View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Kidney International, Vol. 68 (2005), pp. 256–262 Identification of podocin (NPHS2) gene mutations in African Americans with nondiabetic end-stage renal disease JUDITH A. ENGELER DUSEL,1 KATHRYN P. B URDON,1 PAMELA J. HICKS,GREGORY A. HAWKINS, DONALD W. B OWDEN, and BARRY I. FREEDMAN Departments of Internal Medicine, Center for Human Genomics, Biochemistry, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, North Carolina Identification of podocin (NPHS2) gene mutations in African End-stage renal disease (ESRD), with its associated Americans with nondiabetic end-stage renal disease. glomerulosclerosis and interstitial fibrosis, is a major Background. Podocin, encoded by NPHS2 and mapped to cause of morbidity and mortality worldwide. The lead- 1q25.2, is an integral membrane protein exclusively expressed in glomerular podocytes. Mutations in the NPHS2 gene cause ing causes of ESRD are diabetic nephropathy, hy- autosomal-recessive nephrotic syndrome and have been associ- pertension, and chronic glomerular diseases. Alarming ated with proteinuria in several populations. Evidence for link- increases in health care costs and numbers of individu- age of end-stage renal disease (ESRD) to chromosome 1q25-31 als affected by ESRD have recently been reported. More in the region of NPHS2 has been identified in a genome-wide than 96,000 Americans were diagnosed with ESRD, and scan in African American (AA) siblings. Methods. To investigate the potential role of this gene in 364,000 received renal replacement therapy (dialysis) in ESRD, we sequenced all coding regions and approximately 2 kb 2001 (www.usrds.org). These numbers are expected to of upstream promoter sequence of NPHS2 in 96 unrelated AA double within the next decade. While ESRD is clearly a nondiabetic ESRD cases and 96 healthy population-based AA multifactorial disease, several lines of evidence support a controls, and assessed several single nucleotide polymorphisms genetic contribution to its pathogenesis [1–4]. (SNPs) for association in a larger case-control sample. Results. Fifty-five variants were identified with minor allele The glomerular capillaries can become injured in a va- frequencies ranging from <1% to 44%. Twenty-three polymor- riety of systemic and renal-limited diseases [5]. Podocytes, phisms were located in the promoter region, 11 were exonic, visceral epithelial cells, are octopus-like cells containing 13 were intronic, and 8 were in the 5 and 3 - untranslated actin-rich foot processes that sit on the external surface regions. Two novel nonsynonymous coding SNPs were iden- of the glomerular basement membrane (GBM). Each tified (A44E and A61V). An insertion polymorphism in intron 3, IVS3+9insA, was detected in 6 ESRD patients and in no podocyte is attached to its neighboring cell by an inter- controls. This variant, and 4 other common SNPs, were evalu- cellular adherens-type junction termed the slit diaphragm ated in a larger sample of 288 AA ESRD cases and 278 AA (SD) [6], bridging the narrow spaces (30-40 nm) between controls. The overall minor allele frequencies for the insertion cells. The GBM is both a size- and charge-selective bar- allele were 0.018 in cases and 0.002 in controls. Significant evi- rier for plasma proteins, and the SD maintains the bar- dence of association of IVS3+9insA was observed (P = 0.012), and the haplotype containing the insertion allele in cases was rier toward plasma macromolecules. Many proteinuric also associated. glomerular diseases are associated with effacement of the Conclusion. These results suggest that uncommon variants podocyte foot processes, enhancing leakage of proteins of the NPHS2 gene may play a role in the development of non- across the SD into the urine. diabetic ESRD in AAs. Podocin (NPHS2) is a stomatin-like protein that is exclusively expressed in podocytes. It is a membrane- anchored protein that interacts with other key compo- nents such as nephrin, CD2-associated protein, and Kin 1 These authors contributed equally to the preparation of this of Irre-like (KIRREL, NEPH1) at the SD [7–12]. It manuscript. has been suggested that podocin works as a scaffold- ing protein in the SD assembly process, but its precise Keywords: podocin (NPHS2), chronic kidney failure, hypertension, African Americans, genetics. function has not been fully elucidated. Podocin knock- out mice (NPHS2−/−) develop proteinuria during the Received for publication December 23, 2004 antenatal period and die from renal failure shortly af- and in revised form January 31, 2005 Accepted for publication February 11, 2005 ter birth [7]. Mutations in NPHS2 have been associated with autosomal-recessive steroid-resistant nephrotic syn- C 2005 by the International Society of Nephrology drome in children and families [13–15], sporadic focal 256 Engeler and Burdon et al: Podocin gene in ESRD 257 segmental glomerular sclerosis [16–18], proteinuria after and 182 controls using a MassARRAY SNP genotyp- renal transplantation [19], and with