1 Centre for Human Genetics, Edith Cowan University, Joondalup, Perth, WA, 2 University Department of Medicine, University of Western Australia, Medical Research Foundation Building, Perth, WA, 3 Institut für Humangenetik, WW-U, Münster, Germany, 4 Department of Nephrology, Jagiellonian University, Cracow, Poland, 5 Department of Radiology, 6 Department of Nephrology, 7 Department of Biochemistry, Royal Perth Hospital, Perth, WA, 8 Clinic of Nephrology and Hemodialysis, University Hospital, Pleven, Bulgaria, 9 Clinic of Nephrology and Hemodialysis, University Hospital, Plovdiv, Bulgaria, 10 Centre for Clinical Research in Neuropsychiatry, Graylands Hospital, Perth, WA and 11 Western Australian Institute for Medical Research, QEII Medical Centre, Nedlands, Perth, WA, Australia
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Abstract |
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Methods. Blood samples were collected from 307 ADPKD patients (116 Australian, 124 Bulgarian and 67 Polish) for determination of ACE activity levels and I/D genotypes. Chronic renal failure (CRF) was present in 117 patients and end-stage renal failure (ESRF) in 68 patients.
Results. ACE activity was related to the I/D genotype, showing a dosage effect of the D allele (P=0.006). The proportion of variance due to the Alu polymorphism was 14%. No difference in ACE activity and I/D genotype distribution was found between patients with CRF versus normal renal function (P=0.494; P=0.576) or between those with ESRF versus those without ESRF (P=0.872; P=0.825). No effect of the I/D genotype on age at development and progression to renal failure (CRF; ESRF) was detected in the overall group, and in subgroups based on ethnic origin, linkage status and sex.
Conclusion. ACE is not likely to play a role as a determinant of ADPKD phenotype severity.
Keywords: autosomal dominant polycystic kidney disease; angiotensin converting enzyme; chronic renal failure; end-stage renal failure
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Introduction |
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In an attempt to address this problem, we have studied subjects originating from three different populations, and assessed the association between ACE and ADPKD severity in terms of both plasma enzyme activity and I/D genotypes.
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Subjects and methods |
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The diagnosis of ADPKD was based on accepted ultrasonographic criteria [13]. Renal function and rate of deterioration were evaluated by serum creatinine (SCr) values, collected retrospectively over periods ranging from 1 to 7.2 years (mean 6.8±0.31). The number of measurements per patient varied between 1 and 16 (mean 3.4±0.15). Deterioration of kidney function was assessed using two end points: chronic renal failure (CRF), defined as SCr level of 150 µmol/l (2.25 mg/dl), and end-stage renal failure (ESRF). In our patient population, 117 individuals had reached CRF and 68 had ESRF.
The definition of hypertension followed WHO criteria [14]. The overall number of hypertensive individuals was 192. Information on the use of ACE inhibitors at the time of the study was available for both the Australian and Polish patients, where 54 out of 85 Australian and all 41 Polish hypertensive subjects received such treatment.
Based on genetic linkage data, the patients were subdivided into 184 PKD1, 38 PKD2 and 85 subjects where linkage analysis was not feasible. Since the probability of linkage to the PKD1 gene is around 90% in affected individuals of European descent [15], the 85 undefined individuals were included in the PKD1 group, adding to a total of 269 subjects. Statistical analysis was performed separately on the definite and on the expanded PKD1 group.
The information on the affected individuals is summarized in Table 1.
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Methods
Laboratory analyses
Plasma ACE activity was measured in 92 Australian patients using the kinetic method [16], with a reference range of 23100 U/l (covariance 3.2% at 125 U/l and 8.1% at 31 U/l).
The ACE I/D polymorphism was detected by PCR amplification as described [17]. To avoid mistyping, all D/D homozygous samples were re-typed using an insertion-specific PCR primer [18]. PCR products were separated on 3% agarose gels and visualized by ethidium bromide staining.
Statistical analysis
ACE genotype frequencies were compared between countries using the Chi-square test. HardyWeinberg equilibrium (HWE) was assessed with the probability test in the overall sample and with Fisher's exact test within each of the three populations. The relationship between ACE I/D genotypes and plasma ACE activity was assessed by general linear models (GLM). Analysis of covariance (ANCOVA) was used to correct for treatment with ACE inhibitors and analysis of variance (ANOVA) was used to compare the mean values of plasma ACE activity between individuals reaching CRF before or after age 40 years.
Decline in renal function was assessed by linear regression analysis using the least-squares method, with fitting individual regression lines of time vs 1/creatinine values and extrapolating age at CRF. KaplanMeier survival curves were used to calculate cumulative survival to CRF and ESRF. Individuals were grouped according to ACE genotype and compared by means of a two-sided log-rank test.
Differences were considered statistically significant at of 0.05 (P<0.05). All analyses were performed using SPSS 9.0 (SPSS Inc, Chicago III).
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Results |
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Plasma ACE activity and renal function
The relationship between plasma ACE activity and the development of renal failure was tested in the 92 Australian patients (CRF=37; ESRF=27). The mean ACE activity levels were 61.7±3.8 U/l in the group with normal renal function; 59.8±6.4 in the CRF group; and 57.9±5.5 in subjects with ESRF. The difference was not significant (P=0.494 for CRF vs normal renal function and P=0.872 for ESRF vs lack of ESRF). Similar results were obtained after adjustment for treatment with ACE inhibitors.
