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Assessment of 115 Candidate Genes for Diabetic Nephropathy by Transmission/Disequilibrium Test

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Assessment of 115 Candidate Genes for Diabetic Nephropathy by Transmission/Disequilibrium Test
  Original Article  Assessment of 115 Candidate Genes for DiabeticNephropathy by Transmission/Disequilibrium Test Kathryn Gogolin Ewens, 1 Roberta Ann George, 1 Kumar Sharma, 2 Fuad N. Ziyadeh, 3 andRichard S. Spielman 1 Several lines of evidence, including familial aggregation,suggest that allelic variation contributes to risk of diabeticnephropathy. To assess the evidence for specific suscepti-bility genes, we used the transmission/disequilibrium test(TDT) to analyze 115 candidate genes for linkage andassociation with diabetic nephropathy. A comprehensivesurvey of this sort has not been undertaken before. Singlenucleotide polymorphisms and simple tandem repeat poly-morphisms located within 10 kb of the candidate genes were genotyped in a total of 72 type 1 diabetic families of European descent. All families had at least one offspring with diabetes and end-stage renal disease or proteinuria. As a consequence of the large number of statistical testsand modest  P  values, findings for some genes may befalse-positives. Furthermore, the small sample size re-sulted in limited power, so the effects of some tested genesmay not be detectable, even if they contribute to suscepti-bility.Nevertheless,nominallysignificantTDTresults(  P < 0.05) were obtained with polymorphisms in 20 genes, in-cluding 12 that have not been studied previously: aqua-porin 1; B-cell leukemia/lymphoma 2 (bcl-2) proto-oncogene; catalase; glutathione peroxidase 1; IGF1;laminin alpha 4; laminin, gamma 1; SMAD, mothers againstDPP homolog 3; transforming growth factor, beta receptorII; transforming growth factor, beta receptor III; tissueinhibitor of metalloproteinase 3; and upstream transcrip-tion factor 1. In addition, our results provide modestsupportforanumberofcandidategenespreviouslystudiedby others.  Diabetes  54:3305–3318, 2005 D iabetic nephropathy is the most serious long-term complication of diabetes, accounting for   40% of new cases of end-stage renal disease(ESRD) in the U.S. (1). Two lines of evidencesuggest a strong genetic component in susceptibility todiabetic kidney disease.  1 ) Epidemiological studies indi-cate that the prevalence of diabetic nephropathy increasesduring the first 15–20 years after onset of diabetes andthen reaches a plateau, suggesting that only a subset of  patients is susceptible to the development of kidneydisease (2).  2 ) Family studies show clustering of diabeticnephropathy in both type 1 and type 2 diabetes; diabeticsiblings of probands with diabetic nephropathy have a significantly greater risk for developing kidney complica-tions than diabetic siblings of probands without diabeticnephropathy (3–6). In addition, segregation analyses of diabetic nephropathy in both Caucasians and Pima Indianswith type 2 diabetes provide evidence for the presence of a major locus, with a possible role for several minor loci(7,8).Numerous metabolic pathways and associated groups of genes have been proposed as candidates to play a role inthe genetic susceptibility to diabetic nephropathy (9–12).Before onset of overt proteinuria, functional changes areobserved in the kidney (altered glomerular filtration ratesand increasing albumin excretion rates), which arethought to result from the underlying pathological changesthat occur. These changes include thickening of the glo-merular basement membrane and expansion of the mes-angium due to accumulation of extracellular matrix proteins. Products of a wide range of genes might mediatethese renal