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Deletion Polymorphisms Associated With the Development of Posttransplantation Diabetes Mellitus in Renal Allograft Recipients
Angiotensinogen T235 and Angiotensin-Converting Enzyme Insertion/ Deletion Polymorphisms Associated With the Development of Posttransplantation Diabetes Mellitus in Renal Allograft Recipients B.H. Özdemir, F.N. Özdemir, F.B. Ataç, A.A. Özdemir, and M. Haberal ABSTRACT Background. Genetic polymorphisms of the renin-angiotensin system (RAS) have been reported to play an important role in the pathogenesis of diabetes mellitus and hypertension. In addition, a close association has been reported between RAS and the progression of both diabetes and hypertension. But the role of RAS on the development of posttransplantation diabetes mellitus (PTDM) is not known. For this purpose we investigated the association of polymorphisms in the genes for angiotensin-converting enzyme (ACE) and angiotensinogen (AGT) with the development of PTDM. Methods. Genotyping for ACE insertion/deletion (I/D) and AGT M235T polymorphisms was performed in 50 patients who underwent renal transplantation during a 5-year period. Group 1 consisted of 23 recipients who developed PTDM and group 2 consisted of 27 recipients that did not have PTDM. Results. Of 50 patients, 13 (26%) showed the ACE DD, 21 (42%) the ACE ID, and 16 the ACE II genotype. The frequencies of AGT MM, AGT MT, and AGT TT were 0, 54%, and 46%, respectively. Compared with group 2, there were high frequencies of the AGT TT genotype in group 1 recipients (P ⬍ .001). In addition the ACE DD genotype was found significantly higher in group 1 patients compared with group 2 patients (P ⫽ .001). Conclusion. The high frequencies of the AGT TT genotype and ACE DD genotype in recipients may contribute to the high prevalence of PTDM. Our data suggest a synergistic effect between the ACE and AGT polymorphism in the risk of PTDM, but to support this theory a larger patient group must be studied. osttransplantation diabetes mellitus (PTDM) is a frequent and serious complication after kidney transplantation. Its etiopathogenesis is multifactorial and includes the immunosuppressive regimen, ethnicity, older age, and body mass index. Among these, calcineurin inhibitor and steroid use seems to have outstanding relevance.1 The incidence of PTDM related to a cyclosporine (CsA)– and corticosteroidbased regimen has been reported to be as high as 20% among kidney allograft recipients.2,3 Most cases of PTDM occur during the first 3 months after transplantation or after treatment for rejection. Compared with normoglycemic transplant recipients, patients with PTDM have an increased risk of graft loss, cardiovascular morbidity, and premature death with a functioning allograft.4 Impaired insulin secretion and increased insulin resistance are suggested as mechanisms underlying the development of
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0041-1345/11/$–see front matter doi:10.1016/j.transproceed.2011.01.046 572
PTDM. Several genetic polymorphisms have been considered to give rise to the development of DM in general population. Vitamin D receptor and uncoupling protein 2 can influence insulin secretion, and peroxisome proliferators–activated receptor gamma, angiotensin-converting enzyme (ACE), and adiponectin can influence insulin sensitivity.5–7 However, there are no available reports showing an association of From the Department of Pathology (B.H.Ö.), Department of Nephrology (F.N.Ö.), Department of Molecular Biology (F.B.A.), Department of Public Health (A.A.Ö.), and Department of General Surgery and Transplant Surgery (M.H.), Bas¸kent University, Ankara, Turkey. Address reprint requests to Binnaz Handan Özdemir, MD, Professor, Department of Pathology, Bas¸kent University, 12. sokak 7/4, Bahcelievler, 06490, Ankara, Turkey. E-mail: handan27@ hotmail.com © 2011 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 43, 572–574 (2011)
AGT AND ACE MUTATIONS IN RENAL ALLOGRAFT RECIPIENTS
genetic polymorphisms encoding these proteins with the occurrence of PTDM. For that reason, in the present study we investigated the association of polymorphisms in the genes for ACE and angiotensinogen (AGT) with the development of PTDM among patients under a CsA-based immunosuppressive protocol. PATIENTS AND METHODS A total of 50 consecutive patients who received their first kidney transplantation and maintained graft function for a ⱖ12-month posttransplantation period and who used triple therapy including CsA for maintenance immunosuppression were included in the study. All patients were on maintenance immunosuppression including prednisolone and CsA with mycophenolate mofetil. Acute rejection episodes were treated with 3 boluses of 0.5–1 g intravenous methylprednisolone. Patients were eligible to