Adrienne Tin

Genetic association for renal traits among participants of African Ancestry reveals new loci for renal function

Chronic kidney disease (CKD) is an increasing global public health concern, particularly among populations of African ancestry. We performed an interrogation of known renal loci, genome-wide association (GWA), and IBC candidate-gene SNP association analyses in African Americans from the CARe Renal Consortium. In up to 8,110 participants, we performed meta-analyses of GWA and IBC array data for estimated glomerular filtration rate (eGFR), CKD (eGFR <60 mL/min/1.73 m2), urinary albumin-to-creatinine ratio (UACR), and microalbuminuria (UACR >30 mg/g) and interrogated the 250 kb flanking region around 24 SNPs previously identified in European Ancestry renal GWAS analyses. Findings were replicated in up to 4,358 African Americans. To assess function, individually identified genes were knocked down in zebrafish embryos by morpholino antisense oligonucleotides. Expression of kidney-specific genes was assessed by in situ hybridization, and glomerular filtration was evaluated by dextran clearance. Overall, 23 of 24 previously identified SNPs had direction-consistent associations with eGFR in African Americans, 2 of which achieved nominal significance (UMOD, PIP5K1B). Interrogation of the flanking regions uncovered 24 new index SNPs in African Americans, 12 of which were replicated (UMOD, ANXA9, GCKR, TFDP2, DAB2, VEGFA, ATXN2, GATM, SLC22A2, TMEM60, SLC6A13, and BCAS3). In addition, we identified 3 suggestive loci at DOK6 (p-value = 5.3×10−7) and FNDC1 (p-value = 3.0×10−7) for UACR, and KCNQ1 with eGFR (p = 3.6×10−6). Morpholino knockdown of kcnq1 in the zebrafish resulted in abnormal kidney development and filtration capacity. We identified several SNPs in association with eGFR in African Ancestry individuals, as well as 3 suggestive loci for UACR and eGFR. Functional genetic studies support a role for kcnq1 in glomerular development in zebrafish.

Genome-wide association study for serum urate concentrations and gout among African Americans identifies genomic risk loci and a novel URAT1 loss-of-function allele

Serum urate concentrations are highly heritable and elevated serum urate is a key risk factor for gout. Genome-wide association studies (GWAS) of serum urate in African American (AA) populations are lacking. We conducted a meta-analysis of GWAS of serum urate levels and gout among 5820 AA and a large candidate gene study among 6890 AA and 21 708 participants of European ancestry (EA) within the Candidate Gene Association Resource Consortium. Findings were tested for replication among 1996 independent AA individuals, and evaluated for their association among 28 283 EA participants of the CHARGE Consortium. Functional studies were conducted using (14)C-urate transport assays in mammalian Chinese hamster ovary cells. In the discovery GWAS of serum urate, three loci achieved genome-wide significance (P< 5.0 × 10(-8)): a novel locus near SGK1/SLC2A12 on chromosome 6 (rs9321453, P= 1.0 × 10(-9)), and two loci previously identified in EA participants, SLC2A9 (P= 3.8 × 10(-32)) and SLC22A12 (P= 2.1 × 10(-10)). A novel rare non-synonymous variant of large effect size in SLC22A12, rs12800450 (minor allele frequency 0.01, G65W), was identified and replicated (beta -1.19 mg/dl, P= 2.7 × 10(-16)). (14)C-urate transport assays showed reduced urate transport for the G65W URAT1 mutant. Finally, in analyses of 11 loci previously associated with serum urate in EA individuals, 10 of 11 lead single-nucleotide polymorphisms showed direction-consistent association with urate among AA. In summary, we identified and replicated one novel locus in association with serum urate levels and experimentally characterize the novel G65W variant in URAT1 as a functional allele. Our data support the importance of multi-ethnic GWAS in the identification of novel risk loci as well as functional variants.

