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.
Association with nondiabetic ESKD of nonsynonymous SNPs in APOL1, APOL3, and FOXRED2 in the MALD peak and comparison with leading MYH9 SNPs
YRI risk frequencyc (%)
CEU risk frequency (%)
These four variants were genotyped in a previously reported composite sample set of 955 subjects taken from two different populations, namely African American and Hispanic American cases and controls (Behar et al. 2010) (supplementary material). In this composite sample set, subjects with ESKD etiologies designated as MYH9-associated nephropathies as defined above, and notably excluding diabetic nephropathy, which was not previously found to be associated with MYH9 (Behar et al. 2010; Kao et al. 2008; Kopp et al. 2008), were designated as cases (n = 430). Subjects without known kidney disease and a creatinine concentration below 1.7 at age 55 or greater were designated as controls (n = 525). Associations of the candidate mutations with these ESKD phenotypes, previously attributed to MYH9, was determined using logistic regression, with correction for global and local ancestry, and considering three modes of inheritance as previously reported (Behar et al. 2010). It should be noted that the four variants were genotyped for the two population sources using two different techniques each conducted in a separate laboratory, and with sequence validation of the genotyping method (Supplementary Fig. 6). We chose to use two different population sources, African American and Hispanic American, with markedly differing degrees of global African ancestry admixture (Bedoya et al. 2006; Behar et al. 2010), rather than two different collections from the same population, in order to test whether associations were robust to differing global background genomic composition.
Table 1 shows the association results for the combined dataset of the African American and Hispanic American cohorts. For comparison, we include the results from the two previously reported leading MYH9 risk variants as noted above (S-1 rs5750250, F-1 rs11912763) (Nelson et al. 2010). We found that the APOL1 missense variants (rs73885319 and rs60910145) are more strongly associated with ESKD risk than the leading MYH9 risk variants, both in terms of OR and p values (Table 1). In contrast, the lower allele frequency and OR, with a higher p value for the APOL3 nonsense variant, and the weak association for the FOXRED2 missense mutation, render these variants to be very unlikely candidates to explain the risk attributed to this genomic region. For the highly associated APOL1 missense mutations, the risk allele frequency in the African American control cohort is 21% in contrast to 37% in the cases (corresponding values for Hispanic Americans are 6 and 23%, respectively). The most striking difference is in the frequency of the homozygote risk state, with only 3% in controls compared to 18% in cases for African Americans (corresponding values for Hispanic Americans are 0.5 and 11%, respectively) (see also Supplementary Fig. 5). We also show that the results for combined and meta-analysis of the two separate cohort-based results are congruent (Supplementary Table 1). As also evident from Table 1 and Supplementary Table 1, the modes of inheritance for which the associations of the disease risk phenotype with the APOL1 missense mutations are significant is consistent with an additive effect, wherein carrying the missense mutations on a single parental allele is sufficient to confer significantly increased risk, but with a still greater jump in risk conferred by carrying the missense mutations on both parental alleles. As noted above, the two missense mutations are in nearly perfect LD, and therefore, on a population genetics basis alone they can be considered as designating a “missense risk haplotype”. Examination of the predicted effect on protein structure and functional studies with artificial constructs which separate the two missense mutations will determine whether disease risk relates to either or both together as a functional missense haplotype. Therefore, for further analysis in the current study, we go on to use APOL1 SNP rs73885319 as tagging the “missense risk haplotype”. Analysis of deviance of the combined logistic regression indicates that LD with APOL1 SNP rs73885319 accounted for most of the statistical association previously attributed to the leading MYH9 variants with ESKD (supplementary material). In this regard, we also examined two noncoding variants in the APOL1 region which are in high LD with the APOL1 missense mutations, and as expected, both showed significant disease risk association (Supplementary Fig. 2; Supplementary Table 1).
