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Renin-angiotension system plays a major role in the genesis of hypertension and progression of end-stage renal disease in autosomal dominant polycystic kidney disease. The Angiotensin-Converting Enzyme (ACE) is a candidate gene in the aetiology of several disease end points. The present study investigated six ACE tag-single nucleotide polymorphisms (tagSNPs) and I/D in 106 ADPKD patients (46% stage IV or V and 41% in stage II or III of ESRD) and 102 healthy subjects. The tagSNPs were genotyped using FRET-based KASPar method and ACE ID by PCR- electrophoresis. Except rs4293 remaining SNPs were polymorphic and followed Hardy-Weinberg equilibrium. The ACE genotype distribution in different models and Haplotype analysis has shown negative association with ADPKD. The significant LD was observed between SNPs forming two LD blocks. As the age increases progression in the CKD stage was observed (p>0.001). Distribution of different CKD stages among different genotype groups observed positive trend only for rs4362 in recessive model (p=0.034). These results suggest that the tagging single-nucleotide polymorphisms of ACE gene do not confer a substantial risk for ADPKD in our population.
Autosomal dominant polycystic kidney disease (ADPKD) is the most common end stage renal disorder is characterized by the growth of fluid filled cysts in the kidneys leading to renal failure during the fourth and fifth decades of life. ADPKD affects all races, with an estimated frequency one in 400 to 1000 individuals . Several lines of evidence revealed that the mutations in polycystin (PKD) genes are implicated in the pathogenesis of ADPKD. The PKD products are known to exhibit sequence homology and play an important role in cell-cell and cell- matrix interactions that involved in regulating the renal ion transport . The European Polycystic Kidney Disease consortium Mutations in the polycystin-1 (PKD1) gene account for 85-90% cases and polycystin-2 (PKD2) gene accounting for the remaining 10-15% of the cases of ADPKD . Although a clear genotype-phenotype relationship has not been identified so far, studies on mouse and human models suggest that these mutations play a significant role in the differential renal progression that observed in ADPKD . Intra-familial heterogeneity that found in the expression of ADPKD, suggested the involvement of environmental factors along with the genetic factors in the progression ADPKD .
Hypertension is one of the clinical manifestations in majority of ADPKD cases and correlates with the progressive kidney enlargement with high prognostic values. The rennin-angiotensin system (RAS) is known to regulate the blood pressure and fluid balance . The activity of RAS system is regulated by the rate of production of angiotensinogen from renin that mediated by angiotensin-converting enzyme (ACE). In view of the importance of the hypertension on progression of end stage renal disease (ESRD) ADPKD, the gene polymorphisms of ACE are of great interest.
The ACE gene spans over 24 kb with 26 exons and located on chromosome 17. The presence or absence of a 287-bp repeat sequence (I/D polymorphism) at intron 16 has been used as a common marker in susceptibility to various disorders . A deleterious effect of DD genotype of ACE in ADPKD was associated with early hypertension and the activation of RAS. This polymorphism found to account for nearly 50% of variation in the ACE serum activity in 'white' population , but the role of this variant in black populations is still uncertain . In the present study we investigated the ACE ID and six additional tagging-single nucleotide polymorphisms (tagSNPs) that selected based on GIH population to unravel the association between ACE gene and ADPKD in south Indian population.
Materials and methods
A total of 208 individuals of south Indian origin, 106 affected by ADPKD (55.88% men) and 102 controls (60.38% men) were selected from the department of Nephrology, Sri Ramachandra University, Chennai, India. All the ADPKD patients included have fulfilled the standard diagnostic criteria . Upon screening subjects without diabetics, hypertension and kidney related diseases were included as controls. The study was approved by the Institutional Ethics Committee of the Sri Ramachandra University, Chennai, India. A written informed consent was obtained from the each study participant before they were included in it. For each of the study subjects, the basic biochemical and electrolyte variables such as haemoglobin (HB), blood urea nitrogen (BUN), creatinine, sodium, potassium, bicarbonate, calcium were analysed. Furthermore, glomerular filtration rate (eGFR) and chronic kidney disease (CKD) stages were assessed based on Modification of Diet in Renal Disease (MDRD) formula and KDOQI CKD guidelines respectively. In all ADPKD patients we have determined the total number of cysts that can be detected by Ultrasound imaging. Subjects with blood pressure ≥140/90 mm Hg and/or treatment with antihypertensive medication were considered as hypertensive group. Genomic DNA from the samples was extracted by phenol chloroform extraction and ethanol precipitation protocol .
SNP Selection and Genotyping
We selected all tagging SNPs (tSNPs) covering ACE gene from the release 2.0 Phase II data of the HapMap Project (www.hapmap.org) (reference of International HapMap Consortium 2005) using the Tagger Pairwise method (Barrett et al. 2005). The tSNPs were chosen according to the following criteria: r2 ≥0.8 and minor allele frequency of ≥5% in the GIH population. We also included a common well studied I/D polymorphism. Genotyping of the tag-SNPs was carried out by using the FRET-based KASPar SNP genotyping assay method (KBioscience, Herts, UK) on an Applied Biosystems Thermocycler (ABI Prism 9700, Foster City, CA, USA). The genotyping was performed with 7900 SDS software (ABI Prism 7700, Foster City, CA, USA). The genotyping success rate ranged from 99.3% to 100.0%. The ACE gene insertion deletion polymorphism was detected by performing the polymerase chain reaction (PCR) and electrophoresis as described elsewhere . All homozygous deletion samples were further subjected to a second PCR ampli¬cation with insertion-speci¬c primers to prevent mistyping of heterozygous genotypes .
