APOL1 C-Terminal Variants May Trigger Kidney Disease through Interference with APOL3 Control of Actomyosin

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Copy Number Variation at the APOL1 Locus

Two coding variants in the APOL1 gene (G1 and G2) explain most of the high rate of kidney disease in African Americans. APOL1-associated kidney disease risk inheritance follows an autosomal recessive pattern: The relative risk of kidney disease associated with inheritance of two high-risk variants is 7–30 fold, depending on the specific kidney phenotype. We wished to determine if the variability in phenotype might in part reflect structural differences in APOL1 gene. We analyzed sequence coverage from 1000 Genomes Project Phase 3 samples as well as exome sequencing data from African American kidney disease cases for copy number variation. 8 samples sequenced in the 1000 Genomes Project showed increased coverage over a ~100kb region that includes APOL2, APOL1 and part of MYH9, suggesting the presence of APOL1 copy number greater than 2. We reasoned that such duplications should be enriched in apparent G1 heterozygotes with kidney disease. Using a PCR-based assay, we observed the presence of this duplication in additional samples from apparent G0G1 or G0G2 individuals. The frequency of this APOL1 duplication was compared among cases (n = 123) and controls (n = 255) with apparent G0G1 heterozygosity. The presence of APOL1 duplication was observed in 4.06% of cases and 0.78% controls, preliminary evidence that this APOL1 duplication may alter susceptibility to kidney disease (p = 0.03). Taqman-based copy number assays confirmed the presence of 3 APOL1 copies in individuals positive for this specific duplication by PCR assay, but also identified a small number of individuals with additional APOL1 copies of presumably different structure. These observations motivate further studies to better assess the contribution of APOL1 copy number on kidney disease risk and on APOL1 function. Investigators and clinicians genotyping APOL1 should also consider whether the particular genotyping platform used is subject to technical errors when more than two copies of APOL1 are present.     

A Primate APOL1 Variant That Kills Trypanosoma brucei gambiense

Humans are protected against infection from most African trypanosomes by lipoprotein complexes present in serum that contain the trypanolytic pore-forming protein, Apolipoprotein L1 (APOL1). The human-infective trypanosomes, Trypanosoma brucei rhodesiense in East Africa and T. b. gambiense in West Africa have separately evolved mechanisms that allow them to resist APOL1-mediated lysis and cause human African trypanosomiasis, or sleeping sickness, in man. Recently, APOL1 variants were identified from a subset of Old World monkeys, that are able to lyse East African T. b. rhodesiense, by virtue of C-terminal polymorphisms in the APOL1 protein that hinder that parasite’s resistance mechanism. Such variants have been proposed as candidates for developing therapeutic alternatives to the unsatisfactory anti-trypanosomal drugs currently in use. Here we demonstrate the in vitro lytic ability of serum and purified recombinant protein of an APOL1 ortholog from the West African Guinea baboon (Papio papio), which is able to lyse examples of all sub-species of T. brucei including T. b. gambiense group 1 parasites, the most common agent of human African trypanosomiasis. The identification of a variant of APOL1 with trypanolytic ability for both human-infective T. brucei sub-species could be a candidate for universal APOL1-based therapeutic strategies, targeted against all pathogenic African trypanosomes.     

APOL1 G1 genotype modifies the association between HDLC and kidney function in African Americans

Background

Despite evidence of an association between variants at the apolipoprotein L1 gene (APOL1) locus and a spectrum of related kidney diseases, underlying biological mechanisms remain unknown. An earlier preliminary study published by our group showed that an APOL1 variant (rs73885319) modified the association between high-density lipoprotein cholesterol (HDLC) and estimated glomerular filtration rate (eGFR) in African Americans. To further understand this relationship, we evaluated the interaction in two additional large cohorts of African Americans for a total of 3,592 unrelated individuals from the Howard University Family Study (HUFS), the Natural History of APOL1-Associated Nephropathy Study (NHAAN), and the Atherosclerosis Risk in Communities Study (ARIC). The association between HDLC and eGFR was determined using linear mixed models, and the interaction between rs73885319 genotype and HDLC was evaluated using a multiplicative term.