participate in the study if they were the recipients of a kidney allograft with no pretransplantation history of diabetes and a recorded fasting plasma glucose (FPG) level ⬍5.5 mmol/L. Patients were excluded if they had a history of diabetes before transplantation, severe metabolic or infectious disease, or a recorded FPG level ⬎5.5 mmol/L. The Ethics Committee of Baskent University Faculty of Medicine approved the study and each of the patients gave informed consent. The onset of PTDM, clinical characteristics, demographic features (age, gender, dialysis modality, and vintage) and laboratory data were obtained from the patients’ files. The following information was collected for each case: date of transplantation, age and gender of donor and recipient, donor type (living or cadaveric), number of HLA mismatches, body mass index at the time of transplantation (kg/m2), number of acute rejection episodes, and laboratory findings (pretransplantation FPG levels and serum creatinine levels. Diabetes mellitus was diagnosed in accordance with American Diabetes Association/World Health Organization guidelines as symptoms of diabetes plus any time plasma glucose concentrations ⱖ200 mg/dL, or finding of ⱖ2 consecutive fasting glycemia ⬎126 mg/dL or 2-hour plasma glucose ⱖ200 mg/dL during oral glucose tolerance test and the need for insulin or oral antidiabetic therapy.8 of 50 patients, 23 had PTDM (group 1) and the remaining 27 patients did not have PTDM and were used as the control group (group 2). Genotyping was performed for the ACE gene insertion/deletion (I/D) and AGT gene (M¡T, 235) polymorphisms. The patients or patients’ families filled out the consent forms, and venous blood samples anticoagulated with ethylenediaminetetraacetic acid were collected from all patients. Genomic DNA was extracted from peripheral blood leukocytes using the salting-out procedure. The DNA was used as a template for gene polymorphism analysis. All amplifications were performed according to the manufacturer’s recommendations, using polymerase chain reaction sequencespecific primers and appropriate restriction enzymes that have been described previously.9,10
Statistical Analysis Statistical analysis was performed using SPSS software (Statistical Package for the Social Sciences, version 9.05; SSPS, Chicago, IL, USA). For quantitative variables, values are given as mean ⫾ SD. Mean values were calculated for each group and compared using Student t test for normal distribution. The Kruskal-Wallis test was
573 used to compare variables with nonnormal distribution. Categoric data were compared using either chi-square test or Fisher exact probability test, when the expected cells contained ⱕ5 subjects. Results were considered to be statistically significant at a value for P of ⬍.05.
RESULTS
The mean age of all recipients at transplantation was 38 years with a range of 21– 60 years, and the mean posttransplantation follow-up period was 73.8 months. Twenty-three of 50 patients had PTDM according to the above-mentioned diagnostic criteria (group 1). The remaining cases had no PTDM and were used as a control group (group 2). Table 1 shows the demographic, clinical, and laboratory characteristics of 50 renal transplant recipients. There were no significant differences between the patients with or without PTDM regarding gender, dialysis time, donor type, number of mismatches, acute rejection episodes, and duration of transplantation. Univariate analysis showed that there were no differences regarding the risk factors of PTDM between the group with PTDM and the control group (Table 1). Of 50 patients, 13 (26%) showed the ACE DD, 21 (42%) the ACE ID, and 16 the ACE II genotype. The frequencies of AGT MM, AGT MT, and AGT TT were 0, 54%, and 46%, respectively. Compared with group 2 there were high frequencies of the AGT TT genotype in group 1 recipients (P ⬍ .001). In addition, the ACE DD genotype was found significantly higher in group 1 patients compared with group 2 patients (P ⫽ .001; Table 2). DISCUSSION
PTDM is a frequent complication following renal transplantation and serves as a key risk factor for posttransplantation cardiovascular disease. The ability to predict a patient’s risk for developing PTDM would be of considerable benefit in selecting appropriate immunosuppressive regimens for individuals. Although the incidence, risk factors, and clinical relevance of PTDM vary among reports from Table 1. Clinical Characteristics and Demographic Features of Patients With or Without PTDM PTDM No
Age (y) Gender (male/female) Dialysis duration (mo) BMI at the time of transplantation (kg/m2) BMI after the transplantation (kg/m2) Donor source (living/deceased) Number of mismatches [n(%)] Acute rejection [n(%)] Time since transplantation (months) Creatinine at the end of the first year (mg/dL) BMI, body mass index.
