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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Kidney Int. Author manuscript; available in PMC 2009 October 1.
Published in final edited form as:
Published online 2009 January 28. doi:  10.1038/ki.2008.701
PMCID: PMC2698223
NIHMSID: NIHMS118317

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

Abstract

African Americans have high incidence rates of end-stage renal disease (ESRD) labeled as due to hypertension. As recent studies showed strong association with idiopathic and HIV-related focal segmental glomerulosclerosis and non-muscle myosin heavy chain 9 (MYH9) gene polymorphisms in this ethnic group, we tested for MYH9 associations in a variety of kidney diseases. Fifteen MYH9 single-nucleotide polymorphisms were evaluated in 175 African Americans with chronic glomerulonephritis-associated ESRD, 696 African Americans reportedly with hypertension-associated ESRD, and 948 control subjects without kidney disease. Significant associations were detected with 14 of the 15 polymorphisms in all 871 non-diabetic patients with ESRD. In hypertension-associated ESRD cases alone, significant associations were found with 13 MYH9 polymorphisms and the previously reported E1 haplotype. Thus, hypertension-associated ESRD in African Americans is substantially related to MYH9 gene polymorphisms and this may explain the poor response to blood pressure control in those diagnosed with hypertensive nephrosclerosis. It is possible that many African Americans classified as having hypertension-associated ESRD have occult MYH9-associated segmental or global glomerulosclerosis. Our study shows that gene-environment and/or gene–gene interactions may initiate kidney disease in genetically susceptible individuals, because African Americans homozygous for MYH9 risk alleles do not universally develop kidney disease.

Keywords: African American, end-stage renal disease, focal segmental glomerulosclerosis, hypertension, hypertensive nephrosclerosis, MYH9

Hypertensive nephrosclerosis and chronic glomerular diseases were reported as the cause of end-stage renal disease (ESRD) in 35% of incident dialysis patients in 2005.1 Both syndromes are reported more often in African Americans than European Americans, an effect likely due to biologic differences and physician bias.2,3 A causative link between hypertension and nephrosclerosis has not been clearly established.4 Although epidemiologic reports consistently demonstrate graded relationships between serum creatinine concentration and elevated blood pressure, this could be the result of secondary hypertension in the setting of a primary renal disease.5 In spite of renal biopsy studies revealing segmental and global forms of glomerulosclerosis (and other primary glomerular diseases) in those with clinically diagnosed hypertensive nephrosclerosis,6 along with the lack of correlation between systemic blood pressure and renal microvascular changes,7,8 the failure of aggressive blood pressure lowering, even with the use of angiotensin-converting enzyme inhibitors to slow nephropathy progression in hypertensive African Americans with non-diabetic chronic kidney disease,9,10 clinicians commonly diagnose mild-to-moderate essential hypertension as a cause of nephropathy.

Kopp et al.11 demonstrated that the non-muscle myosin heavy chain 9 (MYH9) gene was associated with biopsy-proven focal segmental glomerulosclerosis (FSGS) and HIV-associated nephropathy (HIVAN) in African Americans, using mapping by admixture linkage disequilibrium, an approach suited to diseases with marked ethnic differences in risk.12 The attributable risk due to MYH9 was 70% in FSGS and 100% in HIVAN, with odds ratios (OR) in the 4–5 range.11 MYH9 was associated with FSGS in European Americans, but the attributable risk was smaller due to the low frequency of the MYH9 risk haplotype in European Americans (4% compared with 60% of African Americans). Association between MYH9 was also observed in an extension cohort of 241 African Americans with clinically diagnosed hypertension-associated ESRD (H-ESRD) from North Carolina (OR 1.8, P = 0.003).11 Kao et al.13 replicated these findings by detecting association between MYH9 and several non-diabetic etiologies of ESRD among African Americans in the MALD arm of the ‘Family Investigation of Nephropathy and Diabetes’ (FIND). They estimated that replacing the African American MYH9 risk alleles with European American variants would reduce non-diabetic ESRD by 70% in the African American population. Association was detected separately in FIND cases with FSGS and HIVAN, with weaker association in 347 individuals with H-ESRD.13

The current report evaluates the largest number of unrelated African Americans with H-ESRD to date for association with the MYH9 gene, for the purpose of providing enhanced power to detect additional genomic regions associated with disease. All subjects were born in the southeastern United States of America, where African Americans are diagnosed with H-ESRD 20 times more often than European Americans, the greatest ethnic disparity for this renal disease reported in the nation.

