Understanding co‐activation patterns of the hypothalamic‐pituitary‐adrenal axis (HPA) and sympathetic adrenal medullary (SAM) during early adolescence may illuminate risk for development of ...internalizing and externalizing problems. The present study advances empirical work on the topic by examining SAM‐HPA co‐activation during both the reactivity and recovery phases of the stress response following acute stress exposure. Fourth and fifth grade boys and girls (N = 149) provided cortisol and alpha‐amylase via saliva at seven times throughout a 95‐min assessment in which they were administered the modified Trier Social Stress Test. Parents reported on adolescents’ life stress, pubertal development, medication use, and externalizing problems. Adolescents reported their own internalizing symptoms. Multiple linear regressions tested both direct and interactive effects of SAM and HPA reactivity and recovery on internalizing and externalizing problems. Results from these analyses showed that whereas SAM and HPA reactivity interacted to predict internalizing symptoms, it was their interaction during the recovery phase that predicted externalizing. Concurrent high SAM and HPA reactivity scores predicted high levels of internalizing and concurrently low SAM and HPA recovery scores predicted high levels of externalizing. Implications of the findings for further study and clinical application are discussed.
Investigation of cartilage and chondrocytes has revealed that the osteoarthritis risk marked by the independent DNA variants rs11583641 and rs1046934 mediate their effects by decreasing the ...methylation status of CpG dinucleotides in enhancers and increasing the expression of shared target gene COLGALT2. We set out to investigate if these functional effects operate in a non-cartilaginous joint tissue.
Nucleic acids were extracted from the synovium of osteoarthritis patients. Samples were genotyped, and DNA methylation was quantified by pyrosequencing at CpGs within the COLGALT2 enhancers. CpGs were tested for enhancer effects using a synovial cell line and a reporter gene assay. DNA methylation was altered using epigenetic editing, with the impact on gene expression determined using quantitative polymerase chain reaction. In silico analysis complemented laboratory experiments.
The rs1046934 genotype did not associate with DNA methylation or COLGALT2 expression in the synovium, whereas the rs11583641 genotype did. Surprisingly, the effects for rs11583641 were opposite to those previously observed in cartilage. Epigenetic editing in synovial cells revealed that enhancer methylation is causally linked to COLGALT2 expression.
This is the first direct demonstration for osteoarthritis genetic risk of a functional link between DNA methylation and gene expression operating in opposite directions between articular joint tissues. It highlights pleiotropy in the action of osteoarthritis risk and provides a cautionary note in the application of future genetically based osteoarthritis therapies: an intervention that decreases the detrimental effect of a risk allele in one joint tissue may inadvertently increase its detrimental effect in another joint tissue.
The concept of human dignity lies at the heart of many national and international conventions of human rights. This idea, based on man's rationality, can be found already in Greco-Roman Antiquity, ...was fully developed in Christianity, in its synthesis with the Biblical conception of man as image of God. With the secularization of the European mind from the 18th century onwards, the justification of human dignity becomes problematic. This most influential attempt to justify it by secular rationality came from Kant, who saw man’s dignity as deriving from his capacity for moral reasoning and from it came the notions of autonomy and equality. However, during the last two centuries, secularized cultures produced skeptical attitudes toward both the Judeo-Christian and Kantian concepts of the intrinsic dignity of man, which eventually paved the way for twentieth-century totalitarian-isms. After the horrors of Nazism, concerns about putting human rights in the centre of culture, politics and law compelled a search ―largely impossible― for a common idea of human dignity, shared by different philosophical traditions, both religious and secular. During the years after World War II, especially after the Second Vatican Council, there was a renewed discovery of human rights as based on human dignity by Catholicism, which, in view of the different reduc-tionist or destructive tendencies found in the secularized culture, perhaps is the most satisfactory approach. Finally, the problem of religious freedom is examined as a case study for further reflections on human dignity.
Objective
To identify the functional single‐nucleotide polymorphisms (SNPs) and mechanisms conferring increased risk of hand osteoarthritis (OA) at the ALDH1A2 locus, which is a retinoic acid ...regulatory gene.
Methods
Tissue samples from 247 patients with knee, hip, or hand OA who had undergone joint surgery were included. RNA‐sequencing analysis was used to investigate differential expression of ALDH1A2 and other retinoic acid signaling pathway genes in cartilage. Expression of ALDH1A2 in joint tissues obtained from multiple sites was quantified using quantitative reverse transcription–polymerase chain reaction. Allelic expression imbalance (AEI) was measured by pyrosequencing. The consequences of ALDH1A2 depletion by RNA interference were assessed in primary human chondrocytes. In silico and in vitro analyses were used to pinpoint which, among 62 highly correlated SNPs, could account for the association at the locus.
