A Polynesian-specific SLC22A3 variant associates with low plasma lipoprotein(a) concentrations independent of apo(a) isoform size in males

Lipoprotein(a) (Lp(a)) is a lipoprotein particle that is well established as an independent causal risk factor for multiple cardiovascular diseases (CVDs) [1–4]. Lp(a) is composed of a low-density lipoprotein (LDL)-like particle with the apolipoprotein B100 (apoB100) component covalently attached to apolipoprotein(a) (apo(a)) via a disulphide bond [5]. Plasma Lp(a) concentrations vary enormously between individuals as well as among different ethnic groups [6–8]. Lp(a) concentration is not affected by age, sex, diet, or life styles. There is compelling evidence that genetic factors have the biggest impact on variations in plasma Lp(a) concentrations [1,7,9]. Large-scale genome-wide association studies (GWASs) have identified sets of single-nucleotide polymorphisms (SNPs) (largely in the LPA gene which codes for apo(a)) that are significantly associated with plasma Lp(a) concentrations [10–14]. Several LPA SNPs that associate with elevated Lp(a) concentrations increase the risk of CVDs with some related to poorer outcomes, such as cardiac surgery and severe calcified lesions [14–16]. Apo(a) contains 10 types of Kringle domains, KIV1-KIV10 [17]. Among these, copy number variations in the KIV2 domain determine the size of the LPA gene and correspondingly the size of the apo(a) protein. LPA KIV2 copy numbers are inversely correlated with Lp(a) concentrations [7]. Smaller isoforms of LPA encode smaller apo(a) proteins that are more efficiently secreted from the liver, resulting in higher Lp(a) concentrations in the circulation [18].

A range of SNPs in the LPA gene and adjacent genes have been identified that associate with Lp(a) concentration, one such adjacent gene being the Solute Carrier Family 22 Member 3 (SLC22A3) gene [10,11,19–21]. SLC22A3 codes for a transmembrane protein transporter also known as organic cation transporter 3 (OCT3), which mediates the bidirectional transport of numerous endogenous molecules, bioamines, and drugs such as histamine, serotonin, dopamine, and metformin across the cell membrane [22–24]. The SLC22A3 transporter is widely expressed across different tissues, including heart, skeletal muscle, and aorta [25]. Polymorphisms in the SLC22A3-LPAL2-LPA gene cluster has also been identified associated with CVDs [26–28].

The rs8187715 coding variant (c.131C>T, p.Thr44Met) in SLC22A3 gene was identified in a study which noted that all carriers of the genetic variant were of Polynesian [24] and genome data bases show that this SNP is very rare in other populations [29] (data base accessed in March 2024). Therefore, the objective of the present study was to investigate this Polynesian population specific SLC22A3 genetic variant with the plasma concentration of Lp(a) in a cohort of healthy young male participants.

We hypothesised that the rs8187715 coding variant would act as a biomarker for Lp(a) levels that might be used in future precision medicine strategies to predict or treat cardiovascular disease in Māori and other Polynesian peoples.

Genotype frequency and associations with metabolic parameters were derived from the Māori and Pacific metabolic deep phenotyping cohort that we have described recently [30,31]. The present study recruited young men who had no diagnosed illness and who were not on medications as the aim was to identify penetrant effects of gene variants on metabolic health. A range of metabolic parameters were measured in the present study although family history of cardiovascular disease was not recorded. All participants provided written informed consent for the collection of samples and subsequent analysis. Genotyping was carried out using the Sequenom MassArray as previously described [30,31]. All association analyses were carried out using the SPSS version 29.0.0.0 software as previously described with analyses adjusted for different co-variation factors, including age, BMI, ethnicity, blood lipids, and metabolic variables (HbA1c and blood pressure) [30,31]. Ethical approval was given by the New Zealand Health and Disability Ethics Committee (17STH79). This cohort were all male, aged 18–45 years, identify as being of Māori and/or Pacific descent based on self-reported grandparent ethnicity (with at least one Māori and/or Pacific grandparent), and with BMI between 20 and 50 kg/m². Pairwise linkage disequilibrium measures specific to Māori and Pacific populations were obtained for all genetic variants surrounding rs8187715 (±500 kb) using bcftools and PLINK using low-pass sequencing data from Māori and Pacific people available to us [32]. Frequency of this variant in other populations around the world was assessed using the gnomAD database [29] (data accessed on March 2024).

