A genome-wide association study of copy-number variation identifies putative loci associated with osteoarthritis in Koreans

Osteoarthritis (OA), which is also known as degenerative arthritis, is a disease of mechanical dysfunction involving degradation of joint components, especially articular cartilage and subchondral bone [1]. OA is the most common form of arthritis that affects elderly people and often leads to physical disability and reduced quality of life. OA is a leading cause of impaired mobility in the elderly [2]. The clinical course of OA varies from minimal joint pain, stiffness, and limitations in function to complete disability.

Development of OA is the consequence of interaction between external stimuli, such as mechanical loading and the structure and physiology of the joint [3]. Risk factors of OA include obesity, knee injury, previous knee surgery, and occupational bending and lifting [4]. Symptoms may include joint pain, tenderness, stiffness, locking, and sometimes an effusion from the affected joint. Various causes including genetic predisposition, developmental, metabolic, and mechanical deficits can initiate inflammatory processes that lead to loss of cartilage and clinically overt OA. Not only mechanical and inflammatory stimuli to joint but also genetic susceptibility to such stimuli contribute to the pathogenesis of OA.

Recently, a number of genetic association studies for OA have been performed using candidate gene approaches or genome-wide association studies (GWASs). Several genes have been identified as associated with OA. GDF5 [5,6], DUS4L [7], 7q22 [8], MCF2L [9] and GNL3 [10] meet the strict criteria of genome-wide significance level with a P-value of < 5 ×10^-8. In addition to these genes, several candidate single-nucleotide polymorphisms (SNPs) in FRZB, DIO2, and SMAD2 are regarded as potentially associated genes [11-13]. However, the considerable number of SNPs in OA most likely reflects the polygenic nature of the disease with modest effect sizes for individual genes [14] and may be related to the somewhat inconclusive findings of these studies. Hence, novel approaches beyond GWASs are warrant.

Copy number variations (CNVs) account for a major proportion of human genetic polymorphisms and have been predicted to play an important role in genetic susceptibility to common diseases [15]. CNVs are one type of promising structural variation beyond SNPs, which affect only a single nucleotide. CNVs may range from ~1 kilobase to several megabases in size and can result in cellular dysfunction. CNVs associated with OA have not been elucidated.

The purpose of this study is to identify candidate CNVs associated with OA. To examine the contribution of a CNV region to OA, we carried out a genome-wide association analysis between selected CNV regions and OA. This study revealed potential genetic variants that had not been previously reported from GWASs.

In this study we conducted a genome-wide association study of CNVs in OA to identify genomic regions that confer susceptibility and/or protection. The genome-wide significance level following Bonferroni correction for the 1123 CNV regions in this study was 4.5 × 10^-5. Because not every variant that has a functional impact always satisfies a genome-wide significant level and because relatively small sample size can undermine the reliability of true variants, some variants with evidence obtained from genetic study are suggested to have functional effects, even though none of identified variants meet a genome-wide significance level. Thus, we investigated functional relationships between OA and CNV regions that reached nominal significance (P < 0.05). Consequently, we identified six OA genomic regions that confer susceptibility and/or protection. These regions included TNKS, CA10, POSTN, MAMDC2, KCND3, and ME3 which encompassed or were near CNV regions.

The canonical Wnt/β-catenin signaling pathway has been implicated in the pathogenesis of osteoarthritis [19]. Increased β-catenin accumulations have been observed in degenerative cartilage, suggesting that activated Wnt signaling might contribute to progression of osteoarthritis by downstream target genes such as matrix metalloproteinases (MMPs) [20]. For instance, the secreted Wnt antagonist Dickkopf-1 is related with slowed progression of hip OA in elderly women [21] and the small molecule XAV939, which selectively inhibits β-catenin-mediated transcription, is associated with protecting cartilage degradation in a rat osteoarthritis model [22]. Tankyrase, which is encoded by TNKS, bind directly to axin, a negative regulator of the canonical Wnt/β-catenin signaling pathway, forming a destruction complex with glycogen synthase kinase 3β (GSK-3β) and adenomatous polyposis coli (APC) to degrade β-catenin [23]. Inhibition of TNKS by treating the compound XAV939 or small interfering mediated silencing of TNKS can cause an increase in levels of axin, leading to phosphorylation and degradation of β-catenin, and inhibition of target gene transcription by Wnt signaling. Thus, although further studies are needed to establish the role of TNKS in osteoarthritis through axin-mediated inhibition of Wnt signaling, it is quite possible that TNKS could be risk factor for osteoarthritis.