Results
ALDH1A2 expression was observed across multiple joint tissue samples, including osteochondral tissue from the hand. The expression of ALDH1A2 and of several retinoic acid signaling genes was different in diseased cartilage compared to non‐diseased cartilage, with ALDH1A2 showing lower levels in OA cartilage. Experimental depletion of ALDH1A2 resulted in changes in the expression levels of a number of chondrogenic markers, including SOX9. In addition, reduced expression of the OA risk–conferring allele was witnessed in a number of joint tissues, with the strongest effect in cartilage. The intronic SNP rs12915901 recapitulated the AEI observed in patient tissues, while the Ets transcription factors were identified as potential mediators of this effect.
Conclusion
The ALDH1A2 locus seems to increase the risk of hand OA through decreased expression of ALDH1A2 in joint tissues, with the effect dependent on rs12915901. These findings indicate a mechanism that may now be targeted to modulate OA risk.
Transitioning from a genetic association signal to an effector gene and a targetable molecular mechanism requires the application of functional fine-mapping tools such as reporter assays and genome ...editing. In this report, we undertook such studies on the osteoarthritis (OA) risk that is marked by single nucleotide polymorphism (SNP) rs34195470 (A > G). The OA risk-conferring G allele of this SNP associates with increased DNA methylation (DNAm) at two CpG dinucleotides within WWP2. This gene encodes a ubiquitin ligase and is the host gene of microRNA-140 (miR-140). WWP2 and miR-140 are both regulators of TGFβ signaling.
Nucleic acids were extracted from adult OA (arthroplasty) and foetal cartilage. Samples were genotyped and DNAm quantified by pyrosequencing at the two CpGs plus 14 flanking CpGs. CpGs were tested for transcriptional regulatory effects using a chondrocyte cell line and reporter gene assay. DNAm was altered using epigenetic editing, with the impact on gene expression determined using RT-qPCR. In silico analysis complemented laboratory experiments.
rs34195470 genotype associates with differential methylation at 14 of the 16 CpGs in OA cartilage, forming a methylation quantitative trait locus (mQTL). The mQTL is less pronounced in foetal cartilage (5/16 CpGs). The reporter assay revealed that the CpGs reside within a transcriptional regulator. Epigenetic editing to increase their DNAm resulted in altered expression of the full-length and N-terminal transcript isoforms of WWP2. No changes in expression were observed for the C-terminal isoform of WWP2 or for miR-140.
As far as we are aware, this is the first experimental demonstration of an OA association signal targeting specific transcript isoforms of a gene. The WWP2 isoforms encode proteins with varying substrate specificities for the components of the TGFβ signaling pathway. Future analysis should focus on the substrates regulated by the two WWP2 isoforms that are the targets of this genetic risk.
GDF5 is involved in synovial joint development, maintenance and repair, and the rs143383 C/T single nucleotide polymorphism (SNP) located in the 5′UTR of GDF5 is associated, at the genome-wide ...significance level, with osteoarthritis susceptibility, and with other musculoskeletal phenotypes including height, congenital hip dysplasia and Achilles tendinopathy. There is a significant reduction in the expression of the disease-associated T allele relative to the C allele in synovial joint tissues, an effect influenced by a second SNP (rs143384, C/T) also within the 5′UTR. The differential allelic expression (DAE) imbalance of the C and T alleles of rs143383 varies intra- and inter-individually, suggesting that DAE may be modulated epigenetically. The C alleles of both SNPs form CpG dinucleotides that are potentially amenable to regulation by methylation. Here, we have examined whether DNA methylation regulates GDF5 expression and the allelic imbalance caused by rs143383. We observed methylation of the GDF5 promoter and 5′UTR in cell lines and joint tissues, with demethylation correlating with increased GDF5 expression. The CpG sites created by the C alleles at rs143383 and rs143384 were variably methylated, and treatment of a heterozygous cell line with a demethylating agent further increased the allelic expression imbalance between the C and T alleles. This demonstrates that the genetic effect of the rs143383 SNP on GDF5 expression is modulated epigenetically by DNA methylation. The variability in DAE of rs143383 is therefore partly accounted for by differences in DNA methylation that could influence the penetrance of this allele in susceptibility to common musculoskeletal diseases.