Results

Among individuals homozygous for the risk genotype, a strong inverse HDLC-eGFR association was observed, with a positive association in others (p for the interaction of the rs73885319 × HDLC =0.0001). The interaction was similar in HUFS and NHAAN, and attenuated in ARIC. Given that ARIC participants were older, we investigated an age effect; age was a significant modifier of the observed interaction. When older individuals were excluded, the interaction in ARIC was similar to that in the other studies.

Conclusions

Based on these findings, it is clear that the relationship between HDLC and eGFR is strongly influenced by the APOL1 rs73885319 kidney risk genotype. Moreover, the degree to which this variant modifies the association may depend on the age of the individual. More detailed physiological studies are warranted to understand how rs73885319 may affect the relationship between HDLC and eGFR in individuals with and without disease and across the lifespan.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1645-7) contains supplementary material, which is available to authorized users.     

Oncogene APOL1 promotes proliferation and inhibits apoptosis via activating NOTCH1 signaling pathway in pancreatic cancer

APOL1 encodes a secreted high-density lipoprotein, which has been considered as an aberrantly expressed gene in multiple cancers. Nevertheless, the role of APOL1 in the regulatory mechanisms of pancreatic cancer remains unknown and should be explored. We identified APOL1 was abnormally elevated in human pancreatic cancer tissues compared with that in adjacent tissues and was associated with poor prognosis. The effects of APOL1 in PC cell proliferation, cell cycle, and apoptosis was verified via functional in vitro and in vivo experiments. The results showed that knockdown of APOL1 significantly inhibited the proliferation and promoted apoptosis of pancreatic cancer. In addition, we identified APOL1 could be a regulator of NOTCH1 signaling pathway using bioinformatics tools, qRT-PCR, dual-luciferase reporter assay, and western blotting. In summary, APOL1 could function as an oncogene to promote proliferation and inhibit apoptosis through activating NOTCH1 signaling pathway expression in pancreatic cancer; therefore, it may act as a novel therapeutic target for pancreatic cancer.Subject terms: Oncogenes, Protein-protein interaction networks     

Identifying Darwinian Selection Acting on Different Human APOL1 Variants among Diverse African Populations

Disease susceptibility can arise as a consequence of adaptation to infectious disease. Recent findings have suggested that higher rates of chronic kidney disease (CKD) in individuals with recent African ancestry might be attributed to two risk alleles (G1 and G2) at the serum-resistance-associated (SRA)-interacting-domain-encoding region of APOL1. These two alleles appear to have arisen adaptively, possibly as a result of their protective effects against human African trypanosomiasis (HAT), or African sleeping sickness. In order to explore the distribution of potential functional variation at APOL1, we studied nucleotide variation in 187 individuals across ten geographically and genetically diverse African ethnic groups with exposure to two Trypanosoma brucei subspecies that cause HAT. We observed unusually high levels of nonsynonymous polymorphism in the regions encoding the functional domains that are required for lysing parasites. Whereas allele frequencies of G2 were similar across all populations (3%–8%), the G1 allele was only common in the Yoruba (39%). Additionally, we identified a haplotype (termed G3) that contains a nonsynonymous change at the membrane-addressing-domain-encoding region of APOL1 and is present in all populations except for the Yoruba. Analyses of long-range patterns of linkage disequilibrium indicate evidence of recent selection acting on the G3 haplotype in Fulani from Cameroon. Our results indicate that the G1 and G2 variants in APOL1 are geographically restricted and that there might be other functional variants that could play a role in HAT resistance and CKD risk in African populations.     