PTDM Yes
P Value
38.3 ⫾ 10.9 37.9 ⫾ 10.5 13/7 10/5 18.5 ⫾ 15.2 20.1 ⫾ 16 21.4 ⫾ 2.7 22.9 ⫾ 6.1
NS NS NS NS
23.8 ⫾ 2.8
25.2 ⫾ 6
NS
18/9 2.4 ⫾ 1.2 1.8 ⫾ 1.5 78.9 ⫾ 23
15/8 2.5 ⫾ 1.6 2.1 ⫾ 1.4 63.6 ⫾ 32
NS NS NS NS
1.5 ⫾ 0.4
1.8 ⫾ 0.6
NS
574
ÖZDEMIR, ÖZDEMIR, ATAÇ ET AL
Table 2. Comparison of the ACE and AGT Genotypes of Patients With or Without PTDM
ACE ACE ACE AGT AGT AGT
DD ID II MM MT TT
n
PTDM No
PTDM Yes
P Value
13 21 16 0 27 23
3 (11.1%) 10 (37) 14 (51.9) 0 23 (85.2) 4 (14.8)
10 (43.5) 11 (47.8) 2 (8.7) 0 4 (17.4) 19 (82.6)
.001
⬍.001
clinical studies, the influence of genetic control is not well known. Recently genetic studies have gained potential interest in evaluating the pathogenesis of insulin resistance and DM in the general population, but the influence of genetics on the development of PTDM is not well established. Galanakis et al showed a relationship between the endothelial nitric oxide synthase (eNOS) gene “a” allele and type I and type II DM. They found that having an allele of eNOS intron 4 polymorphism may represent a genetic susceptibility.11 Numakura et al reported that the frequency of PTDM was significantly higher in patients with the vitamin D receptor TaqI t allele.12 Earlier studies have reported the influence of ACE and angiotensin II gene polymorphism on the pathogenesis of the DM in general population. RAS is activated in patients with metabolic syndrome and insulin resistance.6,7 It was reported that individuals with the DD genotype were more insulin sensitive than those with I allele.7,13 But as far as we know, neither study reported a relationship of ACE and angiotensin II gene polymorphism with the development of PTDM. Certain genotypes are associated with higher activity of RAS. D allele (deletion on intron 16 of the ACE) is associated with higher plasma levels of ACE; likewise, T allele (change from methionine to threonine at position 235) of AGT gene is correlated with an increase in plasma AGT concentration.14 In this study, we found that high frequencies of the AGT TT genotype and ACE DD genotype in recipients may contribute to the high prevalence of PTDM. Our data suggest a synergistic effect between the ACE and AGT polymorphism in the risk of PTDM, but to support this theory a larger patient group must be studied.
REFERENCES 1. Hathaway DK, Tolley EA, Blakely ML, et al: Development of an index to predict posttransplant diabetes mellitus. Clin Transplant 7:330, 1993 2. Sumrani NB, Delaney V, Ding Z, et al: Diabetes mellitus after renal transplantation in the cyclosporine era: an analysis of risk factors. Transplantation 51:343, 1991 3. Boudreaux JP, McHugh L, Canafax DM, et al: The impact of cyclosporine and combination immunosuppression on the incidence of posttransplant diabetes in renal allograft recipients. Transplantation 44:376, 1987 4. Kasiske BL, Snyder JJ, Gilbertson D, Matas AJ: Diabetes mellitus after kidney transplantation in the United States. Am J Transplant 3:178, 2003 5. Sasahara M, Nishi M, Kawashima H, et al: Uncoupling protein 2 promoter polymorphism ⫺866G/A affects its expression in -cells and modulates clinical profiles of Japanese type 2 diabetic patients. Diabetes 53:482, 2004 6. Hara K, Boutin P, Mori Y, et al: Genetic variation in the gene encoding adiponectin is associated with an increased risk of type 2 diabetes in the Japanese population. Diabetes 51:536, 2002 7. Daimon M, Oizumi T, Saitoh T, et al: The D allele of the angiotensin converting enzyme insertion/deletion (I/D) polymorphism is a risk factor for type 2 diabetes in a population-based Japanese sample. Endocr J 50:393, 2003 8. Phuong-Thu T, Phuong-Chi T, Gerald SL, Alan HW: New onset diabetes mellitus after solid organ transplantation. Endocrinol Metab Clin N Am 36:873, 2007 9. Broekroelofs J, Stegeman CA, Navis G, et al: Risk factors for long-term renal survival after renal transplantation: a role for angiotensin I– converting enzyme gene (insertion/deletion) polymorphism? J Am Soc Nephrol 9:2075, 1998 10. Abdi R, Huong TTB, Zee R, et al: Angiotensin gene polymorphism as a determinant of posttransplantation renal dysfunction and hypertension. Transplantation 72:726, 2001 11. Galanakis E, Kofteridis D, Stratigi K, et al: Intron 4a/b polymorphism of the endothelial nitric oxide synthase gene is associated with both type 1 and type 2 diabetes in a genetically homogenous population. Hum Immunol 69:279, 2008 12. Numakura K, Satoh S, Tsuchiya N, Horikawa Y, et al: Clinical and genetic risk factors for posttransplant diabetes mellitus in adult renal transplant recipients treated with tacrolimus. Transplantation 80:1419, 2005 13. Panahloo A, Andres C, Mohamed-Ali V, et al: The insertion allele of the ACE gene I/D polymorphism. A candidate gene for insulin resistance? Circulation 92:3390, 1995 14. Rigat B, Hubert C, Alhenc-Gelas F, et al: An insertion/ deletion polymorphism in the angiotensin I– converting enzyme gene accounting for half the variance of serum enzyme levels. J Clin Invest 86:1343, 1990
