We observed that the T allele of rs2187331 residing in a putative regulatory region upstream of GAL is experiment-wide significantly associated with HTG in the combined analysis of FCHL families and combined hyperlipidemia cases/controls. In a non-ascertained population sample, the C allele of rs2187331 was associated with higher levels of TGs. We also provide evidence for the functional relevance of rs2187331 by demonstrating allelic differences in reporter gene expression, nuclear factor binding and expression of lipid metabolism related genes, including LPL, a known downstream target of GAL. These data demonstrate an association to a clinically relevant trait, a plausible mechanism by which this SNP may functionally alter GAL expression, and allelic differential expression of genes that are likely to play a role in the increased susceptibility for HTG.
We observed that LPL
and many other lipid metabolism related genes are differentially expressed based on rs2187331 genotypes. Furthermore, 77% of these genes show higher expression with the TT genotype. This is consistent with data in rats where GAL
was found to regulate LPL
expression both in adipose and muscle3, 4
. Interestingly, not only genes from the anabolic fat storage pathway have higher expression with the TT genotype (CD36
), but also genes from the catabolic pathway (ATGL
). The molecular mechanism(s) underlying the coregulation of these genes will need to be investigated in future studies.
We found the C allele of rs2187331 associated with higher levels of TGs in the population-based METSIM study, whereas in samples ascertained for hyperlipidemia the T allele was associated with HTG. Approximately 10% of the METSIM individuals were taking statins and only 16 of the non-statin users had combined hyperlipidemia. Also 15% of the METSIM study are diabetic. The sample sizes of the HTG (n=2,471) and population (n=4,463) studies make it less likely that either finding is by chance alone. Furthermore, the functional evidence supports a regulative, allele-specific role of rs2187331. Moreover, ultimately an individual's risk to develop a complex cardiovascular trait is a combination of susceptibility variants, environmental factors, behavior and chance. The combination of variants and environmental factors are likely to differ between subjects with HTG and subjects representing the unascertained general population, thus contributing to these types of allelic differences. The difference in direction of the association between the METSIM and hyperlipidemic study samples could be due to these confounding factors (i.e. statins and diabetes status), and will have to be further investigated in independent study samples with refined quantitative phenotypes and highly detailed life style variables. Extensive resequencing of the entire genomic region of GAL in different study populations is also warranted to further explore the possibility that differences in LD structure and/or several functional variants and their interactions with rs2187331 may contribute to the disparity in the association signal.
Originally it was observed that injection of nanomolar levels of GAL peptide onto the paraventricular nucleus (PVN) of the hypothalamus stimulates feeding16
. Subsequent genetic evidence from GAL knockout mice suggests that GAL plays a role in fat intake and preference17-19
. Interestingly, a positive correlation was also observed between plasma glucose and circulating levels of galanin peptide in healthy and type 2 diabetic individuals20, 21
. However, the inhibition of insulin secretion identified in other mammals was not observed in humans given a bolus of either porcine or human galanin22, 23
. Variation near the GAL gene has previously been associated with alcoholism and anxiety24
, whereas obesity studies have not yielded significant findings for GAL25, 26
In normolipidemic individuals plasma TG levels are increased following the consumption of a high fat meal. In response to the elevated plasma TG levels, GAL
expression is up-regulated in the PVN3
, which in turn signals the up-regulation of genes involved in the lipolysis of TGs from triglyceride rich lipoproteins (TRLs) and import of free fatty acids for storage in adipose tissue. A secondary effect of lipolysis of TGs from TRLs is a reduction in the level of plasma TGs due to a relocation of TGs from plasma into another tissue. We hypothesize that in response to postprandial elevated plasma TGs individuals with HTG carrying the C allele of rs2187331 will have decreased expression or reduced upregulation of GAL
in the PVN, which modifies the upregulation of lipolysis and storage related genes in adipose tissue.
In conclusion, our data suggest that rs2187331 residing in a putative regulatory region upstream of GAL contributes to hypertriglyceridemia in FCHL, TG levels in the general population, and has an allele-specific cis-regulatory function.