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Two independent coding variants in the apolipoprotein L1 gene (APOL1), G1 and G2, strongly associate with nephropathy in African Americans; associations with cardiovascular disease are more controversial. Although APOL1 binds plasma high-density lipoproteins (HDLs), data on APOL1 risk variant associations with HDL subfractions are sparse.
Two APOL1 G1 single nucleotide polymorphisms and the G2 insertion/deletion polymorphism were genotyped in 2010 Reasons for Geographic and Racial Differences in Stroke (REGARDS) Study participants with nuclear magnetic resonance spectroscopy-based lipoprotein subfraction measurements. Linear regression was used to model associations between numbers of APOL1 G1/G2 risk variants and HDL subfractions, adjusting for demographic, clinical and ancestral covariates.
Female sex and higher percentage of African ancestry were positively associated with the number of APOL1 G1/G2 risk alleles. In the unadjusted analysis, mean (standard error) small HDL concentrations (μmol/L) for participants with zero, one and two G1/G2 risk alleles were 19.0 (0.2), 19.7 (0.2) and 19.9 (0.4), respectively (P = 0.02). Adjustment for age, sex, diabetes and African ancestry did not change the results but strengthened the statistical significance (P = 0.004). No significant differences in large or medium HDL, very low-density lipoprotein or low-density lipoprotein particle concentrations were observed by APOL1 genotype.
Greater numbers of APOL1 G1/G2 risk alleles were associated with higher small HDL particle concentrations in African Americans. These results may suggest novel areas of investigation to uncover reasons for the association between APOL1 risk variants with adverse outcomes in African Americans.
Two independent coding variants (G1 and G2) in the gene encoding apolipoprotein L1 (APOL1) are strongly associated with the development of non-diabetic chronic kidney disease (CKD) and the progression of CKD to end-stage renal disease (ESRD) among African Americans [1–5]. The association of APOL1 risk alleles with cardiovascular disease is less clear. In a recent report from the Jackson Heart Study (JHS), greater numbers of APOL1 G1/G2 risk alleles were independently associated with higher cardiovascular disease event rates independently of traditional risk factors and baseline kidney function . In contrast, the Systolic Blood Pressure Intervention Trial (SPRINT) failed to detect an association between APOL1 G1/G2 risk alleles and prevalent cardiovascular disease . Furthermore, the African American-Diabetes Heart Study (AA-DHS) demonstrated an inverse association between numbers of APOL1 G1/G2 risk alleles and calcified atherosclerotic plaque —similar to findings in the JHS —as well as an association of greater numbers of APOL1 G1/G2 risk alleles with improved survival. The mechanisms by which APOL1 G1/G2 risk alleles impact kidney and cardiovascular outcomes remain unclear.
The protein product of APOL1 (APOL1) is a member of a family of proteins originally discovered as component elements of high-density lipoprotein (HDL) particles . Subsequent studies identified APOL1 as a key element of HDL molecule complexes known as trypanolytic factors because of their role in clearing trypanosomes from the bloodstream . Given the physiologic relationship between APOL1 and HDL particles, one prior study examined the association of APOL1 G1/G2 risk alleles with HDL subfractions among African Americans and found that greater numbers of risk alleles were associated with lower concentrations of medium-sized HDL particles . Since lower medium HDL concentrations have been linked with excess cardiovascular risk [12–14], these prior findings showing an association of APOL1 G1/G2 risk alleles with HDL subfractions suggest that APOL1 genotypes may impact renal and cardiovascular health, potentially by altering the distribution of HDL subfractions with varying atherogenic potential. However, the prior study was limited by a small sample size and inability to adjust for key confounders. Accordingly, the primary focus of this report was to examine the association of the number of APOL1 G1/G2 risk alleles with HDL subfractions in a large sample of African American participants of the Reasons for Geographic and Racial Differences in Stroke (REGARDS) Study, a cohort of European Americans and African Americans living throughout the USA.