HIVAN has been considered as the most prominent of the nondiabetic forms of kidney disease within what has been termed the MYH9-associated nephropathies (Kopp et al. 2008; Winkler et al. 2010). We have reported absence of HIVAN in HIV infected Ethiopians, and attributed this to host genomic factors (Behar et al. 2006). Therefore, we examined the allele frequencies of the APOL1 missense mutations in a sample set of 676 individuals from 12 African populations, including 304 individuals from four Ethiopian populations (Supplementary Table 2). We coupled this with the corresponding distributions for the African ancestry leading MYH9 S-1 and F-1 risk alleles. A pattern of reduced frequency of the APOL1 missense mutations and also of the MYH9 risk variants was noted in northeastern African in contrast to most central, western, and southern African populations examined (Supplementary Fig. 3). Especially striking was the complete absence of the APOL1 missense mutations in Ethiopia. This combination of the reported lack of HIVAN and observed absence of the APOL1 missense mutations is consistent with APOL1 being the functionally relevant gene for HIVAN risk and likely the other forms of kidney disease previously associated with MYH9.
The APOL1 gene encodes apolipoprotein L-1, whose known activities include powerful trypanosome lysis (Vanhamme et al. 2003), autophagic cell death (Wan et al. 2008), lipid metabolism, cellular senescence, as well as vascular and other biological activities (Monajemi et al. 2002; Vanhollebeke and Pays 2006). Of note, in humans, APOL1 is one of six closely spaced and related APOL genes, respectively, encoding six gene products apolipoproteins L-1 through L-6 (Duchateau et al. 1997, 2001). While APOL3 is thought to have arisen first in genomic evolution of the region, with the others having arisen as a result of duplication events (Monajemi et al. 2002), only apolipoprotein L-1 has a signal peptide which enables it to be both a circulating and intracellular protein (Vanhollebeke and Pays 2006). This latter capacity is of crucial importance to the protective and lytic activity of human serum to many species of trypanosoma. Trypanosoma brucei rhodesiense transmitted by tsetse flies causes human African trypanosomiasis as a result of the expression of serum resistance associated protein (SRA), which interacts with the C-terminal domain of apolipoprotein L-1 and inactivates its lytic function (Lecordier et al. 2009). Apolipoprotein L-1 protein structure is divided into three distinctive structural and functional domains: (1) an anionic pore-forming domain which is thought to be involved in organellar permeation and cell death, (2) a membrane addressing-domain consisting of a pH-sensitive hairpin bridging two alpha helices which facilitates association with the circulating HDL particle at neutral pH and intracellular organellar localization at acidic pH, and 3) a C-terminus amphipathic alpha helix with a leucine zipper for protein–protein interaction (Vanhollebeke and Pays 2006). It should be noted that the APOL1 S342G variant, powerfully associated with kidney disease risk in the current study, is predicted to modify the binding site of the C-terminus domain of the APOL1 gene product (Supplementary Fig. 4).
With respect to kidney disease risk, apolipoprotein L-1 is also prominently involved in autophagic pathways (Zhaorigetu et al. 2008), and a recent study has provided compelling evidence for the role of well-preserved autophagy in the integrity of renal glomerular podocytes (Hartleben et al. 2010). It is thus possible that variation in the C-terminus domain of endogenously expressed or endocytosed apolipoprotein L-1 modifies interaction with an as yet unidentified renal intracellular protein, which regulates the availability of apolipoprotein L-1 in its pore-forming or other functions. Given the numerous known functions of apolipoprotein L-1 noted above, a number of other mechanisms for kidney disease risk are possible, including those related to lipid metabolism or vascular integrity (Monajemi et al. 2002). The involvement of other classes of apolipoproteins in nephropathy has been well documented (Takemura et al. 1993). Moreover, apolipoproteins have also been identified as circulating inhibitors of glomerular proteinuria (Candiano et al. 2001). Remarkably, in this latter study, the amino acid sequence of the apolipoprotein L fraction isolated and studied corresponds exactly to what we now know to be apolipoprotein L-1.
