|Year : 2018 | Volume
| Issue : 1 | Page : 12-16
Association between polymorphisms in genes regulating Vitamin A metabolism and kidney size in Indian Newborns
Ambili Narikot1, Varsha Chotu Pardeshi1, Annes Siji1, Arun George2, Anil Vasudevan3
1 Division of Molecular Medicine, St. John's Research Institute, Bengaluru, Karnataka, India
2 Department of Radiology, St. John's Medical College Hospital, Bengaluru, Karnataka, India
3 Division of Molecular Medicine, St. John's Research Institute; Department of Pediatric Nephrology, St. John's Medical College Hospital, Bengaluru, Karnataka, India
|Date of Web Publication||28-Jun-2018|
Department of Pediatric Nephrology, St. John's Medical College Hospital, Bengaluru, Karnataka
Source of Support: None, Conflict of Interest: None
Introduction: Previous studies suggest that maternal serum vitamin A levels and polymorphisms in genes regulating vitamin A metabolism may impact fetal kidney development. Information on these genetic variations is limited to Caucasian population. Methods: This prospective observational single center study included newborns of 349 pregnant women enrolled at 8 weeks of gestation. Kidney volumes were measured at birth by ultrasound and cord blood was tested for single nucleotide polymorphisms (SNPs) in genes encoding for aldehyde dehydrogenase 1 family member A2rs7169289, cellular retinoic acid binding protein-2rs12724719, receptor tyrosine kinasers1800860, retinol-binding proteinrs11187540, and the Vitamin A receptor stimulated by retinoic acid 6rs17852249. Results: The total (left + right) kidney volume, unadjusted and adjusted for body surface area at birth, was 21.5 ± 4.6 ml and 107.3 ± 20.7 ml/m2, respectively. Single nucleotide polymorphisms (SNPs) in genes encoding for proteins regulating vitamin A metabolism were not associated with kidney volume at birth. Conclusions: Polymorphisms in genes involved in vitamin A metabolism are not associated with kidney size in Indian newborns.
Keywords: Kidney volume, neonatal, retinoic acid
|How to cite this article:|
Narikot A, Pardeshi VC, Siji A, George A, Vasudevan A. Association between polymorphisms in genes regulating Vitamin A metabolism and kidney size in Indian Newborns. Asian J Pediatr Nephrol 2018;1:12-6
|How to cite this URL:|
Narikot A, Pardeshi VC, Siji A, George A, Vasudevan A. Association between polymorphisms in genes regulating Vitamin A metabolism and kidney size in Indian Newborns. Asian J Pediatr Nephrol [serial online] 2018 [cited 2018 Jul 21];1:12-6. Available from: http://www.ajpn-online.org/text.asp?2018/1/1/12/235480
| Introduction|| |
Brenner et al. proposed that individuals born with low nephron number may suffer from essential hypertension and chronic kidney disease later in life. Studies in rats and sheep show that an experimentally induced loss of a critical nephron mass during fetal development or shortly after birth favors the development of hypertension and kidney damage. Various factors such as low birth weight, maternal nutrition, fetal growth environment and prematurity may affect the development of the kidney. Among these factors, there is strong evidence to suggest the involvement of vitamin A in nephrogenesis, and adequate supply of all-trans retinoic acid (ATRA), active metabolite of vitamin A, is crucial in determining final nephron numbers.,,,, Studies in mice show that a mild vitamin A deficiency may cause 20% reduction of nephron number. Apart from nutritional deficiency of vitamin A, perturbations in metabolic pathways regulating intracellular ATRA level may affect renal development. Inactivation of genes in the ATRA pathway causes renal agenesis in mice.