Results from the Atherosclerosis Risk in Communities study suggest that low serum magnesium is associated with incident kidney disease

Low serum magnesium has been associated with kidney function decline in persons with diabetes as well as cardiovascular disease in the general population. Since the association of serum magnesium with incident kidney disease in the general population is unknown, we assessed this in 13,226 participants (aged 45 to 65) in the Atherosclerosis Risk in Communities study with baseline estimated glomerular filtration rate of at least 60 ml/min/1.73m2 in years 1987–89 and followed through 2010. The risks for incident chronic kidney disease (CKD) and end-stage renal disease (ESRD) associated with baseline total serum magnesium levels were evaluated using Cox regression. There were 1,965 CKD and 208 ESRD events during a median follow-up of 21 years. In adjusted analysis, low serum magnesium levels (0.7mmol/L or less) had significant associations with incident CKD and ESRD compared with the highest quartile with adjusted hazard ratio of 1.58 (95% CI: 1.35–1.87) for CKD and 2.39 (95% CI: 1.61–3.56) for ESRD. These associations remained significant after excluding users of diuretics and across subgroups stratified by hypertension, diabetes, and self-reported race. Thus, in a large sample of middle-aged adults, low total serum magnesium was independently associated with incident CKD and ESRD. Further studies are needed to determine whether modification of serum magnesium levels might alter subsequent incident kidney disease rates.

A tripartite complex of suPAR, APOL1 risk variants and αvβ3 integrin on podocytes mediates chronic kidney disease

Soluble urokinase plasminogen activator receptor (suPAR) independently predicts chronic kidney disease (CKD) incidence and progression. Apolipoprotein L1 (APOL1) gene variants G1 and G2, but not the reference allele (G0), are associated with an increased risk of CKD in individuals of recent African ancestry. Here we show in two large, unrelated cohorts that decline in kidney function associated with APOL1 risk variants was dependent on plasma suPAR levels: APOL1-related risk was attenuated in patients with lower suPAR, and strengthened in those with higher suPAR levels. Mechanistically, surface plasmon resonance studies identified high-affinity interactions between suPAR, APOL1 and αvβ3 integrin, whereby APOL1 protein variants G1 and G2 exhibited higher affinity for suPAR-activated avb3 integrin than APOL1 G0. APOL1 G1 or G2 augments αvβ3 integrin activation and causes proteinuria in mice in a suPAR-dependent manner. The synergy of circulating factor suPAR and APOL1 G1 or G2 on αvβ3 integrin activation is a mechanism for CKD.

Explaining the Racial Difference in AKI Incidence

African Americans face higher risk of AKI than Caucasians. The extent to which this increased risk is because of differences in clinical, socioeconomic, or genetic risk factors is unknown. We evaluated 10,588 African-American and Caucasian participants in the Atherosclerosis Risk in Communities study, a community-based prospective cohort of middle-aged individuals. Participants were followed from baseline study visit (1996-1999) to first hospitalization for AKI (defined by billing code), ESRD, death, or December 31, 2010. African-American participants were slightly younger (61.7 versus 63.1 years, P<0.001), were more often women (64.5% versus 53.2%, P<0.001), and had higher baseline eGFR compared with Caucasians. Annual family income, education level, and prevalence of health insurance were lower among African Americans than Caucasians. The unadjusted incidence of hospitalized AKI was 7.4 cases per 1000 person-years among African Americans and 5.8 cases per 1000 person-years among Caucasians (P=0.002). The elevated risk of AKI among African Americans persisted after adjustment for demographics, cardiovascular risk factors, kidney markers, and time-varying number of hospitalizations (adjusted hazard ratio, 1.20; 95% confidence interval [95% CI], 1.01 to 1.43; P=0.04); however, accounting for differences in income and/or insurance by race attenuated the association (P>0.05). High-risk APOL1 variants did not associate with AKI among African Americans (demographic-adjusted hazard ratio, 1.07; 95% CI, 0.69 to 1.65; P=0.77). In summary, the higher risk of AKI among African Americans may be related to disparities in socioeconomic status.