changes. Examples include  1 ) the synthesisand degradation of glomerular basement membrane andmesangial matrix components;  2 ) components of meta-bolic pathways involving glucose metabolism and trans- port;  3 ) blood pressure regulation and the renin-angiotensin system;  4 ) cytokines, growth factors, signalingmolecules, and transcription factors; and  5 ) advancedglycation processes. Many of these candidate genes havebeen tested for association with diabetic nephropathy,typically in case-control studies of only one or a few genes(Table 1). In many instances, initial reports were notconfirmed in follow-up studies.We have carried out family-based studies with simpletandem repeat polymorphisms (STRPs) and single nucle-otide polymorphisms (SNPs) in 83 candidate genes thathave not been studied previously and 32 genes or generegions that have been reported as having significantassociation or linkage with diabetic nephropathy (Table From the  1 Department of Genetics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania; the  2 Department of Medicine, Center for Diabetic Kidney Disease, Division of Nephrology, Thomas JeffersonUniversity, Philadelphia, Pennsylvania; and the  3 Renal-Electrolyte and Hyper-tension Division and Penn Center for Molecular Studies of Kidney Diseases,Department of Medicine, University of Pennsylvania School of Medicine,Philadelphia, Pennsylvania  Address correspondence and reprint requests to Dr. Richard S. Spielman,Department of Genetics, University of Pennsylvania School of Medicine,Philadelphia, PA 19104-6145. E-mail: for publication 1 April 2005 and accepted in revised form 5 August2005. Additional information for this article can be found in an online appendix at a complete list of gene abbreviations, see the  APPENDIX .CEPH, Centre d’Etude du Polymorphisme Humain; ESRD, end-stage renaldisease; HBDI, Human Biological Data Interchange; SNP, single nucleotide polymorphism; STRP, simple tandem repeat polymorphism; TDT, transmis-sion/disequilibrium test; UTR, untranslated region.© 2005 by the American Diabetes Association. The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked “advertisement” in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact. DIABETES, VOL. 54, NOVEMBER 2005 3305  1). No previous studies have undertaken a comprehensiveassessment of the evidence for many candidate genes atonce, applying the same approaches and using a singlesample of patient material. We therefore had two relatedgoals: review briefly all relevant published studies, andcarry out a thorough assessment ourselves. All our resultswere obtained from patients who have both diabeticnephropathy and type 1 diabetes. Consequently, it isformally possible that positive findings are due to diabetesrather than diabetic nephropathy. All of the candidategenes were chosen for a possible role in kidney disease,not in diabetes. Positive results would be of interest ineither case, and the possibilities can be resolved bystudying patients who have long-standing diabetes withoutdiabetic nephropathy.For analysis of our own data, we used the transmission/ disequilibrium test (TDT) in its original form (13). TheTDT tests for the simultaneous presence of linkage andallelic association between a genetic marker and a puta-tive disease susceptibility locus. Because linkage andassociation, when present together, define linkage disequi-librium, we refer to the TDT as a test for linkage disequi-librium. If there is only loose (or no) linkage, or if allelicassociation is only weak or absent, linkage disequilibriumwill not be strong, and the TDT will not detect an effect. RESEARCH DESIGN AND METHODS Forty-three families of European descent were ascertained through an indexcase subject with type 1 diabetes and diabetic nephropathy through