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Exome Sequencing Discerns Syndromes in Patients from Consanguineous Families with Congenital Anomalies of the Kidneys and Urinary Tract

† ‡ Asaf Vivante,* Daw-Yang Hwang,* Stefan Kohl,*§ Jing Chen,* Shirlee Shril,* Julian Schulz,* | Amelie van der Ven,* Ghaleb Daouk,* Neveen A. Soliman, ¶ Aravind Selvin Kumar,** †† ‡‡ Prabha Senguttuvan,** Elijah O. Kehinde, Velibor Tasic, and Friedhelm Hildebrandt*§§

*Department of Medicine, Boston Children’s Hospital, Harvard Medical School, Boston, Massachusetts; †Talpiot Medical Leadership Program, Sheba Medical Center, Tel-Hashomer, Israel; ‡Division of Nephrology, Department of Medicine, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan; §Department of Pediatrics, Cologne Children’s Hospital, Cologne, Germany; |Department of Pediatrics, Kasr Al Ainy School of Medicine, Cairo University, Cairo, Egypt; ¶Egyptian Group for Orphan Renal Diseases, Cairo, Egypt; **Pediatric Nephrology Department, Institute of Child Health and Hospital for Children, Chennai, Tamil Nadu, India; ††Division of Urology, Department of Surgery, Kuwait University, Safat, Kuwait; ‡‡Medical Faculty Skopje, University Children’s Hospital, Skopje, Macedonia; and §§Howard Hughes Medical Institute, Chevy Chase, Maryland

ABSTRACT Congenital anomalies of the kidneys and urinary tract (CAKUT) are the leading renal or urinary anomalies as one of cause of CKD in children, featuring a broad variety of malformations. A monogenic their features.4 Recently, it was shown cause can be detected in around 12% of patients. However, the morphologic clinical that a monogenic cause can be detected phenotype of CAKUT frequently does not indicate specific to be examined. in approximately 12% of patients.2,5–8 To determine the likelihood of detecting causative recessive mutations by whole- The documented mode of inheritance exome sequencing (WES), we analyzed individuals with CAKUT from 33 different in most published pedigrees is compati- consanguineous families. Using homozygosity mapping and WES, we identified the ble with an autosomal dominant inher- causative mutations in nine of the 33 families studied (27%). We detected recessive itance with variable expressivity and mutations in nine known disease–causing genes: ZBTB24, WFS1, HPSE2, ATRX, incomplete penetrance.2 However, sev- ASPH, AGXT, AQP2, CTNS,andPKHD1. Notably, when mutated, these genes cause eral CAKUT–causing genes with autoso- multiorgan syndromes that may include CAKUT as a feature (syndromic CAKUT) or mal recessive mode of inheritance have cause renal diseases that may manifest as phenocopies of CAKUT. None of the been recently identified.5,9,10 Neverthe- above monogenic disease–causing genes were suspected on clinical grounds be- less, in the clinical practice, the genetic fore this study. Follow-up clinical characterization of those patients allowed us to basis of most patients with CAKUT revise and detect relevant new clinical features in a more appropriate pathogenetic is elusive because of a very weak genotype- context. Thus, applying WES to the diagnostic approach in CAKUT provides oppor- phenotype correlation. To determine the tunities for an accurate and early etiology–based diagnosis and improved clinical likelihood of detecting causative recessive management. mutations in CAKUT by whole-exome

J Am Soc Nephrol 28: 69–75, 2017. doi: 10.1681/ASN.2015080962

Received August 28, 2015. Accepted March 16, 2016.

Congenital anomalies of the kidneys and obstruction, , duplex, Published online ahead of print. Publication date urinary tract (CAKUT) are the leading vesicoureteral reflux (VUR), and poste- available at www.jasn.org. 2,3 cause of CKD in children, accounting for rior urethral valves. CAKUT may Correspondence: Prof. Friedhelm Hildebrandt, .40%ofpatients.1 CAKUT features a appear as an isolated feature or part of Division of Nephrology, Department of Medicine, ’ broad variety of structural malforma- systemic conditions that encompass Boston Children s Hospital, 300 Longwood Ave- nue, Boston, MA 02115. Email: friedhelm.hilde- tions, such as , renal hypo- extrarenal manifestations2,3; .200 re- [email protected] dysplasia, multicystic dysplastic , cessive and dominant monogenic syn- Copyright © 2016 by the American Society of hydronephrosis, ureteropelvic junction dromes have been described involving Nephrology

J Am Soc Nephrol 28: 69–75, 2017 ISSN : 1046-6673/2801-69 69 BRIEF COMMUNICATION www.jasn.org sequencing (WES), we have analyzed in- After homozygosity mapping and features pleiotropic clinical presenta- dividuals with CAKUT from 33 consan- WES, we detected recessive mutations tions, which include immunodeficiency guineous families. We used homozygosity in nine known disease–causing genes: of variable extent, developmental mapping combined with WES11 to detect WFS1, ZBTB24, HPSE2, ATRX, ASPH, delay, , and mild recessive monogenic disease–causing AGXT, AQP2, CTNS and PKHD1 (Table facial dimorphism. Immunodeficiency– genes of CAKUT. Using this strategy, we 1, Supplemental Material). Interestingly, centromeric instability facial anomalies identified the causative mutations in nine five of these genes (ZBTB24, WFS1, has also been reported to include of the 33 families studied (27%). In all HPSE2, ATRX,andASPH)ifmutated congenital malformations, including nine families, we established a specific cause multiorgan syndromes that may CAKUT.18 Patient B268 presented with etiologic genetic diagnosis that was not include CAKUT as one of their features isolated CAKUT and was found to made on the basis of the patient’s clinical (syndromic CAKUT) (Table 1, syn- harbor a previously reported truncat- findings. dromes with CAKUT). Four other genes ing mutation in the HPSE2.19 This We have previously shown that re- (AGXT, AQP2, CTNS,andPKHD1) gene is responsible for urofacial syn- cessive-causing mutations can be iden- cause, if mutated, a that drome.19,20 This syndrome, also known tified in a high percentage of individuals may represent a phenocopy of CAKUT as Ochoa syndrome, is characterized by if (1) consanguinity of the parents is pre- (Table 1, phenocopies with CAKUT). typical distorted facial expression with sent and (2) a combination of homozy- In five patients, we identified recessive smiling or laughing expression as well gosity mapping with WES is applied.11,12 mutations that cause syndromic CAKUT as bladder malfunctions leading to void- We, therefore, selected, of approximately (Table 1, syndromes with CAKUT). Pa- ing dysfunction, recurrent urinary tract 700 families with the diagnosis of tient A3857 had ESRD secondary to infections, and other accompanying fea- CAKUT that were referred to us from neurogenic bladder and recurrent tures of CAKUT. After establishment of worldwide sources, 33 unrelated indi- urinary tract infections. This patient the molecular genetic diagnosis, we con- viduals from consanguineous families presented with CAKUT and harbors a tacted the primary nephrologist who for homozygosity mapping and WES. previously reported missense muta- originally recruited this patient at a very All patients were referred to us by a pe- tion in the gene WFS1 responsible to young age and asked him to look for the diatric nephrologist who made a clinical Wolfram syndrome, a diagnosis that typical facial grimacing associated with diagnosis of CAKUTon the basis of renal had not been made in this patient (Table this syndrome. In a follow-up clinical imaging studies. Mutations in all genes 1, syndromes with CAKUT).14 Wolfram examination, the patient was found to k