Polymorphic variation of hypoxia inducible factor-1 A (HIF1A) gene might contribute to the development of knee osteoarthritis: a pilot study

Osteoarthritis (OA) is one of the most common rheumatic disorders; it is characterized by joint disease whose cause is not completely understood. OA has a complex pathogenesis due to the interaction of genetic, mechanical, biochemical, metabolic, endocrine, and environmental factors that result in cartilage breakdown and diverse changes in subchondral bone, which may lead to chronic pain, joint swelling, deformity and whole joint abnormalities resulting in disability [ 1– 3]. This process entails the formation of bony outgrowths (osteophytes) and inflammation of the synovial membrane, thus triggering catabolic and proinflammatory mediators such as cytokines, prostaglandin E2, neuropeptides and nitric oxide [ 4– 7]. Worldwide estimates indicate that 9.6 % of men and 18 % of women ≥ 60 years have symptomatic OA [ 8– 11].

Through inflammatory mediators, biochemical injury and oxidative stress, viability of chondrocytes is compromised. Increased oxidative stress in articular cartilage has been the target of molecular research and therapeutic intervention [ 12– 14]. Healthy articular cartilage is typically an avascular tissue, the oxygen tension ranges from 0.5 to 10 % [ 12]. Therefore, in order to maintain oxygen homeostasis in chondrocytes and cartilage, hypoxia-inducible factor-1 alpha (HIF-1α) is necessary to survive extremely low oxygen tensions. [ 12, 15, 16]. Furthermore, this transcriptional factor also has a role in the regulation of glucose transport, anaerobic energy generation and matrix synthesis by articular chondrocytes. The active site of this protein is an oxygen-dependent degradation domain (ODDD) that functions as an oxygen sensor [ 17, 18].

In the presence of normal oxygen tension levels, HIF-1α is hydroxylated in a proline residue within ODDD; it subsequently binds to ubiquitin and is degraded in the proteasome [ 17– 19]. However, under hypoxic conditions, hydroxylation is inhibited and HIF-1α accumulates in the cytoplasm, is phosphorylated and translocates to the nucleus in order to activate transcription of its target genes [ 18– 22]. Some of these target genes include nitric oxide synthase 2 ( NOS2), vascular endothelial growth factor ( VEGF), erythropoietin ( EPO), some glucose transporters ( GLUT1, GLUT3), Insulin-like growth factor type 2 ( IGF2), which potentially acts in order to maintain the chondroprotective functions challenged by the detrimental conditions occurring in the OA joint environment [ 16, 20– 25]. This relationship among different genes renders the close relation of HIF-1α with several pathologies [ 26– 31].

It have been described polymorphic variants within the HIF1A gene; for example, the rs11549465 (P582S), and rs11549467(A588T) single-nucleotide polymorphisms (SNPs) are located in exon 12, on 14 ^th chromosome and influence the transcriptional activity of this gene to increase dramatically in comparison with the common isoform [ 32, 33].

Due to loss of articular cartilage and with the intention to restore damaged tissue, WNT inducible signaling pathway protein-1 (WISP-1) participates in the development of subchondral bone. This protein plays an important role in the formation, differentiation, growth, and maintenance of bone and articular cartilage. Active Wnt signaling contributes to osteophyte formation and might play an essential role in the anabolic pattern of joint remodeling observed in ankylosing spondylitis and OA [ 34]. Furthermore, it has also been showed that in both OA patients and experimental models that there is an important increase in the Wnt signaling pathway and over expression of the WISP1 gene [ 35– 38]. Likewise, it was discovered that variant rs2929970 (A2364G) of the WISP1 gene (located within of the 8 ^th chromosome) is related with the development of spinal OA in postmenopausal women [ 39].

Because of the genetic variability of OA, the aim of this study was to analyze rs11549465, rs11549467 and rs2057482 SNPs of the HIF1A gene, and rs2929970 SNP of the WISP1 gene in Mexican patients with severe knee OA.

The demographical variables age, gender, weight, BMI, glucose, total cholesterol, and uric acid of our study populations are depicted in Table  2. Cases were older than controls ( P < 0.00001, 55.8 ± 6.7 vs. 47.5 ± 6.6, respectively). The mean BMI of the OA group (29.8 ± 5.1) was significantly higher than the control group (26.9 ± 4.6) ( P = 0.0007), similar with the previous studies that reported high BMI increased the risk of developing OA. None of the individuals belonging to the control group had a BMI characteristic of obesity.