P
0041-1345/11/$–see front matter doi:10.1016/j.transproceed.2011.01.046 572
PTDM. Several genetic polymorphisms have been considered to give rise to the development of DM in general population. Vitamin D receptor and uncoupling protein 2 can influence insulin secretion, and peroxisome proliferators–activated receptor gamma, angiotensin-converting enzyme (ACE), and adiponectin can influence insulin sensitivity.5–7 However, there are no available reports showing an association of From the Department of Pathology (B.H.Ö.), Department of Nephrology (F.N.Ö.), Department of Molecular Biology (F.B.A.), Department of Public Health (A.A.Ö.), and Department of General Surgery and Transplant Surgery (M.H.), Bas¸kent University, Ankara, Turkey. Address reprint requests to Binnaz Handan Özdemir, MD, Professor, Department of Pathology, Bas¸kent University, 12. sokak 7/4, Bahcelievler, 06490, Ankara, Turkey. E-mail: handan27@ hotmail.com © 2011 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 43, 572–574 (2011)
AGT AND ACE MUTATIONS IN RENAL ALLOGRAFT RECIPIENTS
genetic polymorphisms encoding these proteins with the occurrence of PTDM. For that reason, in the present study we investigated the association of polymorphisms in the genes for ACE and angiotensinogen (AGT) with the development of PTDM among patients under a CsA-based immunosuppressive protocol. PATIENTS AND METHODS A total of 50 consecutive patients who received their first kidney transplantation and maintained graft function for a ⱖ12-month posttransplantation period and who used triple therapy including CsA for maintenance immunosuppression were included in the study. All patients were on maintenance immunosuppression including prednisolone and CsA with mycophenolate mofetil. Acute rejection episodes were treated with 3 boluses of 0.5–1 g intravenous methylprednisolone. Patients were eligible to participate in the study if they were the recipients of a kidney allograft with no pretransplantation history of diabetes and a recorded fasting plasma glucose (FPG) level ⬍5.5 mmol/L. Patients were excluded if they had a history of diabetes before transplantation, severe metabolic or infectious disease, or a recorded FPG level ⬎5.5 mmol/L. The Ethics Committee of Baskent University Faculty of Medicine approved the study and each of the patients gave informed consent. The onset of PTDM, clinical characteristics, demographic features (age, gender, dialysis modality, and vintage) and laboratory data were obtained from the patients’ files. The following information was collected for each case: date of transplantation, age and gender of donor and recipient, donor type (living or cadaveric), number of HLA mismatches, body mass index at the time of transplantation (kg/m2), number of acute rejection episodes, and laboratory findings (pretransplantation FPG levels and serum creatinine levels. Diabetes mellitus was diagnosed in accordance with American Diabetes Association/World Health Organization guidelines as symptoms of diabetes plus any time plasma glucose concentrations ⱖ200 mg/dL, or finding of ⱖ2 consecutive fasting glycemia ⬎126 mg/dL or 2-hour plasma glucose ⱖ200 mg/dL during oral glucose tolerance test and the need for insulin or oral antidiabetic therapy.8 of 50 patients, 23 had PTDM (group 1) and the remaining 27 patients did not have PTDM and were used as the control group (group 2). Genotyping was performed for the ACE gene insertion/deletion (I/D) and AGT gene (M¡T, 235) polymorphisms. The patients or patients’ families filled out the consent forms, and venous blood samples anticoagulated with ethylenediaminetetraacetic acid were collected from all patients. Genomic DNA was extracted from peripheral blood leukocytes using the salting-out procedure. The DNA was used as a template for gene polymorphism analysis. All amplifications were performed according to the manufacturer’s recommendations, using polymerase chain reaction sequencespecific primers and appropriate restriction enzymes that have been described previously.9,10
Statistical Analysis Statistical analysis was performed using SPSS software (Statistical Package for the Social Sciences, version 9.05; SSPS, Chicago, IL, USA). For quantitative variables, values are given as mean ⫾ SD. Mean values were calculated for each group and compared using Student t test for normal distribution. The Kruskal-Wallis test was
573 used to compare variables with nonnormal distribution. Categoric data were compared using either chi-square test or Fisher exact probability test, when the expected cells contained ⱕ5 subjects. Results were considered to be statistically significant at a value for P of ⬍.05.
