Ffar2 and Plg etc.) involved in bone resorption, bone formation and cartilage development.

Keywords: Joint destruction; Molecular mechanism; Rheumatoid arthritis; Transcriptomic; Wang-Bi capsule.

Objectives: TNFRSF11B encodes for osteoprotegerin (OPG) and was recently identified as the CCAL1 locus associated with familial calcium pyrophosphate deposition disease (CPDD). While the CCAL1 OPG mutation (OPG-XL) was originally believed to be a gain-of-function, loss of OPG activity causes arthritis-associated osteolysis in mice, likely related to excess subchondral osteoclast (OC) formation and/or activity. The purpose of this work was to further explore the effect of OPG-XL in osteoclastogenesis.

Methods: The effects of recombinant OPG-XL and wild type OPG (wtOPG) were determined in monoculture and co-culture models of RANKL-induced osteoclastogenesis. The effects of OPG-XL on OC survival as well as on TRAIL-induced apoptosis were determined using standard in vitro assays and compared to wtOPG. The ability of OPG-XL and wtOPG to bind to osteoblasts was measured with ELISA and flow cytometry using the osteoblastic cell line MC3T3-E1.

Results: OPG-XL was less effective than wtOPG at blocking RANKL-induced osteoclastogenesis in monoculture and co-culture models. OC survival and inhibition of TRAIL-induced apoptosis were similar in the presence of OPG-XL and wtOPG. Compared to wtOPG, considerably less OPG-XL bound to cells.

Conclusions: We show here that OPG-XL is a loss-of-function mutation as it relates to RANKL-mediated osteoclastogenesis and thus may permit increased OC number and heightened bone turnover. Further studies will be necessary to demonstrate how this mutation contributes to arthritis in individuals carrying this mutation.

Keywords: calcium pyrophosphate deposition disease; osteoclastogenesis; osteoclasts; osteoprotegerin; pseudogout.

OBJECTIVE

To identify risk groups for calcium oxalate urolithiasis among healthy individuals and patients with urolithiasis in the Russian population using molecular genetics.

METHODS

The study comprised 72 patients with calcium oxalate urolithiasis (study group) and 189 healthy adults from the general Russian population (control group). The study group consisted of 39 (54.2%) men and 33 (45.8%) women. The mean age of urolithiasis patients was 41.5+/-12.4 years. Analysis of polymorphic variants of 8 candidate urolithiasis genes: tumor necrosis factor 11B (TNFRSF11B, rs3134057), -subunit of the nuclear estrogen receptor (ESR1, rs851982), Cloto gene (KL, rs526906), vitamin D receptor (VDR, rs1540339 ), an extracellular calcium-sensing receptor (CASR, rs2202127), membrane anion transporter family 26 (SLC26A6, rs2310996), tumor necrosis factor 11 (TNFSF11, rs9525641), the calcium release-activated calcium modulator 1 (ORAI1, rs7135617) in two groups was performed by real-time PCR using Applied Biosystems test. Statistical analysis was performed using Fishers angular transformation and 2.

RESULTS

In the polymorphism of the ORAI1 gene (rs7135617), the differences in the frequencies of the GG genotype and the G allele in the control sample and in the sample of patients with calcium oxalate urolithiasis were significant: p=0.0004 and p=0.001, respectively. No statistically significant differences in the genotype and allele frequencies were found in the remaining seven gene polymorphisms.

CONCLUSIONS

Healthy individuals and patients with urolithiasis in the Russian population who have the GG genotype and/or the G allele of the polymorphism of the ORAI1 gene (rs7135617) represent risk groups for the formation of calcium oxalate stones.

