Abstract
Elevated plasma lipoprotein(a) [Lp(a)] is a major genetically determined risk factor for the
progression of cardiovascular disease. Plasma Lp(a) levels occupy a wide range within the
population, and are predominantly determined by a repeat polymorphism of the KIV-2
domain in LPA, resulting in variable allele sizes of the apolipoprotein(a) [apo(a)] protein.
While a significant inverse correlation exists between the KIV-2 copy number and Lp(a)
levels, some variation in plasma Lp(a) remains unexplained by the size polymorphism.
Additional genetic variation in the form of single nucleotide polymorphisms (SNPs) in LPA
are known to influence Lp(a) levels. A significant subset of the population exhibit no plasma
Lp(a) whatsoever and are termed ‘null’ individuals. So far only two nonsense mutations that
truncate the apo(a) protein are known to cause a null phenotype. It is possible that
nonsynonymous variants also contribute to the null phenotype but the repetitive nature of the
LPA gene makes it difficult to characterize these.
A collection of nonsynonymous SNPs identified in a next-generation sequencing study of the
LPA gene associated with either a null or very low Lp(a) phenotype were investigated for
their potential effects on apo(a) structure. An in silico analysis of the SNPs was performed
using either existing protein structures or homology models of apo(a) kringle domains to
determine whether SNPs were having potential effects on apo(a) structure. Additionally, an
analysis of SNPs associated with null phenotype of the homologous protein plasminogen was
performed using plasminogen kringle structures in order to determine whether these variants
were in similar regions to LPA SNPs.
Two LPA SNPs R990Q and R1771C were identified in individuals with null Lp(a) and were
predicted to potentially abolish a number of biochemical interactions within the kringles they
appeared in. The wildtype arginine residues altered in R990Q and R1771C were also found to
be homologous to each other and conserved between all apo(a) kringles. The plasminogen
analysis revealed that four variants associated with plasminogen deficiency were homologous
to each other as well as the R1771C and R990Q SNPs. A structural analysis on two of the
plasminogen variants also indicated they ablate multiple biochemical interactions within the
protein.
An in vitro analysis was then performed to characterize the effect of the R1771C LPA SNP in
a cell culture model. HEK 293 and human hepatoma cell lines were transiently transfected
with GFP-tagged wildtype and R1771C apo(a) cDNA constructs and the synthesis and
secretion patterns of the expressed apo(a) proteins analysed. Site-directed mutagenesis was
used to construct a mutant apo(a) vector corresponding to the R1771C SNP. The R1771C
apo(a) protein was initially shown to be underexpressed in cell lysate compared to wildtype
apo(a). However, subsequent results showed the R1771C apo(a) was present in cells as a low
molecular weight precursor that is unable to mature to a higher molecular weight form of
apo(a) that is secreted from cells. Immunoprecipitation experiments on cellular media
confirmed that the R1771C apo(a) protein harboured a severe secretion deficit compared to
wildtype apo(a). These results provide evidence that the R1771C SNP in LPA may be
contributing to null allele phenotypes.
To our knowledge this is the first nonsynonymous variant reported to be associated with a
null apo(a) phenotype. It is possible that other nonsynonymous variants underlie unsecreted
apo(a) proteins.