The mean plasma ACE activity was 62.6±4.1 U/l in the group of patients reaching CRF before age 40 years (n=45) and 56.3±5.5 U/l among those reaching CRF after age 40 years. The difference was not significant (P=0.352).
The I/D genotype and ADPKD phenotype severity
Risk of renal failure
The distribution of I/D genotypes among the 307 ADPKD subjects was examined by comparing subjects with normal renal function to those with CRF and with ESRF (Table 3). No significant differences were found between the three groups: P=0.576 in the comparison of CRF vs normal renal function and P=0.825 for ESRF vs lack of ESRF.
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Age at development of renal failure
The relationship between the I/D genotype and development of renal failure was examined in a total of 117 subjects with CRF and 68 with ESRF. No significant differences in the mean age to either CRF and ESRF was found. The mean age at CRF was 40.7±1.9 years for I/I, 39.7±1.1 for I/D and 39.1±1.5 for D/D subjects, P=0.810. The mean age at ESRF was 51.5± 3.0 years for I/I individuals, 55.5±1.6 for I/D and 54.5±1.7 for D/D, P=0.415. This analysis was repeated after grouping the patients according to ethnic origin. Again, no statistically significant differences were found, with P=0.739 and P=0.239 for the Australian patients, P=0.504 and P=0.087 for the Bulgarian and P=0.313 and P=0.184 for the Polish.
Lack of significant effect was observed when the different I/D genotypes were compared, within the entire group of patients using KaplanMeier cumulative survival curves to CRF (P=0.815) and to ESRF (P=0.478) (Figure 1). Similar results were obtained when KaplanMeier survival curves were examined separately for PKD1 and PKD2. Analysis of survival to CRF gave P values of 0.606 in the PKD1 and 0.529 in the PKD2 group. For ESRF, the P values were 0.454 for PKD1 and 0.163 for PKD2. No significant difference was revealed between the definite and expanded PKD1 groups. The separate analysis of male and female individuals with ADPKD also failed to detect significant effects of the I/D genotype on survival to CRF or ESRF.
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Hypertension
The distribution of I/D genotypes was compared between 192 hypertensive and 97 normotensive ADPKD subjects. The I/D distribution for the hypertensive group were: I/I (18%), I/D (51%), D/D (31%), and I/I (12.4%), I/D (57.7%) and D/D (29.9%) for the normotensive group. The analysis failed to reveal a significant difference between the two groups (P=0.384).
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Discussion |
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We have examined the association between the alleles of the Alu polymorphism in the ACE gene and decline in kidney function in 307 ADPKD subjects from three different populations. Inter-population differences did exist in terms of both the frequency of I/D alleles and the ratio of PKD1 to PKD2 patients. However, none of the three populations displayed a significant association between the I/D genotype and ADPKD phenotype severity. Lack of association was also observed for the entire study group. No effect was detected on either the overall risk of renal failure, the age at development of renal failure or the rate of progression from normal function to CRF and to ESRF. The findings were also negative in the separate analysis of patients grouped according to linkage status or sex.
In discussing the results of such association studies, one has to keep in mind their oversimplified design, where a DNA polymorphism in one gene is used to represent the overall effect of an entire pathway which is likely to be under complex control. The approach is simplistic even with the regard to ACE itself. The Alu polymorphism in the ACE gene is considered to be a neutral marker in disequilibrium with another, biologically relevant polymorphism directly affecting ACE activity levels. Strong evidence coming from recent studies suggests that the ACE-linked quantitative trait loci (QTL) are probably located in the 3' region of the gene, where a complexity of intragenic haplotypes can be observed, with the I or D allele of the Alu polymorphism occurring on diverse haplotype backgrounds [20]. The diversity of intragenic ACE haplotypes, their complex additive effects on ACE activity, and the observed inter-population differences [20] could account for the discrepant estimates of the proportion of the variance in ACE activity explained by the Alu polymorphism, where figures range from nil to 47% [9,11,12].
In agreement with previous studies, ACE activity measured in 92 Australian ADPKD patients was related to the I/D genotype, but the proportion of variance due to the Alu polymorphism was only 14%. A similar proportion can be assumed for the patients studied by Baboolal et al. [7], since they share the same ethnic background. The striking effect of the D allele on earlier development of renal failure reported in that study [7] is difficult to interpret: firstly, in view of the modest contribution of the Alu polymorphism to the control of ACE expression and secondly because of the lack of expected dosage effect of the D allele observed by Baboolal et al. [7] and Perez-Oller et al. [8]. Additionally our analysis of plasma ACE activity did not reveal any effect on the development of renal failure, in agreement with Uemaso et al. [9] and Van Dijk et al. [10].
In conclusion, the results from this study suggest that the ACE is not likely to play a role as a determinant of ADPKD phenotype severity. Unless information on the relation between the Alu polymorphism I/D genotypes and ACE activity is available, association studies are neither conclusive nor comparable.
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Acknowledgments |
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Notes |
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References |
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