It is known that the Mediterranean diet is effective in reducing the risk of several chronic diseases, including cancer. A critical component of the Mediterranean diet is olive oil, and the relationship between olive oil consumption and the reduced risk of cancer has been established. Oleuropein (OL) is the most prominent polyphenol component of olive fruits and leaves. This compound has been shown to have potent properties in various types of cancers, including breast cancer. In the present study, the molecular mechanism of OL was examined in two racially different triple-negative breast cancer (TNBC) cell lines-African American (AA, MDA-MB-468) and Caucasian American (CA, MDA-MB-231). The data obtained showed that OL effectively inhibits cell growth in both cell lines, concomitant with S-phase cell cycle arrest-mediated apoptosis. The results also showed that OL-treated MDA-MB-468 cells were two-fold more sensitive to OL antiproliferative effect than MDA-MB-231 cells were. At lower concentrations, OL modified the expression of many apoptosis-involved genes. OL was more effective in MDA-MB-468, compared to MDA-MB-231 cells, in terms of the number and the fold-change of the altered genes. In MDA-MB-468 cells, OL induced a noticeable transcription activation in fourteen genes, including two members of the caspase family: caspase 1 (CASP1) and caspase 14 (CASP14); two members of the TNF receptor superfamily: Fas-associated via death domain (FADD) and TNF receptor superfamily 21 (TNFRSF21); six other proapoptotic genes: growth arrest and DNA damage-inducible 45 alpha (GADD45A), cytochrome c somatic (CYCS), BCL-2 interacting protein 2 (BNIP2), BCL-2 interacting protein 3 (BNIP3), BH3 interacting domain death agonist (BID), and B-cell lymphoma/leukemia 10 (BCL10); and the CASP8 and FADD-like apoptosis regulator (CFLAR) gene. Moreover, in MDA-MB-468 cells, OL induced a significant upregulation in two antiapoptotic genes: bifunctional apoptosis regulator (BFAR) and B-Raf proto-oncogene (BRAF) and a baculoviral inhibitor of apoptosis (IAP) repeat-containing 3 (BIRC3). On the contrary, in MDA-MB-231 cells, OL showed mixed impacts on gene expression. OL significantly upregulated the mRNA expression of four genes: BIRC3, receptor-interacting serine/threonine kinase 2 (RIPK2), TNF receptor superfamily 10A (TNFRSF10A), and caspase 4 (CASP4). Additionally, another four genes were repressed, including caspase 6 (CASP6), pyrin domain (PYD), and caspase recruitment domain (CARD)-containing (PAYCARD), baculoviral IAP repeat-containing 5 (BIRC5), and the most downregulated TNF receptor superfamily member 11B (TNFRSF11B, 16.34-fold). In conclusion, the data obtained indicate that the two cell lines were markedly different in the anticancer effect and mechanisms of oleuropein's ability to alter apoptosis-related gene expressions. The results obtained from this study should also guide the potential utilization of oleuropein as an adjunct therapy for TNBC to increase chemotherapy effectiveness and prevent cancer progression.

Keywords: MDA-MB-231; MDA-MB-468; apoptosis; cell cycle; gene expression; oleuropein; triple-negative breast cancer.

Following a successful renal transplantation circulating markers of inflammation may remain elevated, and systemic inflammation is associated with worse clinical outcome in renal transplant recipients (RTRs). Vitamin D-receptor (VDR) activation is postulated to modulate inflammation and endothelial function. We aimed to explore if a synthetic vitamin D, paricalcitol, could influence systemic inflammation and immune activation in RTRs. Newly transplanted RTRs were included in an open-label randomized controlled trial on the effect of paricalcitol on top of standard care over the first post-transplant year. Fourteen pre-defined circulating biomarkers reflecting leukocyte activation, endothelial activation, fibrosis and general inflammatory burden were analyzed in 74 RTRs at 8 weeks (baseline) and 1 year post-engraftment. Mean changes in plasma biomarker concentrations were compared by t-test. The expression of genes coding for the same biomarkers were investigated in 1-year surveillance graft biopsies (n = 60). In patients treated with paricalcitol circulating osteoprotegerin levels increased by 0.19 ng/ml, compared with a 0.05 ng/ml increase in controls (p = 0.030). In graft tissue, a 21% higher median gene expression level of TNFRSF11B coding for osteoprotegerin was found in paricalcitol-treated patients compared with controls (p = 0.026). Paricalcitol treatment did not significantly affect the blood- or tissue levels of any other investigated inflammatory marker. In RTRs, paricalcitol treatment might increase both circulating and tissue levels of osteoprotegerin, a modulator of calcification, but potential anti-inflammatory treatment effects in RTRs are likely very modest. [NCT01694160 (2012/107D)]; [www.clinicaltrials.gov].