the PennTransplant Center of the University of Pennsylvania Health System. Diabeticindividuals were considered to have diabetic nephropathy if they had ESRD or if their albumin-to-creatinine ratio was   300   g/mg in two of three randomurine samples collected at least 6 weeks apart. When available, diabeticsiblings of the index case subject were phenotyped using the same criteria.Twenty-nine additional families with type 1 diabetes from the Human Biolog-ical Data Interchange (HBDI) collection (14) were also included in this study.These families were contacted in collaboration with HBDI to obtain updatedmedical information, including the presence of ESRD and information onrelevant medications. In the absence of ESRD, diabetic nephropathy statuswas determined as described above. The total family material consisted of 72families with type 1 diabetes: 68 parent-child trios and 4 multiplex families. Among the 77 diabetic offspring in these families, 73 had received a kidneytransplant. The mean  SD age at diagnosis of diabetes was 11.1  6.1 years(range, 1–30), and the mean duration of diabetes before transplant was 23.9  5.9 years (range, 12–42). At the time of enrollment into this study, the meanduration of diabetes was 29.7    8.6 years (range, 17–53). The mean timeelapsed between transplant and enrollment (or until death 8 years after transplant in one case subject) was 6.5  5.5 years (range,  1–30). This studywas carried out in accordance with the protocol and informed consent formsapproved by the Institutional Review Board of the University of Pennsylvania.Thirty-six Centre d’Etude du Polymorphisme Humain (CEPH) families(two parents and three offspring in each family) were studied for transmissiondistortion in nondiabetic control subjects. In these families, we genotyped 29SNP markers that showed nominally significant evidence for linkage disequi-librium with diabetic nephropathy. DNA preparation.  For individuals ascertained through the University of Pennsylvania, total genomic DNA was prepared from peripheral blood leuko-cytes using the PureGene protocol (Gentra Systems). DNA for the HBDI andCEPH families was obtained from the Coriell Cell Repositories (CoriellInstitute for Medical Research). Candidate genes and genotyping.  Candidate genes were chosen because of their role in normal or pathological kidney function and from published TABLE 1Candidate genes (  n  115) for diabetic nephropathy (DN) tested for linkage disequilibrium (LD) Functional categoryGenes (  n  83) not tested previouslyfor LD with DNGenes (  n  32) tested by others for association or linkage with DNGlomerular basement membrane andmesangial matrix components andtheir metabolism; cell adhesionCD36 (58); COL1A1 (59–61); COL4A2 (60,61); COL4A3(60,61); COL4A4 (60,61); FBLN1 (11); FBN1*; FN1(62); HSPG1/SDC2 (62); ICAM1 (63,64); ITGA1 (65);ITGA3 (65); LAMA4*; LAMB1*; LAMC1*; LAMC2*;MMP1 (11,66); MMP2 (11); MMP3 (11,66); NID*;OPN/SPP1 (58); SELE (67); TIMP2 (62); TIMP3 (62)COL4A1 (18,19); HSPG2 (68,69); MMP9(35,36); NPHS1 (27,70,71); SELL (72)Glucose metabolism and transport GLUT2/SLC2A2* AKR1B1 (45–51); GFPT2 (73);GLUT1/SLC2A1 (74–76)Blood pressure regulation and therenin-angiotensin systemEDN1 (77,78); EDN2 (79); EDN3 (79); REN*; SAH (80);UTS2 (81) ACE (37–44); AGT (40–42,82,83); AGTR1 (20–24); NPPA (84–86)Cytokines, growth factors, andreceptors ACVR2 (11); BMP2 (11,71); BMP7 (11,87,88); CCL2(89); CTGF (11,62,90,91); EGF (11); GH1 (62,92);IGF1 (62,92–94); IGF1R (95); IL10*; LTBP1 (96);PDGFB (62,97); PDGFRB (97); TGFB2*; TGFB3*;TGFBR2 (98); TGFBR3*; TNFRSF1A*;TNFSF6/FASLG (11,99); VEGF (11,71,100,101)CCR5 (102); IL1A (103–105); IL1B(103–105); IL1R1 (103–105); IL1RN(103–106); NRP1 (27); TGFB1 (93,107–109)Lipid metabolism APOC2*; APOC4* APOE (25,52–56); LPL (25,55,110)Protein and amino acid metabolism CTSD (111); CTSL*; ECE1*; SGK (112); UBA52 (113) MTHFR (114–116); NOS3 (117–121)Nucleic acid metabolism ANG (10,62,122) ENPP/PC-1 (123–126)Transcription factors and regulatoryand signaling molecules AXL (127); EDNRA (128,129); EDNRB*; FOS*; GAS6(127); MIG6 (130); NFKB1 (89); PRKCA (131,132);SMAD3 (133); UNC13B (134); USF1*; USF2*; VEGFR/KDR (11)BDKRB2 (135–137); CNOT4/D7S500(8); HNF1B/TCF2 (28–32); PPARG(138,139); PRKCB1 (26,93); TSC22(140)Electron transport CAT (141); NOX4 (142) p22phox/CYBA (33,34,142)Transport function AQP1 (71,143); SLC9A1 (93,144,145); SLC12A3 (146);TCN2 (147)Miscellaneous BCL2 (11,148–150); GPX1 (141,151); GREM1/CKTSF1B1(11,152); HSD3B1 (58); LGALS3 (11)CALD1 (153) Underline indicates nominally significant results in this study. *To our knowledge, not previously proposed as candidate gene for diabeticnephropathy. For a complete list of gene abbreviations, see the  APPENDIX . CANDIDATE GENES FOR DIABETIC NEPHROPATHY  3306 DIABETES, VOL. 54, NOVEMBER 2005  reports of candidate gene or expression studies. In the initial phase of thisstudy, linkage disequilibrium with diabetic nephropathy was assessed usingSTRPs mapping in or close to the candidate gene. These markers wereselected from the UCSC Genome Bioinformatics site ( PCR primers were designed from the surrounding sequence, and PCRamplification was carried out by standard methods using fluorescently labeled primers (15). PCR products were electrophoresed on an Applied Biosystems377 DNA Sequencer, and the genotypes were analyzed using Genescan andGenotyper software.SNPs in candidate genes were identified using either dbSNP at NationalCenter for Biotechnology Information ( or  Applied Biosystems/Celera Discovery System ( and Polymorphic markers re- ported by others to be associated with diabetic nephropathy (Table 1) werealso genotyped. (In most cases, the restriction digest assays described in theliterature were converted to Applied Biosystems Taqman Genotyping Assays.)The goal was to genotype one SNP approximately every 20 kb. (Mean spacingof SNPs was 17.3 kb; range, 1.2–88.4 kb; median, 13.4 kb). For genes  20 kbin genomic extent, typically one SNP was typed. When available, SNPs locatedin exons were genotyped in preference to those in introns if the minor allelefrequency exceeded   0.2. Some of the SNP genotyping was carried out byrestriction enzyme digestion, sequencing, or fluorescent polarizatation with AcycloPrime-FP SNP detection assays read on a Victor multilabel reader (Perkin Elmer Life Sciences). For most SNPs, we used Applied BiosystemsTaqman SNP Genotyping Assays and read results on an Applied Biosystems7900HT Sequence Detection System. For specific PCR primer information andinformation on individual SNP locations, see supplemental Tables 1 and 2,respectively, which are presented in the online appendix (available at http:// Statistical analysis.  To assess linkage disequilibrium, differential transmis-sion of polymorphic variants from heterozygous parent to affected child wastested by the TDT (13). TDT for haplotypes was carried out with Genehunter (16). In multiplex families, the TDT is not strictly valid as a test of association.However, in view of the small number of multiplex families (4 of 72), we didnot correct for the small effect of this departure from the assumptions. Themaximum number of transmissions in our sample was 83, and some rarer alleles provided samples of fewer than 30. To avoid compromising statistical power excessively, we restricted analysis to alleles for which the sum of transmissions and nontransmissions from informative parents was 40 or greater. For this minimum sample size of 40, we calculated the power todetect departures from the null hypothesis of 50% transmission