Results

DNA from 871 non-diabetic ESRD cases and 948 unrelated non-renal disease controls were genotyped for 15 MYH9 gene single-nucleotide polymorphisms (SNPs) previously shown to be significantly associated with FSGS and HIVAN.11 On the basis of medical record review, the ESRD case group was classified as containing 696 individuals clinically diagnosed with H-ESRD, 80 with presumed chronic glomerular disease, 15 renal biopsy-proven FSGS, 20 HIVAN, 25 lupus nephritis, and 35 other non-diabetic etiologies of kidney disease. Of the 696 H-ESRD cases, DNA from 262 cases was previously sent to Dr Jeffrey Kopp (NIDDK), and 241 of these samples were included in a prior publication.11 These 262 individuals were retained in our overall analysis to enhance power to detect new polymorphisms independent of the previously reported risk haplotype, and the 434 ‘new’ H-ESRD cases (not previously genotyped) were evaluated separately for replication. Table 1 contains demographic characteristics in study participants. Cases initiated dialysis therapy at nearly 49 years of age, typical of the African-American non-diabetic population in the United States.1 Among the 948 non-renal disease controls, 558 (58.9%) were asked whether they had high blood pressure, and 34% of them (192/558) reported having hypertension, with mean age at onset 44.0±13.0 years (median 45 years). Frequentist estimation of individual ancestry proportion (FRAPPE) was used to calculate the proportion of African ancestry in cases and controls.14 The 70 ancestry informative markers revealed mean African ancestry proportions of 0.79 (s.d. = 0.10) in controls and 0.81 (s.d. = 0.10) in non-diabetic ESRD cases.

Table 1
Demographic characteristics of African American non-diabetic ESRD cases and controls

The 15 SNPs genotyped spanned 49.3 kb of MYH9, encompassing the majority of the coding region (Figure 1). Genotyping success rates for the MYH9 SNPs were 96.2–98.74% in cases and controls. A concordance rate of 99.8% was observed in 465 duplicate samples that were genotyped for quality control purposes. Ancestry informative marker SNPs genotyped at a rate of 93.1–98.5%. Table 2a summarizes allele frequencies and results of the single SNP genotypic association analysis for the non-diabetic ESRD cases and controls. Table 2b contains single SNP genotypic association results for the 696 cases with H-ESRD (excluding the 175 cases with chronic glomerular disease). SNP rs2187776 failed to meet Hardy–Weinberg equilibrium in the control population (P=0.0005) and was removed from further analysis. The initial age, gender, and admixture-adjusted analysis revealed single SNP associations with non-diabetic ESRD, most significantly in the recessive model, with 13 of the 14 SNPs analyzed. These associations were comparably significant without adjustment for admixture (Tables 2a and andbb).

Figure 1
Gene structure and linkage disequilibrium plot of 49 kb of the MYH9 gene
Table 2a
MYH9 single SNP associations in 871 African Americans with non-diabetic ESRDa
Table 2b
MYH9 single SNP associations in 696 African Americans with H-ESRDa

Tables 3a and andbb contain results of a priori MYH9 E1 haplotype associations in all 871 non-diabetic ESRD cases, as well as limited to the 696 cases with H-ESRD, respectively. This analysis was performed to allow for comparison with results previously reported by Kopp et al.11 in FSGS and HIVAN and to allow for the identification of novel risk loci after adjusting for the published risk haplotype. The age, gender, and ancestry adjusted P-value for the E1 risk haplotype determined using a weighted logistic regression analysis in all non-diabetic cases of ESRD vs controls was 1.22 × 10−15, with 71.8% of cases and 57.3% of controls homozygous for the risk haplotype (OR 2.38). Limited to the 696 H-ESRD cases, the ancestry-adjusted E1 haplotype P-value was 4.52 × 10−12 (OR 2.23); a result that survives stringent Bonferroni correction. A novel association in a second haplotype block L1, (1324) consisting of rs7078, rs12107, rs735853, and 5756129, is also displayed.