Recent data indicate that variants in genes involved in vitamin A metabolism such as aldehyde dehydrogenase 1 family member A2 (ALDH1A2) and genes for cytochromes P450 metabolizing retinoic acid such as CYP26A1 and CYP26B1, cellular retinoic acid binding protein-2 (CRABP-II or CRABP2), receptor tyrosine kinase (RET), vitamin A receptor stimulated by retinoic acid 6 (STRA6), and retinol-binding protein (RBP) may be associated with kidney size among normal newborns.,, However, these studies were chiefly conducted in Caucasian populations where the prevalence of maternal vitamin A deficiency is low. Our previous study suggested that maternal vitamin A deficiency is widely prevalent in Indian mothers throughout pregnancy. The implications of polymorphisms in genes regulating vitamin A metabolism in the predominantly vitamin A deficient Indian population remain unknown. The aim of the present study was to investigate the association of common functional single nucleotide polymorphisms (SNPs) in genes involved in vitamin A metabolism (ALDH1A2, RET, CRAPB-II or CRABP2, RBP, and STRA6) with newborn kidney size in Indian population.
| Methods|| |
The study was a prospective observational cohort study approved by Institute's Ethical Board Committee. Pregnant women aged 18–40 years, registered for antenatal screening in the first trimester at the Department of Obstetrics and Gynecology in the hospital, were recruited for the study and followed until delivery. Women with fetal renal anomalies, multiple fetuses, known chronic illness such as diabetes mellitus, hypertension, heart disease or thyroid disease, infection with hepatitis B, human immunodeficiency virus or syphilis, and those who anticipated moving out of the city before delivery, were excluded from the study. Each participant received routine antenatal care along with antenatal supplements of folic acid, iron, and calcium as per the schedule. Informed consent was obtained from each study subject at enrollment. Only mother–baby dyads with complete information were included. The fetal kidney size and volume were measured in the third trimester. After birth, renal scan was performed by one of the two radiologists between 24 and 48 h. All data were collected by trained research assistants.
Sociodemographic, anthropometric, and dietary information
Sociodemographic details and obstetric history were collected at baseline by trained research assistants. Information on maternal anthropometry, dietary intake, clinical status, and routine antenatal blood biochemistry was collected at baseline (11.7 ± 2.2 weeks), second trimester (24.4 ± 1.5 weeks), and third trimester (34.4 ± 1.4 weeks) of pregnancy. A digital balance (Soehnle, Germany) was used to record the weights of all mothers to the nearest 100 g during each antenatal visit. Height was measured with a stadiometer to the nearest 0.1 cm.
Delivery and birth information
At birth, newborn's anthropometric measurements such as baby weight (to the nearest g), length, head circumference and skinfold thickness (to the nearest 0.1 cm) were taken, and body surface area (BSA) was estimated.
The right and left kidney volume of the newborns was measured using renal ultrasonography within the first 48 h of birth by experienced radiologists using a 5 MHz probe. Each measurement was made three times and averaged to minimize measurement error. Renal volume was calculated using an equation of an ellipsoid: length × transverse diameter × anterior-posterior diameter × 0.523 (cm 3 or ml). The combined or total renal volumes were obtained by adding the left and right renal volumes. The corrected kidney volume was obtained by dividing the combined kidney volume by the BSA.
SNP selection and genotyping
At birth, cord blood was obtained and isolation of genomic DNA carried out using the phenol-chloroform method. For genetic screening, SNP in ALDH1A2rs7169289, CRABP2rs12724719, RETrs1800860, STRA6rs17852249 and RBPrs11187540, identified to be associated with newborn kidney size were selected.,,, Primers were designed for Sanger sequencing of each SNP using Primer3plus software (http://primer3plus.com/web_3.0.0/primer3web_input.htm; details available on request). Genotyping was performed by polymerase chain reaction using following conditions: initial denaturation at 95°C for 5 min, followed by 35 denaturation cycles at 94°C for 30 s (60 s for ALDH1A2), annealing at 55°C–60°C for 45 s (60 s for ALDH1A2), extension at 72°C for 45 s (60 s for ALDH1A2), and a final extension at 72°C for 10 min. Bidirectional sequencing of the amplified products was performed using big dye terminator v3.1 cycle sequencing kit (Thermo Fisher Scientific, Life Technologies Corporation, Austin, TX, USA) and sequence analyzed on an ABI 3730xl genetic analyzer (Applied Biosystems, Foster City, CA, USA; Eurofins Genomics India Pvt. Ltd., Bengaluru, Karnataka, India). Sequences were evaluated for variants using the Finch TV software (Geospiza version 1.4.0, PerkinElmer Inc., Waltham, MA, USA), and NCBI BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi).