Inhibition of IRE1 (inositol requiring enzyme-1), the major signaling pathway of endoplasmic reticulm stress, significantly decreases glioma cell proliferation and tumor growth. We have studied the expression of TNFα-related genes and effect of glucose deprivation on these gene expressions in U87 glioma cells over-expressing dominant-negative IRE1 defective in both kinase and endonuclease (dn-IRE1) activity of IRE1 with hopes of elucidating its contribution to IRE1 mediated glioma growth. We have demonstrated that glucose deprivation condition leads to down-regulation of the expression of TNFRSF11B, TNFRSF1A, TNFRSF10D/TRAILR4, and LITAF genes and up-regulation of TNFRSF10B/TRAILR2/DR5 gene at the mRNA level in control glioma cells. At the same time, the expression of TNFRSF21/DR6, TNFAIP1, TNFAIP3, TRADD, and CD70/TNFSF7 genes in control glioma cells is resistant to glucose deprivation condition. The inhibition of IRE1 modifies the effect of glucose deprivation on LITAF, TNFRSF21, TNFRSF11B, and TRADD gene expressions and induces sensitivity to glucose deprivation condition the expression of TNFRSF10B, TNFRSF1A, and CD70 genes. We have also demonstrated that the expression of all studied genes is affected in glioma cells by inhibition of IRE1, except TNFRSF1A gene, as compared to control glioma cells. Moreover, the changes in the expression of TNFRSF1A, TNFRSF10D/TRAILR4, and LITAF genes induced by glucose deprivation condition have opposite orientation to that induced by inhibition of IRE1. The present study demonstrates that fine-tuning of the expression of TNFα-induced proteins and TNF receptor superfamily genes, which related to cell death and proliferation, is regulated by IRE1, an effector of endoplasmic reticulum stress, as well as depends on glucose deprivation in gene specific manner. Thus, the inhibition of kinase and endoribonuclease activity of IRE1 correlates with deregulation of TNFα-induced protein genes and TNF receptor superfamily genes in gene specific manner and thus slower the tumor growth.

Previous research has demonstrated a positive relationship between dietary Metabolisable Energy Intake (MEI) and increased maintenance energy costs associated with the visceral tissues. Limitations in understanding this relationship include a lack of access to samples to assess regulatory control of the putative response gastrointestinal tissues to nutrients. This experiment was conducted with a single nutrient (starch hydrolysate) infused (7 d) directly into the intestine to mimic typical changes in post-ruminal starch delivery in dairy production settings. Duodenal epithelial samples collected via biopsy were evaluated using next-generation sequencing technology (RNA-Seq) to validate the use of this approach for the profiling and comparison of the transcriptome of cattle intestinal epithelial tissues. Samples of intestinal epithelial tissue were collected prior to and during the infusion of starch hydrolysate. Biopsies were collected on day 0 before and day 1, day 3, and day 7 during the infusion. Additionally, samples were collected on day 1 and day 7 after infusion was discontinued (Day 8 and Day14 of the experiment). Evaluation of RNA-seq data revealed dynamic changes in global gene expression during infusion. On day 7 of the infusion, 1490 genes were found to be differentially expressed (DE) compared to the day 0 control samples with FDR p < 0.05, vs. 105 genes on day 1 and 246 genes on Day 3. However, on day 8, after infusion was terminated for 24 h, only 428 genes were identified as differentially expressed compared to day 0 and only 107 genes continued to be identified by Day 14. Thus, the apparent differential expression of these genes is putatively a result of the single nutrient infused. Further, performing function and pathway analysis of the identified DE genes using IPA, we observe changes in digestive system development, and function pathways are among the primary functions of the DE genes, as well as immune response elements. Finally, primary transcription regulators such as PTH, JUN, WNT, and TNFRSF11B were identified as the activated upstream regulators for specific future focus. Using a serial biopsy approach we are able to identify differentially expressed genes from cow duodenal epithelial tissue in response to a short-term perturbation with infused starch hydrolysate.

OBJECTIVE

Search for and identification of possible associations of recurrence-free urolithiasis with polymorphisms of urolithiasis candidate genes in the Russian population.