in a two-sidedtest with     0.05. We used the normal approximation to the binomialdistribution as implemented in SISA (Simple Interactive Statistical Analysis)(17). For a transmission rate of 0.6, power is 0.24; for transmission rate 0.7, power is 0.73. These values are lower limits for the anticipated power. We alsocalculated the corresponding values of power for 60 transmissions: 0.34 and0.89 for transmission rates of 0.6 and 0.7, respectively. For most markers, thesample size was larger than 40, providing greater power to detect the stateddegree of differential transmission.Nominal  P   values for significance of the TDT   2 are reported withoutcorrection for multiple testing, but we indicate here what minimal  P   valueswould be required if Bonferroni correction were used. The number of statistical tests for markers at one candidate gene was typically three to four;for four tests, Bonferroni correction would require a nominal  P   of 0.0125 for adjusted  P     0.05 and 0.0025 for adjusted  P     0.01. The total number of statistical tests was  380. Bonferroni correction would require a nominal  P   of 1.3  10  4 for an adjusted  P   of 0.05 and 2.6  10  5 for an adjusted  P   of 0.01. RESULTS Diabetic nephropathy candidate gene polymorphismsnot previously tested (83 genes).  Of the total of 115genes with results reported here, 83 have not been tested previously, to our knowledge. Among these 83 genes, theTDT was nominally significant (  P   0.05) for 12 (summa-rized individually below and in Table 2). The nonsignifi-cant results for the remaining 71 genes are summarized inTable 3.  B-cell leukemia/lymphoma 2 (bcl-2) proto-oncogene. Ten SNPs in B-cell leukemia/lymphoma 2 (bcl-2) proto-oncogene (BCL2) were genotyped in the 72 diabetic ne- phropathy families. Three gave nominally significantevidence for linkage disequilibrium with diabetic nephrop-athy: rs2062011 (  P     0.001), rs12457700 (  P     0.006), andrs1481031 (  P   0.009). All three SNPs lie in a 24-kb regionin intron 1 (192 kb) of BCL2. Catalase.  We genotyped two SNPs in catalase (CAT).Both were nominally significant: rs1049982, located in the5  -untranslated region (UTR) (  P     0.006); and rs560807,located in intron 1 (  P   0.044).  Laminin, alpha 4.  Eight SNPs and one STRP weregenotyped in laminin, alpha 4 (LAMA4). One SNP,rs3734287, located in an intron, gave a nominally signifi-cant result (  P   0.016). Transforming growth factor, beta receptor II andtransforming growth factor, beta receptor III.  SevenSNPs were genotyped in transforming growth factor, beta receptor II (TGFBR2) and 10 in transforming growthfactor, beta receptor III (TGFBR3). One SNP in each of these unlinked genes gave a nominally significant result:rs6792117, located in an intron of TGFBR2 (  P   0.024); andrs12756024, located in an intron of TGFBR3 (  P   0.018). Glutathione peroxidase 1.  The single SNP we tested inglutathione peroxidase 1 (GPX1), rs1800668, was nomi-nally significant (  P   0.022).  Laminin, gamma 1.  We tested 12 SNPs in laminin,gamma 1 (LAMC1). Significant TDT results were foundacross the entire gene, suggesting strong linkage disequi-librium. We found that the linkage disequilibrium param-eter D   for the mostly widely spaced markers (separatedby 125 kb) ranged from 0.7 to 0.9 (  P     0.01). Thestrongest evidence for linkage disequilibrium with diabeticnephropathy was found with a synonymous SNP, rs20557(Asn837Asn,  P   0.026). There is thus modest evidence for association of diabetic nephropathy with LAMC1; how-ever, the strong linkage disequilibrium across the gene willmake it difficult to narrow the critical region using geneticmeans.  SMAD, mothers against DPP homolog 3.  