Osteoarthritis Susceptibility Research Articles

Correlation between clinical and radiographic classification of osteoarthritis and SNPs linked to osteoarthritis susceptibility

Correlation between clinical and radiographic classification of osteoarthritis and SNPs linked to osteoarthritis susceptibility

Replication study of osteoarthritis susceptibility genes in hand osteoarthritis in Finnish women

ment with all 4 HDIs, consistent with effective suppression of HDAC activity. The most effective specific HDI was MS275, which showed similar A-H3 levels to TSA, but exhibited some undesirable effects of pan-HDI. PCI increased A-H3 levels, though not as effective as MS275, but also reduced the negative effects of TSA, such as decreased COX-2 and MMP-3 expression under treatment with IL1b. Also, the additive effect of TSA and IL1b on the secretion of MMP3was not observed in the case of PCI. Chondrocyte proliferation and survival was unaffected by the tested HDIs after 24 h of culture, with the exception of Droxinostat at concentration over 20 mm(decreased proliferation as shown by MTS assay and increased cell death as shown by Live/Dead assay). However, upon longer incubation (48 h), both TSA (200 nM) and Droxinostat reduced chondrocyte proliferation, while MS275, PCI and Tubostatin A increased it. Conclusions: The HDI, PCI, appears to be a promising candidate for reducing inflammatory and catabolic markers in chondrocytes exposed to pro-inflammatory cytokines. Further studies will investigate the effect of increasing PCI concentrations in two in vitro chondrocytebased OA models: (1) IL1b treatment; and (2) exposure to supraphysiological mechanical loading. In addition, MS275 could be a good alternative to pan-HDAC inhibitors, such as TSA, when longer exposure times are needed. Grant support: Arthritis Foundation, Commonwealth of PA, Dept of Health.

Reproductive status and sex show strong effects on knee OA in a baboon model

We aimed to characterize severity and occurrence of knee osteoarthritis (OA), and effects of age, sex, body mass, and reproductive status on population-level normal variation in this condition in the baboon, a natural model of human knee OA. We visually inspected articular cartilage of distal right femora of 464 baboons (309 females, 155 males) and assigned an OA severity score (comparable to a modified Outerbridge score) from 1 = unaffected to 4 = advanced OA (eburnation). Presence/absence of osteophytes was recorded. We tested for significant effects of age, sex, body mass, and, in females, reproductive status (pre-, peri-, or post-menopausal) on OA. When appropriate, analyses were repeated on an age-matched subset (153 of each sex). Knee OA was more frequent and severe in older animals (P < 0.0001), but significant age variation was apparent in each severity grade. Sex differences within the younger and older age groups suggest that males develop knee OA earlier, but females progress more quickly to advanced disease. There is a strong relationship between reproductive status and OA severity grade in females (P = 0.0005) with more severe OA in peri- and post-menopausal female baboons, as in humans. Idiopathic knee OA is common in adult baboons. Occurrence and severity are influenced strongly by reproductive status in females, and by sex with regard to patterns of disease progression - providing an animal model to investigate sex-specific variation in OA susceptibility in which the environmental heterogeneity inherent in human populations is vastly reduced.

Allelic expression analysis of the osteoarthritis susceptibility gene COL11A1 in human joint tissues

BackgroundThe single nucleotide polymorphism (SNP) rs2615977 is associated with osteoarthritis (OA) and is located in intron 31 of COL11A1, a strong candidate gene for this degenerative musculoskeletal disease. Furthermore, the common non-synonymous COL11A1 SNP rs1676486 is associated with another degenerative musculoskeletal disease, lumbar disc herniation (LDH). rs1676486 is a C-T transition mediating its affect on LDH susceptibility by modulating COL11A1 expression. The risk T-allele of rs1676486 leads to reduced expression of the COL11A1 transcript, a phenomenon known as allelic expression imbalance (AEI). We were keen therefore to assess whether the effect that rs1676486 has on COL11A1 expression in LDH is also observed in OA and whether the rs2615977 association to OA also marked AEI.MethodsUsing RNA from OA cartilage, we assessed whether either SNP correlated with COL11A1 AEI by 1) measuring COL11A1 expression and stratifying the data by genotype at each SNP; and 2) quantifying the mRNA transcribed from each allele of the two SNPs. We also assessed whether rs1676486 was associated with OA susceptibility using a case–control cohort of over 18,000 individuals.ResultsWe observed significant AEI at rs1676486 (p < 0.0001) with the T-allele correlating with reduced COL11A1 expression. This corresponded with observations in LDH but the SNP was not associated with OA. We did not observe AEI at rs2615977.ConclusionsCOL11A1 is subject to AEI in OA cartilage. AEI at rs1676486 is a risk factor for LDH, but not for OA. These two diseases therefore share a common functional phenotype, namely AEI of COL11A1, but this appears to be a disease risk only in LDH. Other functional effects on COL11A1 presumably account for the OA susceptibility that maps to this gene.

