Variations of Wnt/β‐catenin pathway‐related genes in susceptibility to knee osteoarthritis: A three‐centre case‐control study

Abstract Excessive activation of the Wnt signalling pathway in the articular cartilage is demonstrated to be related to the onset and severity of osteoarthritis (OA). However, few studies have investigated the association between variants in Wnt‐pathway‐related genes and the risk of OA by searching Pubmed and EMBASE. Totally, 471 knee OA patients and 532 controls were recruited from three hospitals to evaluate the associations of five genetic variants (rs61735963, rs2908004, rs10795550, rs1799986 and rs1127379) with the risk of knee OA. These polymorphisms were genotyped through polymerase chain reaction and Sanger sequencing. Genetic risk scores (GRSs) were calculated to evaluate the combined effect of these genetic variants. No significant association was found between OA risk and polymorphisms (rs61735963, rs10795550 or rs1127379). However, WNT16 rs2908004 polymorphism was correlated with a decreased risk of OA, especially among females, smokers, non‐drinkers and individuals with age < 60 years or BMI ≥ 25. This SNP was also associated with Kellgren‐Lawrence grade and CRP. Similarly, LRP1 rs1799986 polymorphism decreased the risk of OA among males, smokers, drinkers and individuals with age < 60 years or BMI ≥ 25. TT genotype was more frequent in the group of VAS ≥ 6 versus VAS < 6. A low GRS was positively correlated with a decreased risk of OA. In addition, rs2908004 or rs1799986 polymorphism reduces the expression of WNT16 or LRP1. In conclusion, two SNPs (rs2908004 and rs1799986) are associated with the decreased risk of OA by regulating the Wnt pathway.


| INTRODUC TI ON
Osteoarthritis (OA) is the most prevalent chronic joint disease that contributes to cartilage matrix degradation. 1 Major mechanism of OA is characterized by abnormal joint tissue metabolism and results in cartilage degradation, bone remodelling, joint inflammation, osteophyte formation and loss of normal joint function. 2 About 9.6% of men and 18% of women ≥ 60 years old suffer from symptomatic OA worldwide. 3 Multiple factors, including advanced age, excessive body weight, repeated trauma or surgery to the joint structures, diabetes and hormone-related disorders, contribute to increased risk of OA. 4 Studies provide clear evidence of a heritable component in susceptibility of OA, 2,5,6 but the specific genetic factors are largely unknown. It therefore remains a challenge to identify risk alleles or candidate genes that contribute to OA pathogenesis.
The Wnt/β-catenin signalling pathway plays a crucial role in the regulation, proliferation, differentiation and cellular death processes in numerous anomalies of development, growth and homeostasis in animal organisms. 7 We previously found the Wnt/β-catenin pathway plays a key role in OA by inducing the expression of matrix metalloproteinases. 8 Besides, it contributes to cell differentiation and apoptosis and directly regulates chondrocyte phenotype. 9,10 Once Wnt protein is secreted, it binds to the frizzled receptor and low-density lipoprotein receptor-related protein 5/6 (LRP5/6) in order to stabilize the β-catenin protein and initiate an intricate signalling cascade. 11 Abnormal activation of the Wnt/β-catenin signalling is involved in the OA pathology, but little is known about the association of the Wnt pathway-related gene polymorphisms with OA susceptibility. Thus, this hospital-based case-control study was conducted to investigate the effects of Wnt pathway-related genes (DVL1, WNT16, ITIH5, LRP1 and SFRP1) polymorphisms on the risk of OA in a Chinese population.

| Search strategy
We systematically searched the Pubmed and EMBASE databases up to 12 January 2019 to identify eligible studies using the following keywords: 'osteoarthritis', 'OA', 'wnt signalling pathway', 'Wnt/βcatenin signalling pathway', 'polymorphism', 'SNP' and 'variant'. We also checked references in eligible studies and reviews for other relevant studies. No restriction was set on language or ethnicity.

| Protein-protein interaction network and functional annotation
We constructed protein-protein interaction (PPI) network to identify significant interactions among proteins encoded by Wnt pathwayrelated genes using the search tool STRING (http://string-db.org/). 12 PPI network was constructed by the PPI pairs with protein interaction scores exceeding 0.4. Gene Ontology (GO) is a widely adopted source of gene functional annotation, including biological process, molecular function and cellular component. 13  and Kellgren-Lawrence grade) was collected from all participants by using a written questionnaire and reviewing medical records.

| Study population
Individuals who smoked at least one cigarette daily were considered as smokers. Alcohol usage was defined as consumption of at least three alcoholic drinks one week. This study was approved by the Ethics Committees of the above three hospitals and was consistent with the Declaration of Helsinki. Each subject provided written informed consent prior to participation.

| SNP selection process
Selection process was as follows. First, we identified five OA-related genes (DVL1, WNT16, ITIH5, LRP1 and SFRP1) on the Wnt signalling pathway by reviewing the literature. Second, we found out whether the SNPs on these genes have been studied in the disease. Third, SNPs with minor allele frequency > 0.5, located in the promoter, exon or 3'-UTR were selected. In total, five SNPs in Wnt signalling pathway-related genes were selected, including rs61735963, rs2908004, rs10795550, rs1799986 and rs1127379.