We testedseven SNPs in SMAD, mothers against DPP homolog 3(SMAD3). Linkage disequilibrium with two intronic SNPs,rs12594610 and rs4776890, located 2.9 kb apart, was nom-inally significant (  P   0.033 and 0.046, respectively). Upstream transcription factor 1.  Four SNPs weregenotyped in upstream transcription factor 1 (USF1). Oneof these, rs2516839, located in the 3  -UTR, gave a nomi-nally significant result (  P   0.047).  Aquaporin 1, IGF1, and tissue inhibitor of metallo- proteinase 3.  Nominally significant results were foundfor STRP markers near three genes: aquaporin 1 (AQP1),IGF1, and tissue inhibitor of metalloproteinase 3 (TIMP3).The markers were D7S526 located 2.7 kb 5   of AQP1(125-bp allele,  P   0.027), MFD1 (GDB: 171128) located 0.7kb 5  of IGF1 (209-bp allele,  P   0.047), and D22S280 in the3  -UTR region of TIMP3 (214-bp allele,  P     0.048). For each of these genes, we followed up by testing two or three SNPs in or near the gene and found no evidence tosupport the result from the STRP. We have not pursuedthese genes further.Table 3 presents the results for SNPs and STRPs in 71additional “new” candidate genes (not previously tested)that showed no significant linkage disequilibrium withdiabetic nephropathy. In view of the marker spacing(mean of 17.2 kb) and the modest power of the sample, weconsider the absence of significant linkage disequilibriumto be inconclusive evidence concerning a role for thesegenes. Follow-up of previously reported diabetic nephropa-thy associations (32 genes).  We genotyped SNPs in 32candidate genes that have been studied previously by K.G. EWENS AND ASSOCIATES DIABETES, VOL. 54, NOVEMBER 2005 3307  TABLE 2Candidate genes (  n  12) for diabetic nephropathy not previously tested; nominal  P   0.05 for at least one marker Genesymbol Locus Assay ID dbSNP ID Location Alleles TNotT Total %T    2  P   AQP1 7p14.3 hCV2973378 rs763422 5.1 kb 5   T/C T 33 31 64 0.52 0.1D7S526 2.6 kb 5   125 bp 38 21 59 0.64 4.9 0.027hCV2973385 rs1049305 3  -UTR G/C G 27 24 51 0.53 0.2BCL2 18q21.33 hCV7905447 rs1564483 3  -UTR C/T C 31 29 60 0.52 0.1hCV7905342 rs3943258 Intron T/C T 36 30 66 0.55 0.5hCV8685764 rs1481031 Intron C/T C 39 19 58 0.67 6.9 0.009hCV1408500 rs12457700 Intron C/T C 36 16 52 0.69 7.7 0.006hCV1408502 rs2062011 Intron A/T T 42 17 59 0.71 10.6 0.001hCV1408482 rs8083946 Intron G/A G 40 27 67 0.60 2.5hCV1728132 rs8084922 Intron G/C G 46 31 77 0.60 2.9hCV8687299 rs1381548 Intron G/A G 33 25 58 0.57 1.1hCV2855833 rs11152377 Intron C/T C 32 27 59 0.54 0.4hCV2855835 rs2551402 4.1 kb 5   C/A C 38 30 68 0.56 0.9CAT 11p13 hCV1883211 rs1049982 5  -UTR C/T C 43 21 64 0.67 7.6 0.006hCV3102895 rs560807 Intron A/T A 44 27 71 0.62 4.1 0.044GPX1 3p21.3 hCV7912052 rs1800668 5  -UTR A/G G 29 14 43 0.67 5.2 0.022IGF1 12q23.2 hCV2801121 rs2946834 1.9 kb 3   A/G A 24 22 46 0.52 0.1hCV2801103 rs972936 Intron T/C C 28 28 56 0.50 0.0hCV346219 rs10735380 Intron A/G G 30 27 57 0.53 0.2MFD1 0.7 kb 5   209 bp 15 28 43 0.35 3.9 0.047LAMA4 6q21 hCV2462170 rs1050353 Val(A)1713Val(T) A/T A 30 29 59 0.51 0.0hCV2462178 rs969139 Intron C/T T 44 32 76 0.58 1.9hCV2462186 rs3734287 Intron C/T C 37 19 56 0.66 5.8 0.016hCV2462219 rs11153344 Intron A/G G 35 31 66 0.53 0.2LAMA4-STRP1 Intron 119 bp 27 15 42 0.64 3.4hCV2462251 rs1050348 His(C)491Tyr(T) A/G A 28 24 52 0.54 0.3hCV2462280 rs3777928 Intron A/C A 33 30 63 0.52 0.1hCV2462319 rs2157547 Intron C/G G 20 18 38 0.53 0.1hCV11903282 rs1894682 Intron A/G A 33 23 56 0.59 1.8LAMC1 1q25.3 hCV505167 rs10737236 4 kb 5   C/T T 45 30 75 0.60 3.0hCV26124236 rs10911194 Ala(C)58Ala(T) A/G G 46 31 77 0.60 2.9hCV9066112 rs10797819 Intron G/A A 46 28 74 0.62 4.4 0.036hCV1770066 rs4652775 Intron A/T A 45 29 74 0.61 3.5hCV3127531 rs2296288 Cys(C)182Cys(T) T/C T 46 29 75 0.61 3.9 0.050hCV11632431 rs7556132 Ile(A)458Val(G) A/G A 47 29 76 0.62 4.3 0.039hCV3127590 