Blood samples were collected after an overnight fast. Lipid parameters were measured by standard laboratory techniques at the Liggins Institute, University of Auckland. Lp(a) concentrations were determined using the Tina-quant Lipoprotein(a) Gen.2 (Roche, cat. 05852625) assay, a method assessing the number of Lp(a) particles independent of the apo(a) isoform size. Apolipoprotein A1 and apolipoprotein B concentrations were measured using the Apolipoprotein A1 and Apolipoprotein B assays (Roche Diagnostics, cat. 03032566 and 03032574, respectively). LDL-C, HDL-C, cholesterol, and triglycerides were measured using analysing kits from Roche Diagnostics (cat. 07005717190, 07528566190, 03039773190, and 20767107322, respectively). All these assays were performed on the Cobas c311 platform.

The SPSS Statistics version 29.0.0.0 for Mac (SPSS Inc., U.S.A.) and GraphPad Prism 10 (GraphPad Software Inc., U.S.A.) were used for the statistical analyses. Depending on the nature of the data, results are presented as mean ± standard deviation or median with interquartile range (IQR) in the case of Lp(a). Linear regression model with variety of correction factors was used for the association analysis. We used a cut off of 0.05 for statistical significance. All reported P-values were 2-sided. There were 5 KIV2 copy number variations (CNVs) data, 2 blood pressure data missing from the non-carrier group and 15 CREBRF rs373863828 genotype data missing from the whole cohort.

The basic metabolic characteristics of the study cohort are presented in Table 1. In our study, a total of 302 participants were included, of which 265 had a CC (SLC22A3 Thr44) genotype and 37 had one or two A alleles (SLC22A3 Met44) for rs8187715. Due to the smaller numbers of participants having both risk alleles (AA, n=9), we combined the homozygous genotypes (AA) with the heterozygous genotype (AC) labelled as AX genotype group. The CC and AX genotype groups were similar in body weight, age, height, and BMI. There was no association of the variant with common metabolic parameters including body mass index (BMI), weight, height, blood pressure, or HbA1c. Similarly, there was no association of the variant with blood lipid parameters, including HDL-C, LDL-C, cholesterol, triglycerides, or apolipoprotein A1 and apolipoprotein B.

The median value of Lp(a) concentration was significantly lower in the carrier AX group than in the non-carrier CC group (7.60 nmol/L, IQR: 5.50–12.10 versus 10.60 nmol/L, IQR: 5.40–41.00, P<0.05) (Table 2 and visualised in Figure 1A). There was no difference in copy number of KIV2 repeats in the LPA gene between carriers and non-carriers of the SLC22A3 variant (Figure 1B). When adjusted to KIV2 CNVs, the association of rs8187715 with Lp(a) concentrations remained statistically significant (Table 2). Lp(a) concentrations were inversely correlated to the KIV2 CNV in both CC and AX groups (Figure 1C,D). The difference in Lp(a) concentrations also remained significant when adjusted for metabolic parameters, including age, BMI, metabolic and blood lipid variables, HDL-C, cholesterol, LDL-C, triglyceride, HbA1c, systolic blood pressure, diastolic blood pressure, plasma apo(A1), and apo(B) concentrations as well as genetic admixture (Table 2) and log transformation did not affect this (Table 3).

Genetic variants that associate with LPA expression (cis-eQTL) extend into SLC22A3 [33]. With the availability of genome-wide sequence data for Māori and Pacific people, we also explored whether another variant in linkage disquilibrium (LD) with rs8187715 could be a causal regulatory variant. Ten additional variants were identified as in LD (R² > 0.8) with rs8187715; however, all were non-coding with very little evidence for disruption of regulatory elements. Data from ENCODE indicates that rs8187715 does in fact overlap a large number of ChIP binding sites for transcription factors that are expressed in liver. From this, we conclude that rs8187715 is the most likely causal variant for Lp(a) levels; however, the molecular mechanism of control of Lp(a) levels is unclear and requires further investigation.

We also examined the association of Lp(a) with the CREBRF rs373863828 gene variant that is unique to Māori and Pacific people, since this has been associated with factors that can alter factors regulating CVD risk [34]. There is no association between the Lp(a) levels and this variant (Table 4).

Several studies have shown that there are significant differences in plasma Lp(a) concentrations between ethnic groups [6–8]. Beyond the LPA gene, which has been heavily studied for its strong influence on Lp(a) concentrations [10,12,16,28], SNPs in SLC22A3 gene have been associated with plasma Lp(a) concentrations with majority of them being non-coding variants [11,19,35,36]. The present study demonstrates that the SLC22A3 coding variant rs8187715 that is exclusively found in Polynesian peoples is significantly associated with low circulating concentrations of Lp(a) in a cohort of healthy young Polynesian males.

