The effect of LPA Thr3888Pro on lipoprotein(a) and coronary artery disease is modified by the LPA KIV-2 variant 4925G>A

Background and aims High lipoprotein(a) [Lp(a)] concentrations are associated with increased coronary artery disease (CAD) risk. Lp(a) is regulated mainly genetically by the LPA gene but involved genetic variants have not been fully elucidated. Improved understanding of the entanglements of genetic Lp(a) regulation may enhance genetic prediction of Lp(a) and CAD risk. We investigated an interaction between the well-known LPA missense SNP rs41272110 (known as Thr3888Pro) and the frequent LPA splicing mutation KIV-2 4925G>A. Methods Effects on Lp(a) concentrations were investigated by multiple quantile regression in the German Chronic Kidney Disease (GCKD) study, KORA-F3 and KORA-F4 (ntotal = 10,405) as well as in the UK Biobank (UKB) 200k exome dataset (n = 173,878). The impact of the interaction on CAD risk was assessed by survival analysis in UKB. Results We observed a significant SNP-SNP interaction in all studies (p = 1.26e-05 to 3.03e-04). In quantile regression analysis, rs41272110 as a predictor shows no impact on Lp(a) (β = −0.06 [-0.79; 0.68], p = 0.879), but in a joint model including both SNPs as predictors, rs41272110 is associated with markedly higher Lp(a) (β = +9.40 mg/dL [6.45; 12.34], p = 4.07e-10). Similarly, rs41272110 shows no effect on CAD in UKB (HR = 1.01 [0.97; 1.04], p = 0.731), while rs41272110 carriers not carrying 4925G>A show an increased CAD risk (HR = 1.10 [1.04; 1.16], p = 6.9e-04). This group corresponds to 4% of the population. Adjustment for apolipoprotein(a) isoforms further modified the effect estimates markedly. Conclusions This work emphasizes the complexity of the genetic regulation of Lp(a) and the importance to account for genetic subgroups in Lp(a) association studies and when interpreting genetic cardiovascular risk profiles.


Description of the GCKD study
The GCKD [1,2] study includes German participants from nine recruiting centers suffering of moderate chronic kidney disease (CKD). Moderate CKD is classified as CKD Stage 3 with an estimated glomerular filtration rate (eGFR) according to the CKD_EPI equation [3] of 30-60 mL/min per 1.73 m 2 or overt proteinuria and eGFR >60 mL/min per 1.73 m 2 . Overt proteinuria shows an albumin to creatinine ration of >300 mg/g or a protein to creatinine ratio in 24h urine of >500 mg/g. Individuals with active malignancy, NYHA IV heart failure, renal or any other transplantation, non-Caucasian origin and legal attendance were excluded. Study approval was done by the review boards of the participating institutions and informed consent was obtained from all participants.

Description of the KORA F3 and F4 studies
The KORA [4]

Lp(a) phenotyping
Lp(a) concentration was determined in mg/dL by ELISA [5,6]. A polyclonal affinity-purified rabbit anti-human apo(a) antibody was used for coating and a horseradish peroxidase-conjugated monoclonal anti-apo(a) antibody 1A2 for detection [7]. OD was quantified on two dilutions per sample (1:150 and 1:1500) and measurements within the linear range of the 7-point standard curve were accepted.
Apo(a) isoforms were assessed by Western blotting [6,8]. 150 ng Lp(a) were loaded and separated on a 1.46% agarose gel with 0.08% SDS for 18 h at 0.04 A constant current. A size standard containing apo(a) isoform 13, 19, 23, 27 and 35 KIV repeats (validated by fiber-FISH [9]) was applied in every seventh well of the gel. The gel was semi-dry electro-blotted to a PVDF membrane. The membrane was blocked with 1% BSA, 85 mM NaCl, 10 mM TRIS, 0.2% Triton X-100 for 30 min at 37°C and incubated with horseradish-peroxidase-conjugated 1A2 antibody. Signals were detected with ECL substrate (WesternBright Chemilumineszenz Spray, Biozym, Vienna, AT) and recorded on autoradiography films (Amersham Hyperfilm™ ECL™, GE Healthcare, Chicago, IL, USA). A detailed protocol for ELISA and Western blotting has been published in [8].

Analysis of UK Biobank data
Genotypes of rs41272110 were retrieved from microarray genotyping data and 4925G>A carrier status was retrieved from whole exome sequencing data (n=199,126) using sequencing data reanalysis strategies detailed before [10]. Sequencing data were downloaded as CRAM data (UKB Data Field 23153) and all reads from the LPA KIV-2 region were extracted as defined in the bed files of Ebbert et al [10] (https://github.com/mebbert/Dark_and_Camouflaged_genes). The extracted sequencing reads were realigned to a single KIV-2 copy number as reference [10][11][12] and 4925G>A was called using a standard second-generation variant caller (https://github.com/seppinho/mutserve) capable to detect variants down to 1% mutation level [12].
Data were restricted to Caucasians (British, Irish or any other white ethnic background) with available exome data for 4925G>A and genotype data for rs41272110 (n=186,088). The impact of 4925G>A and rs41272110 on Lp(a) concentrations was investigated by quantile regression in 173,878 participants.
The first occurrence was provided by UKB by mapping self-report at any assessment centre, inpatient hospital data, primary care or death record data. Hazard ratio for CAD was estimated as a function of the carrier status of the two SNPs independently from each other, as well as a joint model (one variant adjusted for the other) and additionally adjusted for sex (n=186,088). Age was used as timescale, meaning that the observation period starts from the year of birth and censored the data as of 1 st of January 2020, including 13,335 incident CAD events, independently of their SNPs carrier status. One individual was excluded due to an implausible date of CAD event. UKB analyses were performed in R version 3.6.3 and the R package survival was used for survival analysis. only, double carriers) in GCKD, KORA F3 and KORA F4. Compared to wild type rs41272110 is significantly associated with increased Lp(a) concentrations, whereas no difference was observed between 4925G>A and double carriers. Since 4925G>A is associated with a defined isoform range [11], its effect becomes appreciable only after isoform stratification (Main Figure 1 Table I