Apolipoprotein L1, cardiovascular disease and hypertension: More questions than answers

Apolipoprotein L1 and Parasites: An Example of Evolutionary Adaption

The Apolipoprotein L1 (APOL1) gene, located on the short arm of chromosome 22, is only found in humans and closely related primate species.¹ As part of the innate immunity mechanism, APOL1 protects humans against African sleeping sickness, which is a protozoan parasitic infection caused by Trypanosoma brucei. This parasite is transmitted by the tsetse fly, and the disease has a prevalence of 11% in sub-Saharan Africa. APOL1 on high-density lipoprotein (HDL) particles is taken up by the trypanosome cells,² causes osmotic swelling of the lysosome by means of chloride influx, then ultimately lysis of the parasite.

There are three components to APOL1 causing trypanolytic activity, a pore-forming domain, a pH-sensitive membrane-addressing domain, and the serum resistance-associated protein (SRA)-interacting domain. However, two species, Trypanosoma brucei rhodesiense in East Africa and Trypanosoma brucei gambiense in West Africa, have separately evolved resistance to the trypanolytic activity of APOL1 through the generation of a virulence factor called SRA that can inactivate APOL1 protein and prevent its trypanolytic activity.³ As a means of evolutionary survival adaptation, variants arose in APOL1, (G1/G2 variants) that encode forms of APOL1 that evade SRA. This likely confers a selective advantage to AA individuals carrying these variants, causing a selection advantage.

Population Genetics and Worldwide Distribution of APOL1

Chromosome 22 has been a genetic region of interest well before the APOL1 risk variants were discovered. Initially, variants of the Myosin Heavy Chain 9 (MYH9), also on chromosome 22, which were in close proximity to APOL1 were considered to be associated with the increased risk of nondiabetic end stage renal disease (ESRD) and focal segmental glomerulosclerosis (FSGS) in AA individuals.⁴ However, a novel form of genetic analysis, called admixture mapping led to the discovery that the increased risk of FSGS initially attributed to MYH9 were likely due to two sequence variants (G1 and G2) in the adjacent APOL1 gene.⁵ G1 risk variant is has 2 missense mutations (rs73885319, rs60910145) in high linkage disequilibrium, whereas G2 has 6 base pair deletions (rs71785313), both resulting in protein alteration.⁶ (Figure 1)

Figure 1.

Description of APOL1 risk variants.

The G1 and G2 risk variants appear to be the most common in western sub-Saharan Africa, as observed in the Human Genome Diversity Project and International HapMap Project. These variants both have highest frequencies Ghana and Nigeria (>40% for G1 and 6–24% for G2). G1 variants are found in 20% of African individuals, whereas G2 variants are found in 15%. Approximately 14–16% of individuals from Africa carry both APOL1 variants (G1/G1, G2/G2 or G1/G2) known as the high-risk genotype.

However, the frequency of these variants differs by countries within the African continent. A study by Behar et al showed that the prevalence of APOL1 variants is much lower than West Africa.⁷ Similarly, a study focusing on South Africans with mixed ancestry found that, risk alleles were carried in ~16 %, with frequency of only 1% for two risk alleles, which is lower than the prevalence in African Americans.⁸ This variability of prevalence of APOL1 risk variants in Africa is likely due to the low prevalence of the tsetse fly in regions outside of West Africa.

On the other hand, individuals who are not from the African continent and those who do not self-identify as African American, may have high frequencies of APOL1 risk variants due to worldwide patterns of migration and the sharing of recent African ancestry. This may be especially true of Hispanic Americans and individuals from Caribbean countries who share recent African ancestry. A recent paper, utilizing linked genetic and demographic data from the Population Architecture using Genetics and Epidemiology-II (PAGE-II) consortium, showed that other populations had elevated frequencies of the APOL1 risk genotype, including Jamaican, Barbadian, Grenadian, and Brazilian from Salvador (>10 to 22%); Trinidadian, Panamanian, Honduran, Haitian, Garifunan, and Palenque (>5 to 10%); and Guyanese, Dominican, Peruvian, Belizean, and Native American (1 to 5%).⁹ A recent tool to estimate frequency of APOL1 risk genotype has been developed at http://APOL1.org. Thus, APOL1 risk variants exist at appreciable frequencies among many populations of persons in the Americas who share Western African genetic ancestry but who may not self-identify as African or African American. Previous studies have also shown that APOL1 risk genotype is also linked to kidney disease outcomes in Hispanic Americans who do not identify themselves as African American.¹⁰ (Figure 1)

