All SNPs tested were in Hardy Weinberg Equilibrium (HWE). LD was assessed for two SNPs in the IL6
promoter region (rs1800795 and rs1800797) as well as two SNPs in the IL6R
gene (rs2228145 and rs4537545), as they demonstrated similar allele frequencies. Consistent with previous reports, 
these two IL6
promoter SNPs were found to be in LD (D’
0.98), as were the two IL6R
Observed allele frequencies for CD patients and HD are depicted in . A significant difference in the frequency of the minor T allele of IL6R SNP rs4537545 was observed between CD patients and HD (0.49 and 0.33, respectively; p<0.018). Similarly, the minor C allele of IL6R SNP rs2228145 was overexpressed in CD patients versus controls (0.42 vs. 0.30, p<0.068). While these two SNPs exhibit high LD, we observed minor discordance in a few subjects, which resulted in the greater p-value for the rs2228145 SNP. No significant differences in allele frequencies were detected in the other SNPs tested.
Levels of sIL6R were measured in the serum (or plasma when serum was not available) of CD patients and controls by ELISA. Individuals possessing one or more copies of the minor C allele of IL6R
SNP rs2228145 produced significantly higher levels of sIL6R (averages 43.98 ng/mL vs. 29.85 ng/mL, p
, ). Furthermore, individuals possessing at least one copy of the minor T allele of the IL6R
SNP rs4537545 expressed higher levels of sIL6R than those homozygous for the major C allele (averages 42.84 ng/mL and 29.36 ng/mL, respectively, data not shown), and this difference was highly significant (p
). For both IL6R
SNPs, this genotype-dependent difference in sIL6R levels was also observed in both the CD patient and control cohorts independently.
Flow cytometry was performed to assess the levels of mbIL6R as well as gp130 on CD patient and HD PBMC. Antibodies to various lymphocyte and monocyte markers were incorporated to determine if changes in mbIL6R/gp130 levels were localized to a particular cell type. Subjects who were heterozygous for the IL6R SNP rs2228145 demonstrated decreased levels of mbIL6R at the cell surface as compared to those who were homozygous for the major A allele. Subjects homozygous for the minor C allele exhibited the lowest levels of mbIL6R. As was done with ELISA data, statistics were calculated by grouping heterozygous and minor-allele homozygous individuals together, and comparing to major-allele homozygous subjects. We observed a genotype-dependent difference in mbIL6R levels on most cell types studied (). Similar results were obtained for the IL6R SNP rs4537545 (data not shown). When mean expression levels for CD patients were compared to HD, we observed that the overall mbIL6R expression is higher on PBMC from HD than CD patients ().
Next, we assessed whether any correlations existed between these SNPs and IL6 levels in CD patients. Given the strong link between IL6 and CRP expression and the observation that patients with elevated serum CRP (>8 mg/L) can display apparently normal serum IL6 levels (< 10 pg/mL), we hypothesized that commonly used IL6 assays were not able to accurately quantify IL6 in all patients. We therefore employed the Panoptic IL6 assay due to its distinct ability to better quantify IL6 levels (complexed and non-complexed) in human blood (manuscript in preparation). Contrary to the observations made by Reich et al. and Rafiq et al., we did not see an association between genotype and IL6 levels 
. Variations among commercially-available IL6 assays in the ability to accurately detect IL6 levels could account for our inability to reproduce the results pertaining to IL6/SNP correlations found in others’ studies.
We found that CD patients express the minor allele of two SNPs in the IL6R
gene (rs4537545 and rs2228145) at higher frequencies than do HD. Our data, similar to previous reports, 
showed that individuals possessing one or more copies of the minor allele of these IL6R
SNPs exhibited increased levels of sIL6R, and these differences were highly significant. Similar findings were reported by our group in MM patients 
. We studied the effect of IL6R
SNPs on levels of mbIL6R, and observed that individuals who are heterozygous for the IL6R
SNPs expressed reduced levels of IL6R at the cell surface, and those homozygous for the minor allele expressed the lowest levels of mbIL6R. This was significant for T cells and monocytes (p
<0.05, ). Further, the mean expression of mbIL6R was greater in HD than CD patients. This data shows that CD patients express IL6R
SNPs rs2228145 and rs4537545 more frequently, which leads to increased shedding of IL6R from the cell surface, increased levels of sIL6R in the serum, and presumably increased levels of IL6 signaling. This study identifies for the first time a potentially predisposing factor for MCD, namely the rs2228145 polymorphism in the IL6R
, in this rare and complex disorder.