Osteoarthritis (OA) is a leading cause of disability in Western society with multiple risk factors, including a complex genetic pattern. Identifying loci involved in the heredity of OA might lead to ...insights into the molecular pathogenesis of this common disorder. A previous genome scan mapped a primary hip OA susceptibility locus to chromosome 2q with a maximum multipoint logarithm of odds score of 1.6 in 378 affected sibling pair families. Here, microsatellite targeting of eight candidate genes in this region from 2q23-2q32 demonstrated significant associations with the tumor necrosis factor α-induced protein 6 gene in all probands and the integrin α6 and frizzled motif associated with bone development (FRZB) genes in female probands. However, genotyping showed lack of association for a nonsynonymous single-nucleotide polymorphism in tumor necrosis factor α-induced protein 6, whereas a single-nucleotide polymorphism in FRZB resulting in an Arg324Gly substitution at the carboxyl terminus was associated with hip OA in the female probands (P = 0.04). This association was confirmed in an independent cohort of female hip cases (n = 338; P = 0.04). In addition, a haplotype coding for substitutions of two highly conserved arginine residues (Arg200Trp and Arg324Gly) in FRZB was a strong risk factor for primary hip OA, with an odds ratio of 4.1 (P = 0.004). FRZB encodes secreted frizzled-related protein 3, which is a soluble antagonist of wingless (wnt) signaling. Variant secreted frizzled-related protein 3 with the Arg324Gly substitution had diminished ability to antagonize wnt signaling in vitro. Hence, functional polymorphisms within FRZB confer susceptibility for hip OA in females and implicate the wnt signaling pathway in the pathogenesis of this disease.
rs143383 is a C to T transition SNP located in the 5'untranslated region (5'UTR) of the growth differentiation factor 5 gene GDF5. The T allele of the SNP is associated with increased risk of ...osteoarthritis (OA) in Europeans and in Asians. This susceptibility is mediated by the T allele producing less GDF5 transcript relative to the C allele, a phenomenon known as differential allelic expression (DAE). The aim of this study was to identify trans-acting factors that bind to rs143383 and which regulate this GDF5 DAE. Protein binding to the gene was investigated by two experimental approaches: 1) competition and supershift electrophoretic mobility shift assays (EMSAs) and 2) an oligonucleotide pull down assay followed by quantitative mass spectrometry. Binding was then confirmed in vivo by chromatin immunoprecipitation (ChIP), and the functional effects of candidate proteins investigated by RNA interference (RNAi) and over expression. Using these approaches the trans-acting factors Sp1, Sp3, P15, and DEAF-1 were identified as interacting with the GDF5 5'UTR. Knockdown and over expression of the factors demonstrated that Sp1, Sp3, and DEAF-1 are repressors of GDF5 expression. Depletion of DEAF-1 modulated the DAE of GDF5 and this differential allelic effect was confirmed following over expression, with the rs143383 T allele being repressed to a significantly greater extent than the rs143383 C allele. In combination, Sp1 and DEAF-1 had the greatest repressive activity. In conclusion, we have identified four trans-acting factors that are binding to GDF5, three of which are modulating GDF5 expression via the OA susceptibility locus rs143383.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The single nucleotide polymorphism (SNP) rs835487 is associated with hip osteoarthritis (OA) at the genome-wide significance level and is located within CHST11, which codes for carbohydrate ...sulfotransferase 11. This enzyme post-translationally modifies proteoglycan prior to its deposition in the cartilage extracellular matrix. Using bioinformatics and experimental analyses, our aims were to characterise the rs835487 association signal and to identify the causal functional variant/s. Database searches revealed that rs835487 resides within a linkage disequilibrium (LD) block of only 2.7 kb and is in LD (r2 ≥ 0.8) with six other SNPs. These are all located within intron 2 of CHST11, in a region that has predicted enhancer activity and which shows a high degree of conservation in primates. Luciferase reporter assays revealed that of the seven SNPs, rs835487 and rs835488, which have a pairwise r2 of 0.962, are the top functional candidates; the haplotype composed of the OA-risk conferring G allele of rs835487 and the corresponding T allele of rs835488 (the G-T haplotype) demonstrated significantly different enhancer activity relative to the haplotype composed of the non-risk A allele of rs835487 and the corresponding C allele of rs835488 (the A-C haplotype) (p < 0.001). Electrophoretic mobility shift assays and supershifts identified several transcription factors that bind more strongly to the risk-conferring G and T alleles of the two SNPs, including SP1, SP3, YY1 and SUB1. CHST11 was found to be upregulated in OA versus non-OA cartilage (p < 0.001) and was expressed dynamically during chondrogenesis. Its expression in adult cartilage did not however correlate with rs835487 genotype. Our data demonstrate that the OA susceptibility is mediated by differential protein binding to the alleles of rs835487 and rs835488, which are located within an enhancer whose target may be CHST11 during chondrogenesis or an alternative gene.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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