Table 3a
Logistic regression results—MYH9 haplotypes (all 871 non-diabetic ESRD cases)a
Table 3b
Logistic regression results—MYH9 haplotypes (696 hypertension-associated ESRD cases)a

Tables 4a and andbb contain the results of single SNP and haplotype association analyses limited to the 434 newly genotyped cases with H-ESRD not analyzed in the earlier report.11 Despite reduced power in the smaller but independent sample, single SNP associations remained highly significant (P-values ranged 0.05–9.1 × 10−11 recessive; admixture adjusted OR 1.35–3.23), and the 3224 and 1324 haplotypes were also strongly associated (P = 2.6 × 10−6 and 3.1 × 10−7, respectively). This analysis replicated the association between MYH9 and H-ESRD in a new sample.

Table 4a
MYH9 SNP associations in 434 new H-ESRD cases (excluding subjects evaluated in the study by Kopp et al.11)
Table 4b
MYH9 haplotype associations in 434 new H-ESRD cases (excluding subjects evaluated in the study by Kopp et al.11)

Tables 5a and andbb contains SNP association analyses, adjusted for the presence of the powerful E1 risk haplotype (Table 5a—E1 additive model, 5b—E1 recessive model). After adjustment for the E1 haplotype, rs5756152, rs12107, rs1005570, rs16996674, and rs16996677 remained independently associated with non-diabetic forms of ESRD (P = 0.047–7.1 × 10−6; OR 1.20–1.50, additive). This suggests that although E1 by itself explained a significant portion of the variation, additional SNPs in the region independently contribute to the risk of non-diabetic ESRD.

Table 5a
Logistic regression results for individual SNPs, adjusting for 3224 haplotype (additive model) in all 871 casesa
Table 5b
Logistic regression results for individual SNPs, adjusting for 3224 haplotype (recessive model) in all 871 casesa

Table 6 contains the results of the multilocus SNP analysis using logistic regression. After adjustment for age, gender, and admixture, three SNPs exhibited independent evidence for association, rs4821480, rs5756152, and rs12107. The estimated OR for an individual SNP in this analysis adjusts for all effects in the model, including all other loci. In addition, only rs4821480 was part of the E1 risk haplotype, associated at a P-value of 7.0 × 10−9. This model demonstrates that after controlling for age, gender, and admixture three SNPs provide discriminating power to distinguish between non-diabetic ESRD cases and non-nephropathy controls.

Table 6
Results of stepwise logistic regression analysis performed on all SNPs

We examined the contribution of the covariates and SNPs to the C-statistic (area under the receiver-operator characteristic curve).15 The C-statistic is a nonparametric measure of the predictive ability of a model and a function of sensitivity and specificity. It is the probability that for a randomly selected pair of subjects, one with non-diabetic ESRD and one without, the individual with ESRD has a higher predicted probability of disease than the individual without ESRD. A C = 0.50 is completely random. Using the same sample for all three estimates, the C-statistic was C = 0.63 for the model with age and gender only, C = 0.64 for age, gender and admixture estimate, and 0.70 for age, gender, admixture, and the three SNPs. Thus, the three SNPs markedly increase the predictive ability of the model in these data, as well as are individually statistically significant.

The individual SNP association analysis and stepwise association analysis (Tables 2a, ,2b2b and and6)6) identified multiple SNPs outside of the original E1 haplotype that predicted risk for non-diabetic ESRD. In particular, the first four SNPs in Figure 1, rs7078, rs12107, rs735853, and rs5756129, potentially explain additional risk and comprise a second haplotype block. The 1324 L1 haplotype in this block was present in 72.2% of ESRD cases and 57.5% of controls. A logistic regression model that adjusted for the original E1 (3224) haplotype under a recessive model, as well as for age, gender, and admixture, provides evidence that the second haplotype (1324) explained independent risk for non-diabetic ESRD (P = 0.00009, additive) (Table 7). Collectively, it is clear from the individual SNP analysis, the multiple locus analysis, and the multiple haplotype analysis that there were several loci with independent evidence for association to non-diabetic ESRD. The relatively high linkage disequilibrium (LD) within the region complicates the elucidation of these effects. However, these multilocus analyses test for associations conditional on the effects at the genotyped loci that are in LD. Finally, rs5756152 remained an independent predictor of risk, even after adjusting for the 1324 and 3224 risk haplotypes (data not shown).

