Rheumatoid arthritis (RA) is a systemic autoimmune inflammatory disorder, affecting approximately 1% in western populations. The disease is primarily characterised by chronic polyarthritis 1 2 . The aetiology of RA remains unknown, although it is estimated that the contribution of genetic factors is about 50 to 60% 3 4 . The strongest genetic association is with polymorphic alleles within the human leukocyte antigen HLA-DRB1 locus on chromosome 6p21.3 and a single nucleotide polymorphism (SNP) in the PTPN22 gene on chromosome 1p13.2 5 . Another proposed potential loci is on chromosome 1q32.1 harbouring the chitinase 3-like 1 (CHI3L1) gene encoding the YKL-40 protein 6 . YKL-40 is a 40 kDa heparin- and chitin-binding glycoprotein, and a member of chitinase like proteins. YKL-40 is expressed by a variety of cells, including macrophages, neutrophils, synovial cells, arthritic chondrocytes and cancer cells 7 8 9 10 . As YKL-40 contains HLA-DR4 binding motifs, it has been suggested to function as an auto antigen in RA 11 12 13 14 15 .

A high serum concentration of YKL-40 is emerging as a new biomarker of severe disease activity and poor prognosis in patients with diseases characterized by inflammation and ongoing tissue remodelling such as RA, inflammatory bowel disease, asthma and cancer 8 10 16 17 18 19 20 21 22 23 24 25 26 . The exact biological function of the YKL-40 protein is still largely elusive. YKL-40 is a trans-membrane protein in which cleavaged components bind to an unidentified receptor and the expression of YKL-40 is regulated by various inflammatory cytokines and hormones 27 28 29 30 . It is suggested that YKL-40 plays a role in cell proliferation, differentiation and protection against apoptotic signals, and has an effect on extracellular tissue remodelling 31 32 . Two recent studies have explored the effect of YKL-40 as a stimulator of angiogenesis in tumours, suggesting that anti-YKL-40 antibodies could have a place in cancer treatment 33 34 .

The proximal promoter region of the CHI3L1 gene contains a highly polymorphic area, suggesting a possibility for several functional variants of the gene. Rehli et al. 35 demonstrated that binding of the SP1 transcription factor to the most proximal part of the CHI3L1 gene affected gene transcription. This finding was supported by Zhao et al. 36 reporting functional variants based on the binding of the MYC/MAX transcription factors to the proximal promoter region. The relationships between CHI3L1 polymorphisms and YKL-40 production have been studied in a small number of patients with various inflammatory disorders, such as sarcoidosis, asthma, hepatitis, schizophrenia and diabetes 37 38 39 40 41 42 43 44 . These studies suggest that serum concentrations of YKL-40 are, at least partly, regulated by polymorphisms in the proximal promotor region. The findings have been somewhat contradictory and the exact position of the regulatory site or sites remains to be demonstrated. Allele frequencies differ significantly between Caucasian, African and Asian populations, and possibly even within these populations, thereby making direct comparison of the reported studies difficult 45 .

Only one small study has evaluated CHI3L1 polymorphisms in patients with RA 46 . In 182 Hungarian patients with RA and 194 healthy controls there were no significant differences in genotype frequencies for the g.-131(C > G) or the g.-329(C > T) polymorphisms between the two groups. This study did not evaluate the functional properties of these polymorphisms. Several questions remain unanswered, namely the relationship between CHI3L1 polymorphisms and serum concentrations of YKL-40 in patients with RA, the association of CHI3L1 promoter genotypes to risk of RA and the Linkage Disequilibrium (LD) properties in different populations.

We aimed to investigate these questions in a cohort of well defined Danish patients with RA and a group of healthy Danish controls. Our hypothesis was that polymorphisms in the proximal promoter region of CHI3L1, most likely the g.-131(C > G) polymorphism (rs4950928), are associated with serum concentrations of YKL-40 in both patients with RA and healthy controls. Moreover, we hypothesized that these polymorphisms could be associated with the risk of developing RA and possibly also associated to IgM rheumatoid factor (RF), since YKL-40 seems to play a role in the pathogenesis and immunomodulation in RA.

No deviations from Hardy-Weinberg equilibrium were found for any of the eight SNPs in the patient or control group. Age stratification into one-year age groups did not reveal deviations from Hardy-Weinberg equilibrium in any of the age groups.

Prior to the SNP association analysis, the effect of age and case-control status on serum YKL-40 was tested using a multiple linear regression model, with serum YKL-40 as dependent variable and case-control status and a non-linear function of age included as covariate. Strong significant association of the serum concentration of YKL-40 with age (P < 2.0e-16) and case-control status (P < 2.0e-16) was observed (Figure 1). Moreover an apparent increase in serum YKL-40 with age was found for the older patients in the case group. To avoid a potential bias due to the high influence of individuals older than 65 years in the RA group, we excluded in all further analysis patients with RA older than 65 years.

