Characterisation of HHIPL1, a Coronary Artery Disease Associated Gene

Genome-wide association studies (GWAS) have produced many single nucleotide polymorphisms (SNPs) significantly associated with coronary artery disease (CAD) in humans. The variant rs10139550 is a CAD-associated SNP within the HHIPL1 gene.

HHIPL1 (hedgehog interacting protein-like 1) is an uncharacterised protein that has homology with the hedgehog (HH) antagonist Hedgehog Interacting Protein (HHIP). This thesis aimed to characterise HHIPL1 within HH signalling pathway. This thesis also aimed to identify causal variants in HHIPL1 and link them to human smooth muscle cell phenotypes. This is based on previous work in the same laboratory using murine models that linked HHIPL1, smooth muscle cell phenotypes, and CAD.

A luciferase assay linked to GLI protein expression in murine Shh-LIGHT2 cells determined the function of HHIPL1 in HH signalling. GLI proteins are transcription factors in the HH pathway. In silico analysis and co-immunoprecipitation studies examined HHIPL1 binding to the HH ligand, sonic hedgehog (SHH). An association study determined if the lead SNP rs10139550 and SNPs in a 100kb area surrounding this variant were significantly associated with human umbilical artery smooth muscle cell phenotypes. Expression analysis determined if there was any link between HHIPL1 variants and altered expression of HHIPL1.

Results of binding studies were suggestive of HHIPL1 binding to SHH. HHIPL1 was found to be a positive modulator of full-length SHH, but a negative modulator with the biologically active SHH-N. No variants were found to be in genome-wide significance with the smooth muscle cell phenotypes studied. One variant was significant at an analysis-wide threshold, but was deemed a false positive. Expression analysis showed no significant difference in HHIPL1 expression.

This study characterises a novel modulator in hedgehog signalling and informs future investigation to further define HHIPL1 binding partners and dynamics, and causal variants in coronary artery disease.