Table 7
Results of logistic regression analysis using both associated haplotypes

Discussion

The present analyses demonstrate that MYH9 gene polymorphisms are associated with non-diabetic etiologies of ESRD in African Americans, particularly with kidney failure that had historically been attributed to essential hypertension. This gene exhibits impressive evidence for association with common, complex kidney diseases, with ORs as high as 3.4 for associated SNPs. This is the largest report containing cases with H-ESRD, and all participants were born in the southeastern United States of America, the region having the highest ESRD incidence rates and the greatest African American/European American disparity in non-diabetic ESRD. We extend the results of earlier reports by replicating association in 434 newly genotyped cases with H-ESRD and by demonstrating independent contributions of MYH9 SNPs and haplotypes in patients with non-diabetic ESRD, beyond the previously described E1 haplotype (although it is conceivable that a causal SNP could be in LD with multiple regions in the gene).11,13 Our findings remain significant after adjusting for age, gender, and multiple testing, factors not previously addressed. The strong MYH9 association observed in these individuals, as well as those in the FSGS cohorts recruited throughout the United States and the FIND sample, mirrors the familial aggregation of non-diabetic forms of ESRD observed in African Americans in the southeastern United States of America, Los Angeles, Cleveland, and Alabama.16 It is clear that MYH9 is a gene with substantial impact on common forms of non-diabetic ESRD in the African-American population.

As all MYH9 risk homozygotes do not develop kidney disease, environmental exposures and/or interactions with other genes are clearly necessary to initiate MYH9-associated nephropathy in genetically susceptible hosts. Although it remains possible that high blood pressure is one such initiating factor, this remains to be proven. HIV infection appears to be a common initiator of renal disease in African Americans and serves as an example of one environmental exposure that can initiate MYH9-associated HIVAN. Other non-HIV viral factors, as well as toxin exposures, may also be initiators of kidney disease in MYH9 risk homozygotes. These important gene–environment and gene–gene interactions require further evaluation.

The recent demonstration that MYH9 gene polymorphisms commonly cause idiopathic and HIV-associated FSGS in the African-American population makes this gene the most common inherited cause of FSGS, far more frequent than polymorphisms in the ACTN4 (α-actinin 4) and TRPC6 (transient receptor potential cation channel 6) genes, or glomerulosclerosis due to NPHS2 (podocin), CD2AP (CD2-associated protein), WT1 (Wilm's tumor-1) genes, and mt DNA tRNA leucine.1721 The MYH9 gene product, myosin-IIA, is a mechanoenzyme localized to the podocyte foot process and is responsible for moving actin filaments in cells. The proteins comprising the filtration slit barrier actively regulate actin dynamics to maintain normal cell structure. Mutations affecting other podocyte proteins rearrange the actin cytoskeleton, ultimately disrupting the filtration barrier and causing renal disease. We hypothesize that polymorphisms in the MYH9 gene may directly cause podocyte injury resulting in either FSGS or global glomerulosclerosis in the absence of hypertension, although this remains to be proven.

The observed ethnic disparities in H-ESRD, FSGS, and HIVAN, all far more common in African Americans, may also be due in part to the lower frequency of MYH9 risk alleles in European Americans, who demonstrate a 4% frequency of the E1 risk haplotype.11,13 This suggests that MYH9, in addition to environmental factors and socioeconomic status, contributes to ethnic disparities in non-diabetic forms of ESRD.

