|Year : 2022 | Volume
| Issue : 6 | Page : 741-746
Early evidence on genetic polymorphisms in conferring a “Two-Hit” propensity to renal injury in Asian Indian children
Suramya Anand1, Minu Bajpai1, Alok Kumar1, Siddharth Kapahtia2
1 Department of Paediatric Surgery, AIIMS, New Delhi, India
2 Division of Noncommunicable Diseases, Indian Council of Medical Research, New Delhi, India
|Date of Submission||14-Jun-2022|
|Date of Decision||02-Jul-2022|
|Date of Acceptance||02-Jul-2022|
|Date of Web Publication||11-Nov-2022|
Department of Paediatric Surgery, AIIMS, New Delhi
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Congenital anomalies of the kidney and urinary tract (CAKUT) are a common cause of end-stage renal disease in children. While certain nephrogenic genes have been incriminated in these malformations, data to identify the frequency of gene polymorphisms in Asian Indian children with CAKUT are scarce. This study was done to identify the effect of polymorphisms in paired-box gene 2 (PAX2), bone morphogenetic protein (BMP)-4, angiotensin-converting enzyme (ACE), and angiotensin II receptor Type 2 (AGTR2) nephrogenic genes on the development of CAKUT.
Materials and Methods: In this prospective cohort study, 158 children <12 years old (86 cases with CAKUT and 72 age-matched controls) were analyzed. DNA from both sets was extracted from peripheral blood using the Keygen DNA extraction kit, and single-nucleotide gene polymorphisms (SNPs) in PAX2, BMP-4, ACE, and AGTR2 nephrogenic genes were detected by polymerase chain reaction (PCR) using previously published primers and PCR conditions.
Results: The presence of A allele SNP for AGTR2 gene at rs3736556 was found to be significantly correlated with the development of ureteropelvic junction obstruction and vesicoureteral reflux (VUR) with the TT allelic genotype having a lower incidence of pelviureteric junction obstruction (odds ratio [OR] 0.18 [95% confidence interval [CI], 0.06–0.55], P = 0.01) and VUR (OR 0.31 [95% CI, 0.11–0.91], P = 0.03). Furthermore, on substratification of the patients with the presence of the A allele of AGTR2, 24 out of 27 patients with scarring were found to harbor the D allele of the ACE gene, thus predisposing them to further renal damage.
Conclusion: This study points to early evidence in the implication of nephrogenic genes in development as well as predisposition to renal injury in Asian Indian patients with CAKUT.
Keywords: Angiotensin II receptor type 2 gene, congenital anomalies of the kidney and urinary tract, nephrogenic genes
|How to cite this article:|
Anand S, Bajpai M, Kumar A, Kapahtia S. Early evidence on genetic polymorphisms in conferring a “Two-Hit” propensity to renal injury in Asian Indian children. J Indian Assoc Pediatr Surg 2022;27:741-6
|How to cite this URL:|
Anand S, Bajpai M, Kumar A, Kapahtia S. Early evidence on genetic polymorphisms in conferring a “Two-Hit” propensity to renal injury in Asian Indian children. J Indian Assoc Pediatr Surg [serial online] 2022 [cited 2022 Dec 7];27:741-6. Available from: https://www.jiaps.com/text.asp?2022/27/6/741/360974
| Introduction|| |
Congenital anomalies of the kidney and urinary tract (CAKUT) are considered a major cause of chronic renal failure in young children and infants. This disease constellation constitutes approximately 20%–30% of all anomalies identified in the prenatal period and has a reported incidence of 0.3–1.6/1000 live and stillborn infants. Because CAKUTs play a causative role in 30%–50% of cases of end-stage renal disease (ESRD) in children, it is vital to ensure early diagnosis and initiation of therapy to minimize renal parenchymal damage and potentially delay the onset of ESRD. CAKUT has also been hypothesized to have a common underlying genetic cause., It has also been reported that those children with CAKUT and polymorphism in nephrogenic genes have a propensity to develop type II diabetes and metabolic syndromes later in life.