Spontaneous osteoarthritis in Str/ort mice is unlikely due to greater vulnerability to mechanical trauma

Relative contributions of genetic and mechanical factors to osteoarthritis (OA) remain ill-defined. We have used a joint loading model found to produce focal articular cartilage (AC) lesions, to address whether genetic susceptibility to OA in Str/ort mice is related to AC vulnerability to mechanical trauma and whether joint loading influences spontaneous OA development. We also develop finite element (FE) models to examine whether AC thickness may explain any differential vulnerability to load-induced lesions. Right knees of 8-week-old Str/ort mice were loaded, AC integrity scored and thickness compared to CBA mice. Mechanical forces engendered in this model and the impact of AC thickness were simulated in C57Bl/6 mice using quasi-static FE modelling. Unlike joints in non-OA prone CBA mice, Str/ort knees did not exhibit lateral femur (LF) lesions in response to applied loading; but exhibited thicker AC. FE modeling showed increased contact pressure and shear on the lateral femoral surface in loaded joints, and these diminished in joints containing thicker AC. Histological analysis of natural lesions in the tibia of Str/ort joints revealed that applied loading increased OA severity, proteoglycan loss and collagen type II degradation. Genetic OA susceptibility in Str/ort mice is not apparently related to greater AC vulnerability to trauma, but joint loading modifies severity of natural OA lesions in the medial tibia. FE modelling suggests that thicker AC in Str/ort mice diminishes tissue stresses and protects against load-induced AC lesions in the LF but that this is unrelated to their genetic susceptibility to OA.

Association study of candidate genes for the progression of hand osteoarthritis

Although a few consistent osteoarthritis (OA) susceptibility genes have been identified, little is known on OA progression. Since OA progression is clinically the most relevant phenotype, we investigate the association between asporin (ASPN), bone morphogenetic protein 5 (BMP5) and growth differentiation factor 5 (GDF5) polymorphisms and progression of hand OA. Single-nucleotide polymorphisms (SNPs) ASPN rs13301537, BMP5 rs373444 and GDF5 rs143383 were genotyped in 251 hand OA patients from the Genetics osteoARthritis and Progression (GARP) study and 725 controls. In a case-control comparison we assessed the association between these SNPs and radiographic progression of hand OA over 6 years, which was based on change in osteophytes or joint space narrowing (JSN), above the smallest detectable change. SNPs with suggestive evidence for association were further analysed for their effect on progression over 2 years, and for the mean change in osteophytes and JSN. The minor allele of ASPN SNP rs13301537 was associated with hand OA progression over 6 years (odds ratio (OR) (95% CI) 1.49 (1.06-2.07); P=0.020). The mean change in osteophytes and JSN was higher in carriers of the minor allele compared to homozygous carriers of the common allele withmean difference of 0.73 (95% CI - 0.07-1.56; P=0.073) and 0.82 (95% CI 0.12-1.52; P=0.022), respectively. Anassociation with similar effect size was found between ASPN SNP rs13301537 and 2-year progression, and the mean change in osteophytes and JSN was significantly higher in homozygotes. ASPN is associated with hand OA progression. This gives insight in the pathogenesis of hand OA progression and identified a potential target for therapeutic approaches.