It is known apo(a) isoform size impacts Lp(a) concentrations due to reductions in producing functional particles as apo(a) molecular mass increases [18,36]. Thus, an inverse correlation between Lp(a) size and the concentration of particles of those sizes is observed [36–38]. Apo(a) isoform size is dictated by the variation in the number of KIV2 repeats in the alleles of the LPA gene [7,39] SLC22A3 gene is adjacent to the LPA gene, one possible explanation for the reduced concentration of Lp(a) in carriers of the SLC22A3 variant is that this variant is linked to alleles of the LPA gene with high copy numbers of KIV2. However, our data indicates there is no difference in copy number variations of KIV2 repeats between carriers and non-carriers of the SLC22A3 variant (Figure 1B). Another hypothesis would be that the SLC22A3 variant might functionally affect apo(a) isoforms differentially by size. However, this does not appear to be the case as plasma Lp(a) concentrations were inversely correlated to the KIV2 CNV in both CC and AX genotype groups. When adjusted to KIV2 CNVs, the association of rs8187715 with Lp(a) concentrations remained statistically significant. Therefore, the association of rs8187715 with low plasma Lp(a) concentration is independent of apo(a) isoform size.

Since Europeans are known to be an ethnic group with a tendency for higher concentrations of Lp(a) than that of southeast Asians [8] where Polynesian populations derive their ancestry [40,41] there are possibilities that the higher plasma concentrations of Lp(a) in non-carriers of rs8187715 variant could be due to ingression of European alleles. However, the significance of the difference in plasma Lp(a) concentrations remained when adjusted for ethnicity.

However, the findings of the present study identify a new biomarker for CVD risk in at least Māori and Pacific males that has not been identified in previous studies in other parts of the world. These findings add to a recent study that identified a coding variant in Cholesterol Ester Transfer Protein (CETP) that is unique to Māori and Pacific peoples and is associated with alterations in HDL levels [42]. This highlights the importance of bespoke genomic studies in isolated populations such as this if we are to achieve equitable outcomes for CVD risk assessment across the world.

Future studies will be required to see whether similar correlation between Lp(a) and this gene variant also occur in women. The findings have potential to be used in future to develop ethnic specific CVD risk and treatment guidelines.

Availability of genetic data used in this study is subject to indigenous data sovereignty protocols.

The authors declare that there are no competing interests associated with the manuscript.

This study was funded by the Health Research Council of New Zealand programme [grant number 18-681] and the Maurice Wilkins Centre for Molecular Biodiscovery (MWC).

Open access for this article was enabled by the participation of The University of Auckland in an all-inclusive Read & Publish agreement with Portland Press and the Biochemical Society under a transformative agreement with CAUL.

Qian Wang: Conceptualization, Formal analysis, Investigation, Writing—original draft, Writing—review & editing. Sally McCormick: Formal analysis, Writing—original draft, Writing—review & editing. Megan P. Leask: Formal analysis, Writing—original draft, Writing—review & editing. Huti Watson: Data curation, Writing—review & editing. Conor O'Sullivan: Data curation, Investigation, Writing—review & editing. Jeremy D. Krebs: Resources, Data curation, Writing—review & editing. Rosemary Hall: Data curation, Writing—review & editing. Patricia Whitfield: Data curation, Investigation, Writing—review & editing. Troy L. Merry: Conceptualization, Data curation, Funding acquisition, Writing—original draft, Writing—review & editing. Rinki Murphy: Data curation, Writing—review & editing. Peter R. Shepherd: Conceptualization, Formal analysis, Funding acquisition, Writing—original draft, Writing—review & editing.

He mihi nui tēnei ki ngā kai tuku taonga (we thank study participants for their time and tissue samples). We also thank Professor Tony Merriman for support in relation to this project.

apo(a)

apolipoprotein(a)

apoB100

apolipoprotein B100

CNV

copy number variation

CVD

cardiovascular disease

GWAS

genome-wide association study

HDL-C

high-density lipid-cholesterol

IQR

interquartile range

LD

linkage disquilibrium

LDL

low-density lipoprotein

Lp(a)

lipoprotein(a)

OCT3

organic cation transporter 3

SLC22A3

Solute Carrier Family 22 Member 3

SNP

single-nucleotide polymorphism