The REGARDS Study is a population-based investigation of stroke incidence in African American and European American adults ≥45 years of age. The study design has been reported elsewhere . In brief, participants were recruited from the 48 contiguous US states and the District of Columbia. The study was designed to provide approximately equal representation of men and women, and oversampled African Americans and persons residing in the stroke belt/buckle regions of the USA. Overall, 30 239 individuals were enrolled between January 2003 and October 2007 (42% African American, 55% women). The REGARDS Study protocol was approved by the Institutional Review Boards governing research in human subjects at the participating centers and all participants provided informed consent.
For the current study, we utilized a case-control sample assembled as part of the Sea Islands Genetics Network (SIGNET), a study of genetic contributors to diabetes and dyslipidemia among African Americans. The SIGNET sample consisted of all African American participants of REGARDS with type 2 diabetes living in South Carolina, Georgia, North Carolina and Alabama (cases), and an equivalent number of race-, sex- and age-strata-matched diabetes-free controls, as well as all African American participants not already selected who were current residents of the 15-county ‘Low Country’ region of South Carolina and Georgia (South Carolina counties Beaufort, Berkeley, Charleston, Colleton, Dorchester, Georgetown, Hampton, Horry, Jasper; Georgia counties Bryan, Camden, Chatham, Glynn, Liberty, McIntosh). Diabetes status was determined at the baseline visit. A total of 2398 African American participants were selected, of whom 1149 had type 2 diabetes and 1249 did not. Of these, 2080 participants had lipoprotein subfraction measurements.
Two single nucleotide polymorphisms (SNPs) in APOL1 (rs73885319 and rs60910145) and a six base-pair insertion/deletion polymorphism (rs71785313) were genotyped in REGARDS participants with lipoprotein subfraction measurements using a custom assay designed in the Center for Genomics and Personalized Medicine Research at the Wake Forest School of Medicine on the Sequenom platform (San Diego, CA, USA). G1 and G2 genotype calls were visually inspected for quality control. Overall, genotyping efficiency for all three SNPs was 97.9–98.2%. African ancestry was estimated in each participant using ancestry informative SNPs from an Affymetrix 6.0 array. For this analysis, ADMIXTURE V1.23 , including HapMap3 CEU (European), YRI (African) and ASN (East Asian), with supervised mode was used to derive the ancestry proportions with K = 3 predicted populations.
Lipoprotein subfractions were measured by LipoScience, Inc. (Raleigh, NC, USA) using nuclear magnetic resonance (NMR) spectroscopy as reported [17, 18]. In brief, lipoprotein subpopulation particle concentrations (VLDL, LDL and HDL) were obtained from the measured amplitudes of their spectroscopically distinct lipid methyl group NMR signals using the LipoProfile-3 spectral deconvolution algorithm. Lipoprotein subclasses were grouped into small, medium and large categories based upon size classifications used in previous studies [13, 19].
Covariates of interest included the following sociodemographic, clinical and laboratory factors: age, sex, annual family income and educational attainment, all determined via self-report; body mass index; waist circumference; systolic and diastolic blood pressure defined as the average of two seated measures taken after a 5 min rest; diabetes defined as fasting serum glucose ≥126 mg/dL, non-fasting serum glucose ≥200 mg/dL or the use of anti-diabetes medications; coronary heart disease (CHD) defined as having any of the following: evidence of myocardial infarction on the baseline ECG, self-report of a prior history of a cardiac procedure (coronary artery bypass surgery or percutaneous angioplasty) or self-reported history of myocardial infarction; stroke ascertained by self-report; estimated glomerular filtration rate (eGFR), urine albumin to creatinine ratio (UACR) and high-sensitivity C-reactive protein. Serum creatinine was calibrated to an international isotope dilution mass spectroscopic (IDMS)-traceable standard, measured by colorimetric reflectance spectrophotometry. eGFR was calculated using the Chronic Kidney Disease Epidemiology Collaboration equation . Albumin and creatinine were measured in a random spot urine specimen by nephelometry (BN ProSpec Nephelometer, Dade Behring, Marburg, Germany) and Modular-P chemistry analyzer (Roche/Hitachi, Indianapolis, IN), respectively. Spot UACR was calculated in mg/g. Prevalent CKD was defined as an eGFR < 60 mL/min/1.73 m2 or a UACR ≥30 mg/g. Serum high-sensitivity C-reactive protein was measured using a high-sensitivity particle-enhanced immunonephelometric assay.