Continuous data are presented as mean ± standard deviation and categorical data as number (%). The data were analyzed using R-software (https://www.r-project.org; version 3.2.3). Genotype frequencies of each SNP were examined for divergence from Hardy–Weinberg equilibrium using Chi-square test. One-way analysis of variance was used to examine association between genotypes and kidney volume normalized to BSA.
| Results|| |
Characteristics of the study cohort
The average age of 356 women, enrolled at 11.7 ± 2.2 weeks of gestation, was 24.3 ± 3.9 years, and 57% of the cohort was primiparous. The average weight, height and body mass index of women were 52.0 ± 9.3 kg, 155.8 ± 5.4 cm, and 21.4 ± 3.6 m 2, respectively. Of the 356 babies enrolled, 48% were female. The gestational age at birth was 38.6 ± 1.2 weeks. The baseline characteristics of the cohort are listed in [Table 1]. The unadjusted left and right kidney volumes, available for 349 babies [Table 1], were used to compute total (left + right) kidney volume unadjusted and adjusted for BSA [Table 1]. A four-fold variation in renal volume persisted despite this adjustment.
Genotyping of ALDH1A2rs7169289, RETrs1800860, and CRAB2rs12724719 SNPs and the association of genotype with newborn kidney volume
The newborns were genotyped for one SNP in each of four genes. Out of 356 newborns, 309, 302, and 284 babies were genotyped to characterize the frequency of ALDH1A2rs7169289, RETrs180086, CRABP2rs12724719, and RBPrs11187540 loci [Figure 1]. Initial screening of 131 newborns for the STRArs17852249 SNP showed that the locus is monomorphic and hence was not screened in remaining newborns. Similarly, out of the 98 subjects screened for RBPrs11187540 SNP, all except one had the common allele. The frequencies of favorable alleles (associated with increase in kidney volume in Caucasian population); ALDH1A2rs7169289 (G) and CRABP2rs12724719 (A) were 31% and 15%, respectively, while the frequency of the unfavorable allele (associated with decrease in kidney volume in Caucasian population) RETrs1800860 (A) was 22%.
|Figure 1: Study flow The last row indicates the number of samples with complete information on anthropometry and renal volume with the member tested for polymorphisms in each of the five genes (shown in parenthesis)|
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The genotype frequencies of ALDH1A2rs7169289 were 8.1% (GG), AG (45%), and AA (46%) in Indian newborn [Table 2]. Total renal volume/BSA among the homozygous ALDH1A2rs7169289 G/G infants was similar to subjects who were homozygous for ALDH1A2rs7169289 A/A and heterozygous for ALDH1A2rs7169289 A/G [Table 2] and [Figure 2]a. No significant association was observed between genotypes and kidney volume (P = 0.35). The RETrs1800860 genotypes GG (61%) and AG (33.7%) were more common, while the AA (5.3%) genotype frequency was low in Indian newborn [Table 2] and [Figure 2]b. Total renal volume/BSA among the homozygous RETrs1800860 A/A infants was similar to that of newborns who were homozygous for the more common RETrs1800860 G/G genotype. Total renal volume/BSA of heterozygous newborns was 4.7% higher than the RETrs1800860 G/G genotype subjects. As observed with ALDH1A2 SNP, there was no significant association between RET genotypes and kidney volume (P = 0.73). The CRABP2rs12724719 genotypes were GG (74%) and AG (22%) were more common, while AA (4%) genotype was less common [Table 2] and [Figure 2]c. Total renal volume/BSA among the more common homozygous CRABP2rs12724719 A/A infants was higher than that of newborns who were homozygous for the CRABP2rs12724719 G/G genotype [Table 2]. Total renal volume/BSA in newborns with heterozygous allele (22% of the cohort) was similar to the CRABP2rs12724719 G/G genotype subjects. The kidney volume was not significantly different between the genotypes (P = 0.35).