A SMAD3 gene polymorphism is related with osteoarthritis in a Northeast Chinese population

Variants in the SMAD family member 3 (SMAD3) have recently been reported to be associated with osteoarthritis (OA) in European populations. However, the results are contestable. To assess the role of such variants in SMAD3 in OA susceptibility in peripheral joints OA, we conducted a case-control study in a Northeast Chinese population. The SMAD3 SNP was genotyped in patients who had primary symptomatic OA with radiographic confirmation and clinical symptom and in controls, and the associations were examined. A total of 111 knee OA patients, 121 hand OA patients and 236 controls were genotyped. Statistically significant difference was detected in genotype and allele frequencies between OA and control groups in the population. There were significant association for knee OA OR = 3.68 (95 % CI 2.03-6.70; p < 0.001) and for hand OA OR = 3.60 (95 % CI 2.01, 6.44; p < 0.001). The association was also positive even after stratification by sex except for male population of knee OA. Our data indicated that genetic variation in the SMAD3 gene is involved in pathogenesis of both knee OA and hand OA in Northeast Chinese population, which is consistent with in European populations.

Synonymous SNP influences Adam12 mRNA expression level in synovial tissue

Synonymous SNP influences Adam12 mRNA expression level in synovial tissue

ADAMTS14 gene polymorphism associated with knee osteoarthritis in Thai women

Osteoarthritis (OA) is the most prevalent form of arthritis in the elderly. This disease is characterized by breakdown and loss of articular cartilage due to genetic, mechanical and environmental factors. Although the pathophysiology of OA is not completely known, several candidate genes have been reported to be associated with OA susceptibility. We assessed the association between genetic variation in the ADAMTS14 region and knee osteoarthritis susceptibility in the Thai population. The rs4747096 SNP was genotyped by PCR-RFLP on genomic DNA extracted from peripheral blood of 108 OA patients and 119 controls. The PCR product (196 bp) was digested with BspEI. A sample with the GG genotype showed two band sizes of 158 and 38 bp, while a sample with the AA genotype showed a single band size of 196 bp. Heterozygotes with the AG genotype showed all three corresponding bands. Genotype distributions, allele frequencies and model of inheritance in patients and controls were compared. In females, the frequency of the AA genotype and the A allele were significantly higher in knee OA patients than in controls [odds ratio (OR) = 2.79, 95% confidence interval (CI) = 1.05-7.59 and OR = 1.58, 95%CI = 1.00-2.45, respectively]. Moreover, genotypic AA and AG were associated with significantly increased risk for knee OA when compared to GG (OR = 2.72, 95%CI = 1.10-6.87). No significant associations were observed in males. In conclusion, the nsSNP rs4747096 in ADAMTS14 was associated with knee OA in female Thai patients; therefore, the role of ADAMTS14 in OA seems to be gender-dependent.

OPN gene polymorphisms influence the risk of knee OA and OPN levels in synovial fluid in a Chinese population

IntroductionA body of studies suggests the role of osteopontin (OPN) in onset and development of osteoarthritis (OA), however, the association between OPN polymorphisms and OA susceptibility as well as its clinical features has not been reported.MethodsA total of 750 patients with primary knee OA and 794 healthy volunteer were enrolled as controls. Both OA and control groups were interviewed to obtain demographic and clinical data. Three polymorphisms of OPN gene, namely, -156GG/G, -443C/T and -66T/G were determined. The levels of the full length and the thrombin-cleaved OPN in synovial fluid (SF) from OA subjects were measured.ResultsWe found the polymorphisms of the -443C/T and the -66/T/G were significantly associated with the OA risk and the radiographic severity. The -443TT and -66GG showed protective effect against developing OA and were associated with lower Kellgren-Lawrence grade. Besides, the polymorphisms of -443C/T and -66T/G significantly affected the thrombin-cleaved OPN levels in SF from OA subjects. Subjects with -443TT and -66GG genotypes had lower thrombin-cleaved OPN levels in SF. The thrombin-cleaved OPN levels in SF were positively correlated to the radiographic severity of OA.ConclusionsOur findings suggest that certain OPN gene polymorphisms may be used as molecular markers for the susceptibility and severity of OA.

The genetics and functional analysis of primary osteoarthritis susceptibility

Recent genome-wide association scans (GWASs) along with several adequately powered candidate gene studies have yielded a number of risk alleles for osteoarthritis (OA). This number is now sufficiently large to allow conclusions to be drawn regarding the nature of genetic susceptibility, including the fact that the risk alleles have variable effects depending on sex, ethnicity and on the skeletal site of the disease. Several of the alleles that have emerged from the GWASs are within or close to highly plausible candidate genes, including RUNX2 and CHST11. However, the majority of risk alleles do not map to genes previously reported to play a role in musculoskeletal biology, indicating that the GWAS datasets are telling us something new about the OA disease process. Functional studies have so far revealed that effects on gene expression are likely to be one of the main mechanisms through which OA susceptibility is acting. Epigenetic mechanisms such as DNA methylation also influence OA risk, and integration of genetic, transcriptomic and epigenetic data will allow us to use the genetic discoveries for informed development of new OA biological treatments.