Association of Estimated Glomerular Filtration Rate and Urinary Uromodulin Concentrations with Rare Variants Identified by UMOD Gene Region Sequencing

Background Recent genome-wide association studies (GWAS) have identified common variants in the UMOD region associated with kidney function and disease in the general population. To identify novel rare variants as well as common variants that may account for this GWAS signal, the exons and 4 kb upstream region of UMOD were sequenced. Methodology/Principal Findings Individuals (n = 485) were selected based on presence of the GWAS risk haplotype and chronic kidney disease (CKD) in the ARIC Study and on the extremes of of the UMOD gene product, uromodulin, in urine (Tamm Horsfall protein, THP) in the Framingham Heart Study (FHS). Targeted sequencing was conducted using capillary based Sanger sequencing (3730 DNA Analyzer). Variants were tested for association with THP concentrations and estimated glomerular filtration rate (eGFR), and identified non-synonymous coding variants were genotyped in up to 22,546 follow-up samples. Twenty-four and 63 variants were identified in the 285 ARIC and 200 FHS participants, respectively. In both studies combined, there were 33 common and 54 rare (MAF<0.05) variants. Five non-synonymous rare variants were identified in FHS; borderline enrichment of rare variants was found in the extremes of THP (SKAT p-value = 0.08). Only V458L was associated with THP in the FHS general-population validation sample (p = 9*10−3, n = 2,522), but did not show direction-consistent and significant association with eGFR in both the ARIC (n = 14,635) and FHS (n = 7,520) validation samples. Pooling all non-synonymous rare variants except V458L together showed non-significant associations with THP and eGFR in the FHS validation sample. Functional studies of V458L revealed no alternations in protein trafficking. Conclusions/Significance Multiple novel rare variants in the UMOD region were identified, but none were consistently associated with eGFR in two independent study samples. Only V458L had modest association with THP levels in the general population and thus could not account for the observed GWAS signal.

Association between Mitochondrial DNA Copy Number in Peripheral Blood and Incident CKD in the Atherosclerosis Risk in Communities Study

Mitochondrial dysfunction in kidney cells has been implicated in the pathogenesis of CKD. Mitochondrial DNA (mtDNA) copy number is a surrogate measure of mitochondrial function, and higher mtDNA copy number in peripheral blood has been associated with lower risk of two important risk factors for CKD progression, diabetes and microalbuminuria. We evaluated whether mtDNA copy number in peripheral blood associates with incident CKD in a population-based cohort of middle-aged adults. We estimated mtDNA copy number using 25 high-quality mitochondrial single nucleotide polymorphisms from the Affymetrix 6.0 array. Among 9058 participants, those with higher mtDNA copy number had a lower rate of prevalent diabetes and lower C-reactive protein levels and white blood cell counts. Baseline eGFR did not differ significantly by mtDNA copy number. Over a median follow-up of 19.6 years, 1490 participants developed CKD. Higher mtDNA copy number associated with lower risk of incident CKD (highest versus lowest quartile: hazard ratio 0.65; 95% confidence interval, 0.56 to 0.75; P<0.001) after adjusting for age, sex, and race. After adjusting for additional risk factors of CKD, including prevalent diabetes, hypertension, C-reactive protein level, and white blood cell count, this association remained significant (highest versus lowest quartile: hazard ratio 0.75; 95% confidence interval, 0.64 to 0.87; P<0.001). In conclusion, higher mtDNA copy number associated with lower incidence of CKD independent of traditional risk factors and inflammation biomarker levels in this cohort. Further research on modifiable factors influencing mtDNA copy number may lead to improvement in the prevention and treatment of CKD.

Hemostatic Factors, APOL1 Risk Variants, and the Risk of ESRD in the Atherosclerosis Risk in Communities Study

BACKGROUND AND OBJECTIVES Hemostatic factors have been associated with kidney function decline, and evidence suggests stronger effects among African Americans. The presence of APOL1 renal risk variants, common in African Americans, might partly underlie this risk difference. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS A total of 13,337 participants in the Atherosclerosis Risk in Communities study were followed from 1987-1989 until 2010. Participants were categorized into three groups by ancestry and APOL1 risk status: European Americans (n=10,206), African Americans with zero or one APOL1 risk allele (n=2,733), and African Americans with two APOL1 risk alleles (n=398). ESRD events were ascertained through linkage to the US Renal Data System. Cox regression was used to estimate the risk for ESRD associated with hemostatic factors (factor VIIc, factor VIIIc, fibrinogen, von Willebrand factor, protein C, and antithrombin III). RESULTS There were 232 cases of ESRD over 21.5 years (European Americans, 119; African Americans with zero or one APOL1 risk allele, 94; African Americans with two APOL1 risk alleles, 19). In unadjusted and adjusted analysis of the overall population, higher levels of all hemostatic factors, except antithrombin III, were significantly associated with ESRD (all P<0.05). Factor VIIc had the strongest association (per one interquartile range; adjusted hazard ratio, 1.46; 95% confidence interval, 1.21 to 1.76). With the exception of fibrinogen, the risk associated with each hemostatic factor was stronger in African Americans with two APOL1 risk alleles compared with the other two groups. Statistically significant interactions were seen for factor VIIIc and protein C (interaction between those with two APOL1 risk allele and the other two groups: P<0.02 for factor VIIIc and <0.04 for protein C). CONCLUSIONS Higher levels of factor VIIc, VIIIc, fibrinogen, von Willebrand factor, and protein C were associated with ESRD risk, with a significantly stronger association of factor VIIIc and protein C in African Americans with two APOL1 risk alleles.