METHODS

The study involved 43 patients with recurrence-free urolithiasis, 13 (30.2%) women and 30 (69.8%) of men (main group) from Central Russia, and 189 healthy adults (control group) from the same region. The mean age of the main group was 42.5+/-13 years. The venous blood samples were used for the evaluation. Real-time PCR using the "Applied Biosystems" test systems was performed to determine the spectrum and frequency of eight urolithiasis candidate genes polymorphisms: tumor necrosis factor receptor gene (TNFRSF11B (rs3134057), vitamin D receptor gene (VDR, (rs1540339), extracellular calcium-sensing receptor gene (CASR, (rs2202127), calcium release-activatedcalcium modulator 1 gene (ORAI1, rs7135617), Klotho gene (KL, rs526906), nuclear estrogen receptor alpha-subunit gene (ESR1, rs851982), tumor necrosis factor 11 gene (TNFSF11, rs9525641), and 26 gene family anionic membrane transporter gene (SLC26A6, rs2310996). Fishers angular transformation and 2 test were used for statistical analysis of the data.

RESULTS

For ORAI1 gene, differences in genotype and allele frequencies in the control and main groups are significant: p=0.0001 and p=0.013, respectively. For polymorphisms of the other seven genes studied, the differences in genotype and allele frequencies are non-significant. The results indicate the presence of the association between recurrence-free urolithiasis and calcium release-activatedcalcium modulator 1 gene polymorphism (ORAI1, rs7135617), both in genotypes and alleles.

CONCLUSIONS

In Russian population, genetic factors, in particular, ORAI1 (rs7135617) gene polymorphism, can play the role in the development of recurrence-free urolithiasis.

We examined single-nucleotide polymorphisms (SNPs) across the tumour necrosis factor receptor superfamily member 11B (TNFRSF11B) gene and knee OA. We identified alleles in a VNTR region in intron 3 that was observed exclusively in women OA cases (P = 0.007, Pc = 0.042). Our results reveal that a previously unreported association between a VNTR genotype in TNFRSF11B and knee OA in women.

Juvenile Paget's disease (JPD) is a rare recessively-inherited bone dysplasia. The great majority of cases described to date have had homozygous mutations in TNFRSF11B, the gene encoding osteoprotegerin. We describe a boy who presented with recurrent clavicular fractures following minor trauma (8 fractures from age 2 to 11). He was of normal height and despite mild lateral bowing of the thighs and anterior bowing of the shins he remained physically active. Abnormal modelling was noted in ribs and humeri on clavicular radiographs, and a skeletal survey at the age of 7 showed generalised diaphyseal expansion of the long bones with thickening of the periosteal and endosteal surfaces of the cortices. On biochemical evaluation, serum alkaline phosphatase was noted to be persistently elevated. The diagnosis of JPD was confirmed by the finding of compound heterozygous mutations in TNFRSF11B: a maternally-inherited A > G missense mutation at position 1 of the first amino acid codon (previously reported) and a paternally-inherited splice acceptor site mutation in intron 3 at a highly conserved position (not previously reported). Bioinformatics analysis suggested both mutations were disease-causing. Compound heterozygote mutations in TNFRSF11B causing JPD have been previously reported only once - in a boy who also had a relatively mild skeletal phenotype. The milder features may lead to delay in diagnosis and diagnostic confusion with other entities, but the extraskeletal features of JPD may nonetheless develop.

Abstract Introduction: To test whether genetic variants of osteoprotegerin gene (TNFRSF11B) affect metabolic traits (body mass index [BMI], glucose, triglyceride, total cholesterol) and bone mass traits.

METHODS

We conducted a population based association study to investigate associations of eight tagging single nucleotide polymorphisms (tSNPs) of the TNFRSF11B gene with the aforementioned traits in a Chinese Han population and an ethnic group admixed with Caucasians and Asians - Uyghur. The associations between the tSNPs and bone mass density (BMD) were also tested in Han population.

