nature publishing group
original contributions
Association of ACE Gene Insertion/Deletion Polymorphism With Birth Weight, Blood Pressure Levels, and ACE Activity in Healthy Children Aline R. Ajala1, Sandro S. Almeida2, Marina Rangel1, Zaira Palomino1, Maria Wany L. Strufaldi3, Rosana F. Puccini3, Ronaldo C. Araujo2, Dulce Elena Casarini1 and Maria C.P. Franco1 Background The human angiotensin-converting enzyme (ACE) gene contains a polymorphism consisting of either an insertion (I) or a deletion (D) of a 287 bp Alu repetitive sequence in intron 16. The potential role of ACE polymorphism in the risk of developing hypertension or other cardiovascular disorders has not been determined in relation to birth weight (BW). Methods The ACE genotype and plasma ACE activity were determined in 167 children. Among these children, 60 were identified with low BW (LBW), and 107 were of normal BW (NBW). Results ACE activity levels were significantly elevated in LBW children compared with the NBW group (P < 0.001). There was a significant association of the ACE activity with systolic blood pressure (SBP) levels in our population (P < 0.001). Among the ACE genotypes, no
Growing clinical evidences have emerged that adulthood hypertension, type 2 diabetes, obesity, and coronary heart disease are associated with low birth weight (LBW) .1–3 It has been suggested that this association may be a result of an adverse uterine environment, that induces metabolic adaptations in physiological systems, leading to altered programming of the fetal key organs.1–3 There is an increasing need to better understand the factors that influence this correlation between chronic diseases and LBW. Although the mechanistic hypothesis is unknown, the gene variants and their interaction with the fetal environment may act by predisposing or modulating risk for the development of adult diseases such as hypertension. The contribution of renin–angiotensin system (RAS) gene polymorphisms in the development of hypertension and 1Division of Nephrology, School of Medicine, Federal University of São Paulo, São Paulo, Brazil; 2Department of Biophysics, Federal University of São Paulo, São Paulo, Brazil; 3Department of Pediatrics, School of Medicine, Federal University of São Paulo, São Paulo, Brazil. Correspondence: Maria C.P. Franco (
[email protected])
Received 13 December 2011; first decision 1 February 2012; accepted 22 March 2012. © 2012 American Journal of Hypertension, Ltd. AMERICAN JOURNAL OF HYPERTENSION
significant differences were found with respect to BW (P = 0.136). However, our results revealed that LBW children had a higher D allele frequency than NBW children (P = 0.036). When analyzed by quartiles of SBP or ACE activity, we found a greater frequency of both the LBW children and those carrying the DD genotype in the highest quartiles of these parameters, whereas the NBW children tended to be in the lowest quartile (P < 0.001). Similar results were observed with the heterozygote ID children after categorization by quartiles of both SBP (P < 0.001) and ACE activity (P = 0.004). Conclusions The ACE I/D polymorphism, especially the DD genotype, can be interpreted as a major factor in association between LBW and high BP levels. Keywords: ACE activity; ACE I/D polymorphism; birth weight; blood pressure; children; hypertension American Journal of Hypertension, advance online publication 31 May 2012; doi:10.1038/ajh.2012.50
c oronary heart diseases has been recognized.4–8 The RAS plays an important role in the regulation of blood pressure (BP) levels and volume homeostasis.9,10 Angiotensin-converting enzyme (ACE) is the rate-limiting enzyme of this system; its actions include catalysis of the conversion of angiotensin I (Ang I) to angiotensin II (Ang II) and inactivation of the vasodilator bradykinin.9 This enzyme is not only a key component in the regulation of BP levels, but it is also an important risk factor for cardiovascular disease (CVD).4–8,11 Recent studies have demonstrated that ACE levels are significantly elevated in both children and infants with a history of LBW.12,13 Thus, the ACE gene is a relevant candidate for elucidating a possible link between LBW and CVDs in later life, as the presence of different genetic profiles due to allelic variants could explain the occurrence of ACE overactivity in children with LBW. The ACE gene is known to contain polymorphisms consisting of either the insertion (I) or deletion (D) of a 287-bp sequence inside intron 16. It has been demonstrated that allelic ACE variation is responsible for 47% of the variance of plasma ACE activity.14 Notably, the D allele and the DD genotype are associated with elevated levels of ACE and a higher risk of 1
original contributions adverse cardiovascular events and end-organ damage.4,5,15–17 The correlation between the DD genotype and increased propensity for the development of hypertension can be attributed to elevated Ang II conversion, suggesting that in these homozygotes, the elevated ACE levels modulate RAS function. A relationship between the allelic variation in ACE polymorphism and BW has been suggested.18–21 A few studies found a significant correlation between the presence of the ACE I allele and increased indexes of insulin resistance in adults with LBW.18,19 The potential role of I/D ACE polymorphism in influencing the risk of developing hypertension or other negative cardiovascular outcomes has not been determined in relation to BW. In this cross-sectional study, we aimed to investigate possible interactions between the ACE I/D polymorphism and BW as well as their effects on BP levels and RAS components in healthy children. Methods