APOL1 and the Spectrum of Kidney Disease

It has been known through several epidemiological studies that African Americans have two-fold higher risk of end stage renal disease (ESRD) compared to European Americans, even when accounting for environmental, socioeconomic, and comorbid risk factors.^11–13

Since the seminal paper showing the association of APOL1 risk variants with FSGS,⁵ a plethora of literature has shown its consistent association with kidney disease phenotypes. The association with FSFS was confirmed by Kopp et al who found that those with APOL1 high risk genotype had a younger age of onset of disease by almost 6 years.¹⁴ Additionally, they showed that individuals with APOL1 high risk genotype had 29 fold odds of develop Human Immunodeficiency Virus Associated Nephropathy (HIVAN). This suggests that HIVAN in African ancestry individuals is unlikely to develop in the absence of APOL1 high risk genotype. This is further supported by the fact that individuals with Ethiopian ancestry and HIV have a low prevalence of HIVAN and they have an absence of APOL1 risk variants.⁷

There have been several studies now linking APOL1 high risk genotype with various kidney phenotypes including CKD,¹⁵ earlier age of dialysis initiation,^16,17 and proteinuria. A seminal study by Parsa et al in two separate cohorts (the African American Study of Kidney Disease and Hypertension [AASK] and Chronic Renal Insufficiency [CRIC] showed that APOL1 high-risk genotype was strongly associated with CKD progression, regardless of baseline proteinuria or history of type 2 diabetes.¹⁸ The summary of kidney phenotypes and histopathology findings associated with APOL1 are shown in Figure 2.

Figure 2.

The Spectrum of Kidney Disease in APOL1.¹⁹

This epidemiological evidence is further bolstered by several elegant mechanistic studies on how APOL1 causes kidney injury. The putative mechanisms include activation of stress kinases and AMP-activated protein kinase,²⁰ endolysosomal trafficking/autophagic flux,²¹ mitochondrial dysfunction,²² and outside-in cellular signaling.²³

APOL1 and Cardiovascular Disease: The Plot Thickens

As opposed to the clear links between APOL1 and kidney disease, the relationship with cardiovascular disease (CVD) is much more convoluted and controversial. Again it is well known that African Americans are known to have higher incidence of CVD compared to European Americans.²⁴ Although the overall rate of death attributable to CVD has decreased from 2003 to 2013 by 28.8%, the rates have been much higher in African American males compared to white males (356 vs. 270 per 100 000 Americans, respectively).²⁵ Thus it stands to reason that ethnic specific genetic factors contribute and thus APOL1 might play a role.

Initial evidence for this association was found in 2014. Ito et al utilized longitudinal clinical and APOL1 genotype data from the Jackson Heart Study (JHS) (1959 African Americans) and found that there was a two-fold increased risk for CVD events in patients with APOL1 high risk genotype even after correcting for both traditional CVD risk factors and CKD. This was replicated with the Women’s Health Initiative (WHI) cohort, finding an increased risk in APOL1 high-risk genotype.²⁶ Another study analyzing the Cardiovascular Heart Study (CHS), which showed that the risk genotype is associated with APOL1 genotype is associated subclinical atherosclerosis, incident myocardial infarction, and mortality in older African Americans >65 years of age.²⁷ Additional evidence was provided by a autopsy study which showed that the risk genotype was associated with an early age at cardiovascular death independent of nephrosclerosis.²⁸ Finally, recently in the REGARDS (Reasons for Geographic and Racial Differences in Stroke) cohort, APOL1 high risk genotype was found to be associated with a greater risk of composite cardiovascular endpoint in non-diabetic AAs, which was predominantly driven by stroke and small vessel disease.²⁹

However, there is conflicting evidence provided by two recent studies. Chen, et al analyzed the AASK cohort and found no significant association for increased risk for CVD events, such as cardiovascular death or hospitalization although there may be a weak association with cardiovascular mortality.³⁰ In recent analysis of the Coronary Artery Risk Development in Young Adults (CARDIA) study, In the CARDIA study population, coronary artery calcification and carotid artery intimal thickness and left ventricular hypertrophy did not differ according to APOL1 genotype.³¹ Finally, in the post-hoc analysis of Systolic Blood Pressure Intervention Trial (SPRINT) trial, there were no differences by APOL1 genotype in the composite outcome or in any of its components.³² These studies are summarized in Table 1.