Figure 1 shows the chromosomal position of the copy number deletion of third intron of TNKS. Interestingly, several transcription factor binding sites as determined by ENCODE of UCSC genome browser (http://​genome.​ucsc.​edu/​ENCODE) overlapped with this CNV region (Figure 1B). We speculated that copy number deletion of this region may directly or indirectly influence the binding of transcription factors, possibly including JunD, which belongs to the Jun family of activator protein 1 transcriptional regulators. A recent study reported that JunD-deficient immortalized fibroblast exhibit increased proliferation [24]. JunD also regulates interleukin-6 and matrix metalloproteinase-9 transcription in hepatic stellate cells, indicating that JunD may be a key profibrogenic regulator [25]. Copy number deletion of TNKS was associated with a 1.37-fold decreased risk for OA (Table 2).

CA10 gene encodes a protein that belongs to the carbonic anhydrase family of zinc metalloenzymes, which catalyze the reversible hydration of carbon dioxide [26]. The protein encoded by CA10 is an acatalytic member of the alpha-carbonic anhydrase subgroup, which includes major players in many physiological processes including renal and male reproductive tract acidification and bone resorption. Carbonic anhydrases are thought to be involved in bone mineral solubilization and digestion of organic bone matrix by acid proteases. The rapid conversion of carbon dioxide to bicarbonate and protons occurs while maintaining a normal intracellular pH in osteoclasts [27]. The use of an inhibitor of carbonic anhydrase is associated with a bone-sparing effect, as shown by measurement of spinal bone mineral density [28]. Moreover, the intronic variants, rs2106329 in CA10 shows strong association with osteoporosis in Japanese women [29]. A homozygous A allele at rs2106329 is a protective factor for osteoporosis. In our study, copy number deletion on CA10 was associated with a 1.69-fold decrease risk for hand OA as compared with a normal copy number (Table 3). Copy number deletion can also be a protective factor for OA.

POSTN encodes periostin, an osteoblast-specific factor that functions as a ligand for αV/β3 and αV/β5 integrins to support adhesion and migration of epithelial cells [30]. Periostin is a matricellular glutamate-containing protein that is expressed in connective tissues including bone, periosteum, and ligament [31]. Periostin is believed to function in the regulation of bone formation and, with its preferential localization in the periosteum, may play a role in fracture repair during the early process of bone regeneration [31]. In response to mechanical stress, periostin expression is up-regulated and collagen fibrillogenesis and matrix organization occur to preserve tissue integrity and function [32]. Periostin knock-out mice show altered cortical bone microarchitecture and lower bone mineral density [33]. Periostin appears to influence the properties of bone materials and damage accumulation and repair, including local modeling/remodeling processes in response to fatigue [34]. Periostin deficiency may influence the propensity for fatigue fractures [34], and may accelerate the pathogenesis of OA in cooperation with an imbalance in bone repair mechanisms. Similar to previously reported study, in our study, copy number deletion downstream of POSTN was associated with an increase in the risk for hand and/or knee OA compared with the normal copy number. However, we did not find a relationship between this CNV region and POSTN.

Also, we could not find a biological role of MAMDC2, KCND3, and ME3 in the pathogenesis of OA.

The present study has some limitations. First, diagnosis of OA was radiologically based (i.e., using X-ray) without assessment of symptoms. Different methods of assessment such as self-reported and symptomatic OA could not be available in population-based study samples. This limitation has resulted in small sample size of discovery stage and a difficulty of collecting samples for replication analysis. Second, because of first limitation which was mentioned above, we did not success to find radiographic case samples in independent general population samples. Eventually, we did not conduct a replication study, which is highly desirable with association studies. This limitation can be solved through a meta-analysis with other data in the future. To reduce heterogeneity of meta-analysis, we provide our cohort information such as exact phenotype definition, cut-off, and number of cases of each OA type. Third, knee and hand OA were analyzed together. OA of the knee can be a component of a generalized diathesis, including OA of the hand, which may be inherited. The OA phenotype is heterogeneous and occurs at specific joint locations, in a more generalized form, or with and without clinical presentation. Each phenotype has its own heritability estimate [35]. Fourth, the sample size was relatively small, and this reduced the power of the analysis. Fifth, six novel CNVs were located not in exons but in introns or were intergenic. Despite these limitations, our study has the following strengths. First, the present study describes the first CNVs to be reported regarding OA using high-resolution chip. Second, we were able to define precise disease phenotype using X-rays of all the participants.

We identified several previously unreported CNV loci that predisposed to or provided protection from OA. Identification of the associated variants at these genomic loci and their potential functional consequences will lead to better understanding of the pathogenesis of OA. Although this study has some limitations, further functional investigations of these genes are warranted to fully characterize the association.