Between March and November, 2008, 765 children were evaluated during an anthropometric census performed by the Pediatric Department of the Medical School of the Federal University of São Paulo—UNIFESP. The selection criteria were the following: BW≤ 2,500 g or 3,000 g, no clinical signs of hypertension, CVD, diabetes, renal or chronic illness and adequate amount of blood or DNA for evaluation of the biomarkers and ACE I/D genotyping. After applying these criteria, a total of 167 children were selected (83 girls and 84 boys). Among these children, 60 (31 girls and 29 boys) were identified with LBW (children: BW ≤2.5 kg), and 107 (52 girls and 55 boys) had normal birth weights (NBW children: BW ≥3.0 kg). As part of this study, we took detailed personal and family medical histories by questionnaire. The BW or gestational age data were derived from maternal recall. No child had any clinical signs of hypertension, endocrinopathy, renal or chronic illness. The present study conforms to the principles outlined in the Declaration of Helsinki. This study was approved by the Research Ethics Committee of the UNIFESP, and written informed consent was obtained from the parents of the children enrolled in the study. The procedures followed were in accordance with our institutional guidelines. Anthropometry. Body weight and height were measured in light clothing without shoes during the anthropometric census using a standard balance beam scale. Abdominal circumference was measured at a level midway between the lower rib margin and iliac crest to the nearest 0.5 cm. Measurement of BP levels. BP was measured between 9:00 AM and 4:00 PM in accordance with standard procedures and recommendations.22 Systolic BP (SBP) and diastolic BP (DBP) were measured by auscultation after the child was seated for 10 min. Three measurements were made at 2-min intervals, and the mean value was used in the analysis. Height, age, and sexspecific z-scores were also used for analysis of BP.22 The mean arterial pressure (MAP = (SBP − DBP/3) + DBP) and pulse pressure (PP = SBP − DBP) were calculated. The same nurse, blinded to the clinical data, examined and evaluated all the children. 2
ACE I/D Polymorphism in LBW Children
Blood sample collection. Blood venous samples (5 ml) were collected from each child by venipuncture of a forearm vein into separate vacutainer tubes containing ethylenediaminetetraacetic acid or heparin after overnight fasting. Blood samples were centrifuged within 1 h (1,500g for 5 min) and aliquots of heparin plasma were stored at −80 °C until assay. Plasma ACE activity. Measurement of ACE activity using ippuryl-histidine-leucine as a substrate was performed using h the fluorometric method in heparin plasma samples.13 This assay was performed by the same researcher, who used a blind analysis technique. Plasma renin concentration. Renin concentration was determined with commercially available ELISA kits (DIAsource Renin Kit; DIAsource, Nivelles, Belgium) in ethylenediaminetetraacetic acid plasma samples. The intra-assay accuracy of the method was assessed from control samples (DIAsource), which were included in each assay. The control samples were aliquoted and stored frozen with the plasma samples until assay. The concentrations of the control samples were determined for each assay, and the coefficient of variation was 2.7%. This assay was performed in duplicate, and to remove any bias, the researcher devised blind analysis techniques. DNA isolation. For DNA isolation, ethylenediaminetetraacetic acid tubes were centrifuged and the buffy coat (the white layer between red blood cells and plasma) was extracted and stored at −80 °C until analysis. Isolation of genomic DNA from buffy coats was performed using the QIAmp DNA Mini kits (Qiagen, Valencia, CA). The integrity of the isolated DNA was checked on a 1% agarose gel. ACE I/D genotyping. The ACE I/D polymorphism (NCBI ref. SNP ID: rs1799752) was determined by PCR. Briefly, PCR primers (ECAS, 5′-CTGGAGACCACTCCCATCCTTTCT-3′ and ECAR, 5′-GATGTGGCCATCACATTCGTCAGAT-3′) flanking the polymorphic region outside of the Alu insert in intron 16 were used to amplify a portion of the ACE gene, and the amplified product was analyzed by gel electrophoresis and ethidium bromide staining to determine the I/D pattern. Because the D allele in heterozygotes is preferentially amplified, each DD genotype was confirmed by a second independent PCR with another primer pair (ECAint, 5′-GTCTCGATCTCCTGACCTCGTG-3′ and ECAR, 5′-GATGTGGCCATCACATTCGTCAGAT-3′) that amplifies the insertion-specific sequence. To eliminate any possible bias, the same researcher devised blind analysis techniques; therefore, our results are free of genotyping errors or mistakes in data manipulation. Statistical analysis. Categorical variables were compared using the χ2 test. Student’s t-test was used to compare the mean values of continuous variables between the two groups. All continuous variables were examined for normality with the Kolmogorov– Smirnov test. Analysis of covariance was used to compare the mean values of ACE activity and BP levels between BW groups, AMERICAN JOURNAL OF HYPERTENSION
original contributions
ACE I/D Polymorphism in LBW Children
adjusting for potential confounding variables. Haploview software was used to analyze the ACE I/D alleles and to determine their Hardy–Weinberg equilibrium status.23 Comparisons of the frequency of different ACE genotypes were made by χ2 analyses. The values of continuous variables are expressed as the mean ± s.e.m. In an additive model, SBP and ACE activity were also assessed by χ2 after categorization by quartiles. Quartiles of the SBP (Q1: ≤90, Q2: 91–93, Q3: 93–99, and Q4: ≥100 mm Hg) and ACE activity (Q1: ≤63.8, Q2: 63.9–77.4, Q3: 77.5–94.2, and Q4: ≥94.3 nmol/ml/min) were computed within each ID/ DD genotype and according to BW groups. The comparisons of SBP levels or ACE activity between the NBW and LBW groups and between genotypes were performed by two-way analysis of variance. Statistical tests were two-tailed, and the significance level was set at P < 0.05. Statistical analyses were conducted using SPSS version 13.0 (SPSS, Chicago, IL).
Table 1 | Main characteristics of the study population according to birth weight groups NBW children (n = 107)
LBW children (n = 60)
P value
Perinatal data Birth weight (g)
3,386.0 ± 21.9
2,324.5 ± 20.4
