Polymorphism in rs2229783 of the Alpha 1(XI(2)

Following collection, the cartilage tissues were immediately ?xed in 4% (w/v) paraformaldehyde, washed in phosphate-buffered saline (PBS), decalcified, embedded in paraffin, and cut into 5-8-μm-thick slices for immunohistochemistry and hematoxylin and eosin (HE) staining. Immunochemical identification was performed using the streptavidin-peroxidase (SP) method. Briefly, after deparaffinization, endogenous peroxidase was blocked with 3% H2O2 for 15 min, after which the slides were washed with PBS. The slides were then predigested using a digestive complex followed by rinsing with PBS. After blocking using 10% normal goat serum, the sections were incubated with a primary antibody recognizing COL11A at a 1:100 dilution (polyclonal rabbit anti-COL11A, Bioss Co, Beijing, China) or with PBS as a negative control, at 4 °C overnight. Next, the sections were incubated with 1:200 biotinylated goat anti-rabbit IgG (ZSGB-Bio Co, Beijing, China) at 37 °C for 20 min, followed by incubation with horseradish peroxidase-labeled streptavidin solution at 37 °C for 15 min. Color development was continued for 5 min at room temperature using diaminobenzidine followed by rinsing with distilled water. Counterstaining was performed with hematoxylin. The positive cells were counted at 40× magnification in six randomly selected fields; an average of six fields was observed for each tissue. A comparison of the positive rate of COL11A-stained cells between the two groups showed that the percentage of stained cells in the KBD group was 18.53% ± 8.41%, significantly lower than that of 26.58% ± 11.47% in the controls (t = 2.637, P < 0.05).

The Hardy-Weinberg equilibrium (HWE) of each SNP was tested by the goodness-of-?t χ2 test to compare the expected and observed genotype frequencies in the control group; SNPs with P > 0.05 were considered to be in HWE. An unconditional logistic regression analysis model was used to evaluate the relationships between different genotypes and disease risk [odds ratios(OR), 95% confidence intervals (95% CI)] adjusted by age and gender[9]. To account for multiple testing, a Bonferroni correction was applied, and a significant association was defined at P < 0.002 (0.05/22)[9].

A case-control comparison of both genotype and allele frequencies for the 22 SNPs is presented in Table 1. All tested SNPs were in HWE (P = ). When the allele frequencies were compared between the KBD patients and controls, a significant χ2 value was detected for rs (P = 0.006). The distribution frequencies of rs genotypes was significantly different between the two groups (P = 0.0003). However, the significant association of allele frequency of rs did not persist following Bonferroni correction (P > 0.002).

Odds ratios and 95% CIs for KBD were calculated from an unconditional logistic regression model to evaluate relative risk (Table 2). Weakly increased KBD risks were observed among individuals with the homozygote mutant genotype AA at rs (OR = 1.96, P = 0.03) and CC at rs (OR = 1.84, P = 0.04), compared with the homozygous wild type GG (rs) and TT (rs). The two SNPs also showed an elevated KBD risk in the recessive model (OR = 2.13, 95% CI = and OR = 1.81, 95% CI = ), but the significance did not remain after Bonferroni correction. However, rs was associated with a decreased risk of KBD in the dominant model (OR = 0.49, 95% CI = , P = 0.0001), which remained significant after Bonferroni correction for multiple testing. These results suggest that polymorphism in COL11A1 is associated with susceptibility to KBD: carriers of the mutant allele ‘A’ in rs have a decreased risk for KBD relative to those homozygous for the wild type allele ‘G’.

To explore whether rs contributes to the severity of KBD, the effect of rs on the clinical stage of KBD was further observed in the KBD group. When the allele and genotype frequencies were compared according to clinical stage of KBD, no significant association was detected. Dominant and recessive models were also applied to rectify this, and the results showed a lack of association between polymorphisms of rs and the severity of KBD (Table 3).

KBD is an endemic OA with a specific geographical distribution. In the present study, we first investigated the associations between 22 SNPs in the COL11A1 gene and the corresponding risk of KBD in a Han Chinese population. A coding region single nucleotide polymorphism (cSNPs) rs (Ile1602Ile) showed a significant association with the risk of KBD. The results suggested that polymorphisms in the COL11A1 gene play an important role in the risk of KBD in the Han Chinese population. Subjects who carry allele ‘A’ in rs have a lower risk of KBD than those who do not. The polymorphic locus rs, which is located in exon 62 of COL11A1, has been reported to be a susceptibility locus for LDH[4]. However, there are limited studies on the function of rs in osteoarthropathy. The effects of rs on clinical characteristics of KBD (clinical stages of KBD) were further observed in the KBD group in our study; dominant and recessive models were also applied to rectify it. However, our data showed a lack of association between COL11A1 gene polymorphism and severity of KBD in the northwest Han Chinese population. These results suggest a role of COL11A1 in the susceptibility to but not in the severity of KBD. Polymorphisms of the COL11A1 gene may play an important role in determining the expression of COL11A and should be correlated with cartilage destruction via changes in the expression of COL11A related to KBD. In the present study, the expression of COL11A in articular cartilage of the knee was further analyzed by immunohistochemistry. Our results showed that the percentages of cells that stained positive for COL11A was lower in the KBD group than in the controls. COL11A is a quantitatively minor component of cartilage collagen fibrils, but it is essential for the interaction between proteoglycan (PG) aggregates and collagens, and the highly oriented network of the fibrillar collagens offers tensile strength[10]. However, the expression of COL11A was lower in knee articular cartilage of KBD, and the reduction in COL11A, the critical organizer of ECM, ultimately causes disintegration of the ECM and joint degeneration related to KBD. Therefore, we believe that the A allele of rs may induce a relative increase in COL11A expression compared with the G allele, and this increase may be associated with a reduced risk of KBD; further studies should be carried out to explore it.

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