These results demonstrate that two variants (CCR5-1835T and CCR5-Δ32) in the gene encoding CCR5 are associated with JRA, especially in children with disease onset before the age of 6 years. Both variants are believed to have functional consequences, and it should be noted that both effects on JRA were in the same direction, i.e., protective against JRA.
Case-control association studies are commonly used to investigate associations between phenotypes of interest and genetic variants. However, case-control studies can produce spurious associations due to population stratification. Several approaches have been suggested to account for population stratification.25
Many of these approaches use family-based controls to reduce the effect of population stratification. The TDT provides a joint test of linkage and association and eliminates the effects of stratification when applied to probands and parents. Although TDT based methods are more robust to population stratification, they are thought to be less powerful than conventional case-control studies at detecting associations.25
While failure to detect statistically significant associations by TDT could be due to type 2 statistical errors, positive results obtained by TDT are meaningful. We think that the demonstration of significant deviation of transmission of two CCR5
variants to children with JRA likely represents a true association. This conclusion is further supported by the observation that there is no deviation of transmission of these alleles to unaffected children in these families, although ideally a larger cohort of unaffected siblings would have likely given more robust results.
To minimize statistical artifacts, our strategy was to first test the 5’ cis-
variants in only the multiplex cohort, and then to confirm the significant results by genotyping a large cohort of simplex families. Thus, the only SNP we tested in the entire cohort was C-1835T
, and we demonstrated a significant association not only with the entire JRA cohort, but also some JRA subtypes, which had been designated a priori
. The other polymorphism tested, CCR5-Δ32
was chosen based on its known functional consequence and confirmed associations in adults with RA. Again we had designated the subtypes to be tested a priori
. While the JRA subtypes have unique clinical and genetic features, they cannot be thought of as being entirely independent. Similarly variants in LD with one another are also not independent. For these reasons, we did not think it appropriate to apply the conventional Bonferroni correction to our results. Moreover, the Bonferroni correction tends to be very conservative, increasing the possibility of a type-2 statistical error. Our combined JRA cohort is one of the largest reported among studies looking at genetic associations in JRA, likely further minimizing type-2 statistical errors. Our study had sufficient power (> 0.8) to detect an association under most models for a marker allele frequency of 0.1, both for JRA as a whole, and for most sub-phenotypes examined.26
Ultimately, like other genetic association studies the present observations need to be validated by replication in independent cohorts.
The SNP CCR5 C-1835T
, located in the 5’ cis
-regulatory region of CCR5 has been shown to result in loss of novel nuclear factor binding.27
Investigations of the transcriptional activity of different haplotypes in the cis
-regulatory region of CCR5 have demonstrated that CCR5-haplotypes containing -1835T (HHF) have significantly different promoter efficiency compared to other haplotypes.27
Haplotypes containing −1835T are associated with different disease modifying effects among patients with acquired immune deficiency syndrome.28
These data suggest the possibility that −1835T is protective against developing early onset JRA, conceivably by altering the expression level of CCR5. Although one would expect these haplotypes to decrease the expression of CCR5, haplotypes containing −1835T appear to increase the efficiency of the CCR5
promoter in vitro
. It is possible that −1835T may have other effects in vivo
. Also, this effect of −1835T is on gene expression, and the effect if any, of −1835T on cell surface expression of CCR5 is unknown. Furthermore the effects observed could be due to complex interactions with other polymorphisms on these haplotypes. Ideally, experiments addressing the genotype-phenotype correlations of CCR5 variants and CCR5 expression in subjects with JRA would better address the role played by this polymorphism and others in the development or phenotype of inflammatory arthritis. It is also possible that another variant in LD with this SNP influences the protective effect observed here. For instance −1835T has been reported to be in LD with a SNP in the coding region of another chemokine receptor gene, CCR2
), located ~9kb 5’ of the CCR5
gene on chromosome 3p21.31.22
allele has also been shown to have protective influences against human immunodeficiency virus (HIV) disease progression.29
It has been proposed that genetic variants subjected to natural selection are likely to harbor functional polymorphisms and hence are good candidates for association studies.30
The 5’ cis-
regulatory region of CCR5
has been demonstrated to be under balancing selection, where diverse haplotypes have been maintained.22
-regulatory region of CCR5
has several polymorphisms that regulate the expression of CCR5, and different CCR5
SNPs and haplotypes have been associated with phenotypic differences in HIV infection.28
It has also been suggested that the CCR5-Δ32
allele has been subjected to recent positive selection.31, 32
(But see 33
). The fact that our associations have been discovered in regions affected by natural selection strengthens the evidence that they might have functional significance.
Studies investigating the association between CCR5-Δ32
and RA in adults have reported conflicting results.17, 20
However, a meta-analysis of the published association studies between CCR5-Δ32
and RA suggests that some of these studies were underpowered, and confirms that this polymorphism is strongly and negatively associated with RA (p < 0.0001).21
This suggests that CCR5-Δ32
is associated with protection against the development of RA. Until now, this polymorphism has not been formally investigated in association studies of individuals with JRA, although two children with JRA who were homozygous for this mutation have been described.7, 9
The demonstration of under-transmission of CCR5-Δ32
to probands with early onset JRA is consistent with these observations and suggests that JRA and RA share some genetic risk factors.
To answer the question whether these association results could be due to LD between CCR5 - 1835T
, we examined the haplotypes and the LD pattern in CCR5. The minor alleles, −1835T
were not in LD, and they occur on two different haplotypes. Examination of the published data on Great Apes, and Old and New world monkeys, shows that neither allele is present in these species and that both are newly derived in humans, suggesting that both of these variants are derived from the ancestral haplotype of CCR5-1835C-CCR5 WT
Thus, the protective effects observed at these loci appear to be independent of each other and not due to LD. As anticipated, we did not find haplotypes that had the minor alleles at both loci. However, the haplotype containing CCR5 −1835C and CCR5 WT
(i.e., lacking the protective variants) did show evidence of excessive transmission to all children with JRA and those with early onset JRA by TDT, further strengthening our results. Together these observations support the notion that CCR5 might play important roles in the pathogenesis of inflammatory arthritis, suggesting that CCR5 blockade could modify the phenotype of inflammatory arthritis.