RESULTS

We found that SNP rs3102727, located in the first intron of the TNFRSF11B gene, was significantly associated with triglyceride levels in Uyghur population and Han population simultaneously. T allele of the rs3102727 variant was associated with a 0.10 mmol/L and 0.09 mmol/L lower level of triglyceride than C allele in Uyghur (p = 0.019) and Han subjects (p = 0.037), respectively. In addition, the T allele is also associated with a lower level of hip BMD (p = 0.025) and total BMD (p = 0.048). Further, we found significant associations between SNP rs11573869 and BMI in Uyghur subjects and SNP rs3134062 with hip BMD in Han sbujects. Rs11573869-T allele was associated with a 0.81 kg/m(2) lower level of BMI than C allele (p = 0.002) and the hip BMD decreases with the copy of rs3134062-T allele increases (p = 0.002).

CONCLUSIONS

We detected novel associations between TNFRSF11B polymorphisms and metabolic traits in Uyghur and Han populations. In addition, we found associations between TNFRSF11B polymorphisms and bone mass traits in Han population.

Osteoprotegerin is involved in the progression of atherosclerosis. This study aimed to determine whether TNFRSF11B polymorphisms are associated with prognosis of large artery atherosclerosis (LAA) stroke. Three TNFRSF11B polymorphisms (rs2073617, rs2073618 and rs3134069) were genotyped in 1,010 patients with LAA stroke. Short-term outcome was evaluated using the modified Rankin Scale score at 3-month after stroke onset. Long-term outcome was assessed using the stroke recurrence. We found that rs2073617G was associated with an increased risk of poor outcome of LAA stroke (additive model: odds ratio (OR) = 1.35, 95% confidence interval (CI) = 1.06-1.73). This association was also observed in rs3134069C (additive model: OR = 1.53, 95% CI = 1.10-2.12). Furthermore, when we combined these two polymorphisms according to the numbers of risk alleles (rs2073617G and rs3134069C), we found that the patients with 3-4 risk alleles were statistically significantly associated with an increased risk of poor outcome of LAA stroke (OR = 1.90, 95% CI = 1.10-3.28) compared with 0-2 risk alleles, and this increased risk was more evident among those with hypertension (OR = 2.02, 95% CI = 1.04-3.91), those without diabetes (OR = 2.02, 95% CI = 1.02-4.01) and those with smoking (OR = 2.43, 95% CI = 1.09-5.42). In silico analysis showed that rs2073617 and rs3134069 are located in various histone modification marked regions, DNase I hypersensitive sites and can change the binding of regulatory motifs. Moreover, rs2073617 is also located in the binding site of transcription factors. Our findings suggested that TNFRSF11B polymorphisms may be associated with an increased risk of short-term poor outcome of LAA stroke.

Introduction: Juvenile Paget's Disease (JPD) is an ultra-rare inherited osteopathy featuring markedly accelerated bone turnover. Several clinical characteristics have been reported, including bone deformities developing in childhood and hearing loss.

Case report: We report the case of a 2 ¾-year-old girl that presented with progressive bowing of both legs since the age of 2, lower limb pain and frequent falls with one consequent femur fracture. Plain radiographs revealed osteoectasia of the long bone's diaphysis, and laboratory tests showed extremely high serum total alkaline phosphatase levels. A missense mutation on the gene TNFRSF11B was identified in homozygosity, and the diagnosis of JPD was made.Treatment with bisphosphonates was initiated early and markedly improved lower limb bowing and pain. The patient reached adulthood with normal height, minor bone deformities, and no functional impairment. Despite the good skeletal symptom's response, bisphosphonates failed to prevent or improve sensorineural hearing loss.

Conclusions: In this clinical case, early treatment with bisphosphonates was effective for the treatment of JPD skeletal deformities. New therapeutic strategies need to be developed to better control the extraskeletal manifestations of JPD.

Keywords: Juvenile Paget's Disease; bisphosphonates; case report.