The primary independent variable of interest was the number of APOL1 G1/G2 alleles (risk status). The two SNPs comprising the G1 locus are reported to be in near perfect linkage disequilibrium in African American populations , thus either can be used to assess risk status. G1/G2 risk was defined according to the number of copies of the high-risk haplotypes—zero, comprising individuals lacking either the high-risk G1or G2 haplotypes; one copy, comprising individuals with either one high-risk G1 haplotype or one high-risk G2 haplotype but not both; and two copies, comprising individuals with two copies of high-risk haplotypes (either G1/G1, G1/G2 or G2/G2). The primary dependent variables of interest were HDL particle subfractions (total, large, medium and small). Associations of APOL1 genotype with VLDL subfractions (total, large, medium and small) and LDL subfractions (large and small) were also examined. Intermediate density lipoprotein results did not pass quality control testing, and were not included in the analysis. Since intermediate density lipoprotein concentrations were used to calculate total LDL concentrations, total LDL concentrations were also not included in the analysis. Characteristics of the study population at the baseline visit were compared across categories of APOL1 risk status using standard descriptive statistics. Generalized linear models were used to examine lipoprotein subfraction concentrations and particle size by APOL1 G1/G2 risk status (zero versus one versus two G1/G2 risk alleles). Initial models were unadjusted and subsequent models were adjusted for age, sex, diabetes and covariates significantly associated with APOL1 risk status on univariable analysis (Table 1). In sensitivity analyses, the analyses were repeated using recessive models comparing participants with two copies of risk haplotypes (G1/G1, G2/G2 or G1/G2) to individuals with one or zero risk haplotypes and dominant models comparing individuals with at least one risk haplotype (G1/referent, G2/referent, G1/G1, G2/G2 or G1/G2) to individuals with zero risk haplotypes. Analyses were performed using SAS version 9.4 (Cary, NC, USA).
Of the 2080 REGARDS participants with lipoprotein subfraction measurements, 70 were excluded for missing data on G1 or G2 APOL1 risk alleles. This left a total of 2010 participants in the final analyzed sample. Of these, 853 had zero APOL1 risk alleles, 906 had one risk allele (560 had the G1/referent genotype and 346 the G2/referent genotype) and 251 had two risk alleles (102 were G1 homozygotes, 37 were G2 homozygotes and 112 were compound G1/G2 heterozygotes). The 12.5% frequency of participants possessing two APOL1 risk alleles is consistent with prior reports.
Table 1 compares demographic, clinical and laboratory characteristics of participants by APOL1 G1/G2 risk status. Female sex and greater percentage African ancestry were the only variables associated with greater numbers of G1/G2 risk alleles. There was no statistically significant difference in the prevalence of CKD among those with zero, one and two G1/G2 risk alleles (27, 29 and 30%, respectively, P = 0.59). When stratified by diabetes status, the prevalence of CKD among non-diabetic participants with zero, one and two G1/G2 risk alleles was 15, 18 and 21%, respectively (P = 0.27), whereas the prevalence of CKD among diabetic participants with zero, one and two risk alleles was 39, 39 and 38%, respectively (P = 0.79). The differences in baseline prevalence of CKD when recessive or dominant models were examined are presented in Supplementary Table S1. Among participants without diabetes, the risk of ESRD was significantly higher in those with two APOL1 nephropathy risk alleles when compared with those with zero or one risk allele (hazard ratio 6.08, 95% confidence interval 1.18, 31.45).