Biogenesis and cytotoxicity of APOL1 renal risk variant proteins in hepatocytes and hepatoma cells

Two APOL1 gene variants, which likely evolved to protect individuals from African sleeping sickness, are strongly associated with nondiabetic kidney disease in individuals with recent African ancestry. Consistent with its role in trypanosome killing, the pro-death APOL1 protein is toxic to most cells, but its mechanism of cell death is poorly understood and little is known regarding its intracellular trafficking and secretion. Because the liver appears to be the main source of circulating APOL1, we examined its secretory behavior and mechanism of toxicity in hepatoma cells and primary human hepatocytes. APOL1 is poorly secreted in vitro, even in the presence of chemical chaper­ones; however, it is efficiently secreted in wild-type transgenic mice, suggesting that APOL1 secretion has specialized requirements that cultured cells fail to support. In hepatoma cells, inducible expression of APOL1 and its risk variants promoted cell death, with the G1 variant displaying the highest degree of toxicity. To explore the basis for APOL1-mediated cell toxicity, endoplasmic reticulum stress, pyroptosis, autophagy, and apoptosis were examined. Our results suggest that autophagy represents the predominant mechanism of APOL1-mediated cell death. Overall, these results increase our understanding of the basic biology and trafficking behavior of circulating APOL1 from the liver.     

Epistatic Role of the MYH9/APOL1 Region on Familial Hematuria Genes

Familial hematuria (FH) is explained by at least four different genes (see below). About 50% of patients develop late proteinuria and chronic kidney disease (CKD). We hypothesized that MYH9/APOL1, two closely linked genes associated with CKD, may be associated with adverse progression in FH. Our study included 102 thin basement membrane nephropathy (TBMN) patients with three known COL4A3/COL4A4 mutations (cohort A), 83 CFHR5/C3 glomerulopathy patients (cohort B) with a single CFHR5 mutation and 15 Alport syndrome patients (cohort C) with two known COL4A5 mild mutations, who were categorized as “Mild” (controls) or “Severe” (cases), based on renal manifestations. E1 and S1 MYH9 haplotypes and variant rs11089788 were analyzed for association with disease phenotype. Evidence for association with “Severe” progression in CFHR5 nephropathy was found with MYH9 variant rs11089788 and was confirmed in an independent FH cohort, D (cumulative p value = 0.001, odds ratio = 3.06, recessive model). No association was found with APOL1 gene. Quantitative Real time PCR did not reveal any functional significance for the rs11089788 risk allele. Our results derive additional evidence supporting previous reports according to which MYH9 is an important gene per se, predisposing to CKD, suggesting its usefulness as a prognostic marker for young hematuric patients.     

APOL1 Null Alleles from a Rural Village in India Do Not Correlate with Glomerulosclerosis

Background

Among African-Americans, genome wide association revealed a strong correlation between the G1 and G2 alleles of APOL1 (apolipoproteinL1, also called trypanolytic factor) and kidney diseases including focal and segmental glomerulosclerosis, HIV-associated nephropathy and hypertensive nephrosclerosis. In the prevailing hypothesis, heterozygous APOL1 G1 and G2 alleles increase resistance against Trypanosoma that cause African sleeping sickness, resulting in positive selection of these alleles, but when homozygous the G1 and G2 alleles predispose to glomerulosclerosis. While efforts are underway to screen patients for G1 and G2 alleles and to better understand “APOL1 glomerulopathy,” no data prove that these APOL1 sequence variants cause glomerulosclerosis. G1 and G2 correlate best with glomerulosclerosis as recessive alleles, which suggests a loss of function mutation for which proof of causality is commonly tested with homozygous null alleles. This test cannot be performed in rodents as the APOL gene cluster evolved only in primates. However, there is a homozygous APOL1 null human being who lives in a village in rural India. This individual and his family offer a unique opportunity to test causality between APOL1 null alleles and glomerulosclerosis.