microalbuminuria in ing system (Sequenom, Inc., San Diego, CA, USA) as the general population [20]. previously described [22]. This genotyping system uses In light of the role of podocin in the glomerular filtra- single base extension reactions to create allele specific tion barrier and the association of genetic variants of the products that are separated and automatically scored in a NPHS2 gene with several renal disorders, this gene is a MALDI-TOF mass spectrometer. Primers for PCR am- functional candidate gene for ESRD. Our prior genome- plification and extension reactions were designed using wide scan of nondiabetic ESRD in AA sibling pairs re- MassARRAY assay design software (Sequenom, Inc.) vealed suggestive evidence for linkage at the NPHS2 gene and are available on request. locus on chromosome 1q25-31 [21]. Hence, podocin is also a positional candidate gene for ESRD in AAs. We investigated the role of the NPHS2 gene in AA non- Statistical analysis diabetic ESRD in a case-control study. We directly se- Clinical characteristics were compared between the re- quenced the gene in affected and unaffected individuals sequenced sample and the remainder of ESRD affected in order to identify genetic variants contributing to non- participants using Student t test for comparison of means diabetic ESRD. and a chi-square test for comparison of proportions. SNPs were tested for Hardy-Weinberg Equilibrium (HWE) us- × METHODS ing the normal 2 3 chi-squared 1 degree of freedom (df) test of goodness-of-fit to HW proportions. The pairwise Participants linkage disequilibrium statistic (D) was calculated using We recruited 288 unrelated AAs born in North Haploview software with the solid spine of LD algorithm Carolina reportedly with hypertension-associated ESRD with a threshold of D = 0.7 [23]. Pearson chi-square test from dialysis facilities located in the northwestern region was used to assess allele and genotype frequency distribu- of the state. These individuals were all nondiabetic and tion differences between individuals affected by ESRD had hypertension listed as the etiology of ESRD on their and unaffected controls. If any cell contained fewer than CMS 2728 form. In addition, 278 reportedly healthy (non- 5 counts, Fisher exact test was employed. Genotypic as- renal diseased) AAs also born in North Carolina were sociation was also assessed under a priori genetic mod- recruited as population-based controls. This study was els (dominant and recessive). Haplotypes were estimated approved by the Institutional Review Board at the Wake and assessed for association using DANDELION, which Forest University School of Medicine, and all cases and implements an expectation-maximization algorithm [24]. controls provided written informed consent [22]. The empirical P value was calculated from 10,000 permu- tations. P values < 0.05 were considered significant in all Sequencing of the podocin gene analyses. All 8 exons, including the 3 and 5 untranslated regions, and 2.2 kb of upstream promoter sequence, were directly sequenced in 96 AA ESRD cases and 96 healthy AA RESULTS population-based controls. The sequence of primers used We evaluated a total of 288 AA nondiabetic ESRD for PCR amplification of target sequences is available on cases and 278 AA population-based controls. Clinical request. Fragments were amplified in 20-lL reaction vol- characteristics of the study group are shown in Table 1. umes using standard polymerase chain reaction (PCR) There was a slight excess of males in the overall study, techniques, and the products were purifed with filter as is also present in the United States Renal Data Sys- plates (Eppendorf) according to manufacturer’s proto- tem (USRDS) database. The mean ± SD age at ESRD cols. Cleaned PCR products were then directly sequenced onset was 48.1 ± 15.7 years, with a mean duration of hy- using Big Dye Ready Reaction Mix (Applied Biosystems, pertension before ESRD of 14.0 ± 13.3 years. The rese- Foster City, CA, USA), and the sequence determined quenced subset was slightly younger than the remainder on an ABI 3730xl (Applied Biosystems). All sequences of the sample (P = 0.04), and the mean age at ESRD were compared with reference sequence NT 004487, and onset in the resequenced subset was also lower than the with each other, for the detection of variations using remainder of the sample, although this did not reach sta- Sequencher software (GeneCodes, Ann Arbor, MI, tistical significance (P = 0.07). The mean body mass index USA). (BMI) did not differ between the resequenced subsample and the remainder of the sample, nor did the presence of Genotyping hypertension, its mean age at onset, or the duration of Five SNPs were chosen for further analysis in the hypertension prior to ESRD onset. larger sample: IVS+9insA, C725T, T954C, C1206G, and Sequencing of 96 ESRD cases and 96 population con- A1410G. These were genotyped in an additional 192 cases trols revealed 55 variable locations within the coding 258 Engeler and Burdon et al: Podocin gene in ESRD Table 1.