Maria Beatriz S. Freire1,3, David J. van Dijk2, Arie Erman2, Geoffrey Boner2, James H. Warram1 and. Andrzej S. ... between 30 and 300 mg/24 h were considered microalbumin- ..... Quinn M, Angelico MC, Warram JH, Krolewski AS. Familial.
Nephrol Dial Transplant (1998) 13: 2553–2558
Nephrology Dialysis Transplantation
Original Article
DNA polymorphisms in the ACE gene, serum ACE activity and the risk of nephropathy in insulin-dependent diabetes mellitus Maria Beatriz S. Freire1,3, David J. van Dijk2, Arie Erman2, Geoffrey Boner2, James H. Warram1 and Andrzej S. Krolewski1 1Research Division, Joslin Diabetes Centre and Department of Medicine, Harvard Medical School Boston, Massachusetts, USA, 2Institute of Hypertension and Kidney Diseases, Rabin Medical Centre, Beilinson Medical Centre, Petach Tikwa and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel, and 3Currently at Faculdade de Medicina de Jundiai, Jundiai, Brazil
Abstract Background. To determine the relationship between DNA polymorphisms in the angiotensin I converting enzyme (ACE) gene, serum ACE activity and the risk of diabetic nephropathy. Methods. A case-control study was carried out in a population of Jewish insulin-dependent diabetes mellitus (IDDM ) patients. Cases (77 IDDM patients with diabetic nephropathy) and controls (89 IDDM patients with normoalbuminuria) were genotyped with PCR protocols for detecting two DNA polymorphisms in the ACE gene: one in intron 7 detected with the restriction enzyme PstI and the other in intron 16 identified as an insertion/deletion (I/D). Results. The risk of nephropathy was increased only in patients homozygous for the allele with the PstI site. These homozygotes had a nephropathy risk that was 2.3 times (95% C.I.: 1.2–4.5) that of the other genotypes. Furthermore, these individuals did not have elevated serum ACE activity. Conclusions. The results of this study are evidence that the risk of diabetic nephropathy in IDDM is influenced by genetic variability at the ACE locus, but the responsible variant is not the I/D polymorphism in intron 16. Our findings require further studies in other populations.
Introduction Only a subset of insulin-dependent diabetes mellitus (IDDM ) patients (about 30%) is susceptible to diabetic nephropathy [1], and familial clustering of this complication points to genetic factors as the determinants of susceptibility [2–4]. So far, genes encoding for proteins of the renin-angiotensin system have been Correspondence and offprint requests to: Andrzej S. Krolewski, MD, PhD, Joslin Diabetes Centre, One Joslin Place, Boston, MA 02215, USA.
examined extensively, with the angiotensin I converting enzyme (ACE) gene being the favourite candidate gene for nephropathy [5,6 ]. This has occurred for several reasons. First, it was reported a long time ago that IDDM patients with diabetic nephropathy have elevated serum levels of ACE [7–9]. Second, carriers of certain allele(s) in the ACE gene were found to have high levels of serum ACE [10]. The same individuals were found in some studies to be at increased risk of diabetic nephropathy [11,12]. Third, clinical trials have demonstrated the effectiveness of ACE inhibition in retarding the progression of diabetic nephropathy [13,14]. ACE is an ectoenzyme anchored to the plasma membrane with the bulk of its mass exposed on the extracellular surface of endothelial, epithelial and neuronal cells [15,16 ]. It cleaves the carboxyl-terminal dipeptide from angiotensin I to produce angiotensin II, and inactivates bradykinin (the most favoured substrate) by the sequential removal of two carboxylterminal dipeptides [15,16 ]. Cloning of ACE cDNA revealed that the enzyme is composed of two homologous domains: the amino and carboxyl domain [17]. Each of these domains contains enzymatically active sites that catalyse physiological substrates at slightly different rates [16 ]. ACE is encoded by a single gene located on the long arm of chromosome 17 [17,18]. The gene has 26 exons: exons 1–12 encode for the amino domain and exons 13–26 encode for the carboxyl domain. Several different DNA polymorphisms have been identified in this gene [10,19,20]. A frequent insertion/deletion (I/D) polymorphism, located in intron 16 has been the one most studied [10]. The D allele is associated with higher plasma levels of ACE than the I allele, and the D allele also seems to be associated with increased myocardial infarction risk in diabetic and non-diabetic individuals [10,21–23]. It is not clear, however, that the increased risk of myocardial infarction is related to the elevated plasma ACE levels [24]. In some
© 1998 European Renal Association–European Dialysis and Transplant Association
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populations, the association of the D allele with myocardial infarction has not been confirmed [25]. The role of the I/D polymorphism at the ACE gene has also been evaluated in the development of kidney diseases. The presence of the D allele predisposes to progression of IgA nephropathy [26,27], but findings with regard to diabetic nephropathy have been contradictory. Whereas Marre et al. [11,12] reported that the I/D polymorphism was associated with the development of diabetic nephropathy, several other studies did not confirm this finding [28,29]. In our research in Boston, while we found an effect of sequence differences at the ACE locus on the risk of diabetic nephropathy, the effect was associated with a polymorphism in intron 7 of the gene rather than the I/D in intron 16 [30]. The present study was undertaken to study the relationship between polymorphisms in the ACE gene in a Jewish population of IDDM patients and to examine whether the association between the specific polymorphisms in the ACE gene and diabetic nephropathy can be accounted for by elevated serum ACE activities.
Research design and methods Study population Patients selected for this study included IDDM patients attending the outpatient clinic at the Rabin Medical Centre, Beilinson Campus, a tertiary referral centre in Petach Tikwa, Israel. This group of patients includes some of the patients with IDDM described previously [31]. For the present study only patients with at least 10 years duration of IDDM were selected. The study protocol was approved by the Human Subjects Committee at the Rabin Medical Centre.
Patient examination Each patient had a physical examination performed that included height, weight and blood pressure measurements. A standardized questionnaire was used to obtain the medical history and demographic information. In addition retinopathy status was assessed by reviewing medical records. Proliferative retinopathy was diagnosed if a patient had laser treatment or had proliferative retinopathy diagnosed by a retinal specialist. A blood sample was drawn for biochemical determinations and for isolation of DNA.
Determination of the albumin excretion rate (AER) The albumin excretion rate was determined in three 24-h urine collections performed at least 1 month apart. The urine samples were stored in polystyrene tubes at 4°C and assayed within 1 week. Urinary albumin was determined by RIA as previously described [32]. Human albumin antibodies were purchased from Bioyeda (Rehovot, Israel ). Human serum albumin was obtained from Sigma (Holon, Israel ). 125ILabelled human albumin (specific activity 10 mCi/mg) was purchased from the Radiochemical Centre (Negev, Israel ). Measurements of the AER were classified into three categories: values