Towards elucidating the role of SirT1 in osteoarthritis

Osteoarthritis (OA) is a degenerative joint disease particularly affecting the elderly population. Although several genetic features have been characterized as risk factors for OA susceptibility, a growing body of evidence indicates that epigenetic effectors may also modulate gene expression and thus contribute to OA pathology. One such epigenetic regulator of particular relevance to OA is Silent Information Regulator 2 type 1 (SirT1) which has been linked to aging and caloric intake, Consistently, SirT1 has been also connected with various age-associated diseases such as diabetes type II, Alzheimers and osteoporosis. Recent reports show that OA is linked to changes in SirT1 activity or levels within cartilage. In human chondrocytes, SirT1 plays a role in cartilage extracellular matrix (ECM) synthesis and promotes cell survival, even under proinflammatory stress. It appears that SirT1 fine tunes many cellular biochemical processes through its capacity to interact and modify various histone and non-histone proteins. Taken together these investigations demonstrate that SirT1 is involved in cartilage biology and could potentially serve as novel drug target in treating OA even at its premature stages, thereby possibly reversing mechanical-stress induced cartilage degeneration.

Association of a functional polymorphism in the promoter region of TLR‐3 with osteoarthritis: A two‐stage case–control study

Recent studies have suggested that polymorphisms in toll-like receptor 9 (TLR-9), an endosomal TLR, are associated with knee osteoarthritis (OA). TLR-3, -7, and -8 are also found on the surface of endosomes and to investigate whether similar associations exist with polymorphisms in these TLR genes we performed a two-stage case-control study and genotyped 11 TLR single nucleotide polymorphisms (SNPs) in 823 OA cases and 594 healthy controls by polymerase chain reaction restriction fragment length polymorphism assays. Real-time PCR was performed to assess the functional expression of an identified promoter polymorphism in TLR-3 following dexamethasone stimulation of articular chondrocytes. An association between TLR-3 SNPs at rs3775296 and rs3775290 and OA was identified in both populations. In males the allelic frequencies of TLR-7 rs179010 and TLR-8 rs5744080 were significantly different between OA cases and healthy controls. The ATCA, CTCA, and CCTA haplotypes of TLR-3 were associated with OA susceptibility. A significant difference in TLR-3 gene expression following dexamethasone treatment was seen among the various genotypes of rs3775296 (p = 0.004). Our findings indicate that a SNP in the promoter region of TLR-3 is associated with elevated TLR-3 gene expression and susceptibility to knee OA in a Chinese Han population.

New tools for studying osteoarthritis genetics in zebrafish

SummaryObjectiveIncreasing evidence points to a strong genetic component to osteoarthritis (OA) and that certain changes that occur in osteoarthritic cartilage recapitulate the developmental process of endochondral ossification. As zebrafish are a well validated model for genetic studies and developmental biology, our objective was to establish the spatiotemporal expression pattern of a number of OA susceptibility genes in the larval zebrafish providing a platform for functional studies into the role of these genes in OA.DesignWe identified the zebrafish homologues for Mcf2l, Gdf5, PthrP/Pthlh, Col9a2, and Col10a1 from the Ensembl genome browser. Labelled probes were generated for these genes and in situ hybridisations were performed on wild type zebrafish larvae. In addition, we generated transgenic reporter lines by modification of bacterial artificial chromosomes (BACs) containing full length promoters for col2a1 and col10a1.ResultsFor the first time, we show the spatiotemporal expression pattern of Mcf2l. Furthermore, we show that all six putative OA genes are dynamically expressed during zebrafish larval development, and that all are expressed in the developing skeletal system. Furthermore, we demonstrate that the transgenic reporters we have generated for col2a1 and col10a1 can be used to visualise chondrocyte hypertrophy in vivo.ConclusionIn this study we describe the expression pattern of six OA susceptibility genes in zebrafish larvae and the generation of two new transgenic lines marking chondrocytes at different stages of maturation. Moreover, the tools used demonstrate the utility of the zebrafish model for functional studies on genes identified as playing a role in OA.