Table 1:

Studies of APOL1 Association with CVD

Study Year	Cohort	N	Clinical Outcome	Adjusted Effect Size (CI 95%)	Overall Conclusion	References
2014	Jackson Heart Study	1959	Incident CVD	OR 2.17 (1.34–3.48)	APOL1 high risk genotype associated with higher atherosclerotic CVD risk	Ito, et al. 26
	Women’s Health Initiative	749		OR 1.98 (1.17–3.31)
2016	CHS	798	Incident CVD	HR 1.8 (1.1–3.0)	APOL1 high risk genotype associated with subclinical atherosclerosis, incident myocardial infarction, and mortality in older African Americans	Mukamal, et al.27
2017	AASK	693	CVD event	HR 1.16 (0.77–1.76)	APOL1 high risk genotype not associated with an overall risk for CVD in AA with hypertension-attributed CKD	Chen, et al.30
2017	SPRINT	2571	CVD	HR 1.2 (0.76–1.92)	APOL1 high risk genotype not associated with incident CVD in high-risk hypertensive patients	Freedman, et al. 32
2017	Autopsy data	298	CAD	OR 1.33 (1.09–1.64)	APOL1 high risk genotype are associated with earlier age deaths due to coronary artery disease and cardiomyopathy	Hughson, et al. 28
2018	CARDIA	1315	Prevalence coronary artery calcification	RR 1.04 (0.70–1.54)	APOL1 high risk genotype is not associated with subclinical atherosclerotic disease or LVH	Gutiérrez, et al. 31
			LV hypertrophy	RR 0.96 (0.70–1.33)
2018	REGARDS	10605	Incident CVD	HR 1.67 (1.12–2.47)	APOL1 high risk genotype is associated with CVD events in community-dwelling AAs	Gutiérrez, et al. 29

CVD= Cardiovascular Disease, CHS=Cardiovascular Health Study, AASK=African American Study of Kidney; SPRINT= Systolic Blood Pressure Intervention Trial; CARDIA= The Coronary Artery Risk Development in Young Adults (CARDIA) Study, REGARDS= REasons for Geographic And Racial Differences in Stroke Study

So how do we interpret the conflicting evidence from these studies? Differences in study design, study populations, and the handling of confounding by kidney function may potentially account for these differing conclusions. Additionally, survival bias in these cohorts may play a major role in these discrepant findings. Thus, the issue of cardiovascular disease and APOL1 remains very much an open question and should be addressed by both longitudinal, carefully designed studies and mechanistic experiments.

APOL1 and Hypertension: The Plot Thickens Further

Essential hypertension is a multifactorial disorder and significant ethnic differences both in the incidence and prevalence exist. African Americans with either treated or untreated hypertension had significantly higher mean systolic blood pressure than non-Hispanic white adults.³³ Hypertension has been diagnosed earlier in life and at higher frequencies in African Americans compared to other ethnicities.³⁴ Hypertension has a known to be highly heritable. Thus, African Americans not only have environmental and socioeconomic factors contributing to hypertension, but also genetics, which plays a role to this disparity.³⁵

There have been two studies looking at blood pressure, APOL1 and hypertension. Using genetic data linked to electronic medical record phenotypic data from several biobanks in the Electronic Medical Records and Genomics (eMERGE) Network for 10,000 individuals, a study found that APOL1 high-risk genotype was associated with earlier onset of hypertension and increased blood pressure after adjusting for kidney function. Increased blood pressure was evident in individuals aged 20–29 years, much before kidney function decline started. Importantly, the increment in blood pressure was linked to per risk variant, rather than the risk genotype. These associations replicated across three biobanks.³⁶

In contrast to these findings Chen, et al, conducted a longitudinal study over 25 years with the CARDIA cohort of 1330 black patients.³⁷ They found that African American patients had overall significant higher blood pressures than whites. However, they were unable to find a statistically significant association of hypertension between patients with APOL1 high risk and low risk.

