The primary hypothesis of our study is that variations in MBL2 genetic background alter susceptibility to colon cancer. Our results suggest that variations in both the secretor haplotype and 3′UTR region of the gene increase colon cancer risk in African Americans; whereas similar observations were not seen in Caucasians. We investigated whether plasma MBL levels and activity were altered in relation to the variants associated with increased risk of colon cancer. Our results suggest that the secretor and 3′UTR region haplotypes are associated with altered MBL plasma levels; however the results observed for plasma activity were less consistent. Furthermore, we showed evidence that one SNP in the 3′-UTR (rs10082466) may alter predicted miRNA binding, thus accounting for the lower plasma MBL levels.
Our results for the 3′-UTR region and secretor haplotypes are consistent with previously published studies that show these haplotypes modify serum levels (9
). Additionally, we did confirm that genetic variation in the 5′-region of the gene is associated with both altered MBL plasma levels and activity in both Caucasians and African Americans. Interestingly, the median MBL plasma levels for haplotypes LXPA, LYPB, HYPD, and LYQC are lower in African American cases when compared with Caucasian cases. This correlates with previous reports that low serum MBL levels are more prevalent in populations from Africa. It has been suggested that low serum MBL levels have been selected for in African countries due to the protection that the lower levels provide against the deleterious effects of certain infections by intracellular bacteria and parasites (9
It is possible that higher MBL plasma levels enhance immunological surveillance against colon neoplasia and, in turn, provide protection against the onset of disease. It has been shown that MBL recognizes and binds to oligosaccharide ligands expressed on the surfaces of human tumor cells, including a human colorectal carcinoma cell line SW1116 (29
). Furthermore, MBL has been shown to inhibit tumor progression by MBL-dependent cell-mediated cytotoxicity (30
). Thus, those individuals carrying the HYPA haplotype, which is associated with the high serum concentration of the protein, may be less susceptible to the onset of colon cancer. Our study results are supported by a previous study which found an association between an MBL2
haplotype associated with lower serum concentrations and increased risk of stomach cancer in a population-based case-control study in Warsaw, Poland (12
We only observed evidence for increased susceptibility to colon cancer in African Americans. It has been shown that MBL serum concentrations correlate with the MBL2
secretor haplotypes in distinct populations (31
) and we observed a similar correlation with MBL plasma levels in both Caucasians and African Americans in our study. Additionally, we observed differential levels of MBL activity correlating with the secretor haplotypes in both groups. These results suggest that similar functional consequences of the serum/plasma levels should be observed between races. However, we also observed that those African American and Caucasian cases carrying the haplotypes associated with low MBL serum levels differed in MBL plasma levels. Furthermore, the African American cases had lower MBL plasma levels when compared with Caucasian cases for these specific haplotypes. We did not see similar associations when Caucasian controls were compared with African American controls. This may account in part for the increased risk we see associated with colon cancer in those African Americans carrying specific haplotypes. It should be noted that although the cases have overall higher MBL levels and activity when compared with controls, this does not negate the possibility that the lower levels seen in African Americans could be associated with risk of developing cancer. Higher MBL levels in cases have been previously reported in the literature (32
); and it has been suggested that this observation may be because MBL is a marker of inflammatory response associated with having the disease.
It is also plausible to hypothesize that the serum/plasma MBL levels may interact with additional genetic and/or environmental risk factors in which African Americans are exposed that we have not identified in our study. It has been shown that Caucasian individuals carrying the HYD haplotype, which is a truncated version of the HYPD haplotype, and a variant of IL-1β at position -511 have an increased risk of stomach cancer (12
). Furthermore, it is possible that the colonic microbiota differ between African Americans and Caucasians. Evidence suggests that diet directly influences the bacterial diversity of the microbiota (34
), therefore differences in diet between Caucasian and African American populations could influence the colonic microbiota. Thus, the increased susceptibility to colon cancer seen in African Americans in this study may be due to the response this specific population has to the lower levels of MBL when compared with Caucasians.
We also investigated the potential functional implications of the observed association of the 3′UTR region genotypes with increased risk of colon cancer and found that one of the associated SNPs leads to differential miRNA binding. Specifically, we found that more efficient binding of hsa-miR-27a to the risk-associated allele of MBL2
is consistent with lower levels and activity of MBL found in rs10082466C carriers. Of note, it was indicated in an earlier report investigating the association of MBL2
3′-UTR haplotypes and breast cancer risk in African Americans that carriers of the risk haplotype had lower MBL serum levels, although the association was only borderline significant (15
). In our study, we observed a similar trend in cases and controls, regardless of race; therefore, the question of how this functional association explains the robustly increased colon cancer risk in African Americans still remains. Interestingly, evidence for differential miRNA expression associated with race is starting to emerge (35
). Therefore, it is possible that expression of hsa-miR-27a in liver tissues, which is where MBL is mainly expressed, differs in African Americans when compared with Caucasians. When colon cancer tissue was examined, we could not show differential expression of mature hsa-miR-27a between African Americans and Caucasians; however we were able to demonstrate the fold increase of hsa-miR-27a precursor to be significantly higher in African Americans when compared with Caucasians (data not shown). A limitation of our study is that we have been unable to reliably quantify MBL2
protein expression in cultured cells or conditioned media, thus the correlation between rs10082466 and MBL2
expression levels could not be readily tested in vitro
In addition to their functional association with miRNAs, it cannot be ignored that the 3′-UTR SNPs may be in linkage disequilibrium with another functional SNP not identified in our study. Bernig, et al.
have shown that the CGGG 3′-UTR haplotype is most frequently seen in linkage with the truncated secretor haplotype 0, which also includes the secretor haplotype LYQC (28
). We have observed similar results in that the CGGT 3′-UTR haplotype is most frequently seen in linkage with the truncated secretor haplotype 0 (data not shown). Therefore, the associations of the secretor haplotypes (LYPA, LYQC) and at the 3′-end of the gene could also represent an epistatic effect.
The main limitation of this study is the small sample size; therefore the observed associations will need to be replicated in a larger study. Additionally, further studies to elucidate the underlying biological mechanisms associated with MBL levels and the differences between colon cancer susceptibility in African Americans and Caucasians are warranted.
In conclusion, our study supports the hypothesis that MBL2 genetic variation in both the 3′- and 5′-region of the gene increase susceptibility of colon cancer, but only in African Americans. We also show that the genetic variation observed alters plasma MBL levels and activity. In the 3′UTR region, we showed that hsa-miR-27a more efficiently binds to rs10082466C which is consistent with the lower plasma MBL levels and activity observed. However, further studies are warranted to validate the associations we have observed in our study.