Association of ADAMTS5 gene polymorphisms with osteoarthritis in Chinese Han population: a community-based case–control study

Aggrecanase-2 (ADAMTS5) is reported to play essential roles in the pathophysiology of osteoarthritis (OA). To explore the relationship between ADAMTS5 gene polymorphisms and primary OA, we conducted a community-based case-control study. A total of 732 community residents aged 40-84 years participated in the community-based study in Northeast China. After taking physical examination and radiographic examination, 420 persons of the residents were diagnosed OA (216 women and 204 men). The other 312 individuals without any symptoms of osteoarthritis or signs in the radiographs (156 women and 156 men) were considered as healthy controls. After obtaining the DNA of patients and control group, genotypes of the ADAMTS5 gene polymorphisms were determined by polymerase chain reaction (PCR) followed by restriction enzyme digestion (HAEIII for P692L in exon 7 and BSRBI for R614H in exon 5). The numbers of patients with different OA subtypes were also calculated. The genotype and allele frequency of for the exon 5C/T BSRBI polymorphism was significantly different between OA patients and control individuals (P = 0.001, OR = 0.701, 95% CI = 0.569-0.863). This difference was more obvious in cervical OA patients (P = 0.001, OR = 0.664, 95% CI = 0.521-0.847). The mutation type of exon 5C/T BSRBI polymorphism would be a protective factor for OA especially for cervical OA. Our results suggest that the ADAMTS5 gene polymorphisms may contribute to the susceptibility of osteoarthritis in the Chinese Han population.

A rare variant in the osteoarthritis-associated locus GDF5 is functional and reveals a site that can be manipulated to modulate GDF5 expression

Osteoarthritis (OA) is a polygenic disease characterized by cartilage loss, with the single-nucleotide polymorphism (SNP) rs143383 (C/T) influencing OA susceptibility across a range of ethnic groups. The SNP resides within the 5′-UTR of the growth and differentiation factor 5 gene (GDF5), with the OA-associated T-allele mediating reduced GDF5 expression. As GDF5 codes for a cartilage anabolic protein, this reduced expression may explain why the T-allele of rs143383 is an OA risk factor. Our deep sequencing of GDF5 identified a C/A transversion located −41 bp relative to the gene's transcription start site. This promoter variant is predicted to affect transcription factor binding and it may therefore highlight a regulatory site that could be exploited to manipulate GDF5 expression and alleviate the detrimental effect mediated by the T-allele of rs143383. Here, we describe our functional assessment of the −41 bp variant. Using reporter constructs we demonstrated that the transversion leads to increased gene expression to such a degree that the A-allele is able to compensate for the reduced expression mediated by the T-allele of rs143383. Using electrophoretic mobility shift assays we identified YY1 as a trans-acting factor that differentially binds to the alleles of the −41 bp variant, with more avid binding to allele A. Knockdown of YY1 led to a significant reduction in GDF5 expression, supporting YY1 as a GDF5 activator. In conclusion, we demonstrated that the −41 bp variant is functional and we have identified a regulatory region of GDF5 that can be exploited to overcome the OA genetic deficit mediated by the T-allele of rs143383.

Gene expression analysis reveals HBP1 as a key target for the osteoarthritis susceptibility locus that maps to chromosome 7q22

ObjectivesAn osteoarthritis (OA) susceptibility locus has been mapped to chromosome 7q22, to a region of high-linkage disequilibrium encompassing six genes: PRKAR2B, HBP1, COG5, GPR22, DUS4L and BCAP29. The authors assessed...