The aim was to study the prevalence of Bone morphogenetic protein 4 (BMP-4), Paired Box gene 2 (PAX2), Angiotensin-converting enzyme (ACE), and Angiotensin II Receptor Type 2 (AGTR2) gene polymorphism in Indian children with congenital uropathies and their association with renal outcomes. The presence of both an allele predisposing to the development of a certain urological anomaly and also an allele conferring renal damage, as shown by scarring, could be useful as a pointer to the patients who are in need of a closer follow-up right from the inception of the disease.
Data from genetic studies to identify the frequency of gene polymorphisms in Indian children with CAKUT are scarce, and this study was done to identify gene polymorphisms as risk factors for renal parenchymal damage in patients with congenital urological abnormalities.
| Materials and Methods|| |
In this prospective cohort study, medical records of 86 children <12 years old with congenital uropathies who presented to the pediatric urology clinic at our institute were reviewed. Patients not willing for follow-up, syndromic children with other congenital anomalies, and those who refused consent were excluded from the study.
A standardized data collection form developed for reviewing records was used. Patient history and investigations, including serum creatinine, renal ultrasound, dimercaptosuccinic acid (DMSA) radionuclide scan, and renal dynamic scan were recorded. The study was done according to the ethical committee guidelines, informed consent was obtained from participants' legal guardians, and assent was also taken from children. The patients were telephonically contacted and asked to make a targeted visit, and blood samples for these patients were obtained. Blood samples from sex- and age-matched children from the same geographical area with no urological complaints who presented to our clinic were also obtained as controls. All controls were normotensive and had serum creatinine within the normal range at the time of blood sampling. Family history of renal disease, urinary tract malformation, or urinary tract infection was ruled out.
After informed consent, all cases and controls were submitted to an intravenous puncture to collect 10 mL of peripheral blood samples. DNA was extracted from peripheral blood using the Keygen DNA extraction kit. The amplification-refractory mutation system (ARMS) polymerase chain reaction (PCR) primers to detect point mutations, as described in the table, were used [Table 1].
The primer sequences were 5'CTG AGA CCA CTC CCA TC 3' and 5'GAT GTG GCC ATC ACA TTC GTC AGA T 3' for ACE polymorphism (ACE gene I/D polymorphism is characterized by the presence or absence of a 287 bp Alu repeat sequence within intron 16). Amplification with this primer pair produces 490 bp and 190 bp products corresponding to the I allele and D allele, respectively.
Normal and mutant ARMS reaction premixes were prepared according to previously published ARMS PCR guidelines, and 40 μL aliquots of the premix were dispensed into reaction tubes suitable for use in a thermal cycler. After adding Taq DNA polymerase, a conventional PCR device was used according to the following protocol: samples were exposed to 94°C for 2 min, then 35 cycles at 94°C for 15 s, 59°C for 10 s, and 72°C for 30 s. Case and control samples were randomly arranged in well plates with at least 20% of genotypes retyped as quality control. The PCR products were run on a 2% agarose gel containing ethidium bromide and visualized by ultraviolet illumination.
Statistical comparisons of genotype distributions and allelic frequencies were compared between groups by the Chi-square test. The odds ratios (OR) with the 95% confidence interval (CI) were calculated by comparing the allelic distributions in the study groups. A P < 0.05 was considered statistically significant. Data analysis was carried out using Stata 12.0 (College Station, Texas, USA).
| Results|| |
The sample for analysis consisted of 158 patients, 86 cases and 72 controls without the urological disease. Within the CAKUT group, there were 64 (74.4%) males and 22 (25.6%) females with a median age of 32 months. Demographic characteristics of patients within the CAKUT group, as well as the distribution of patients by their presenting uropathy, are displayed in [Table 2].
|Table 2: Demographic characteristics of patients with congenital anomalies of the kidney and urinary tract|
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For the purpose of this study, the following genes and their alleles were analyzed: BMP4 (GG, GA, AA); PAX2 (GG, GT, TT); AGTR2 (AA, AT, TT), and ACE (II, ID, DD). Initial analysis showed that the expression of these genes did not signify an increased risk for the development of CAKUT in the patient group compared to the control group [Table 3].
