Siglec-8 is expressed exclusively on human eosinophils, mast cells, and basophils.2
It has been demonstrated that Siglec-8 engagement induces eosinophil apoptosis in vitro
by triggering the ‘intrinsic' stress-mediated apoptotic pathway through sequential ROS production, mitochondrial dysfunction, and caspase cleavage.3
Of interest, murine studies targeting Siglec-F, the closest functional paralog to Siglec-8, also demonstrated a significant role in regulating the pathogenesis of eosinophil-mediated disorders.8, 9, 10
As eosinophils and their many biologically active mediators are associated with allergic diseases and other chronic inflammatory disorders, this suggests that Siglec-8 could be a candidate gene for human eosinophilic disorders like asthma and EE. In this study, we tested the hypotheses that genetic polymorphisms in Siglec-8 are associated with asthma and EE, and that mutations in the glycan-binding extracellular domain of Siglec-8 could disrupt the affinity of Siglec-8 for its ligand, which might then lead to loss of ligand-induced apoptosis in vivo
, resulting in exaggerated eosinophilic inflammation.
We took advantage of genome-wide association studies (GWAS) originally performed to identify susceptibility genes for asthma in a total of 935 African Americans.19
Seven Siglec-8 SNPs were included in the GWAS final data analysis after all the filtering processes were performed for quality control. In parallel with this analysis, an additional seven SNPs were genotyped. Of these, two common non-synonymous variants (rs3829659 Arg388Gly and rs10409962 Ser170Pro) were located along the functional regions of the Siglec-8 gene in the glycan-binding extracellular domain (Ser170Pro) and the cytoplasmic negative signaling domain (Arg388Gly). Among all the SNPs genotyped, the strongest evidence for association with asthma was observed for the promoter SNP rs36498 (-4354C/T), and suggestive evidence for the non-synonymous coding SNP rs10409962 (Ser170Pro) on exon-2, where both mutant alleles were associated with reduced risk of asthma. When replicating these findings in two additional independent populations, similar associations were found, namely SNP rs36498 was significantly, and rs10409962 was suggestively, associated with current asthma as documented by a combination of wheezing in the past 12 months and lifetime asthma among Brazilian families. In contrast, SNP rs10409962, but not rs36498, was significantly associated with asthma in the Japanese case–control analysis. These findings suggest that genetic variants in the Siglec-8 gene may be associated with asthma. Although we did not observe SNP-for-SNP replication of findings in these populations, this was not unexpected. As highlighted in Supplementary Table S1, the allele frequencies of all the SNPs genotyped differed across ethnic groups, suggesting there was considerable genetic heterogeneity among these populations. It is possible that variants other than those tested here are causal and directly alter Siglec-8's function, and the observed statistical associations in this study are merely due to strong LD between markers and these unobserved causal variants. Heterogeneity of the phenotype is another commonly cited explanation for failure to replicate positive associations across independent populations. However, it is important to note that SNPs in each population showed associations with asthma, providing a convincing basis for further investigation of the role of Siglec-8 in the pathogenesis of asthma. In addition, although SNP rs10409962 failed to pass the correction for Bonferroni Multiple testing, the most stringent test of all, the associated SNP, rs10409962, is located two amino acids away from a putative carbohydrate-binding domain. It is still possible that this SNP results in aberrations in Siglec-8 structure and ligand binding, and subsequently leads to alterations in Siglec-8's function. As a result, we investigated the effect of this coding SNP on Siglec-8 ligand-binding activity using a synthetic polymer decorated with 6′-sulfo-sLex
when tested on HEK293 cells stably transfected with the rs10409962 risk allele or the WT allele. We found no difference in binding to 6′-sulfo-sLex
ligand-containing polymers between transfectants with or without the risk allele, suggesting that this point mutation does not alter its ligand-binding activity. However, it is possible that the interaction between Siglec-8 and its natural ligand is different and not adequately reflected by the artificial polymer ligand. Alternatively, the assay used for testing interactions might not be sensitive enough to detect the potential differences between Siglec-8 molecules with 170S versus 170P. However, this is the best way to currently explore this issue because the natural Siglec-8 ligand remains to be determined. Another possibility is that it could alter the levels of Siglec-8 surface expression on eosinophils. Further studies are clearly needed to elucidate the mechanisms underlying the role of rs10409962 in the pathogenesis of asthma.
We recognized that, after Bonferroni Multiple testing correction, the SNP rs36498 was the only SNP significantly associated with asthma in the African American case–control group (adjusted P
=6.29 × 10−4
). Bonferroni correction offers the most conservative approach to control for false positives, and we therefore believe that the association with asthma for SNP rs36498 is more likely to be true among African Americans. Interestingly, the haplotype centered on rs36948 showed enhanced association with asthma and in particular, a haplotype (TA) carrying two promoter SNPs of Siglec-8 (rs36498 and rs11672925) demonstrated increased association when compared with either SNP individually. These findings suggest that SNP rs36498 and others in the promoter region of Siglec-8 may be critical in conferring susceptibility to asthma. Hence, more SNPs with comprehensive coverage within the promoter region of Siglec-8 are clearly needed to be genotyped, with the aim of identifying the variant(s) influencing transcriptional regulation. Also, we observed a significant association for SNP rs36498 and haplotypes carrying rs36948 among the Brazilian families, but the global P
-value from the strongest haplotype is not as impressive as the single SNP rs36498. It can be speculated that the LD-genotyped SNPs are not great in coverage (2.6
kb/SNP) and the signal flagged by the single promoter SNP is washed out by the SNPs surrounding it. An additional association was observed for SNP rs6509541 as an individual or in combination with other SNPs in the 3′-UTR and downstream from Siglec-8 and tIgE in the African American population, and replicated in the Brazilian population. This finding suggests a role for SNP rs6509541 via Siglec-8 in allergic diseases because tIgE is a useful marker for assessing allergic diseases.35
Given that Siglec-8 is a key molecule involved in facilitating eosinophil apoptosis and that mutations in Siglec-8 may also be involved in other allergic and eosinophilic disorders, we investigated whether Siglec-8 SNPs are associated with other human eosinophilic disorders like EE. We selected eight SNPs encompassing the Siglec-8 gene with a coverage of 2
kb/SNP, including those associated with asthma. However, none of the SNPs were significantly associated with EE among the Caucasian population. Of these, four SNPs (rs10406692, rs3829659, rs39711, rs10420357) were further replicated among 68 EE families, but no significant association was observed (data not shown). The results suggest that Siglec-8 does not associate with susceptibility to EE at least in Caucasians. However, further studies may be needed to get a comprehensive coverage of genetic variants in and around the Siglec-8 gene in order to fully exclude Siglec-8 as a candidate gene for EE.
In summary, we tested for associations between Siglec-8 SNPs and asthma, tIgE, and EE in diverse populations. We are able, for the first time, to provide evidence of association between Siglec-8 SNPs and asthma and tIgE in different racial populations. The strongest association was seen for SNP rs36498 in the promoter region of Siglec-8, even after correction for multiple tests, suggesting that the Siglec-8 gene may be associated with susceptibility to asthma. The finding that Siglec-8 SNPs were not associated with EE suggests that the association is not simply due to atopy, which is found in similar percentages in these otherwise unrelated disorders. Further studies are needed to identify additional genetic variants, with a particular focus on the promoter region of the Siglec-8 gene. As interaction assay failed to show any changes in ligand-binding activity on HEK293 cells stably transfected with the rs10409962 risk allele or the WT allele, additional studies are needed to determine exactly how this SNP might affect the biology of Siglec-8 and contribute to the pathophysiology of asthma.