This disease association may alter our understanding of the factors that ‘initiate’ H-ESRD. Several studies revealed that high blood pressure does not commonly lead to progressive nephropathy in African-American patients.22,23 The few hypertensive African Americans who demonstrate progressive kidney disease are typically labeled as having ‘hypertensive nephrosclerosis,’ which is presumed to progress to H-ESRD. It is possible that individuals harboring MYH9 risk alleles may be more likely to develop renal failure due to a primary renal disease process, although the role of high blood pressure requires further study. The frequency of the MYH9 E1 risk haplotype was similar in hypertensive African Americans from HyperGEN and in the general community.24 This result may lessen the likelihood that high blood pressure per se is a proximate cause of MYH9-associated nephropathy. We further hypothesize that the MYH9 association with H-ESRD could explain the failure of intensive blood pressure reduction, including with the use of ACE inhibition, to slow nephropathy progression in hypertensive African Americans with chronic kidney disease.9,10,25 Prior to these reports, MYH9 was associated with rare autosomal dominant forms of macrothrombocytopenia, often with variable degrees of sensorineural deafness, cataracts, neutrophil inclusions, and glomerular disease.26

African American patients clinically labeled with ‘hypertensive nephrosclerosis’ and with non-nephrotic proteinuria have been shown to have FSGS on renal biopsy.6 In contrast, renal biopsies from a small subset of African American Study of Kidney Disease and Hypertension (AASK) participants were interpreted as being ‘consistent with hypertension-associated kidney disease.’ Renal arteriolar changes that were historically ascribed to high blood pressure did not correlate with measured blood pressure in AASK participants.7 The majority of subjects in AASK with renal biopsies had global glomerulosclerosis with interstitial fibrosis. It is possible that global glomerulosclerosis could result from polymorphisms in MYH9, and be part of a disease spectrum linking FSGS with global glomerulosclerosis. We propose that DNA samples from AASK participants be tested for association with MHY9 to address this intriguing possibility.

African Americans with nonspecific and incompletely characterized causes of ESRD, typically attributed to hypertension and chronic glomerular diseases, were thought to be a heterogeneous group consisting of several different diseases.4,27,28 The MYH9 association with H-ESRD in these 696 patients (262 previously sent to the Laboratory of Genomic Diversity at the National Cancer Institute and 434 newly genotyped cases), coupled with the results in 347 FIND participants with H-ESRD from Maryland reveal a unifying major genetic etiology for this syndrome. Although African Americans develop ESRD attributed to diabetes mellitus four times more often than European Americans, MYH9 associations have not yet been observed in African Americans with type 2 diabetes-associated ESRD, suggesting that this gene relates to non-diabetic forms of nephropathy.11,13 One limitation of our report is the potential for recruitment bias; however, similar results seen in the FIND appear to make this less likely. In addition, individuals classified as having H-ESRD in the absence of quantified proteinuria may actually have had proteinuria and underlying FSGS. This difficulty plagues all existing genetic and epidemiologic analyses involving H-ESRD. Phenotype information in African Americans labeled as having H-ESRD is notoriously poor, often a result of late referral to nephrologists and a paucity of kidney biopsies. The difficulty classifying causes of non-diabetic ESRD in African Americans, coupled with strong familial aggregation, were reasons for initiating a search for causative genetic factors. Regardless of the underlying histology, which will never be available in these 696 subjects with H-ESRD, these association results demonstrate that African Americans who are labeled with H-ESRD by their treating nephrologists often have MYH9-associated kidney diseases. Evaluation of renal histology in those with the early stages of MYH9-associated nephropathy will clarify this issue.

There are many remaining questions in this rapidly evolving story. Most importantly, are African Americans with MYH9-associated renal disease responsive to immunologic (or other) commonly applied therapies as prescribed for FSGS? Should African-American subjects with non-nephrotic proteinuria undergo renal biopsy, as well as testing for MYH9? Should African Americans with newly diagnosed hypertension undergo MYH9 testing and those found to be homozygous for the risk haplotypes be treated to lower blood pressure targets before the development of proteinuria? Beyond HIV infection, what environmental factors trigger progressive nephropathy in MYH9 risk homozygotes? Prospective studies and clinical trials will be required to answer these questions. We need to examine the role of MYH9 in other kidney diseases disproportionately impacting African Americans, such as lupus nephritis where affected individuals often have relatives with non-lupus forms of kidney disease, and the role of MYH9 gene polymorphism in kidney diseases involving native African, Aboriginal and Afro-Caribbean populations. The causative SNP and mechanism of action of MYH9-associated kidney disease has not yet been fully elucidated, although a new and exciting era has dawned with this important discovery. MYH9 is the first major gene to be identified using mapping by admixture disequilibrium. It is also a gene with pronounced effect in common and complex kidney disease.