Inhibition of asporin signaling is critical in the prevention of cartilage damage by physiotherapies

Purpose. Physiotherapies are the most frequently recommended non-invasive treatment options recommended for arthritic diseases. Here, we examined the signaling pathways responsible for the beneficial effects of exercise on various stages of monoiodoacetate-induced arthritis (MIA). We demonstrate that both success and failure of the physiotherapy is mediated via discrete signaling pathways involving Asporin, a susceptibility gene in osteoarthritis (OA). Asporin-mediated regulation of matrix synthesis in turn may regulate Transforming Growth Factor-β (TGF-β) networks essential for cartilage matrix synthesis. Methods. Treadmill walking (TW), used as a means of exposing MIA afflicted knees to physiotherapy, was initiated in rats at different stages of MIA induced cartilage damage and compared to unexercised MIA afflicted knees. TW started at 1 day post-MIA induction, or after cartilage damage had progressed to Grade I or Grade II. The cartilage specimens were analyzed by macroscopic, microscopic, μCT imaging, transcriptome-wide gene expression analysis and activation/suppression of signaling networks. Data was further analyzed by Ingenuity Pathways Analysis to construct molecular functional networks and signaling pathways to dissect pathways regulated by exercise. One-way ANOVA and a post hoc Tukey test were used for statistical analysis. Results. Treadmill walking immediately following initiation of MIA (1 day post-MIA induction) demonstrated a significant prevention of MIA progression. However, the efficacy of this intervention was significantly reduced when implemented on knees showing close to Grade I or greater cartilage damage. On the contrary, TW accelerated damage in the knees with close to Grade II cartilage pathologies. Transcriptome-wide gene expression analysis revealed that exercise intervention started 1-day post-MIA inception significantly suppressed signaling networks that inhibit matrix synthesis. In parallel, TW upregulated gene networks associated with matrix synthesis. However, TW intervention following Grade I damage was less effective in preventing cartilage damage and regulating matrix synthesis. Interestingly, Asporin and networks associated with TGF-β expression and signaling were the major gene products that were regulated by TW to control matrix synthesis (Aggrecan, Collagen type II, Matrilin, FRZB, Col 9A2 and Col9A3) and prevent cartilage damage as evident by microscopic grading of cartilage. Conclusions. The findings demonstrate that Asporin might be one of the critical genes controlled by TW, that in turn controls expression of TGF-β family of molecules, essential for cartilage matrix synthesis. The findings underscore the importance of physiotherapies by showing suppression of Asporin, an OA susceptibility gene by TW. Nevertheless, the extent of cartilage damage at the initiation of physiotherapy is an important determinant for the effectiveness of TW.

CpG sites of osteoarthritis susceptibility gene DIO2 are differentially methylated in arthritic compared to preserved cartilage

CpG sites of osteoarthritis susceptibility gene DIO2 are differentially methylated in arthritic compared to preserved cartilage

Allelic expression analysis of the osteoarthritis susceptibility locus that MAPS to MICAL3

Background: A genome-wide association scan with subsequent replication study that involved over 67,000 individuals of European ancestry has produced evidence of association of single nucleotide polymorphism rs2277831 to primary osteoarthritis (OA) with a P-value of 2.9 × 10 -5 . rs2277831, an A/G transition, is located in an intron of MICAL3. This gene is located on chromosome 22q11.21 and the association signal encompasses two additional genes, BCL2L13 and BID. It is becoming increasingly apparent that many common complex traits are mediated by cis-acting regulatory polymorphisms that influence, in a tissue-specific manner, gene expression or transcript stability. Methods: We used total and allelic expression analysis to assess whether the OA association to rs2277831 is mediated by an influence on MICAL3, BCL2L13 or BID expression. Using RNA extracted from joint tissues of 60 patients who had undergone elective joint replacement surgery, we assessed whether rs2277831 correlated with allelic expression of either of the three genes by: 1) measuring the expression of each gene by quantitative PCR and then stratifying the data by genotype at rs2277831 and 2) accurately discriminating and quantifying the mRNA synthesised from the alleles of OA patients using allelic-quantitative PCR. Results: We found no evidence for a correlation between gene expression and genotype at rs2277831, with Pvalues of 0.09 for BCL2L13, 0.07 for BID and 0.33 for MICAL3. In the allelic expression analysis we observed several examples of significant (p < 0.05) allelic imbalances, with an allelic expression ratio of 2.82 observed in BCL2L13 (P = 0.004), 2.09 at BID (P = 0.001) and the most extreme case being at MICAL3, with an allelic expression ratio of 5.47 (P = 0.001). However, there was no correlation observed between the pattern of allelic expression and the genotype at rs2277831. Conclusions: In the tissues that we have studied, our data do not support our hypothesis that the association between rs2277831 and OA is due to the effect this SNP has on MICAL3, BCL2L13 or BID gene expression. Instead, our data point towards other functional effects accounting for the OA associated signal.