To test the effect of genotypes on serum concentrations of YKL-40 in the RA group (age 65 and below) and control group, a multiple linear regression model including serum YKL-40 as dependent variable and a non-linear function of age, case-control status, genotypes and gender as well as the interaction between case-control status and genotype with age as independent variables was applied (Table 2). From this analysis a strong association was observed with case-control status (P < 2.0e-16) (as before), age (P < 2.0e-16) (as before) and genotype (P < 2.0e-16).

Regarding the age-dependent increase in the serum concentrations of YKL-40, no significant difference was found between a non-linear and a linear model for the age-dependence in both the case group (age 65 and below) and control group (P = 0.19) suggesting that the linear model can be used for age adjustment of the serum concentrations of YKL-40 in both groups. The linear model was fitted and depicted in Figure 2.

Serum concentrations of YKL-40 were not associated with gender (P = 0.16). There were no interaction effects between case-control status or genotype and age (P = 0.89) and no association between serum YKL-40 and the interaction effect between genotype and case-control status (P = 0.16) (Table 2). This suggests that age, case-control status and genotypes are all strong independent factors affecting serum concentrations of YKL-40.

To test the association of each SNP on age-adjusted serum YKL-40 in the RA group (age 65 and below) and control group, a linear age-adjustment was applied and genotypes were included one-by-one as dependent variables in a multiple linear regression analysis. The g.-131(C > G) genotype was found to be most strongly associated with age-adjusted serum concentrations of YKL-40 in both the patient (P = 2.4e-08) and control group (P < 2.2e-16) (Table 3). Consistently within both groups, the rare GG genotype was associated with low serum YKL-40, the CG genotype with intermediate serum concentrations of YKL-40, and the common CC genotype with high serum YKL-40 (Figure 3). With respect to genotypes, the RA patients had significantly higher serum YKL-40 than controls for both the CC and CG group. For the rare GG group, the difference was not significant, most likely because of low statistical power due to the limited number of individuals in the GG groups.

When the g.-131 C/G was used as a covariate to determine the influence of the remaining seven SNPs on serum concentrations of YKL-40 none of the other SNPs contributed significantly to the association supporting the isolated highly significant effect of the g.-131 C/G polymorphism on serum concentrations of YKL-40 (Table 4).

Haplotype analysis did not add further information as all the haplotypes associated with low serum concentrations of YKL-40 carried the g.-131G allele and no further increase in association was seen with any of the haplotypes (data not shown). LD analysis of the eight genotyped SNPs revealed that both the proximal promoter and the distal part of the gene contained blocks of high or moderate LD (Figure 4) explaining the effect of all the included polymorphisms on serum YKL-40 when analysed individually. In particular the -131 C/G polymorphism displayed moderate LD with g.-329C/T (R² 0.78) indicating that the effect on serum concentrations of YKL-40 with g.-329C/T is caused by LD. These findings are in line with CEU HapMap data (Figure 5).

To investigate the association of the eight SNPs with case-control status, allelic and genotypes were tested for association with RA using Fishers exact test. No association was found with alleles or genotypes for any of the eight SNPs (Table 5) indicating that these SNPs do not confer risk to the development of RA itself.

The high producer genotypes were not more frequent in the IgM-RF positive subgroup and no difference was found in geno- or phenotype distribution between seropositive and seronegative patients with RA (data not shown).

This study aimed to investigate eight polymorphic sites in the CHI3L1 gene with possible functional properties in both patients with RA and healthy individuals. We focused on the g.-131(C > G) allele and closely related polymorphisms described in Caucasian populations 26 36 37 38 39 43 44 46 . The g.1219(G > A) polymorphism was also included as one study reported an individual functional property of this polymorphism 43 . Serum concentrations of YKL-40 were strongly associated with age and case-control status. After adjustment of the serum concentrations of YKL-40 for these two variables, serum YKL-40 was found to be significantly associated with SNPs in the CHI3L1 gene. The strongest association was with the g.-131(C > G) promoter polymorphism. The association of serum YKL-40 with the remaining SNPs could be explained by LD. Our findings indicate that serum concentrations of YKL-40 are under the influence of genetic variability in the CHI3L1 gene in both patients with RA and healthy controls, and the effect of genotypes seems to be the same in both groups. Though our results indicate that CHI3L1 polymorphisms are not involved in the pathogenesis of RA, we do not know if high producer genotypes results in a more severe clinical phenotype.

Several other studies have suggested the g.-131(C > G) is a strong candidate for a functional promoter polymorphism influencing the serum concentrations of YKL-40 36 42 43 44 45 . The promoter SNP g.-131(C > G) in the CHI3L1 gene was associated with elevated serum YKL-40, asthma, bronchial hyper responsiveness and pulmonary function 44 45 , and with elevated serum YKL-40 and the severity of hepatitis C virus-induced liver fibrosis 43 . This indicates a functional role of YKL-40 in these diseases. An association is also found between schizophrenia and haplotypes within the promoter region of the CHI3L1 gene suggesting that polymorphisms in an area starting from base pair position -180 could have functional properties 36 42 . Our findings support these earlier studies.