|Table 2: Genotype and allele frequencies for genes associated with vitamin A metabolism tested in newborn cohort, in relation to total kidney volume adjusted for body surface area (KV/BSA), presented as mean±SD, ml/m2|
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|Figure 2: The association of (a) Aldehyde dehydrogenase 1 family, member A2 (ALDH1A2), (b) receptor tyrosine kinase (RET), and cellular retinoic acid binding protein-2 (CRAB p 2) variants with newborn renal volume. (a) ALDH1A2 rs7169289 (G) variant, (b) RET rs1800860(A) variant, and (c) CRABP2rs12724719(A) variant did not show any significant correlation with newborn kidney volume when adjusted with body surface area KV/BSA, ml/m2|
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| Discussion|| |
The complex interaction between metanephric blastema and the ureteric bud plays an important role in nephrogenesis and in determining the final nephron endowment in humans. There was fourfold variation in kidney volume even after correcting for BSA in this study, possibly reflecting the wide variation in nephron numbers (0.4–1 million) observed in healthy subjects. The mean kidney size among healthy Indian newborns reported in this study is significantly lesser than in newborns from developed countries (114 ± 33 ml/m 2 versus. 184 ± 44 ml/m 2). Studies indicate that paired bo x2 and RET genes regulate nephron number at birth by influencing the branching morphogenesis, and mutations in these genes lead to hypoplasia or agenesis of the kidney.,, Since vitamin A is an important modulator of nephrogenesis, variation in genes of ATRA metabolism might also influence nephrogenesis. El Kares et al. identified an SNP ALDH1A2 variant, rs7169289 (G), that is associated with 22% increase in mean newborn total kidney volume/BSA. The same group of investigators also identified an SNP in the gene regulating the tyrosine kinase receptor, RET, that is associated with 10% decrease in newborn kidney volume. Results from this prospective cohort study show that the frequency of the favorable genotypes (ALDH1A2 [G] [31%] and CRABP2 [G] [78%]) was high in Indian population compared to the Caucasian population (ALDH1A2 [G] [20%]) and European population (CRABP2 [G] [74%]., Newborns with favorable ALDH1A2 variant showed the expected direction, i.e., increase in kidney volume; however, the increase was statistically not significant compared to subjects carrying more common variant. These results indicate that there is no association of ALDH1A2 and CRABP2 gene variants and kidney volume [Table 2]. The higher frequency of favorable alleles in Indian population indicates that evolutionary processes may have contributed for the favorable alleles in Indian population to compensate for the maternal vitamin A deficiency. The frequency of unfavorable RETrs1800860 (A) allele was 22% in our cohort which was similar to Canadian population (25%). However, in Indian newborns, this allele did not show any effect [Table 2]. It is likely that other gene variants are compensating for the dysfunctional RET variant in Indian population. It is also possible that yet unknown genetic variants are possibly influencing the kidney volume in Indian population. Surprisingly, heterozygous carriers showed 4.7% increase in kidney volume compared to subjects who were homozygous for the wild-type allele (RETrs1800860[G]). The reason for this observation is not clear.