RESULTS
The mean age of all recipients at transplantation was 38 years with a range of 21– 60 years, and the mean posttransplantation follow-up period was 73.8 months. Twenty-three of 50 patients had PTDM according to the above-mentioned diagnostic criteria (group 1). The remaining cases had no PTDM and were used as a control group (group 2). Table 1 shows the demographic, clinical, and laboratory characteristics of 50 renal transplant recipients. There were no significant differences between the patients with or without PTDM regarding gender, dialysis time, donor type, number of mismatches, acute rejection episodes, and duration of transplantation. Univariate analysis showed that there were no differences regarding the risk factors of PTDM between the group with PTDM and the control group (Table 1). Of 50 patients, 13 (26%) showed the ACE DD, 21 (42%) the ACE ID, and 16 the ACE II genotype. The frequencies of AGT MM, AGT MT, and AGT TT were 0, 54%, and 46%, respectively. Compared with group 2 there were high frequencies of the AGT TT genotype in group 1 recipients (P ⬍ .001). In addition, the ACE DD genotype was found significantly higher in group 1 patients compared with group 2 patients (P ⫽ .001; Table 2). DISCUSSION
PTDM is a frequent complication following renal transplantation and serves as a key risk factor for posttransplantation cardiovascular disease. The ability to predict a patient’s risk for developing PTDM would be of considerable benefit in selecting appropriate immunosuppressive regimens for individuals. Although the incidence, risk factors, and clinical relevance of PTDM vary among reports from Table 1. Clinical Characteristics and Demographic Features of Patients With or Without PTDM PTDM No
Age (y) Gender (male/female) Dialysis duration (mo) BMI at the time of transplantation (kg/m2) BMI after the transplantation (kg/m2) Donor source (living/deceased) Number of mismatches [n(%)] Acute rejection [n(%)] Time since transplantation (months) Creatinine at the end of the first year (mg/dL) BMI, body mass index.
