CLINICAL RESEARCH www.jasn.org Polymorphisms in a-Defensin–Encoding DEFA1A3 Associate with Urinary Tract Infection Risk in Children with Vesicoureteral Reflux † ‡ Andrew L. Schwaderer,* Huanyu Wang,* SungHwan Kim, Jennifer M. Kline, Dong Liang,§ | † Pat D. Brophy, Kirk M. McHugh,¶ George C. Tseng, Vijay Saxena,* Evan Barr-Beare,* †† ‡ Keith R. Pierce,§ Nader Shaikh,** J. Robert Manak, Daniel M. Cohen, Brian Becknell,* ‡‡ || John D. Spencer,* Peter B. Baker, Chack-Yung Yu,§§ and David S. Hains§ *The Centers for Clinical and Translational Medicine and §§Molecular and Human Genetics, The Research Institute at Nationwide Children’s Hospital, Columbus, Ohio; Departments of †Biostatistics and **Pediatrics, University of Pittsburgh, Pittsburgh, Pennsylvania; ‡Emergency Medicine and ‡‡Department of Pathology, Nationwide Children’s Hospital, Columbus, Ohio; §Innate Immunity Translational Research Center, Children’s Foundation Research Institute at Le Bonheur Children’s Hospital, Memphis, Tennessee; |Division of Nephrology, Department of Pediatrics, University of Iowa Children’s Hospital, Iowa City, Iowa; ¶Division of Anatomy, The Ohio State University, Columbus, Ohio; ††Departments of Biology and Pediatrics, University of Iowa, Iowa; and ||Department of Pediatrics, University of Tennessee Health Science Center, Memphis, Tennessee ABSTRACT The contribution of genetic variation to urinary tract infection (UTI) risk in children with vesicoureteral reflux is largely unknown. The innate immune system, which includes antimicrobial peptides, such as the a-defensins, encoded by DEFA1A3, is important in preventing UTIs but has not been investigated in the vesicoureteral reflux population. We used quantitative real–time PCR to determine DEFA1A3 DNA copy numbers in 298 individuals with confirmed UTIs and vesicoureteral reflux from the Randomized Interven- tion for Children with Vesicoureteral Reflux (RIVUR) Study and 295 controls, and we correlated copy numbers with outcomes. Outcomes studied included reflux grade, UTIs during the study on placebo or antibiotics, bowel and bladder dysfunction, and renal scarring. Overall, 29% of patients and 16% of con- trols had less than or equal to five copies of DEFA1A3 (odds ratio, 2.09; 95% confidence interval, 1.40 to 3.11; P,0.001). For each additional copy of DEFA1A3, the odds of recurrent UTI in patients receiving antibiotic prophylaxis decreased by 47% when adjusting for vesicoureteral reflux grade and bowel and bladder dysfunction. In patients receiving placebo, DEFA1A3 copy number did not associate with risk of recurrent UTI. Notably, we found that DEFA1A3 is expressed in renal epithelium and not restricted to myeloid-derived cells, such as neutrophils. In conclusion, low DEFA1A3 copy number associated with recurrent UTIs in subjects in the RIVUR Study randomized to prophylactic antibiotics, providing evidence that copy number polymorphisms in an antimicrobial peptide associate with UTI risk. J Am Soc Nephrol 27: 3175–3186, 2016. doi: 10.1681/ASN.2015060700 Vesicoureteral reflux (VUR), which is present in Received June 25, 2015. Accepted January 13, 2016. – 1% 2% of children, is a risk factor for the develop- A.L.S. and H.W. contributed equally to this work. ment of urinary tract infections (UTIs).1 The Ran- Published online ahead of print. Publication date available at domized Intervention for Children with Vesicoureteral www.jasn.org. Reflux (RIVUR) Trial recently randomized children Correspondence: Dr. David S. Hains, Children’sFoundation with a history of UTI and VUR to daily antibiotic Research Institute, Innate Immunity Translational Research Center, 50 prophylaxis or placebo and followed them for 2 years North Dunlap Street, Memphis, TN, 38103. Email: [email protected] to monitor for UTI recurrence risk. Additional details Copyright © 2016 by the American Society of Nephrology J Am Soc Nephrol 27: 3175–3186, 2016 ISSN : 1046-6673/2710-3175 3175 CLINICAL RESEARCH www.jasn.org about the RIVUR Study design and outcomes have been pre- DEFA1A3 DNA copy numbers, contribute to UTI risk in pa- viously reported as well as our methods.2–4 The RIVUR Study tients with VUR. The objectives of this study included (1) showed that 25.4% of children with VUR treated with placebo comparison of DEFA1A3 CNP in children from the RIVUR experience UTI recurrence over a 2-year period, whereas anti- Trial who had a history of VUR and one or two UTIs with biotic prophylaxis reduces the UTI recurrence risk to 13%.3 healthy controls and (2) determination of the biologic rele- These recurrent UTIs can lead to renal scarring, hypertension, vance of DEFA1A3 expression in the kidney.2,3 and CKD.5,6 However, the broad application of antibiotic pro- phylaxis to all patients with VUR remains controversial, because only 30% of children with VUR will experience an initial UTI.7,8 RESULTS Therefore, strategies are needed to identify a subset of patients with VUR who will experience UTIs and benefit from antibiotic Patient Cohorts Analyzed Were Matched for