|Table 3: Study gene genotypes and analysis with congenital anomalies of the kidney and urinary tract cases versus controls|
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On further analysis, when polymorphisms of the AGTR2 gene were evaluated based on their presenting phenotype (pelviureteric junction obstruction [PUJO], vesicoureteral reflux [VUR], PUV, etc.), children expressing the TT allele genotype of AGTR2 gene were seen to have a lower incidence of PUJO (OR 0.18 [95% CI, 0.06–0.55], P = 0.01) as well as VUR (OR 0.31 [95% CI, 0.11–0.91), P = 0.03) [Table 4].
|Table 4: Angiotensin II receptor type 2 genotype analysis and associations with different variants of congenital anomalies of the kidney and urinary tract patients versus controls|
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Polymorphisms for the genes, i.e., BMP-4, AGTR2, and PAX2, were not found to be associated with an increased incidence of renal scarring in the CAKUT group. The D allele of the ACE I/D gene was present in 25 out of 30 of the patients who developed scarring. Thus, the presence of D allele was found to be significantly associated with the development of renal scarring.
Since expression of the D allele of the ACE gene was seen to increase the propensity of renal parenchymal damage, we decided to perform subgroup analysis of patients with CAKUT expressing the A allele of the AGTR2 gene. This analysis revealed a significant association with expression of the D allele of ACE in the subgroup who demonstrated renal parenchymal scarring (24/27 patients with renal scarring, 36/49 patients without renal scarring-[Table 5]).
|Table 5: Sub-stratification of all patients with the presence of into A allele in angiotensin II receptor type 2 (76), i.e., all patients with proposed increased predisposition to development of congenital anomalies of the kidney and urinary tract and presence or absence of angiotensin-converting enzyme insertion/deletion polymorphism|
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| Discussion|| |
CAKUT is one of the common causes of end-stage renal disease in children, leading to considerable morbidity and mortality. In our study, of the 86 children presenting to the outpatient clinic and diagnosed with CAKUT, PUV was the most commonly witnessed pathology, with an incidence of 30 (34.8%) patients. Male children (74.4%) had a significantly higher incidence of CAKUT than females (25.6%). Evidence of the male gender is an important predisposing factor in the development of CAKUTs has been documented in previous studies. Li et al. found that male neonates were more likely to present with CAKUTs than female neonates. Other studies have shown that male children are between 1.3 and 1.9 times more likely to be affected by CAKUT than female children.
The genetic polymorphisms chosen for this study have been shown to be associated with certain CAKUT pathologies in previous studies. It is well known that the BMP4 family takes part in the ureteric bud development., Miyazaki et al. demonstrated that mice with reduced expression of BMP4 demonstrated three different types of malformations – hydronephrosis with hypo/dysplastic kidneys, ureterovesical junction obstruction, and duplex kidney with the bifid ureter, which led to the hypothesis that BMP4 is a fine-tuning protein that modulates the number of functional nephrons and ureteric branching. In our study, the presence of BMP4 polymorphisms was not seen to correlate with an increased risk of developing CAKUT, which is in contrast to a previous study comprising a sample of Brazilian patients conducted in 2014, wherein Reis et al. found the presence of allele A at rs17563 to be a risk factor for anomalies of CAKUT. Reis et al. found that when the AA genotype for BMP-4 was present, the risk of developing congenital uropathies was 2.49 times higher than when the GG genotype was present.
PAX2 polymorphism is associated with reduced kidney size in neonates. In this study, PAX2 polymorphisms of the alleles studied rs4244351 (G/T alleles) did not show a significant association with the development of CAKUT. This is in contrast to de Miranda et al., who studied PAX2 polymorphisms in 241 cases versus 259 controls in a Brazilian pediatric population. The authors evaluated associations with five single-nucleotide polymorphisms covering the entire PAX2 gene. In the subgroup of patients with VUR, frequencies of monozygotic ancestral alleles significantly differed at the markers rs11190693 (A/T alleles) and rs4244351 (G/T alleles) in comparison with controls, whereas no changes were detected in cases of PUJO or MCDK.