PTDM Yes
P Value
38.3 ⫾ 10.9 37.9 ⫾ 10.5 13/7 10/5 18.5 ⫾ 15.2 20.1 ⫾ 16 21.4 ⫾ 2.7 22.9 ⫾ 6.1
NS NS NS NS
23.8 ⫾ 2.8
25.2 ⫾ 6
NS
18/9 2.4 ⫾ 1.2 1.8 ⫾ 1.5 78.9 ⫾ 23
15/8 2.5 ⫾ 1.6 2.1 ⫾ 1.4 63.6 ⫾ 32
NS NS NS NS
1.5 ⫾ 0.4
1.8 ⫾ 0.6
NS
574
ÖZDEMIR, ÖZDEMIR, ATAÇ ET AL
Table 2. Comparison of the ACE and AGT Genotypes of Patients With or Without PTDM
ACE ACE ACE AGT AGT AGT
DD ID II MM MT TT
n
PTDM No
PTDM Yes
P Value
13 21 16 0 27 23
3 (11.1%) 10 (37) 14 (51.9) 0 23 (85.2) 4 (14.8)
10 (43.5) 11 (47.8) 2 (8.7) 0 4 (17.4) 19 (82.6)
.001
⬍.001
clinical studies, the influence of genetic control is not well known. Recently genetic studies have gained potential interest in evaluating the pathogenesis of insulin resistance and DM in the general population, but the influence of genetics on the development of PTDM is not well established. Galanakis et al showed a relationship between the endothelial nitric oxide synthase (eNOS) gene “a” allele and type I and type II DM. They found that having an allele of eNOS intron 4 polymorphism may represent a genetic susceptibility.11 Numakura et al reported that the frequency of PTDM was significantly higher in patients with the vitamin D receptor TaqI t allele.12 Earlier studies have reported the influence of ACE and angiotensin II gene polymorphism on the pathogenesis of the DM in general population. RAS is activated in patients with metabolic syndrome and insulin resistance.6,7 It was reported that individuals with the DD genotype were more insulin sensitive than those with I allele.7,13 But as far as we know, neither study reported a relationship of ACE and angiotensin II gene polymorphism with the development of PTDM. Certain genotypes are associated with higher activity of RAS. D allele (deletion on intron 16 of the ACE) is associated with higher plasma levels of ACE; likewise, T allele (change from methionine to threonine at position 235) of AGT gene is correlated with an increase in plasma AGT concentration.14 In this study, we found that high frequencies of the AGT TT genotype and ACE DD genotype in recipients may contribute to the high prevalence of PTDM. Our data suggest a synergistic effect between the ACE and AGT polymorphism in the risk of PTDM, but to support this theory a larger patient group must be studied.
REFERENCES 1. Hathaway DK, Tolley EA, Blakely ML, et al: Development of an index to predict posttransplant diabetes mellitus. Clin Transplant 7:330, 1993 2. Sumrani NB, Delaney V, Ding Z, et al: Diabetes mellitus after renal transplantation in the cyclosporine era: an analysis of risk factors. Transplantation 51:343, 1991 3. Boudreaux JP, McHugh L, Canafax DM, et al: The impact of cyclosporine and combination immunosuppression on the incidence of posttransplant diabetes in renal allograft recipients. Transplantation 44:376, 1987 4. Kasiske BL, Snyder JJ, Gilbertson D, Matas AJ: Diabetes mellitus after kidney transplantation in the United States. Am J Transplant 3:178, 2003 5. Sasahara M, Nishi M, Kawashima H, et al: Uncoupling protein 2 promoter polymorphism ⫺866G/A affects its expression in -cells and modulates clinical profiles of Japanese type 2 diabetic patients. Diabetes 53:482, 2004 6. Hara K, Boutin P, Mori Y, et al: Genetic variation in the gene encoding adiponectin is associated with an increased risk of type 2 diabetes in the Japanese population. Diabetes 51:536, 2002 7. Daimon M, Oizumi T, Saitoh T, et al: The D allele of the angiotensin converting enzyme insertion/deletion (I/D) polymorphism is a risk factor for type 2 diabetes in a population-based Japanese sample. Endocr J 50:393, 2003 8. Phuong-Thu T, Phuong-Chi T, Gerald SL, Alan HW: New onset diabetes mellitus after solid organ transplantation. Endocrinol Metab Clin N Am 36:873, 2007 9. Broekroelofs J, Stegeman CA, Navis G, et al: Risk factors for long-term renal survival after renal transplantation: a role for angiotensin I– converting enzyme gene (insertion/deletion) polymorphism? J Am Soc Nephrol 9:2075, 1998 10. Abdi R, Huong TTB, Zee R, et al: Angiotensin gene polymorphism as a determinant of posttransplantation renal dysfunction and hypertension. Transplantation 72:726, 2001 11. Galanakis E, Kofteridis D, Stratigi K, et al: Intron 4a/b polymorphism of the endothelial nitric oxide synthase gene is associated with both type 1 and type 2 diabetes in a genetically homogenous population. Hum Immunol 69:279, 2008 12. Numakura K, Satoh S, Tsuchiya N, Horikawa Y, et al: Clinical and genetic risk factors for posttransplant diabetes mellitus in adult renal transplant recipients treated with tacrolimus. Transplantation 80:1419, 2005 13. Panahloo A, Andres C, Mohamed-Ali V, et al: The insertion allele of the ACE gene I/D polymorphism. A candidate gene for insulin resistance? Circulation 92:3390, 1995 14. Rigat B, Hubert C, Alhenc-Gelas F, et al: An insertion/ deletion polymorphism in the angiotensin I– converting enzyme gene accounting for half the variance of serum enzyme levels. J Clin Invest 86:1343, 1990