Methods and Findings

We obtained clinical data, blood and urine from this APOL1 null patient and 50 related villagers. Based on measurements of blood pressure, BUN, creatinine, albuminuria, genotyping and immunoblotting, this APOL1 null individual does not have glomerulosclerosis, nor do his relatives who carry APOL1 null alleles.

Conclusions

This small study cannot provide definitive conclusions but the absence of glomerulosclerosis in this unique population is consistent with the possibility that African-American glomerulosclerosis is caused, not by loss of APOL1 function, but by other mechanisms including a subtle gain of function or by the “genetic hitchhiking” of deleterious mutations in a gene linked to APOL1 G1 and G2.     

Coiled-coil binding of the leucine zipper domains of APOL1 is necessary for the open cation channel conformation

Apolipoprotein L-I (APOL1) is a channel-forming effector of innate immunity. The common human APOL1 variant G0 provides protection against infection with certain Trypanosoma and Leishmania parasite species, but it cannot protect against the trypanosomes responsible for human African trypanosomiasis. Human APOL1 variants G1 and G2 protect against human-infective trypanosomes but also confer a higher risk of developing chronic kidney disease. Trypanosome-killing activity is dependent on the ability of APOL1 to insert into membranes at acidic pH and form pH-gated cation channels. We previously mapped the channel’s pore-lining region to the C-terminal domain (residues 332–398) and identified a membrane-insertion domain (MID, residues 177–228) that facilitates acidic pH-dependent membrane insertion. In this article, we further investigate structural determinants of cation channel formation by APOL1. Using a combination of site-directed mutagenesis and targeted chemical modification, our data indicate that the C-terminal heptad-repeat sequence (residues 368–395) is a bona fide leucine zipper domain (ZIP) that is required for cation channel formation as well as lysis of trypanosomes and mammalian cells. Using protein-wide cysteine-scanning mutagenesis, coupled with the substituted cysteine accessibility method, we determined that, in the open channel state, both the N-terminal domain and the C-terminal ZIP domain are exposed on the intralumenal/extracellular side of the membrane and provide evidence that each APOL1 monomer contributes four transmembrane domains to the open cation channel conformation. Based on these data, we propose an oligomeric topology model in which the open APOL1 cation channel is assembled from the coiled-coil association of C-terminal ZIP domains.     

Differential effects of MYH9 and APOL1 risk variants on FRMD3 Association with Diabetic ESRD in African Americans

Single nucleotide polymorphisms (SNPs) in MYH9 and APOL1 on chromosome 22 (c22) are powerfully associated with non-diabetic end-stage renal disease (ESRD) in African Americans (AAs). Many AAs diagnosed with type 2 diabetic nephropathy (T2DN) have non-diabetic kidney disease, potentially masking detection of DN genes. Therefore, genome-wide association analyses were performed using the Affymetrix SNP Array 6.0 in 966 AA with T2DN and 1,032 non-diabetic, non-nephropathy (NDNN) controls, with and without adjustment for c22 nephropathy risk variants. No associations were seen between FRMD3 SNPs and T2DN before adjusting for c22 variants. However, logistic regression analysis revealed seven FRMD3 SNPs significantly interacting with MYH9—a finding replicated in 640 additional AA T2DN cases and 683 NDNN controls. Contrasting all 1,592 T2DN cases with all 1,671 NDNN controls, FRMD3 SNPs appeared to interact with the MYH9 E1 haplotype (e.g., rs942280 interaction p-value = 9.3E⁻⁷ additive; odds ratio [OR] 0.67). FRMD3 alleles were associated with increased risk of T2DN only in subjects lacking two MYH9 E1 risk haplotypes (rs942280 OR = 1.28), not in MYH9 E1 risk allele homozygotes (rs942280 OR = 0.80; homogeneity p-value = 4.3E⁻⁴). Effects were weaker stratifying on APOL1. FRMD3 SNPS were associated with T2DN, not type 2 diabetes per se, comparing AAs with T2DN to those with diabetes lacking nephropathy. T2DN-associated FRMD3 SNPs were detectable in AAs only after accounting for MYH9, with differential effects for APOL1. These analyses reveal a role for FRMD3 in AA T2DN susceptibility and accounting for c22 nephropathy risk variants can assist in detecting DN susceptibility genes.     