Mean HDL subfraction concentrations by APOL1 G1/G2 risk status are presented in Table 2. There was a statistically significant increase in mean total HDL concentrations with increasing numbers of APOL1 risk variants in the unadjusted analysis (P = 0.004) and after further adjustment for age, sex, diabetes and percentage African ancestry (P = 0.02). This association was primarily due to differences in mean concentration of small HDL subfractions based on APOL1 G1/G2 risk status. In the unadjusted analysis, mean (standard error) small HDL concentrations (μmol/L) for participants with zero, one and two risk alleles were 19.0 (0.2), 19.7 (0.2) and 19.9 (0.4), respectively (P = 0.02). After adjustment for age, sex, diabetes and percent African ancestry, the results were minimally changed and remained statistically significant (P = 0.004). The distribution of small HDL particles by numbers of APOL1 risk alleles is presented graphically in Figure 1. There were no statistically significant associations of total number of APOL1 G1/G2 risk variants with large HDL or medium HDL subfractions in either unadjusted or multivariable adjusted analyses. When models were stratified by diabetes status, the results were qualitatively unchanged (data not shown). When examined using a recessive model, there were no statistically significant differences in any HDL subfraction in multivariable adjusted models (Supplementary Table S2). In contrast, in both unadjusted and multivariable adjusted dominant models, total HDL and small HDL particle concentrations were significantly higher in participants with at least one G1 or G2 risk allele when compared with participants with zero risk alleles.
Table 3 presents associations of APOL1 genotype with apolipoprotein B particle concentrations and particle sizes. There were no statistically significant associations of the number of APOL1 G1/G2 risk alleles with very low-density lipoprotein (VLDL) or low-density lipoprotein (LDL) mean particle concentrations or particle size, or with NMR-derived total triglyceride concentrations. Although greater numbers of APOL1 G1/G2 risk alleles were associated with greater NMR-derived HDL cholesterol concentrations (P = 0.02), this association was attenuated with adjustment for sex (P = 0.16).
In this cross-sectional study of African Americans living in southeastern USA, greater numbers of APOL1 G1/G2 risk alleles were associated with higher circulating concentrations of small-sized HDL particles independently of age, sex, diabetes and percentage of African ancestry. This is the largest study examining lipid subfractions by APOL1 genotype and the first to show an association of increasing numbers of APOL1 G1/G2 risk alleles with small HDL particle concentrations. Given the known associations of small HDL with established kidney and cardiovascular disease risk factors, these findings may suggest novel areas of investigation to determine possible mechanisms underlying the associations of APOL1 risk variants with kidney and cardiovascular outcomes.
Only one prior study reported associations between APOL1 genotype and NMR-derived lipid subfractions in African Americans . In 73 Natural History of APOL1-associated Nephropathy study participants, greater numbers of APOL1 G1/G2 risk alleles were associated with decreased medium HDL concentrations and no significant differences in total or small HDL concentrations, in contrast to the results in the current study. There may be several reasons for this. The small sample size of the prior study may have resulted in insufficient statistical power to detect an association of APOL1 nephropathy risk variants with small HDL subfraction concentrations. In addition, the study populations were markedly different—the prior study consisted of first-degree relatives of individuals with non-diabetic forms of ESRD whereas the current study consisted of community-dwelling adults living in southeastern USA with a higher proportion of individuals with type 2 diabetes (50 versus ~10%).
It is unclear why greater numbers of APOL1 G1/G2 risk alleles were positively associated with small HDL particle concentrations. Prior studies showed that higher small HDL particle concentrations were closely linked with markers of cardiometabolic disease including increased central adiposity, insulin resistance, inflammation and higher risk of incident diabetes [21–26]. Importantly, there were no differences in available markers of cardiometabolic health including waist circumference, body mass index, blood pressure, diabetes, dyslipidemia or high-sensitivity C-reactive protein by APOL1 G1/G2 risk status in this study. This makes it unlikely that indices of poor metabolic health could explain the observed association of APOL1 G1/G2 risk alleles with small HDL particle concentrations. Since APOL1 circulates in low concentrations in plasma , it is unlikely that the associations between APOL1 G1/G2 risk status and small HDL were due to direct interactions between these proteins. Notably, prior studies have linked higher small HDL concentrations with kidney disease [27, 28]; thus, it is conceivable that our findings may reflect the known link between these risk variants and kidney disease. Although this seems unlikely given that there were no differences in the prevalence of CKD across APOL1 risk categories in the additive genetic model, serum creatinine and urine albumin excretion are relatively insensitive markers of very early kidney disease . Finally, since the kidney plays an important role in the catabolism of small HDL particles , it is possible that the findings of this study may be due to alterations in renal catabolic pathways for small HDL.