Genome-wide significant locus of beta-trace protein, a novel kidney function biomarker, identified in European and African Americans

BACKGROUND Beta-trace protein (BTP), measured in serum or plasma, has potential as a novel biomarker for kidney function. Little is known about the genes influencing BTP levels. METHODS We conducted a genome-wide association study of log-transformed plasma BTP levels in 6720 European Americans (EAs) and replicated the significant associations in 1734 African Americans (AAs) from the Atherosclerosis Risk in Communities (ARIC) study. RESULTS We identified a genome-wide significant locus in EA upstream of Prostaglandin D2 synthase (PTGDS), the gene encoding BTP. Each copy of the A allele at rs57024841 was associated with 5% higher BTP levels (P = 1.2 × 10(-23)). The association at PTGDS was confirmed in AAs (6% higher BTP for each A allele at rs57024841, P = 1.9 × 10(-7)). The index single nucleotide polymorphisms (SNPs) in EAs and AAs explained ∼1.1% of the log(BTP) variance within each population and explained over 30% of the difference in log(BTP) levels between EAs and AAs. The index SNPs at the PTGDS locus in the two populations were not associated with the estimated glomerular filtration rate (eGFR) or the urine albumin creatinine ratio (P > 0.05). We further tested for the associations of BTP with 16 known loci of the eGFR in EA, and BTP was associated with 3 of 16 tested. CONCLUSIONS The identification of a novel BTP-specific (non-renal related) locus and the confirmation of several genetic loci of the eGFR with BTP extend our understanding of the metabolism of BTP and inform its use as a kidney filtration biomarker.

The Association Between APOL1 Risk Alleles and Longitudinal Kidney Function Differs by HIV Viral Suppression Status

BACKGROUND Existing data suggest that human immunodeficiency virus (HIV)-infected African Americans carrying 2 copies of the APOL1 risk alleles have greater risk of kidney disease than noncarriers. We sought to determine whether HIV RNA suppression mitigates APOL1-related kidney function decline among African Americans enrolled in the Multicenter AIDS Cohort Study. METHODS We genotyped HIV-infected men for the G1 and G2 risk alleles and ancestry informative markers. Mixed-effects models were used to estimate the annual rate of estimated glomerular filtration rate (eGFR) decline, comparing men carrying 2 (high-risk) vs 0-1 risk allele (low-risk). Effect modification by HIV suppression status (defined as HIV type 1 RNA level <400 copies/mL for >90% of follow-up time) was evaluated using interaction terms and stratified analyses. RESULTS Of the 333 African American men included in this study, 54 (16%) carried the APOL1 high-risk genotype. Among HIV-infected men with unsuppressed viral loads, those with the high-risk genotype had a 2.42 mL/minute/1.73 m(2) (95% confidence interval [CI], -3.52 to -1.32) faster annual eGFR decline than men with the low-risk genotype. This association was independent of age, comorbid conditions, baseline eGFR, ancestry, and HIV-related factors. In contrast, the rate of decline was similar by APOL1 genotype among men with sustained viral suppression (-0.16 mL/minute/1.73 m(2)/year; 95% CI, -.59 to .27; P for interaction <.001). CONCLUSIONS Unsuppressed HIV-infected African Americans with the APOL1 high-risk genotype experience an accelerated rate of kidney function decline; HIV suppression with antiretroviral therapy may reduce these deleterious renal effects.