The HWE demonstrated no differences in the distribution of all polymorphisms studied in both groups (Table  3). Table  4 shows the gene and allele frequencies of the polymorphisms studied in patients in comparison with the control group. Only rs11549465 polymorphism of the HIF1A gene was found to be significantly associated with increased risk of OA. The OA group exhibited an increased frequency of the CC genotype and the C allele in comparison with the control group ( P = 0.003, 94.2 vs. 74.2 %, OR = 5.7, 95 % CI = 1.7–21.6; and P = 0.004, 97.0 vs. 87.1 %, OR = 5.03, 95 % CI = 1.53–18.2, respectively). For the same polymorphism, CT genotype and the T allele frequencies were higher in the control group in comparison with the knee OA group ( P = 0.003, 25.8 vs. 5.8 %, OR = 0.2, 95 % CI = 0.05–0.60; and P = 0.004, 12.9 vs. 3.0 %, OR = 0.2, 95 % CI = 0.05–0.65, respectively). Distribution of rs11549467 and rs2057482 polymorphisms of the HIF1A gene and the rs2929970 polymorphism of the WISP1 gene in both study groups were statistically nonsignificant.

After adjustment of age, gender, weight, BMI, glucose, total cholesterol, and uric acid, no statistically significant associations with OA were found. Additionally, we investigate the relationship between the radiological grade of OA and the rs11549465 polymorphism showing that the grade 2 maintains a relationship with the polymorphism ( CC genotype: P = 0.009, OR = 5.0; C allele: P = 0.01, OR = 4.4; CT genotype: P = 0.009, OR = 0.2; T allele: P = 0.01, OR = 0.2); whereas the grade 4 did not showed any relationship (Additional file 1).

As is the case of other ethnic groups, OA is the most common human arthritis in Mexicans [ 10], its incidence is increasing due to the current pandemic of overweight and obesity in this ethnic group. Several risk factors have been described for the development, progression and severity of OA; however, at present it is not possible to explain interindividual variability in destruction of joint cartilage. We studied Mexican patients with knee OA, some SNP in HIF1A and WISP1 genes related with oxygen homeostasis in joint cartilage and bone development, and their relationship with biochemical parameters.

Our results show that the BMI in the patients with OA was higher with respect the healthy control group. This is in line with previous studies that reported how a higher BMI is a risk factor for developing knee OA, probably induced by the biomechanical joint stress [ 41].

Interestingly, our results showed a significant decreased level of uric acid in the OA patients with respect the healthy control. This is in line with the study of Mishra et al. [ 42], who attributed this phenomenon to the antioxidant properties of uric acid since they found an inverse correlation between uric acid and the presence of malondialdehyde (pro-oxidant agent). This induces to consider a possible link between the reduction of both the content and the antioxidant property of uric acid and the inflammatory process of OA.

Regarding the analysis of polymorphic variants of HIF1A, we found that the rs11549465 SNP located in the exon 12 within the HIF1A gene was associated in Mexican patients with knee OA. Our results show that the presence of the CC homozygous variant or C allele represent potential risk factors for development of knee OA; contrarily, we detected that the heterozygous variant of CT or T allele of the rs11549465 polymorphism of the HIF1A gene (in comparison with the homozygous carriers) play a protector role against the disease. This phenomenon may be explained by the fact that the presence of this polymorphism confers greater stability to the HIF-1α protein, as demonstrated by Tanimoto et al. [ 22]. In their study it has been demonstrated that the substitution of proline by serine in the 582 (P582S) position enhances its transcriptional activity due to an alteration in the characteristics and properties of the binding sites with the target genes [ 43]. This permit supposes a beneficial effect in maintaining cartilage homeostasis and therefore avoiding joint damage. A similar phenomenon was also obtained in other immunological disease such as type 2 diabetes mellitus [ 23].

Additionally, endochondral ossification process is triggered by the development of osteophytes. WISP-1 is an osteogenic factor and an osteoblast differentiation factor that has been studied in OA models, in which an increase in its expression was reported [ 32, 33]. WISP-1 is a member of the CCN family of connective tissue growth factors, which also includes WISP-2 and WISP-3. Members of the CCN family have been implicated in developmental processes such as chondrogenesis, osteogenesis, and angiogenesis. French et al. [ 44] could show that WISP-1 functions as osteogenesis potentiating factor promoting mesenchymal cell proliferation while repressing chondrocytic differentiation in vitro. This would suggest that the CCN family play a critical role in cartilage homeostasis. Urano et al. [ 39] analyzed the rs2929970 (A2364G) 3′UTR polymorphism in Japanese women with vertebral OA, demonstrating an increase in the GG genotype in the sub-group with end-plate sclerosis. This polymorphism is located in the 3′ untranslated region, within a region of splicing variation, and it may influence messenger RNA stability, translational efficiency or gene expression, and subsequently contribute to the risk of OA. Under the same context, and based on the frequency of polymorphism in our population, we decided use the same variant of the WISP1 gene in Mexican patients with knee OA, but in our study we did not find any association. Whatsoever, it is possible that the evaluated polymorphism, do not play a major role in the development of knee OA in our population, so interesting it would be to explore other polymorphic sites within the WISP1 gene. It will be important to replicate this study in distinct ethnic groups to better understand the mechanisms whereby gene interacts with environmental factors to develop OA.