Urinary Tract Anomalies Associated with MTHFR Gene Polymorphism C677T in Girls

Background/Aims: Periconceptional folate has a preventive effect not only on neural tube defects, but possibly also on other birth defects such as urinary tract anomalies (UTA), orofacial clefts and conotruncal heart defects. Folate metabolism gene variants are therefore being investigated as potential susceptibility factors. Methods: We assessed the methylenetetrahydrofolate reductase (MTHFR) gene C677T and A1298C genotypes in 132 UTA patients and 290 controls, also with respect to sex. Results: We found a significantly higher incidence of the T allele/TT genotype of the C677T polymorphism in UTA patients compared with controls (p = 0.019/p = 0.044). In the individual sexes, the T allele frequency in UTA girls versus control girls was 42.6 versus 21.7%, p ! 0.0001 (OR = 2.68; 95% CI: 1.63–4.40), and the frequency of TT genotypes was 19.2 versus 5.6%, p = 0.02 (OR = 4.0; 95% CI: 1.26–12.69); no difference was observed between the boys’ groups. Conclusion: The higher incidence of the C677T MTHFR gene polymorphism in girls with UTA could point to a developmental difference between the sexes that might be related to sexual dimorphism in methylation due to the Received: November 29, 2010 Accepted: June 1, 2011 Published online: August 10, 2011 Jana Behunova, MD, PhD 1st Department of Pediatrics, Children’s University Hospital University of P.J. Safarik (UPJS) , Tr. SNP 1 SK–040 66 Kosice (Slovakia) Tel. +421 55 640 4129, E-Mail jana.behunova @ upjs.sk © 2011 S. Karger AG, Basel 1420–4096/11/0346–0465$38.00/0 Accessible online at: www.karger.com/kbr 1 Given its frequent use in the text, we opted for the shorter, more practical abbreviation ‘UTA’ for the group of all congenital urinary tract anomalies, though the meaning is equivalent to ‘CAKUT’ (congenital anomalies of kidney and urinary tract), an acronym used in some of the related literature. D ow nl oa de d by : 54 .7 0. 40 .1 1 11 /7 /2 01 7 2: 11 :2 5 A M Behunova /Klimcakova /Podracka Kidney Blood Press Res 2011;34:465–471 466 being among the most significant (but also the most investigated) according to reviews/meta-analyses [15, 16] . The C677T polymorphism (rs1801133, coding position after a new renumbering c.665C 1 T, but the original position c.677C 1 T is still widely used) leads to a change in one amino acid in protein p.A222V and results in MTHFR enzyme thermolability, a reduction of its activity by up to 35–50% and higher concentrations of homocysteine in homozygotes [17, 18] . Depending on the C677T genotype, TT homozygotes require a higher intake of folate to reach the same homocysteinemia [19] . Similarly, the A1298C polymorphism (rs1801131, coding position after a new renumbering c.1286A 1 C) leads to a change in one amino acid in protein p.E429A and slightly lowers MTHFR activity in the mutant allele in homozygotes by up to 60% [20] . The MTHFR A1298C polymorphism is not associated with hyperhomocysteinemia in homozygotes, but it becomes important in combination with the C677T polymorphism; heterozygotes for both polymorphisms have higher plasmatic concentrations of homocysteine and lower MTHFR activity compared with heterozygotes for only one polymorphism [20, 21] . Despite definite evidence that folate has a preventive effect regarding some birth defects, the exact mechanism of this effect is still not fully clear. Recent findings emphasize the importance of methylation capability due to DNA methylation and gene expression regulation [22– 24] . In this context, MTHFR activity is of significant importance as it serves as a checkpoint between methylation capabilities on the one hand and thymidine or purine synthesis on the other. As it is known that gene methylation processes are considerably sex-specific – concerning either physiologic dimorphism or complex diseases [25– 27] – we can assume that it would be of interest to follow sex-related differences within various groups of nonsyndromic birth defects. Our objectives were to determine the C677T and A1298C MTHFR gene polymorphisms in patients with UTA (also with respect to sex). Subjects and Methods Cases and Controls UTA Group. This group included 132 patients: 85 boys (64.4%) and 47 girls (35.6%; boys to girls: p = 0.009, OR = 1.76; the sex ratio in our UTA group was consistent with generally reported data, with a predominance of male gender). The patients were recruited from 2006 to 2009 from the inpatient pediatric department and the outpatient nephrological clinic of a University children’s hospital, a tertiary center in Kosice, East Slovakia. The major anomalies (52%) were obstructive anomalies (hydronephrosis, megaureter), while nonobstructive kidney anomalies (unilateral agenesis, hypoplasia, non-PKD cysts) represented 25%. The remaining 23% of cases were defects of the ureter-bladder connection, mostly vesicoureteral reflux or a double ureter. The average age of the patients was 6.2 years (range: 0–19). Children with multiple birth defects or suspected monogenic disorders were not included. Population Control Group. This group consisted of 290 unselected and unrelated newborns, consecutively born within 4 months at the Faculty Hospital Kosice, Slovakia: 147 boys (50.7%) and 143 girls (49.3%). A capillary dry blood spot was taken together with newborn blood screening to minimize the trauma. The parents of both patients and control newborns signed an informed consent form, and the entire clinical study was approved by the Ethics Committee of Safarik University School of Medicine. Genotyping and Statistics Genomic DNA from patients was extracted from whole venous blood in EDTA using the Promega Wizard Genomic DNA Purification Kit (Promega Corp., Madison, Wisc., USA). DNA from control newborns was extracted from dry blood spots using the method described previously [28] . All samples were genotyped by real-time PCR for both C677T and A1298C using a LightCycler 1.5 Instrument Roche (F. Hoffmann-La Roche Ltd., Basel, Switzerland) and commercial kits from Roche and Tib Molbiol (Berlin, Germany), following the manufacturers’ instructions. Genotype frequencies in the patient and control groups were compared using Pearson’s  2 test or (if the number of samples did not fulfill its criteria) Fisher’s exact test. The OR was rated using Gart’s exact method. The statistical significance level for all tests was determined ( = 0.05). We used Arcus QuickStat Biomedical statistical software ver. 1.1 (Arcus Statistical Software, Research Solutions, Addison Wesley Longman Ltd. USA) and Statistica Cz. 6.1 (Statsoft s.r.o., Prague, Czech Republic).