rs2296292 Ala(C)592Ala(A) A/C A 45 28 73 0.62 4.0 0.047hCV3127518 rs20557 Asn(C)837Asn(T) T/C T 46 27 73 0.63 4.9 0.026hCV3127512 rs7410919 Leu888Pro T/C T 47 29 76 0.62 4.3 0.039LAMC1-STRP1 Intron 215 bp 31 20 51 0.61 2.4hCV3127470 rs4651146 Arg(C)1376Arg(T) T/C C 42 28 70 0.60 2.8hCV3127469 rs3818419 Ala(A)1433Ala(G) G/A G 33 32 65 0.51 0.0hCV3127459 rs1547715 3  -UTR A/G A 47 30 77 0.61 3.8SMAD3 15q22.33 hCV9707890 rs1498506 Intron A/C A 28 18 46 0.61 2.2hCV2113018 rs4776890 Intron C/G T 40 24 64 0.63 4.0 0.046hCV11306173 rs12594610 Intron G/A G 36 20 56 0.64 4.6 0.033hCV2112975 rs11631380 Intron C/T T 32 19 51 0.63 3.3hCV2112965 rs745103 Intron A/G A 29 29 58 0.50 0.0hCV1044749 rs731874 Intron A/G G 31 23 54 0.57 1.2hCV2112907 rs2289791 Intron G/T T 29 19 48 0.60 2.1TGFBR2 3p24.1 hCV3158972 rs13081419 Intron A/C C 41 31 72 0.57 1.4hCV11565979 rs1431131 Intron A/T T 34 30 64 0.53 0.3hCV1612549 rs1155705 Intron A/G G 34 32 66 0.52 0.1hCV972343 rs1078985 Intron A/G G 24 22 46 0.52 0.1hCV8778179 rs995435 Intron A/G G 27 21 48 0.56 0.8hCV1612506 rs6792117 Intron A/G G 41 23 64 0.64 5.1 0.024hCV1612480 rs744751 2.8 kb 3   A/G A 29 25 54 0.54 0.3TGFBR3 1p22.1 hCV945103 rs284878 Thr(C)746Thr(T) A/G A 10 5 15 0.67 1.7hCV1931721 rs1805113 Phe(C)673Phe(T) A/G G 38 30 68 0.56 0.9hCV3130156 rs284180 Intron A/C A 38 32 70 0.54 0.5hCV3130147 rs284190 Intron A/T T 37 29 66 0.56 1.0hCV3130125 rs12756024 Intron A/C C 42 23 65 0.65 5.6 0.018hCV11643684 rs5019497 Intron A/C A 38 34 72 0.53 0.2 Continued on following page CANDIDATE GENES FOR DIABETIC NEPHROPATHY  3308 DIABETES, VOL. 54, NOVEMBER 2005  others. Table 4 shows results from our TDT studies for 11of these genes. In eight of these, we found nominallysignificant results. Table 4 also includes results for SNPs inthree genes (ACE, aldose reductase [AKR1B1], and apoli- poprotein E [APOE]) that deserve attention because theyhave been the subject of numerous diabetic nephropathyassociation studies. For these genes, we found a trend thatsupports published results, although our results were notsignificant, perhaps because of the small sample size. Thenonsignificant results for the remaining 21 genes aresummarized in Table 5. Collagen, type IV, alpha 1.  Nine SNPs and one STRPwere genotyped in collagen, type IV, alpha 1 (COL4A1).Two SNPs in intron 1 showed significant association withdiabetic nephropathy: rs614282 (  P     0.002) and rs679062(  P     0.0002). Because of the strong evidence with thelatter SNP, we looked for nearby coding SNPs. We se-quenced a 700-bp region that included all of exon 2(located  4 kb from rs614282) in two sets of pooled DNA samples: 16 diabetic nephropathy and 42 CEPH individu-als. No sequence variants were found, suggesting that nocommon disease-associated variant is located in thisnearby exon.Two studies of COL4A1 by others (18,19) led to contra-dictory conclusions that have not been followed up since.The region of association we found in intron 1 lies  100 kb5   to a polymorphic  Hin dIII restriction site found byKrolewski et al. (19) to be associated with increased riskfor progression to overt nephropathy. Chen et al. (18)failed to confirm this finding with a larger sample (  n  116diabetic nephropathy and 91 individuals with long-stand-ing diabetes but no evidence of kidney disease [diabeticnephropathy negative]). In our studies, SNP rs1133219,located only 8 kb from the site first tested by Krolewski etal. (19), provided no significant evidence (55 transmis-sions,  P   0.53).  AngiotensinIIreceptor,type1region. Moczulski et al.