| Genotyping
Peripheral blood (2 mL) was collected from each subject. Genomic

| Reverse transcription-polymerase chain reaction
Total RNA was isolated from the whole blood from the OA patients and healthy controls using the Trizol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions.

| Statistical analysis
Continuous variables were expressed as mean ± standard deviation and analysed using Student's t test or Mann-Whitney U test.
Categorical variables were reported as frequency and percentage and analysed using chi-squared test.

| Bioinformatics analysis
We constructed the PPI network of the Wnt pathway genes associated with OA reported in the literature (Figure 1). Three genes (WNT16, LRP5 and LRP6) were considered as key genes in the PPI module. GO analysis indicated these genes were significantly related to cellular process (biological process), cell part (cellular component) and protein binding (molecular function) (Figure 2).

| Clinical parameters of the study population
The OA patients and controls were aged 57.87 and 57.47 years on average respectively (Table 3). About 54% of the participants in the two groups were females. The average BMIs did not differ significantly between groups (P = .632). The majority of OA patients were assigned with Kellgren-Lawrence grade III + IV. Clinical characteristics (eg ESR, CRP, VAS and Lequesnes' index) of patients were also collected to investigate the pathology of OA.

| Wnt pathway-related genes variant analyses
The genotype and allele distributions of five SNPs are shown in  (Table 5). For rs2908004, the decreased effect was more evident in the subgroups of females, age < 60 and BMI ≥ 25.
In addition, the decreased association between LRP1 rs1799986 polymorphism was stronger in the subgroups of males, smokers, non-drinkers, age < 60 and BMI ≥ 25. Subsequently, we investigated the possible association between the two above polymorphisms and clinical characteristics (CRP, ESR, VAS, Lequesnes' index and K-L grade) of OA patients (Table 6). GG + AA genotype carriers of rs2908004 polymorphism had lower CRP expression and K-L grade compared to the GG genotype. For rs1799986, the TT genotype was more frequent in the group of VAS ≥ 6 versus VAS < 6.
Weighted GRS revealed that individuals in the third/second versus the first quartile had a lower risk of developing OA (Table 7). This relationship remained true after adjusting for sex and age.
The mRNA expression of WNT16 or LRP1 was regulated by the genotypes of rs2908004 or rs1799986 polymorphism ( Figure 3).
Our results indicated that WNT16 and LRP1 were upregulated in OA compared to that in healthy controls. There existed a significant difference in the mRNA expression for rs2908004 or rs1799986 polymorphism (P < .05). suggested as susceptibility factors for OA development. 16 Some SNPs of Wnt-related genes influence the genetic predisposition to OA. Therefore, 13 studies changed their focus to the association of polymorphisms in pathway-related genes with risk of OA. Careful reviewing suggests these 13 articles involved 20 genes and 127 SNPs.

| D ISCUSS I ON
Twelve of the 127 SNPs were associated with OA among Caucasians.
However, no study uncovered any significant association between these SNPs and OA risk among Asians.
As reported, WNT16 supported the homeostasis of progenitor cells in OA, while WNT16-deficient mice developed more severe OA with lubricin downregulation and increased chondrocyte apoptosis. 17 The expression of inter-alpha inhibitor H5 (ITIH5) differed significantly between OA synovial tissues and healthy tissues. 18 Lipoprotein receptor-related protein 1 (LRP1) could inhibit tumour necrosis factor (TNF)-α-induced apoptosis and inflammation in chondrocytes. 19 The reduced expression of secreted frizzled related protein 1 (SFRP1) increased the activation of the Wnt/β-catenin signalling and rendered the articular cartilage prone to premature. 20 72 0.63 (0.42-0.94) bone mineral density (BMD), cortical bone thickness and osteoporotic fracture risk. 23 Radiographic OA was associated with high BMD and increased rate of bone loss. 24  distribution between cases and controls. LRP1 dictates physiological and pathological catabolism of aggrecan by regulating extracellular activity of ADAMTS5. 25 Inhibited shedding of low-density LRP1 reversed the cartilage matrix degradation in OA. 26 The role of LRP1 rs1799986 polymorphism was studied in various diseases, including cardiovascular disease 27 and Alzheimer's disease (AD). 22 LRP1 rs1799986 polymorphism was associated with lower risk of AD among Asians and could reduce risk of late onset of AD, as revealed by a meta-analysis containing 6455 AD cases and 6304 controls. 22 However, little is known about their association with OA susceptibility. We found TT genotype or T allele significantly increased risk of OA in a Chinese population. Furthermore, the mRNA expression of WNT16 or LRP1 in mutant genotype carriers was significantly lower than that of wild genotype carriers. We hypothesized that rs2908004 or rs1799986 polymorphism conferred susceptibility to OA by influencing its production and function.
Several limitations of this study should be addressed. First, other confounders (eg occupation and diet) were not considered. Second, we recruited cases and controls only from hospitals, which may lead to selection bias during the collection process. Third, we may obtain the falsenegative or false-positive results due to limited sample size. Fourth, two positive SNPs in exons may affect the protein expression, leading to OA susceptibility. However, this was not verified by experiments.
This study confirms that WNT16 rs2908004 polymorphism plays a protective role in the pathology of knee OA. Additionally, LRP1 rs1799986 polymorphism is associated with a decreased risk of OA. Nevertheless, larger-size and well-designed case-control studies among other populations are warranted to identify the association between these polymorphisms and knee OA susceptibility.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.