Functional assays of the effect of the APOL1 missense variants described herein in appropriate experimental model systems will be needed to link the strong and biologically plausible association to a functional pathogenic pathway in kidney disease and to the possible selective factors which contributed to the observed African allele frequency distribution.
It is noteworthy that this entire region of the genome shows a high degree of LD and evidence of strong evolutionary selection, which may well explain why MYH9 yielded such a strong association signal in prior studies due to a hitchhiking effect of MYH9 with variants in the APOL gene family that have conferred adaptive advantage (Grossman et al. 2010; Smith and Malik 2009; Stephan et al. 2006). It should also be noted that the expression of apolipoproteins L in vascular and immune cells is greatly increased by viral infections, interferons, and inflammatory mediators (Vanhollebeke and Pays 2006), any or all of which might constitute the basis for a second “trigger” needed to induce the actual clinical manifestation of nephropathy in a genotypically “at risk” individual. Moreover, given the close evolutionary relationship of the six members of the human apolipoprotein L family and their manifold and overlapping functions, it is conceivable that additional rare or common variants might also interact and be involved in kidney disease risk (Supplementary Table 3).
The current findings strongly suggest that the intensive efforts (http://www3.niddk.nih.gov/fund/other/MYH9KidneyDisease/) currently underway to identify the ESKD disease phenotype risk causative variant in the chromosome 22 MALD peak should certainly be extended beyond the MYH9 locus. In particular, a strong emphasis should also be placed on APOL1 mutations with strong association and functional plausibility.
Methods and supplementary information and any associated references are available on the Human Genetics website: http://www.springer.com/biomed/human+genetics/journal/439.
We thank Steve Smith, Susan Kirby, Rhian Gwilliam, Daniella Magen and Liat Linde for excellent technical assistance. This study was supported by the Canadian and American Technion Societies (Eshagian Estate Fund, Dr. Sidney Kremer Kidney Disease Research Fund), the Israel Science Foundation (890015), and Legacy Heritage Fund to KS and by the European Research Commission (MIRG-CT-2007-208019) and the Israel Science Foundation (1227/09) to SR. DMB thanks the European Commission, Directorate-General for Research for FP7 Ecogene grant 205419.
Conflict of interest statement
The authors declare no competing financial interests.
This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.
- Bedoya G, Montoya P, Garcia J, Soto I, Bourgeois S, Carvajal L, Labuda D, Alvarez V, Ospina J, Hedrick PW, Ruiz-Linares A (2006) Admixture dynamics in Hispanics: a shift in the nuclear genetic ancestry of a South American population isolate. Proc Natl Acad Sci USA 103:7234–7239CrossRefPubMedGoogle Scholar
- Behar DM, Rosset S, Tzur S, Selig S, Yudkovsky G, Bercovici S, Kopp JB, Winkler CA, Nelson GW, Wasser WG, Skorecki K (2010) African ancestry allelic variation at the MYH9 gene contributes to increased susceptibility to non-diabetic end-stage kidney disease in Hispanic Americans. Hum Mol Genet 19:1816–1827CrossRefPubMedGoogle Scholar