(20) reported linkage and association studies in discordantsibpairs and parent-offspring trios with a diabetic nephrop-athy or diabetic nephropathy–negative offspring. Theyfound linkage with the STRPs ATCA (located near theangiotensin II receptor, type 1 [AGTR1 gene]) andD3S1308 (located 575 kb telomeric to AGTR1), but noassociation was found with six SNPs in AGTR1 or with anyalleles of ATCA. (No association results were reported for D3S1308.) We tested these two STRPs, plus three addi-tional SNPs in AGTR1. These included the A1166C SNPreported previously (21–24). We also tested 11 SNPslocated in the 1-Mb region telomeric to AGTR1 (summa-rized in Table 4). The only significant evidence for linkagedisequilibrium with diabetic nephropathy is seen atD3S1308 itself (allele 2 [106 bp],  P     0.001; and allele 3[108 bp],  P     0.009; alleles named as in GDB allele set:63031,  Lipoprotein lipase.  Five SNPs in lipoprotein lipase(LPL) were tested. Three of the SNPs, located in a 5.4-kbregion near the 3   end of the gene, had nominally signifi-cant TDT results: rs320 (  P   0.005), rs326 (  P   0.011), andrs13702 (  P     0.004). In a study of Caucasian type 1diabetic patients, Orchard et al. (25) reported an associa-tion between rs320 (a   Hin dIII restriction site) and in-creased albumin-to-creatinine ratio.  Protein kinase C, beta 1.  Eleven SNPs and one STRP inor near protein kinase C, beta 1 (PRKCB1) were geno-typed. Only SNP rs1015408, located in intron 4, wasnominally significant (  P     0.025). Two of the SNPs wegenotyped were previously found to be associated withdiabetic nephropathy (26): rs3760106 (C-1504T) andrs2575390 (G-546C). However, in our families, there wasno significant evidence for linkage disequilibrium witheither of these SNPs.  Neuropilin 1.  Iyengar et al. (27) found linkage betweenD10S1654 and diabetic nephropathy in Caucasian sibpairswith type 2 diabetes. Because this marker maps in anintron of neuropilin 1 (NRP1), we tested seven SNPs in thisgene. Two of these, rs869636 and rs2804495, located 40 kbapart in intron 2, were nominally significant (  P   0.047 and0.027, respectively).  HNF1B1/transcription factor 2, hepatic (MODY5). Several studies have reported that rare mutations inHNF1B1 are associated with renal dysfunction in Japaneseand Caucasian maturity-onset diabetes of the young fam-ilies (28–31). However, no HNF1B1 mutations were foundamong 63 German and Czech type 2 diabetic patients withdiabetic nephropathy (32). In our type 1 diabetic familieswith diabetic nephropathy, we found nominally significantevidence with an SNP located in the 3  -UTR (rs2688,  P   0.029), but three SNPs in introns of HNF1B1 and onelocated 2.2 kb 3  of the gene failed to support this finding.  p22phox/cytochrome b-245,    -polypeptide.  ThreeSNPs were genotyped in p22phox, including rs4673(C242T, His72Tyr) previously studied for association withdiabetic nephropathy in Caucasians with type 1 diabetes(33) and Japanese with type 2 diabetes (34). In our type 1 TABLE 2— Continued Genesymbol Locus Assay ID dbSNP ID Location Alleles TNotT Total %T    2  P  hCV11643667 rs10783040 Intron A/G G 38 28 66 0.58 1.5hCV1931638 rs11165595 Intron A/G A 34 30 64 0.53 0.3hCV3130092 rs1192524 Intron A/G A 32 32 64 0.50 0.0hCV3181378 rs7550034 Intron A/G A 37 35 72 0.51 0.1D1S1588 Intron 132 bp 17 28 45 0.38 2.7TIMP3 22q12.3 hCV8712827 rs135025 Intron A/G A 38 26 64 0.59 2.3D22S280 Intron 214 bp 32 18 50 0.64 3.9 0.048hCV3294872 rs242075 Intron A/G G 39 37 76 0.51 0.1hCV8712964 rs1065314 3  -UTR T/C C 26 25 51 0.51 0.0USF1 1q23.3 hCV1459759 rs3737787 3  -UTR A/G G 25 24 49 0.51 0.0rs2073658 rs2073658 Intron C/T C 22 19 41 0.54 0.2hCV15949520 rs2073656 Intron C/G G 22 21 43 0.51 0.0hCV1839183 rs2516839 5  -UTR C/T T 45 28 73 0.62 4.0 0.047 T, number of transmissions in the TDT analysis. For a complete list of gene abbreviations, see the  APPENDIX . K.G. EWENS AND ASSOCIATES DIABETES, VOL. 54, NOVEMBER 2005 3309
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