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. 2021 Aug 3;78(5):437-449.
doi: 10.1016/j.jacc.2021.05.037.

Frequent LPA KIV-2 Variants Lower Lipoprotein(a) Concentrations and Protect Against Coronary Artery Disease

Johanna F Schachtl-Riess  1 , Azin Kheirkhah  1 , Rebecca Grüneis  1 , Silvia Di Maio  1 , Sebastian Schoenherr  1 , Gertraud Streiter  1 , Jamie Lee Losso  1 , Bernhard Paulweber  2 , Kai-Uwe Eckardt  3 , Anna Köttgen  4 , Claudia Lamina  1 , Florian Kronenberg  5 , Stefan Coassin  6 ; GCKD Investigators
Collaborators, Affiliations

Frequent LPA KIV-2 Variants Lower Lipoprotein(a) Concentrations and Protect Against Coronary Artery Disease

Johanna F Schachtl-Riess et al. J Am Coll Cardiol. .

Abstract

Background: Lipoprotein(a) (Lp(a)) concentrations are a major independent risk factor for coronary artery disease (CAD) and are mainly determined by variation in LPA. Up to 70% of the LPA coding sequence is located in the hypervariable kringle IV type 2 (KIV-2) region. It is hardly accessible by conventional technologies, but may contain functional variants.

Objectives: This study sought to investigate the new, very frequent splicing variant KIV-2 4733G>A on Lp(a) and CAD.

Methods: We genotyped 4733G>A in the GCKD (German Chronic Kidney Disease) study (n = 4,673) by allele-specific polymerase chain reaction, performed minigene assays, identified proxy single nucleotide polymorphisms and used them to characterize its effect on CAD by survival analysis in UK Biobank (n = 440,234). Frequencies in ethnic groups were assessed in the 1000 Genomes Project.

Results: The 4733G>A variant (38.2% carrier frequency) was found in most isoform sizes. It reduces allelic expression without abolishing protein production, lowers Lp(a) by 13.6 mg/dL (95% CI: 12.5-14.7; P < 0.0001) and is the strongest variance-explaining factor after the smaller isoform. Splicing of minigenes was modified. Compound heterozygosity (4.6% of the population) for 4733G>A and 4925G>A, another KIV-2 splicing mutation, reduces Lp(a) by 31.8 mg/dL and most importantly narrows the interquartile range by 9-fold (from 42.1 to 4.6 mg/dL) when compared to the wild type. In UK Biobank 4733G>A alone and compound heterozygosity with 4925G>A reduced HR for CAD by 9% (95% CI: 7%-11%) and 12% (95% CI: 7%-16%) (both P < 0.001). Frequencies in ethnicities differ notably.

Conclusions: Functional variants in the previously inaccessible LPA KIV-2 region cooperate in determining Lp(a) variance and CAD risk. Even a moderate but lifelong genetic Lp(a) reduction translates to a noticeable CAD risk reduction.

Keywords: Mendelian randomization; cardiovascular disease; cohort study; copy number variation; genetic variability; lipoprotein(a).

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Conflict of interest statement

Funding Support and Author Disclosures The study was supported by the Austrian Science Fund (FWF) project P31458-B34 and the D•A•CH Advancement Award Lipidology 2015 (supported by the Christine Katharine Schmitz Foundation) of the D•A•CH-Society for Prevention of Cardiovascular Diseases (to S.C.). The GCKD study is supported by the German Ministry of Education and Research (Bundesministerium für Bildung und Forschung, grants 01ER 0804, 01ER 0818, 01ER 0819, 01ER 0820, and 01ER 0821) and the KfH Foundation for Preventive Medicine (Kuratorium für Heimdialyse und Nierentransplantation e.V.–Stiftung Präventivmedizin) and corporate sponsors (see the GCKD website). Ms Schachtl-Riess has received support from the Dr Legerlotz Foundation. Dr Köttgen has received support from the German Research Foundation (grant KO 3598/5-1). Dr Kronenberg has received support from the Austrian Science Fund (project W-1253DK HOROS); has received lecture fees from Novartis, Amgen, and Kaneka; and has served on the advisory boards of Amgen and Kaneka. Drs Kronenberg and Coassin have received support from the Lipoprotein(a) Center And Research InstitutE [Lp(a)CARE] for their lipoprotein(a) research. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose.

Figures

Graphical abstract
Graphical abstract. CENTRAL ILLUSTRATION Frequent LPA KIV-2 Variants Lower Lipoprotein(a) and Coronary Artery Disease Risk
The frequent LPA kringle IV type 2 (KIV-2) variant 4733G>A and compound heterozygosity for 4733G>A and 4925G>A are associated with lower lipoprotein(a) (Lp(a)) concentrations and a lower risk for coronary artery disease (CAD).
Figure 1
Figure 1. Lp(a) Concentrations by Carrier Status and Isoform 1 in GCKD
The 4733G>A variant lowers lipoprotein(a) (Lp(a)) over the complete isoform range. Scale restricted to <210 mg/dL for better representation. Isoform grouping was done to have $20 in each group. Number of carriers per group are given in Supplemental Table 6. The same figure restricted to individuals that do not carry 4925G>A is shown in Supplemental Figure 3. GCKD = German Chronic Kidney Disease study; WB = Western blot.
Figure 2
Figure 2. Distribution of Isoform 1 by 4733G>A Carrier Status in GCKD
The 4733G>A variant is predominantly expressed in isoforms 24 to 33. Plot is restricted to individuals who express 2 isoforms in plasma. Abbreviations as in Figure 1.
Figure 3
Figure 3. KIV-2 4733G>A Modifies Splicing, Causing Deletion of 1 Structure-Determining Cysteine
(A) Representative gel of the minigene reverse-transcriptase polymerase chain reaction products (wild type [WT], mutant [mut], · puromycin) from 5 biological replicates with 2 technical replicates each (Supplemental Figure 6), showing different splicing behavior as described in the text. (B) Kringle IV type 2 (KIV-2) structure according to Guevara et al (37) with the amino acids and the disulfide bond (magenta) abolished by activation of splice site 3 (Supplemental Figure 8). bp = base pair(s).
Figure 4
Figure 4. Lp(a) Concentrations in Carriers of 4733G>A and 4925G>A in GCKD
(A) The graph shows Lp(a) concentrations in the 4 groups. (B) The graph shows Lp(a) concentrations that are additionally grouped by the isoform 1. Both variants lower Lp(a) but virtually no variability is left in the double carriers. Isoform grouping was done to have $5 per group. Scale restricted to <210 mg/dL for better representation. Numbers per isoform stratum are given in Supplemental Table 8. IQR = interquartile range; other abbreviations as in Figure 1.
Figure 5
Figure 5. HR for CAD Riskin UK Biobank
(A) Model is adjusted for sex. (B) Model is adjusted for sex and inverse-normal transformed lipoprotein(a) (Lp(a)) concentration. Plot is restricted to individuals with Lp(a) measurements available. Status refers to the presence (yes) or absence (no) of the proxy single nucleotide polymorphisms (SNPs) rs6938647/rs75692336 for 4733G>A/4925G>A. The noncarriers of proxy SNPs of both variants make up the reference group. Age is taken as time scale (model with time-on-study shown in Supplemental Figure 10). HR for Lp(a) concentration is given for a 1-unit increase of the inverse-normal transformed Lp(a) concentration. CAD = coronary artery disease.
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Comment in

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