In conclusion, MYH9 underlies a significant percentage of non-diabetic etiologies of ESRD in African Americans, particularly hypertension-associated ESRD. MYH9 is also associated with disparate kidney diseases, such as HIVAN and FSGS, which along with H-ESRD have been shown to aggregate in single African-American families.3 The identification of a gene expressed in podocytes and involved in the motor regulation of cytoskeleton components, promises new breakthroughs that may substantially reduce the burden of ESRD in non-diabetic African Americans.

Materials and Methods

Participants

Self-reported African Americans born in North Carolina, South Carolina, Georgia, Virginia, or Tennessee formed the study population. Peripheral blood specimens for DNA extraction were collected from unrelated, prevalent hemodialysis, and peritoneal dialysis patients in these states. Medical records were reviewed by a single investigator (BIF). Subjects who had ESRD attributed to H-ESRD, idiopathic, or secondary glomerular diseases on their CMS 2728 form were recruited. The majority of cases (696/871 or 79.9%) were diagnosed as having H-ESRD by virtue of high blood pressure preceding initiation of renal replacement therapy with hypertensive target organ damage (retinopathy or left ventricular hypertrophy) and low level proteinuria (≤30 mg/100ml on urine dipstick, <0.5 g protein/24 h on timed urine collection, or urine protein/creatinine ratio <0.5 g/g), or in the absence of proteinuria measurements (review of dialysis records in 50 randomly selected H-ESRD cases revealed that only 16% had quantitated urinary protein excretion). In the presence of renal biopsy evidence of a primary glomerular disease (for example, FSGS or membranous glomerulonephritis), proteinuria ≥0.5g/24h or ≥100mg/100 ml on urinalysis, non-diabetic subjects were diagnosed as having chronic glomerular disease-associated ESRD. Individuals with diabetic causes of ESRD (type 1 or type 2) were excluded from these analyses, as were those with renal cystic diseases, hereditary nephritis, or urologic causes of ESRD. Diabetes-associated ESRD was diagnosed in patients with either renal histologic evidence of diabetic nephropathy or with diabetes ≥5 years before the initiation of renal replacement therapy in the presence of diabetic retinopathy and/or proteinuria >500 mg/24h in the absence of other known causes of kidney disease. Two hundred and forty one of the H-ESRD cases were previously evaluated in the Kopp et al.11 report, demonstrating association with the MYH9 E1 risk haplotype (P = 0.003 recessive model, OR 1.8). These 241 individuals with H-ESRD (a subset of the 262 H-ESRD participant DNA samples submitted) were retained in this analysis, and we included an additional 434 unrelated African Americans with H-ESRD.

Unrelated, non-diabetic African Americans born in the same five southeastern states served as controls. Control subjects denied a personal or family history (in first degree relatives) of diabetes or kidney disease (kidney disease was defined as kidney failure, dialysis, or kidney transplantation); however, most did not have direct measurement of renal function or blood pressure. Among the last 200 African American controls recruited, 0.5% (1/200) had a non-fasting blood sugar >150 mg/100ml (value was 152 mg/100 ml) and 2% (4/200) had a serum creatinine concentration ≥1.5 mg per 100ml (maximum = 1.85 mg/100 ml). The presence of occult kidney disease in the control group would bias against association and could deflate significance. The study was approved by the Institutional Review Board at the Wake Forest University School of Medicine and met criteria outlined in the Declaration of Helsinki Principles. All participants provided written informed consent. DNA from these participants was extracted using Puregene (Gentra, Minneapolis, MN, USA).