Fibrosis is defined by excessive formation and accumulation of extracellular matrix proteins, produced by myofibroblasts, that supersedes normal wound healing responses to injury and results in progressive architectural remodelling. Fibrosis is often detected in advanced disease stages when an organ is already severely damaged and can no longer function properly. Therefore, there is an urgent need for reliable and easily detectable markers to identify and monitor fibrosis onset and progression as early as possible; this will greatly facilitate the development of novel therapeutic strategies. Osteoprotegerin (OPG), a well-known regulator of bone extracellular matrix and most studied for its role in regulating bone mass, is expressed in various organs and functions as a decoy for receptor activator of nuclear factor kappa-B ligand (RANKL) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Recently, OPG has been linked to fibrosis and fibrogenesis, and has been included in a panel of markers to diagnose liver fibrosis. Multiple studies now suggest that OPG may be a general biomarker suitable for detection of fibrosis and/or monitoring the impact of fibrosis treatment. This review summarizes our current understanding of the role of OPG in fibrosis and will discuss its potential as a biomarker and/or novel therapeutic target for fibrosis.

Keywords: Fibroblast; Lung; RANKL; TGFβ; TNFRSF11B; TRAIL.

Exosomes are a type of extracellular vesicle that act as shuttles, transporting certain genetic information to other cells. MiRNA cargo within exosomes can regulate gene expression at the transcriptional level. The objective of this study was to investigate the exosomal miRNAs that regulate lipopolysaccharide (LPS)-induced inflammation in dairy cow mammary alveolar (Mac-T) cells. We found two exosome miRNAs upregulated and five exosomal miRNAs downregulated, respectively, in the LPS-stimulated Mac-T cells. MiR-193b-5p was upregulated 6.3-fold in the LPS-stimulated cell-derived exosome. Target prediction results showed that nuclear factor kappa B (NF-κB) inhibitor delta (NFKBID), transforming growth factor-beta 1 induced transcript 1 (TGFB1I1), interleukin 22 (IL-22), TNF receptor superfamily member 11b (TNFRSF11B), and Janus kinase 3 (JAK3) might be the main target genes of miR-193b-5p. After treatment of Mac-T cells with the miR-193b-5p mimic, the phosphorylation levels of inhibitor of nuclear factor-kappa Bα (IκBα) and p65 were upregulated, the level of IL-6 mRNA was upregulated, and IL-1β, TNF-α, and TGF-β mRNA levels were downregulated. After treatment of Mac-T cells with miR-193b-5p inhibitor, the phosphorylation levels of IκBα and p65 were downregulated. In summary, these findings provide strong evidence that exosomal miR-193b-5p could be a regulator of LPS-induced inflammation in Mac-T cells and reveal a new role of exosomal miRNAs in regulating dairy cow mastitis.

Keywords: Dairy cow; Exosome; Inflammation; Mammary epithelial cells; miRNA.

Objective: This study aimed to investigate genetic variations in the osteoprotegerin-encoding gene (TNFRSF11B) in patients with temporomandibular joint ankylosis (TMJA).

Study design: The sample comprised 17 patients diagnosed with TMJA, of both sexes with ages ranging from 6 to 57 years old. TNFRSF11B mutational analysis was performed using the Sanger sequencing method with DNA extracted from oral cells, and the functional impact prediction of the variants was assessed using bioinformatic analysis.

Results: Sequencing analysis identified 15 (88.23%) patients that presented at least 1 genetic variant in TNFRSF11B. The mutation rs202090603 (p.E33K) was found in 6 individuals, and rs140782326 (p.V281M), rs11573942 (p.L295), and rs1375250340 (p.I389T) were identified in 1 subject each. According to the pathogenicity potential of mutations, 3 variants were considered of low impact (rs2073618, rs202090603, and rs2228568) and 3 as disease causing (rs140782326, rs11573942, and rs1375250340). The variant rs202090603 (p.E33K) was found in the first cysteine domain with differences in the loop positions of p.E33K mutated the 3D structure of osteoprotegerin.

Conclusion: Two polymorphisms (rs2073618 and rs2228568) and the mutations rs202090603 (p.E33K), rs140782326 (p.V281M), rs11573942 (p.L295), and rs1375250340 (p.I389T) in the TNFRSF11B gene may be associated with TMJA.

MSC, mesenchymal stem cells; OPG, osteoprotegerin; RUNX2, Run-trelated transcription factor 2.

Keywords: P63; breast; mesenchymal stem cells; osteogenic differentiation; p53 family; prostate cancer.