ACE insertion/deletion (ACE I/D) gene polymorphism is a risk factor for renal parenchymal damage in patients with congenital urologic abnormalities and seems particularly relevant in children with VUR, where it is an independent predisposing factor. Previous studies have indicated that the ACE I/D polymorphisms may be associated with diabetic nephropathy, IgA nephropathy, nephritic syndrome, or focal segmental glomerulosclerosis.,,,,, The serum ACE level has been found to vary with I versus D polymorphism of the ACE gene. It has been reported that the DD genotype of this polymorphism is associated with higher levels of plasma and tissue ACE activity, as described by Rigat et al.
In our study, the analysis showed that expression of ACE gene polymorphisms did not signify an increased risk for the development of CAKUT in the patient group as compared to the control group though the presence of the D polymorphism (DD or ID allele) was seen to be associated with a significantly increased incidence of renal parenchymal damage, as assessed by renal scarring (on DMSA scan) in children with CAKUT. This agrees with the previous study conducted in Italian children by Rigoli et al., who compared the incidence of the ACE I/D polymorphism in 102 children with CAKUT, leading to varying degrees of renal impairment and in 92 healthy controls with a similar ethnic background and found that ACE genotype distribution did not differ between the CAKUT patients and normal control subjects. The findings of our study are also in keeping with the findings of a previous study by Bajpai et al. who evaluated the role of ACE I/D polymorphism as a risk factor for progressive renal damage in Indian children with congenital uropathies.
Studies of the angiotensin II receptor 2 gene (AGTR2) in patients with CAKUT have historically shown controversial results. Nishimura et al. observed an increased A–G transition in the AT2R gene in male Caucasian American and German patients with multicystic dysplastic kidney (MCDK) and PUJO, while Hiraoka et al. reported that there is no evidence for this AT2R gene derangement in human urinary tract anomalies in Japanese patients.
In our study, the presence of the A allele at rs3736556 of the AGTR2 gene was significantly correlated with the development of PUJO and VUR but not PUV. Our study has a few findings similar to that by Miranda et al., where the authors studied 290 pediatric patients with CAKUT and 262 healthy controls from the same geographic area for AGTR2 gene polymorphisms at rs1403543, rs3736556, rs35474657, rs5193, and rs5194 and found that the diagnosis of PUJO was significantly associated with AGTR2 gene polymorphisms at rs3736556 and rs5194. On the other hand, the diagnoses of VUR and of multicystic dysplastic kidney were not associated with AGTR2 gene polymorphisms.
On further stratification of the patients of CAKUT who expressed the A allele (AGTR2), a significant correlation was found between the expression of the D allele (ACE I/D) and renal parenchymal damage. Although limited by the sample size in this study, further studies confirming this trend would be beneficial in establishing the association between multiple mutations and worsening renal injury. This would, in turn, assist in closer follow-up of children with the presence of at least one polymorphism, as inheriting additional mutations or “second hits” would confer them with increased propensity to renal injury. Besides close clinical monitoring of high-risk children, this information would aid in genetic counseling.
Although being a pilot study in Asian Indian children, the primary limitation of our study is the small sample size rendering definitive conclusions regarding the general population difficult. Further, adequately powered studies are needed to explore the association between gene polymorphisms and the development of congenital uropathies in this patient population.
| Conclusion|| |
In our study, a significant association was found between the presence of the A allele of the AGTR2 gene at rs3736556 with the development of PUJO and VUR. Furthermore, these children were more likely to develop renal scarring if they were found to harbor the D allele (ACE I/D polymorphism) as well. As congenital uropathies are quite common and also known to lead to end-stage renal disease, early identification of children at risk and also segregation of them into those who would need closer follow-up due to additional genetic polymorphisms could lead to significant improvement in their management.
The authors are grateful to Prof. Balram Bhargava-Secretary (Director General-Indian Council of Medical Research (ICMR)) and Dr. R S Dhaliwal (Scientist 'G at ICMR) for providing support for this project. The authors are thankful to ICMR for funding the project.
Financial support and sponsorship
Indian Council of Medical Research.
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]