SNP selection and genotyping

Fifteen SNPs in MYH9 were chosen for genotyping, based on those previously selected by Kopp et al.11 for pronounced frequency differences between reference Yoruban and European (CEU) populations, possible functional significance, or strong LD with SNPs typed in the original African American cohort with FSGS that remained significantly associated after adjustment for admixture. Tested SNPs included rs7078; rs12107; rs735853; rs5756129; rs5756130; rs2187776; rs4821480; rs2032487; rs4821481; rs3752462; rs5756152; rs1557539; rs1005570; rs16996674; and rs16996677 (Figure 1). The MYH9 E1 risk haplotype containing rs4821480, rs2032487, rs4821481, and rs3752462, previously associated with FSGS and HIVAN, was evaluated.11 SNP genotyping was performed on a Sequenom Mass Array Genotyping System (Sequenom, San Diego, CA, USA).

Seventy di-allelic ancestry informative markers were genotyped to determine whether population substructure biased our conclusions. The African American cases and control DNA samples were genotyped using either Illumina Inc's Custom Genotyping Services (San Diego, CA, USA) or using the Sequenom Mass Array (San Diego). Thirty nine unrelated European American controls were recruited, as described for the African American controls, and DNA was obtained from 44 Yoruba Nigerians (YRI) from the National Institute of General Medicine Sciences (NIGMS) Human Variation Collection (Coriell Repositories, Camden, NJ, USA).

Statistical analyses

Each SNP was tested for departures from Hardy–Weinberg equilibrium expectations by a χ2 goodness of fit test.29 LD was estimated using the classic D′ and r2 statistics as implemented in Dprime (http://www.phs.wfubmc.edu/public/bios/gene/downloads.cfm) and Haploview 3.32.30 All tests of association are adjusted for age, gender, and admixture proportions. The individual admixture proportions were estimated by the expectation–maximization algorithm implemented in the software FRAPPE.31 To test for an association between each SNP and non-diabetic ESRD, we computed the overall genotypic test of association and the three a priori genetic models (dominant, additive, and recessive). We tested for departures from additivity and computed the allelic and two- and three-marker haplotypes. These tests were computed using SNPGWA and Dandelion (http://www.phs.wfubmc.edu/public/bios/gene/downloads.cfm); both programs use the expectation–maximization algorithm for haplotype estimation. Both the large sample test distribution and permutation methods were used to estimate statistical significance. As anticipated from the large-sample tests, the most significant SNPs did not have a more extreme test statistic in 10 million permutations of the data. The C-statistic was also computed. For binary outcomes, the C-statistic is identical to the area under the receiver–operating characteristic curve. It estimates the probability of correctly identifying the case individual from a randomly selected pair of individuals (1 case, 1 control) given the genotype data and the estimated logistic regression model. The C-statistic parameter varies between 0.5 (random prediction) and 1.0 (perfect prediction).

To examine the effect of SNPs outside the four marker E1 haplotypes, we estimated each person's haplo–genotype probabilities in Dandelion and computed a weighted logistic regression analysis. Specifically, the logistic regression model contained age, gender, admixture proportion, genetic model for the four marker haplotypes (for example, dominant model for 3224 haplotype defined as presence of the 3224 haplotype vs all other haplotypes), where the weights are the corresponding haplo–genotype probabilities. Stepwise methods (entry and exit criteria of 0.05) were used to examine the independent contribution to risk of the individuals SNPs outside the haplotype while adjusting for age, gender, admixture proportion, and haplotype. In addition to the goal of testing for replication of the haplotype previously reported, the independent contribution to risk of all SNPs was of interest. Last, the stepwise logistic regression analysis was repeated adjusting for age, gender, and admixture proportion, without forcing the haplotype into the model.

Acknowledgments

This study was supported in part by NIH grants RO1 DK 070942 (BIF) and RO1 DK53591 (DWB), and by the NIDDK and NCI Intramural Research Programs. We are indebted to the local nephrology community, including all physicians and their patients who participated, as well as to our study coordinators Joyce Byers, Carrie Smith, Mitzie Spainhour, Cassandra Bethea, and Sharon Warren. This project has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under contract N01-CO-12400 and HHSN261200800001E. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government. This research was supported (in part) by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.

Footnotes

Disclosure: All the authors declared no competing interests.

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