Investigation of a nonsense mutation located in the complex KIV-2 copy number variation region of apolipoprotein(a) in 10,910 individuals

Abstract

Background The concentrations of the highly atherogenic lipoprotein(a) [Lp(a)] are mainly genetically determined by the LPA gene locus. However, up to 70% of the coding sequence is located in the complex so-called kringle IV type 2 (KIV-2) copy number variation, a region hardly accessible by common genotyping and sequencing technologies. Despite its size, little is known about genetic variants in this complex region. The R21X variant is a functional variant located in this region, but it has never been analyzed in large cohorts. Methods We typed R21X in 10,910 individuals from three European populations using a newly developed high-throughput allele-specific qPCR assay. R21X allelic location was determined by separating the LPA alleles using pulsed-field gel electrophoresis (PFGE) and typing them separately. Using GWAS data, we identified a proxy SNP located outside of the KIV-2. Linkage disequilibrium was determined both statistically and by long-range haplotyping using PFGE. Worldwide frequencies were determined by reanalyzing the sequencing data of the 1000 Genomes Project with a dedicated pipeline. Results R21X carriers (frequency 0.016–0.021) showed significantly lower mean Lp(a) concentrations (− 11.7 mg/dL [− 15.5; − 7.82], p = 3.39e−32). The variant is located mostly on medium-sized LPA alleles. In the 1000 Genome data, R21X mostly occurs in Europeans and South Asians, is absent in Africans, and shows varying frequencies in South American populations (0 to 0.022). Of note, the best proxy SNP was another LPA null mutation (rs41272114, D ′ = 0.958, R 2 = 0.281). D ′ was very high in all 1000G populations (0.986–0.996), although rs41272114 frequency varies considerably (0–0.182). Co-localization of both null mutations on the same allele was confirmed by PFGE-based long-range haplotyping. Conclusions We performed the largest epidemiological study on an LPA KIV-2 variant so far, showing that it is possible to assess LPA KIV-2 mutations on a large scale. Surprisingly, in all analyzed populations, R21X was located on the same haplotype as the splice mutation rs41272114, creating “double-null” LPA alleles. Despite being a nonsense variant, the R21X status does not provide additional information beyond the rs41272114 genotype. This has important implications for studies using LPA loss-of-function mutations as genetic instruments and emphasizes the complexity of LPA genetics.

Genome-Wide Characterization of a Highly Penetrant Form of Hyperlipoprotein(a)emia Associated With Genetically Elevated Cardiovascular Risk

“…1,13 In addition, specific SNP haplotypes associate with Lp(a) concentrations that can be much lower or much higher than what would be expected from the sole size of the isoforms. 11,12 Some examples of such variants have been reported, [14][15][16] but many more probably exist. Genome-wide association studies (GWAS) have identified hundreds of variants associated with Lp(a) levels.…”

Protein-coding repeat polymorphisms strongly shape diverse human phenotypes

“…3). Stepwise conditional analyses within this framework identified an allelic series including 18 protein-altering variants and 3 variants in the 5’ UTR of LPA that each appeared likely to causally influence Lp(a) levels (based on achieving top or near-top association strengths in successive steps of analysis); 2 additional protein-truncating variants within KIV-2 exons had effects mostly masked by linkage disequilibrium with a canonical splice site variant 64 (Supplementary Table 3 and Supplementary Note). Second, we created an intuitive model that accurately predicted Lp(a) as a sum of allelic contributions determined by KIV-2 length and the combination of alleles of the 23 likely-causal LPA SNP and indel variants carried on each haplotype.…”

“…3). Stepwise conditional analyses within this framework identified an allelic series including 18 protein-altering variants and 3 variants in the 5' UTR of LPA that each appeared likely to causally influence Lp(a) levels (based on achieving top or near-top association strengths in successive steps of analysis); 2 additional protein-truncating variants within KIV-2 exons had effects mostly masked by linkage disequilibrium with a canonical splice site variant 64 (Supplementary Table 3 We compared the above model of Lp(a) to two simpler models corresponding to standard analyses: a model predicting Lp(a) from KIV-2 length alone (used in Fig. 2b) and a linear model a change in the log-ratio of observed vs. predicted Lp(a); if so, we exponentiated this change (and its 95% CI) and subtracted 1 to obtain non-log-scale changes in Lp(a).…”

Lipoprotein(a)

“…It is a likely causal SNP resulting in a nonsense mutation, which leads to a truncated protein that is rapidly degraded (Parson et al 2004). We recently developed a highly sensitive allele-specific qPCR assay and genotyped R21X in 10,910 individuals from three populations and showed that R21X carriers have significantly lower (À11.7 mg/dL) mean Lp(a) concentrations (Di Maio et al 2020). A study by Morgan and colleagues investigated two very rare variants, R990Q (rs41259144) located in the KIV type 4 and R1771C (rs139145675) located in the kringle V which were present in four null Lp(a) individuals.…”

“…Later approaches that investigated LPA variants on the DNA level either by the number of KIV repeats identified by pulsed-field gel electrophoresis or by the sum of KIV repeats of the two alleles by quantitative PCR or by the investigation of SNPs that are associated with high Lp(a) concentrations revealed the same finding (Clarke et al 2009;Kamstrup et al 2009;Kraft et al 1996). On the other hand, genetic variants that are associated with low Lp(a) concentrations are obviously protective from CVD (Coassin et al 2017;Di Maio et al 2020;Lim et al 2014). These strong associations make Lp(a) probably the most important genetic risk factor for CVD if we keep in mind the high frequency of small apo(a) isoforms or variants which go along with high Lp(a) concentrations in the population (Kronenberg 2016b).…”