HDL consists of a heterogeneous group of lipoprotein particles with a variety of biological functions that vary according to particle size, such that in general small, dense, lipid-poor HDL particles demonstrate greater cholesterol efflux capacity and more potent anti-inflammatory, anti-oxidant and insulin sensitizing effects than larger-sized HDL particles in vitro [30, 31]. However, these properties do not seem to translate into expected associations with atherogenic risk as some, but not all, studies have shown a link between higher small-sized HDL particle concentrations and greater risk of cardiometabolic disease [31, 32]. The reasons for these findings are unclear, but have been attributed to the formation of functionally defective small HDL particles with compromised anti-atherogenic properties that contribute to cardiovascular disease . In this context, the findings of the current study may suggest important new avenues of investigation as to why APOL1 risk variants contribute to excess renal disease—potentially by facilitating the formation of plasma HDL subpopulations with pro-atherogenic properties.
The implications of our findings for the association of APOL1 G1/G2 risk status with cardiovascular disease are more complicated. A recent study showed an independent association of APOL1 G1/G2 risk variants with increased cardiovascular disease risk . However, other studies showed either null or opposite findings [7, 8]. Given current uncertainties as to the role of small HDL subfractions in cardiovascular and metabolic disease [33–35], future studies will need to delineate whether the direct association of APOL1 risk variants with small HDL concentrations has beneficial or harmful effects and to what extent this may help to explain reported links between APOL1 G1/G2 risk variants and cardiovascular disease.
The results of the current study should be considered in the context of its limitations. The difference in mean small HDL concentrations across APOL1 risk categories was relatively modest. Nonetheless, comparably sized differences in mean small HDL particle concentrations were associated with differences in urinary albumin excretion in a prior study , suggesting that while modest, these differences may be clinically meaningful. NMR-derived lipoprotein analyses do not distinguish between subdivisions of small HDL particles such as α-3, α-4 and pre-β-1 particles . Given important differences in the structure and function of these latter particles, future studies will need to investigate whether concentrations of these particles also differ by APOL1 risk allele status. Furthermore, we did not have measurements of other apolipoproteins associated with HDL such as apoA1. In addition, we did not have any measures of HDL cholesterol efflux capacity which may be a better marker of cardiovascular disease risk than HDL particle concentrations alone . Although there were no differences in available measures of metabolic health such as waist circumference and hsCRP across APOL1 G1/G2 risk status, we did not have more specific measures of insulin sensitivity or body mass partitioning which may provide important insights into potential reasons why the concentrations of small HDL particles increased with increasing APOL1 G1/G2 risk alleles. We had very few cardiovascular disease events and so were unable to examine associations of APOL1 G1/G2 risk alleles with these outcomes. Additionally, we were unable to examine what role, if any, the differences in HDL subfractions by APOL1 genotype may have on innate immunity.
In conclusion, greater numbers of APOL1 G1/G2 risk alleles were associated with higher small HDL concentrations in community-dwelling African Americans. Future studies will need to determine the reasons for these findings and what role, if any, these associations have in explaining the associations of APOL1 G1/G2 risk status with alterations in cardiovascular and renal outcomes among African Americans.
The results of this paper have not been published previously in whole or in part.
(See related article by Hu and Ray. How complicated can it be? The link between APOL1 risk variants and lipoprotein heterogeneity in kidney and cardiovascular diseases. Nephrol Dial Transplant 2016; 31: 509–511)
The authors thank the other investigators, the staff and the participants of the REGARDS Study for their valuable contributions. A full list of participating REGARDS investigators and institutions can be found at http://www.regardsstudy.org. This study was supported by a cooperative agreement U01 NS041588 from the National Institute of Neurological Disorders and Stroke, National Institutes of Health. O.M.G. was supported by R03DK095005 and R01NS080850 and B.I.F. was supported by R01DK070941 and R01DK084149 from the National Institutes of Health. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Neurological Disorders and Stroke, the National Institute of Diabetes and Digestive and Kidney Diseases or the National Institutes of Health. Representatives of the funding agency have been involved in the review of the manuscript but not directly involved in the collection, management, analysis or interpretation of the data. Additional funding was provided by an investigator-initiated grant-in-aid from Amgen Corporation. Amgen did not have any role in the design and conduct of the study, the collection, management, data analysis or interpretation of the data, or the preparation of the manuscript.