Although the kidney volume observed in our cohort is lesser than that reported in other studies, the common polymorphic variants of genes in vitamin A pathway that regulate renal development did not explain the difference in a large cohort of babies who were genotyped., The effect size of change in kidney volume due to the presence of favorable or unfavorable alleles in ALDH1A2 and RET genes in Caucasian newborns is small (10–20% change in kidney volume). It is possible that widely prevalent subclinical maternal vitamin A deficiency may have masked the effect of the polymorphic variants on the kidney size. It is also be possible that smaller kidney volume observed in the study may be due to the presence of yet to be identified genetic or environmental factors.
| Conclusions|| |
Observations from the prospective study suggest that common variants of the ALDH1A2, CRABP2, and RET genes do not affect the kidney volume in Indian newborns. Future research involving larger sample sizes, diverse ethnic background and genome-wide screening is essential to identify the genetic determinants that affect kidney volume in Indian newborns.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
The study was supported by a grant from the Department of Biotechnology, Division of Science and Technology, Government of India (BT/PR15353/MED/12/506/2011).
| References|| |
Brenner BM, Garcia DL, Anderson S. Glomeruli and blood pressure. Less of one, more the other? Am J Hypertens 1988;1:335-47.
Sammut S, Behr L, Hekmati M, Gubler MC, Laborde K, Lelièvre Pégorier M, et al.
Compensatory renal growth after unilateral or subtotal nephrectomy in the ovine fetus. Pediatr Res 2013;74:624-32.
Luyckx VA, Bertram JF, Brenner BM, Fall C, Hoy WE, Ozanne SE, et al.
Effect of fetal and child health on kidney development and long-term risk of hypertension and kidney disease. Lancet 2013;382:273-83.
Clark AT, Bertram JF. Molecular regulation of nephron endowment. Am J Physiol 1999;276:F485-97.
Serluca FC, Fishman MC. Pre-pattern in the pronephric kidney field of zebrafish. Development 2001;128:2233-41.
Cartry J, Nichane M, Ribes V, Colas A, Riou JF, Pieler T, et al.
Retinoic acid signalling is required for specification of pronephric cell fate. Dev Biol 2006;299:35-51.
Mendelsohn C, Lohnes D, Décimo D, Lufkin T, LeMeur M, Chambon P, et al.
Function of the retinoic acid receptors (RARs) during development (II). Multiple abnormalities at various stages of organogenesis in RAR double mutants. Development 1994;120:2749-71.
Rosselot C, Spraggon L, Chia I, Batourina E, Riccio P, Lu B, et al.
Non-cell-autonomous retinoid signaling is crucial for renal development. Development 2010;137:283-92.
Lelièvre-Pégorier M, Vilar J, Ferrier ML, Moreau E, Freund N, Gilbert T, et al.
Mild vitamin A deficiency leads to inborn nephron deficit in the rat. Kidney Int 1998;54:1455-62.
Zhang Z, Quinlan J, Hoy W, Hughson MD, Lemire M, Hudson T, et al.
Acommon RET variant is associated with reduced newborn kidney size and function. J Am Soc Nephrol 2008;19:2027-34.
El Kares R, Manolescu DC, Lakhal-Chaieb L, Montpetit A, Zhang Z, Bhat PV, et al.
Ahuman ALDH1A2 gene variant is associated with increased newborn kidney size and serum retinoic acid. Kidney Int 2010;78:96-102.
Manolescu DC, El-Kares R, Lakhal-Chaieb L, Montpetit A, Bhat PV, Goodyer P, et al.
Newborn serum retinoic acid level is associated with variants of genes in the retinol metabolism pathway. Pediatr Res 2010;67:598-602.
Goodyer P, Kurpad A, Rekha S, Muthayya S, Dwarkanath P, Iyengar A, et al.
Effects of maternal vitamin A status on kidney development: A pilot study. Pediatr Nephrol 2007;22:209-14.
Mosteller RD. Simplified calculation of body-surface area. N
Engl J Med 1987;317:1098.
Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 1988;16:1215.
Amengual J, Zhang N, Kemerer M, Maeda T, Palczewski K, Von Lintig J, et al.
STRA6 is critical for cellular vitamin A uptake and homeostasis. Hum Mol Genet 2014;23:5402-17.
Shah MM, Sampogna RV, Sakurai H, Bush KT, Nigam SK. Branching morphogenesis and kidney disease. Development 2004;131:1449-62.
[Figure 1], [Figure 2]
[Table 1], [Table 2]