These findings need to be interpreted in light of their differences in the studies. The eMERGE cross-sectional study with a large cohort comprised of several age groups of over 9000 participants had a large number of blood pressure measurements per participant. Thus, was powered to detect weaker associations of blood pressure with APOL1 risk alleles and showed that each copy of the APOL1 risk variant was associated with an increment in systolic blood pressure. However, all participants were all hospital-based and may have had higher comorbid disease and did not include ambulatory blood pressure measurements. Although the Chen et al study was smaller, it was longitudinal and in an observational cohort. The study was also underpowered to detect associations of APOL1 risk alleles with blood pressure, thus may require a much larger sample size, especially as the differences in blood pressure were very small for each risk variant. Additionally, there was no replication cohort, which is important for studies of associations between genotype and phenotype

Hypertension, Kidney Disease and APOL1: Inextricably Connected?

The relationship between hypertension and CKD is complicated where both perpetuate each other. The etiology of hypertensive renal disease is multifactorial. Long term uncontrolled hypertension causes arteriolar nephrosclerosis leading to glomerular ischemia. As renal function declines, hypertension usually worsens. Limited salt excretion, salt sensitivity, and ECF volume expansion are key mechanisms for poor blood pressure control in CKD patients. These patients also have increased oxidative stress and worse atherosclerotic disease. Hypertension accelerates kidney disease with a vicious cycle of RAAS activation, systemic vasoconstriction, and glomerular hypertension.³⁸

The association of APOL1 and hypertension is not completely understood. Individuals with risk variants do have higher blood pressures and higher risk of CKD. Does the kidney disease in these patients cause the higher blood pressures or do the higher blood pressures cause the kidney disease or are these two entities caused by separate mechanisms? As not everyone with APOL1 high risk variants develop kidney disease, there may also be a question of a “second hit” that triggers CKD and progression (Figure 3).³⁹

Figure 3:

The possible relationships between APOL1 risk alleles, CKD, and hypertension. APOL1 risk alleles cause CKD causing hypertension. Data from Nadkarni GN, Coca SG. APOL1 and blood pressure changes in young adults. Kidney Int 2017;92(4):793–5.

What does the future hold for APOL1?

APOL1 risk variants are common in persons of African ancestry (including African Americans and Hispanic Americans) and are one of the most powerful disease variants identified to date in terms of frequency and effect size for kidney disease. This is an important discovery for nephrology and has helped further our understanding of disparities. There are efforts to incorporate genetic testing in routine clinical settings including in pre-transplant evaluation and as a therapeutic target.^40,41

The association of APOL1 with other linked diseases such as cardiovascular disease and hypertension is still controversial. The evidence from epidemiological and observational studies is conflicting and there is no mechanistic evidence yet. However, APOL1 is broadly expressed in human tissues, raising the question of whether the APOL1 risk allele toxicity is restricted to kidney diseases only. Additionally, genetic variants usually exhibit pleiotropy i.e. association with many phenotypes.⁴² Additionally, it has been proposed that, there may be a possible unifying mechanism of endothelial dysfunction leading to both kidney and cardiovascular disease.⁴³ However, definitive answers are lacking, and thus the association with cardiovascular disease and hypertension should still be considered an open question.

Regardless, the role of APOL1 genetic testing in clinical medicine needs to studied. Clinical trials such as the Genetic Testing to Understand and Address Renal Disease Disparities (GUARDD) are ongoing, where patients will be randomized to undergo genotyping for the APOL1 risk variants with clinical decision support or to usual care.⁴⁴ These studies will be essential to understanding the role that testing will play in clinical outcomes, and ultimately what the benefit to patients is, the ultimate goal of translational research.

Synopsis.

Ethnic disparities in health care exist among multiple complex diseases especially cardiovascular disease, hypertension and kidney disease. Recent discoveries in genetics have taught us that these disparities go beyond environmental and socioeconomic factors. The discovery of ethnic specific risk variants in the Apolipoprotein L1 (APOL1) gene on chromosome 22 seen only in individuals of recent African ancestry explains a large proportion of kidney disease disparities. In addition, recent large-scale genotype-phenotype association studies have identified associations with cardiovascular disease and hypertension. This review aims to review the recent literature in this field and point towards future directions for research.

Key Points:

• APOL1 risk variants are common in persons of African ancestry (including African Americans and Hispanic Americans) and are one of the most powerful disease variants identified to date in terms of frequency and effect size for kidney disease.

• The association of APOL1 with other linked diseases such as cardiovascular disease and hypertension is still controversial.

• These studies will be essential to understanding the role that testing will play in clinical outcomes, and ultimately what the benefit to patients is, the ultimate goal of translational research.