Functional Validation of a Common Nonsynonymous Coding Variant in ZC3HC1 Associated With Protection From Coronary Artery Disease

Abstract

Background— Although virtually all coronary artery disease associated single-nucleotide polymorphisms identified by genome-wide association studies (GWAS) are in noncoding regions of the genome, a common polymorphism in ZC3HC1 (rs11556924), resulting in an arginine (Arg) to histidine (His) substitution in its encoded protein, NIPA (Nuclear Interacting Partner of Anaplastic Lyphoma Kinase) is linked to a protection from coronary artery disease. NIPA plays a role in cell cycle progression, but the functional consequences of this polymorphism have not been established. Methods and Results— Here we demonstrate that total ZC3HC1 expression in whole blood is similar across genotypes, despite expression being slightly biased toward the risk allele in heterozygotes. At the protein level, the protective His363 NIPA variant exhibits increased phosphorylation of a critical serine residue (Ser354) and higher protein expression as compared with the Arg363 variant. Binding experiments indicate that neither SKP1 (S-phase kinase-associated protein 1) nor CCNB1 binding were affected by the polymorphism. Despite similar nuclear distribution, NIPA His363 exhibits greater nuclear mobility. NIPA suppression results in a modest reduction of proliferation in vascular smooth muscle cells, but given low proliferative capacity, a significant effect of the variant was not noted. By contrast, we demonstrate that the protective variant reduces cell proliferation in HeLa cells. Conclusions— These findings extend the genetic association between rs11556924 and coronary artery disease risk by characterizing its effects on the encoded protein, NIPA. The resulting amino acid change Arg363His is associated with increased expression and nuclear mobility, as well as lower rates of cell growth in HeLa cells, further supporting a role for cell proliferation in atherosclerosis and its clinical consequences.

ZC3HC1 Is a Novel Inherent Component of the Nuclear Basket, Resident in a State of Reciprocal Dependence with TPR

“…In fact, in the course of our experiments, initially all performed with cells in which ZC3HC1 had been knocked down by RNAi, we had realised that this protein was most likely neither essential for cell cycle progression nor cellular housekeeping activities in interphase (Figure S14). However, even though these early results turned out to be in accordance with the finding that mice in which the ZC3HC1 gene had been knocked out by homologous recombination are viable [104,105], the alleged role for ZC3HC1 in regulating cell cycle progression via controlling cellular levels of CCNB1 was recurrently reported to manifest itself in a range of aneuploid human tumour cell lines and other types of immortalised human cells [104,[106][107][108]. HeLa cells were actually suggested to be amongst those cells in which such ZC3HC1 knockdown phenotypes were most pronounced [104], ranging from most cells of a ZC3HC1-deficient HeLa population being no longer capable of cell cycle progression and entering apoptosis instead [104], to hyperproliferation of another ZC3HC1-deficient HeLa population and conspicuous increase in its cell numbers [108], when using the one or other ZC3HC1 siRNA.…”

Deficiency of ZC3HC1 modulates vascular smooth muscle cell phenotype and increases neointima formation

“…Because eQTL effects are often cell type specific, we tested whether an eQTL was present in aortic SMCs from 151 heart transplant donors 16 , finding that SMCs from donors carrying the rs11556924-T allele have lower ZC3HC1 expression and migrate faster than SMCs from donors carrying the rs11556924-C allele. The absence of a significant association between ZC3HC1 expression and rs11556924 in blood samples 9 , monocytes/macrophages 28 and aortic endothelial cells 29 suggests that the regulatory impact of the variant at the ZC3HC1 locus is specific to SMCs.…”

“…The ubiquitously expressed ZC3HC1 gene encodes the cell cycle protein NIPA, which has been found first to be associated with CAD in several independent GWASs [2][3][4][5][6][7][8] . The CADassociated rs11556924-C/T SNP in ZC3HC1 is functional, leading to an amino acid change from arginine to histidine (p.Arg363His) 4,7,9 . Analysis of the publicly available GTEx dataset eQTL (V8) showed that the genetic variant rs11556924-T results in reduced ZC3HC1 gene expression in heart (atrial appendage and left ventricle) and skin samples.…”

“…conditions NIPA, an essential part of the SCF-type E3 ubiquitin ligase complex, activates the degradation of cyclin B1, which reduces SMC migration and proliferation (left). However, ZC3HC1 modulation (right), by reducing the amount of NIPA or increasing NIPA phosphorylation 7,9 , is accompanied by cyclin B1 accumulation and increased SMC migration and proliferation. This in turn leads to injury-induced neointima formation and progression of early-stage atherosclerosis 34,[52][53][54] .…”

2018 George Lyman Duff Memorial Lecture

“…In functional studies, we demonstrated that the ensuing cellular phenotype of the protective variant is one of increased NIPA expression and nuclear mobility and lower rates of cell growth. 67…”