Mutational landscape of TRPC6, WT1, LMX1B,APOL1, PTPRO,PMM2, LAMB2 and WT1 genes associated with Steroid resistant nephrotic syndrome

Molecular Biology Reports - Nephrotic syndrome appears as a group of symptoms like proteinuria, edema and hyperlipidemia. Identification of monogenic forms revealed the physiology and pathogenesis...     

Missense mutations in the APOL1 gene are highly associated with end stage kidney disease risk previously attributed to the MYH9 gene

MYH9 has been proposed as a major genetic risk locus for a spectrum of nondiabetic end stage kidney disease (ESKD). We use recently released sequences from the 1000 Genomes Project to identify two western African-specific missense mutations (S342G and I384M) in the neighboring APOL1 gene, and demonstrate that these are more strongly associated with ESKD than previously reported MYH9 variants. The APOL1 gene product, apolipoprotein L-1, has been studied for its roles in trypanosomal lysis, autophagic cell death, lipid metabolism, as well as vascular and other biological activities. We also show that the distribution of these newly identified APOL1 risk variants in African populations is consistent with the pattern of African ancestry ESKD risk previously attributed to MYH9. Mapping by admixture linkage disequilibrium (MALD) localized an interval on chromosome 22, in a region that includes the MYH9 gene, which was shown to contain African ancestry risk variants associated with certain forms of ESKD (Kao et al. 2008; Kopp et al. 2008). MYH9 encodes nonmuscle myosin heavy chain IIa, a major cytoskeletal nanomotor protein expressed in many cell types, including podocyte cells of the renal glomerulus. Moreover, 39 different coding region mutations in MYH9 have been identified in patients with a group of rare syndromes, collectively termed the Giant Platelet Syndromes, with clear autosomal dominant inheritance, and various clinical manifestations, sometimes also including glomerular pathology and chronic kidney disease (Kopp 2010; Sekine et al. 2010). Accordingly, MYH9 was further explored in these studies as the leading candidate gene responsible for the MALD signal. Dense mapping of MYH9 identified individual single nucleotide polymorphisms (SNPs) and sets of such SNPs grouped as haplotypes that were found to be highly associated with a large and important group of ESKD risk phenotypes, which as a consequence were designated as MYH9-associated nephropathies (Bostrom and Freedman 2010). These included HIV-associated nephropathy (HIVAN), primary nonmonogenic forms of focal segmental glomerulosclerosis, and hypertension affiliated chronic kidney disease not attributed to other etiologies (Bostrom and Freedman 2010). The MYH9 SNP and haplotype associations observed with these forms of ESKD yielded the largest odds ratios (OR) reported to date for the association of common variants with common disease risk (Winkler et al. 2010). Two specific MYH9 variants (rs5750250 of S-haplotype and rs11912763 of F-haplotype) were designated as most strongly predictive on the basis of Receiver Operating Characteristic analysis (Nelson et al. 2010). These MYH9 association studies were then also extended to earlier stage and related kidney disease phenotypes and to population groups with varying degrees of recent African ancestry admixture (Behar et al. 2010; Freedman et al. 2009a, b; Nelson et al. 2010), and led to the expectation of finding a functional African ancestry causative variant within MYH9. However, despite intensive efforts including re-sequencing of the MYH9 gene no suggested functional mutation has been identified (Nelson et al. 2010; Winkler et al. 2010). This led us to re-examine the interval surrounding MYH9 and to the detection of novel missense mutations with predicted functional effects in the neighboring APOL1 gene, which are significantly more associated with ESKD than all previously reported SNPs in MYH9.