Allele frequencies were calculated by the gene-counting method, Genotypes at each SNP were tested for Hardy-Weinberg equilibrium by means of a χ2 test with one degree of freedom. Genotypic effects were evaluated for ADPKD. Logistical regression analyses were used to calculate the odds ratios (OR), 95% confidence intervals (CI), and corresponding p-values for each SNP and haplotype. Pairwise linkage disequilibrium (LD) measures (D' and r2) and haplotype blocks were assessed using the default setting of the Haploview software . Haplotype frequencies were also estimated from polymorphic SNPs within the linkage disequilibrium block using Haploview software .
The study included a total of 106 patients with ADPKD and 102 control subjects. According to the KDOQI CKD guidelines 49 (46%) patients with ADPKD were in stage IV or V and 43 (41%) in stage II or III of ESRD. The mean age of selected controls is 53.27 ± 12.43 and patients with ADPKD 46.89 ± 11.38yrs. The basic biochemical stricture of kidney function information of controls and patients with ADPKD are described in table 1. For ACE gene GIH population yielded six tagSNPs (rs4293, rs4309, rs4311, rs4325, rs4329 and rs4362). The allele and genotype frequencies of the six tagSNPs along with Insertion deletion polymorphisms were shown in table 2. Except rs4293 all polymorphisms followed Hardy-Weinberg equilibrium, SNPs of the ACE gene were statistically not associated with ADPKD when comparing genotype and allele distributions between control and patients with ADPKD groups (Table-2). The OR (Odds Ratio) and 95% confidence interval (CI) calculated for the heterozygous and high risk homozygous genotypes and no significant differences was found in controls and patients with ADPKD (Table -2). The dominant and recessive models of genotype also were calculated but none of these models has shown significant association between controls and patients with ADPKD (data not shown). As the age increases progression in the CKD stage was observed (trend p>0.001) (Table S1). Distribution of different CKD stages among different genotype groups of the studied polymorphisms revealed positive trend only for rs4362 in recessive model (trend p=0.034) (Table S2). Analysis of LD showed strong and significant LD between the markers by forming two LD blocks (Table 3). The ¬rst LD block included rs4311 and rs4325, which were separated by 2.4 kb in intron 9 and intron 13 respectively. The second LD block included rs4329, InDel and rs4362, which encompassed intron 13, intron 16 and exon 21 respectively. The rs4309 SNP located in exon 8 remained outside the LD blocks. Haplotypes were constructed using the SNPs located in individual LD blocks is presented in Table 4. Comparison of haplotypes between ADPKD and control groups also revealed no signi¬cant association with ADPKD.
Analysis of tagSNPs within the ACE gene in 106 ADPKD and 102 control subjects did not show significant association with ADPKD. Strong LD was found among all SNPs studied, covering a region of about 13.8 kb within the ACE gene. Comparison of haplotypes between ADPKD and control groups also revealed no significant association with ADPKD. In ADPKD, hypertension is an early sign that occur even before reduction in glomorular filtration rate. Multiple lines of evidence indicated that inappropriate activation of RAS is the major cause in the pathogenesis of hypertension. Although there is no clear understanding of the underlying mechanisms for the rise in hypertension in individuals with ADPKD, pharmacological blockade of the RAS using ACE inhibitors has significantly reduced CKD progression . ACE is suspected to play a key role and is the first candidate modifier gene to be investigated in ADPKD .
Extrapolation of these results to examine the impact of the ACE gene ID polymorphism on ADPKD has rendered multiple studies reporting conflicting results. An association between D allele of ACE ID in among ADPKD patients was detected in different populations, Australia , Netherland , Japan , Italy , Caucasians of Belgium and France , Spain . In contrast to this no association was found in Japan , Spain , Korea , UK , Australia, Bulgaria and Poland , Czech Republic , USA , Poland . These discrepancies may be explained by underpowered studies and by population stratification because of a heterogeneous genetic heterogeneity. In a meta-analysis that increase power to detect associations by reducing heterogeneity, D-allele did not reveal a significant association with the risk of ADPKD patients when compared with I-allele carriers . Analysis of HapMap population for these polymorphisms showed variations among the populations. African population has showed fairly lesser frequency for all studied SNPs except rs4329 (Figure S1) (www.hapmap.org).
Correlation of ACE ID polymorphisms with serum ACE levels revealed that the DD genotype have unanimously showed higher serum ACE levels while II and ID genotypes produces low and intermediate levels of proteins respectively . But ACE ID polymorphism did not show evidence for Transcriptional regulation in vitro is further supported by mRNA expression in human heart tissue . The ACE mRNA expression in human heart tissues correlated with the rs7213516, rs7214530, and rs4290 SNPs residing in 2−3 kb upstream conserved regions of ACE gene . As the minor allele frequencies of these SNPs differed significantly between African Americans, Hispanics and Caucasians, population specific SNP scanning can then be exploited in a search for regulatory polymorphisms. However, ACE gene has been comparatively less studied and most of the studies on these variants lack in vitro evidences. Therefore, more in vitro investigations on ACE ID are anticipated before the results of various association studies can be analyzed.
Although, the direct biological mechanism by which ACE polymorphisms might influence serum ACE levels remains unclear, immunohistochemical studies observed ACE staining in many tubules and in 30% of the cysts of ARPKD kidney than the normal control kidney . In conclusion the genotyping of tagSNPS of ACE gene in south Indian patients with ADPKD did not allow us to detect any allelic, genotypic, or haplotypic associations with the ADPKD.
Conflict of interest: There are no conflicts of interests.
Acknowledgements: Dr. L. V. K. S. Bhaskar greatly thank Sri Ramachandra University providing facilities to conduct this work and Dr. S. P. Thiyagarayan, prochancellor research, Sri Ramachandra University his valuable suggestions.