- Bostrom MA, Freedman BI (2010) The spectrum of MYH9-associated nephropathy. Clin J Am Soc Nephrol 5(6):1107–1113Google Scholar
- Candiano G, Musante L, Carraro M, Faccini L, Campanacci L, Zennaro C, Artero M, Ginevri F, Perfumo F, Gusmano R, Ghiggeri GM (2001) Apolipoproteins prevent glomerular albumin permeability induced in vitro by serum from patients with focal segmental glomerulosclerosis. J Am Soc Nephrol 12:143–150PubMedGoogle Scholar
- Duchateau PN, Pullinger CR, Orellana RE, Kunitake ST, Naya-Vigne J, O’Connor PM, Malloy MJ, Kane JP (1997) Apolipoprotein L, a new human high density lipoprotein apolipoprotein expressed by the pancreas. Identification, cloning, characterization, and plasma distribution of apolipoprotein L. J Biol Chem 272:25576–25582CrossRefPubMedGoogle Scholar
- Freedman BI, Hicks PJ, Bostrom MA, Comeau ME, Divers J, Bleyer AJ, Kopp JB, Winkler CA, Nelson GW, Langefeld CD, Bowden DW (2009a) Non-muscle myosin heavy chain 9 gene MYH9 associations in African Americans with clinically diagnosed type 2 diabetes mellitus-associated ESRD. Nephrol Dial Transplant 24:3366–3371CrossRefPubMedGoogle Scholar
- Freedman BI, Hicks PJ, Bostrom MA, Cunningham ME, Liu Y, Divers J, Kopp JB, Winkler CA, Nelson GW, Langefeld CD, Bowden DW (2009b) Polymorphisms in the non-muscle myosin heavy chain 9 gene (MYH9) are strongly associated with end-stage renal disease historically attributed to hypertension in African Americans. Kidney Int 75:736–745CrossRefPubMedGoogle Scholar
- Hartleben B, Godel M, Meyer-Schwesinger C, Liu S, Ulrich T, Kobler S, Wiech T, Grahammer F, Arnold SJ, Lindenmeyer MT, Cohen CD, Pavenstadt H, Kerjaschki D, Mizushima N, Shaw AS, Walz G, Huber TB (2010) Autophagy influences glomerular disease susceptibility and maintains podocyte homeostasis in aging mice. J Clin Invest 120:1084–1096PubMedGoogle Scholar
- Kao WH, Klag MJ, Meoni LA, Reich D, Berthier-Schaad Y, Li M, Coresh J, Patterson N, Tandon A, Powe NR, Fink NE, Sadler JH, Weir MR, Abboud HE, Adler SG, Divers J, Iyengar SK, Freedman BI, Kimmel PL, Knowler WC, Kohn OF, Kramp K, Leehey DJ, Nicholas SB, Pahl MV, Schelling JR, Sedor JR, Thornley-Brown D, Winkler CA, Smith MW, Parekh RS (2008) MYH9 is associated with nondiabetic end-stage renal disease in African Americans. Nat Genet 40:1185–1192CrossRefPubMedGoogle Scholar
- Kopp JB, Smith MW, Nelson GW, Johnson RC, Freedman BI, Bowden DW, Oleksyk T, McKenzie LM, Kajiyama H, Ahuja TS, Berns JS, Briggs W, Cho ME, Dart RA, Kimmel PL, Korbet SM, Michel DM, Mokrzycki MH, Schelling JR, Simon E, Trachtman H, Vlahov D, Winkler CA (2008) MYH9 is a major-effect risk gene for focal segmental glomerulosclerosis. Nat Genet 40:1175–1184CrossRefPubMedGoogle Scholar
- Sekine T, Konno M, Sasaki S, Moritani S, Miura T, Wong WS, Nishio H, Nishiguchi T, Ohuchi MY, Tsuchiya S, Matsuyama T, Kanegane H, Ida K, Miura K, Harita Y, Hattori M, Horita S, Igarashi T, Saito H, Kunishima S (2010) Patients with Epstein-Fechtner syndromes owing to MYH9 R702 mutations develop progressive proteinuric renal disease. Kidney Int 78:207–214CrossRefPubMedGoogle Scholar
- Vanhamme L, Paturiaux-Hanocq F, Poelvoorde P, Nolan DP, Lins L, Van Den Abbeele J, Pays A, Tebabi P, Van Xong H, Jacquet A, Moguilevsky N, Dieu M, Kane JP, De Baetselier P, Brasseur R, Pays E (2003) Apolipoprotein L-I is the trypanosome lytic factor of human serum. Nature 422:83–87CrossRefPubMedGoogle Scholar