Notch receptors maintain skeletal homeostasis. NOTCH1 and 2 have been studied for their effects on bone remodeling. Whereas NOTCH3 plays a significant role in vascular physiology, knowledge about its function in other cellular environments, including bone, is limited. The present study was conducted to establish the function of NOTCH3 in skeletal cells using models of Notch3 misexpression. Microcomputed tomography (μCT) demonstrated that Notch3 null mice did not have appreciable bone phenotypes. To study the effects of the NOTCH3 activation in the osteoblast lineage, BGLAP-Cre or Dmp1-Cre transgenics were crossed with Rosa^Notch3 mice, where the NOTCH3 intracellular domain is expressed following the removal of a loxP-flanked STOP cassette. μCT demonstrated that BGLAP-Cre;Rosa^Notch3 and Dmp1-Cre;Rosa^Notch3 mice of both sexes exhibited an increase in trabecular bone and in connectivity, with a decrease in cortical bone and increased cortical porosity. Histological analysis revealed a decrease in osteoclast number and bone resorption in trabecular bone and an increase in osteoclast number and void or pore area in cortical bone of Rosa^Notch3 mice. Bone formation was either decreased or could not be determined in Cre;Rosa^Notch3 mice. NOTCH3 activation in osteoblasts inhibited Alpl (alkaline phosphatase) and Bglap (osteocalcin) and induced Tnfsf11 (RANKL) and Tnfrsf11b (osteoprotegerin) mRNA, possibly explaining the trabecular bone phenotype. However, NOTCH3 induced Tnfsf11 and suppressed Tnfrsf11b in osteocytes, possibly explaining the cortical porosity. In conclusion, whereas basal NOTCH3 is dispensable for skeletal homeostasis, activation of NOTCH3 in osteoblasts/osteocytes inhibits osteoclastogenesis and bone resorption in cancellous bone, but increases intracortical remodeling and causes cortical porosity.

Keywords: Notch receptor; bone; cortical porosity; osteoblast; osteoclast; osteocyte.

Objectives: Osteoarthritis (OA) is a complex genetic disease with different risk factors contributing to its development. One of the genes, TNFRSF11B, previously identified with gain-of-function mutation in a family with early-onset OA with chondrocalcinosis, is among the highest upregulated genes in lesioned OA cartilage (RAAK-study). Here, we determined the role of TNFRSF11B overexpression in development of OA.

Methods: Human primary articular chondrocytes (9 donors RAAK study) were transduced using lentiviral particles with or without TNFRSF11B. Cells were cultured for 1 week in a 3D in-vitro chondrogenic model . TNFRSF11B overexpression was confirmed by RT-qPCR, immunohistochemistry and ELISA. Effects of TNFRSF11B overexpression on cartilage matrix deposition, matrix mineralization, and genes highly correlated to TNFRSF11B in RNA-sequencing dataset (r>|0.75|) were determined by RT-qPCR. Additionally, glycosaminoglycans and collagen deposition were visualized with Alcian blue staining and immunohistochemistry (COL1 and COL2).

Results: Overexpression of TNFRSF11B resulted in strong upregulation of MMP13, COL2A1 and COL1A1. Likewise, mineralization and osteoblast characteristic markers RUNX2, ASPN and OGN showed a consistent increase. Among 30 genes highly correlated to TNFRSF11B, expression of only 8 changed significantly, with BMP6 showing highest increase (9-fold) while expression of RANK and RANKL remained unchanged indicating previously unknown downstream pathways of TNFRSF11B in cartilage.

Conclusion: Results of our 3D in vitro chondrogenesis model indicate that upregulation of TNFRSF11B in lesioned OA cartilage may act as a direct driving factor for chondrocyte to osteoblast transition observed in OA pathophysiology. This transition does not appear to act via the OPG/RANK/RANKL triad common in bone remodeling.

Ethics approval and consent to participate: The Medical Ethics Committee of the LUMC gave approval for the RAAK study (P08.239). Written informed consent was obtained from all donors.

Keywords: CCAL1; TNFRSF11B; cartilage hypertrophy; chondrogenesis; matrix mineralization; osteoarthritis.