Zhao et al. 36 investigated Chinese patients with schizophrenia and found lower activity of the transcription factor MYC/MAX and decreased CHI3L1 gene expression related to the low frequency G allele for the g.-131(C > G) SNP. Ober et al. 44 studied 443 patients with asthma and 491 healthy controls from a genetically preserved group of Americans of European descent. They found that serum concentrations of YKL-40 were associated with many alleles in the promoter region including the g.-131(C > G) and g.-329(C < T) polymorphisms. This supports the g.-131(C > G) polymorphism as a site of genetic regulation in both healthy controls and patients with asthma. Conclusions were complicated by strong LD in the promoter region in the population studied. They also showed a strong association to the g.-1219(G > A) polymorphisms, which was not in LD with the promoter polymorphisms. This individual effect on serum YKL-40 with g.-1219(G > A) was not supported in our study as we found this phenomena related to LD in the Danish population. In contrast, Sohn et al. 40 demonstrated a functional effect of the g.-247 (G > A) polymorphisms in a study of 295 atopic children and 180 healthy controls from a Korean population. They concluded that this polymorphism was responsible for most of the genetic effects on YKL-40 production, and that the g.-131C allele was associated to low promoter activity. These results are complicated by the fact that the g.-131(C > G) and g.-247(G > A) polymorphisms showed no LD in the Asian populations, contradictory to our finding which suggest a high degree of LD in this part of the proximal promoter. In the Danish population the region on chromosome 1 bearing the g.-131(C > G) polymorphism was in strong LD, illustrated by the occurrence of just 8 frequent haplotypes (f > 1%). The g.-131(G > C) allele was found to be in LD with several other loci in the CHI3L1 gene, and three haplotypes could be defined as low producer haplotypes, all including the g.-131G allele. It must be emphasized that ethnicity seems to play an important role in the genetic regulation of YKL-40 production, and results from our and similar studies can only be considered valid in ethnically similar populations. Further studies in different populations are awaited.

Serum concentrations of YKL-40 increased with increasing age in both healthy controls and patients with RA making age a possible confounding variable. The cause of this remains unknown, but the phenomena has been explained by a higher level of general inflammation and apoptosis in the elderly, which is well known for other inflammatory mediators 53 . Similar increases in plasma YKL-40 with age have recently been described in a large group of 8,899 subjects from the general Danish population 20 . We initially decided to fit a non-linear model to explain the effect of age on serum concentrations of YKL-40. Our control group did not include any persons above the age of 65, but below this age we were able to fit a linear model explaining the relationship between age and serum concentrations of YKL-40. This supports the findings by Kruit et al. 38 , who suggested a linear relationship between age and serum YKL-40. In the patients with RA it seems as if serum concentrations of YKL-40 rise more rapidly with age above age 65, indicating that elevated serum YKL-40 in this age group needs careful interpretation. It is possible that high serum YKL-40 is associated to co-morbidity or a latent malignant disease 10 20 21 22 23 24 25 26 53 54

It remains unknown whether high serum YKL-40 affects a person:s risk of autoimmune disease in the long term. YKL-40 expression is stimulated by the inflammatory cytokines TNF-α, IL-6 30 and IL-1β, whereas YKL-40 inhibits cellular responses induced by IL-1 and TNF-α, suggesting an autocrine feed-back mechanism 9 28 . YKL-40 is strongly expressed by macrophages in the synovial membrane of RA patients possibly activated by a pro-inflammatory IFNγ-mediated immune response, and elevated YKL-40 can stimulate local production of anti-inflammatory IL-10 32 . In inflammatory diseases such as RA, the excessive YKL-40 production may also have the opposite effect stimulating a continuous pro-inflammatory state and stimulation of VEGF and angiogenesis 32 33 34 .

In conclusion, this study reports a strong association between the g.-131(C > G) allele and serum concentrations of YKL-40 in both patients with RA and healthy controls. Our findings indicate that the g.-131(C > G) polymorphism is the main contributor to the inter-individual variation of serum YKL-40 in Caucasian patients with RA, and that the effect of other polymorphic sites in this region is related to a high degree of LD in this area of the genome.

CHI3L1: chitinase 3-like 1 gene; CV: coefficient of variations; LD: linkage disequilibrium; RA: rheumatoid arthritis; RF: IgM rheumatoid factor; RS: RefSNP number; RT-PCR: real-time polymerase chain reaction; SNP: single nucleotide polymorphism

The authors declare that they have no competing interests.

KRN was involved in all aspects of study conception, design, analysis, interpretation and report generation and provided final approval of the version of the submitted manuscript. RS, JB, SLC, MLH, SK, HEJ and JSJ were involved in data acquisition, analysis and report drafting and provided final approval of the submitted manuscript. MB and MN were involved in statistical analysis and linkage analysis and provided final approval of the submitted manuscript.