Sex and prophylaxis. Ethnic/Racial Category The innate immune system is largely responsible for the DNA was obtained on 298 white girl patients in the RIVUR body’s defense against UTIs. Innate immune effectors include Trial. Two hundred ninety-five white girl controls with no antimicrobial peptides (AMPs) that rapidly destroy microbes history of UTI or VUR were enrolled between 2010 and 2014. and chemokines that result in inflammatory cell migration to The mean age of the patients in the RIVUR Trial was 22.7 the affected site and the inflammatory cells.9–11 AMPs, such as months (range =2–71) at the start of the 2-year study. defensins, have a broad range of antimicrobial activity The mean age of the control patients was 129.8 months against a wide variety of microbes, including Gram-negative (range =3.5–215). The patients in the RIVUR Trial were and -positive bacteria.12 Defensins are categorized into two matched with controls the same age or older to have equal or families, the a-defensins and the b-defensins, depending on more years to acquire a UTI. Complete clinical characteristics their disulfide-bridging pattern. b-Defensins are widely ex- of our cohorts are summarized in Table 1. pressed by epithelial cells, whereas a-defensins are expressed by neutrophils or in the case of a-defensin-5 (DEFA5), the Patients in the RIVUR Study Have Decreased DNA epithelia of the gastrointestinal tract, the genitourinary tract Copy Number of DEFA1A3 Compared with Controls in girls, and the urinary tract.13–15 We determined DEFA1A3 gene copy number per diploid ge- Neutrophil-associated DEFA1 differs from an adjacent nome using quantitative real–time PCR and verified it in a gene, DEFA3, by a single nucleotide. This gene locus has col- subset of samples using pulsed–field gel electrophoresis. Pa- lectively been renamed DEFA1A3, and it encodes the protein tients in the RIVUR Trial and matched controls were copy Human Neutrophil Peptide 1–3(HNP1–3).16 Historically, typed. The DEFA1A3 copy number in the patients in the myeloid-derived cells were the only cells thought to produce RIVUR Trial varied from three to 13, with a mean copy num- HNP1–3.17–19 HNP1–3 possesses broad spectrum antimicro- ber of 6.361.4. The DEFA1A3 copy number for controls bial activity against a range of bacteria, including Escherichia ranged between four and 18, with a mean copy number 762.0. coli, and urine levels of HNP1–3increaseeightfoldwith The distributions of copy numbers were significantly pyelonephritis versus controls.18,20,21 However, DEFA1A3/ different between the two groups (P,0.001) (Figure 1). HNP1–3 has not been extensively evaluated in the kidney. Overall, a higher percentage of patients in the RIVUR Trial Genetic variations in humans range from single-nucleotide (n=86; 29%) had five or fewer copies of DEFA1A3 compared changesto large microscopically visible chromosome defects.22 with controls (n=48; 16%; odds ratio [OR], 2.09; 95% confi- Duplications, deletions, or insertions of $1000 bp have been dence interval, 1.40 to 3.11; P,0.001). Conversely, a higher identified in humans and designated DNA copy number var- percentage of controls (n=93; 31%) had eight or more copies iations (CNVs).23 Some genes, including DEFA1A3, exist as compared with patients in the RIVUR Trial (n=55; 18%; OR, multiallelic genes, meaning that they exist in multiple copies 0.50; 95% confidence interval, 0.34–0.73); P,0.001). A similar (more than two per diploid genome). Because 70% of CNVs statistically significant difference was seen in white boys in span genes, they likely affect genetic networks. Thus, although single-nucleotide polymorphisms and point mutations occur Table 1. Clinical characteristics of subjects and controls more frequently, CNVs are more likely to be important com- Characteristics RIVUR, N=298 Control, N=295 ponents of genomic diversity and phenotypic variation by – – their ability to generate significant gene dosage effects.24 Mean age, mo (range) 22.7 (2 71) 129.8 (3.5 215) White, % 100 100 CNVs that occur in .1% of the population have been Girls, % 100 100 termed copy number polymorphisms (CNPs). CNPs may rep- Antibiotic group, n (%) 154 (52) N/A fi resent excellent candidates for disease risk modi ers. For in- Breakthrough UTI 23 (15) N/A stance, DEFA1A3 CNPs have been associated with various Placebo group, n (%) 144 (48) N/A inflammation and autoimmune diseases, including sepsis, Breakthrough UTI 47 (33) N/A Crohn disease, and Behcet disease.25–27 We hypothesize that Acquired scar, n (%) 22 (9) N/A deficiencies in the host innate immune defense, such as low N/A, not applicable. 3176 Journal of the American Society of Nephrology J Am Soc Nephrol 27: 3175–3186, 2016 www.jasn.org CLINICAL RESEARCH the RIVUR Trial compared with control white boys (Supple- group. Patients treated with antibiotic prophylaxis who expe- mental Figure 1). Thus, a lower copy number of DEFA1A3 was rienced breakthrough UTIs (5.82 copies) averaged a 0.5-copy found in patients in the RIVUR Trial (VUR and UTI).