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Renal Hypodysplasia Associates with a Wnt4 Variant That Causes Aberrant Canonical Wnt Signaling

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Renal Hypodysplasia Associates with a Wnt4 Variant That Causes Aberrant Canonical Wnt Signaling BRIEF COMMUNICATION www.jasn.org Renal Hypodysplasia Associates with a Wnt4 Variant that Causes Aberrant Canonical Wnt Signaling † † ‡ † † Asaf Vivante,* Michal Mark-Danieli, Miriam Davidovits, Orit Harari-Steinberg, Dorit Omer, † ‡ ‡| Yehudit Gnatek, Roxana Cleper, Daniel Landau,§ Yael Kovalski, Irit Weissman,¶ †† ‡‡ || Israel Eisenstein,** Michalle Soudack, Haike Reznik Wolf, Naomi Issler,§§ Danny Lotan, †|| Yair Anikster,¶¶ and Benjamin Dekel *Department of Pediatrics, Talpiot Medical Leadership Program, Sheba Medical Center, Tel Hashomer, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; †Pediatric Stem Cell Research Institute, Edmond and Lily Safra Children’s Hospital, Sheba Medical Center, Tel Hashomer, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; ‡Institute of Pediatric Nephrology, Schneider Children’s Medical Center of Israel, Petah Tiqwa, Israel; §Department of Pediatrics A and Pediatric Nephrology, Saban Children’s Hospital, Soroka Medical Center, Ben Gurion University, Beer Sheva, Israel; |Children’s Medical Center, Ramla, Israel; ¶Nephrology Unit, Western Galilee Hospital, Nahariya, Israel; **Pediatric Nephrology Unit, Meyer Children’s Hospital, Rambam Health Care Campus, Faculty of Medicine, Technion, Israel Institute of Technology, Haifa, Israel; ††Pediatric Imaging, Edmond and Lily Safra Children’s Hospital, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; ‡‡Genetic Institute, Sheba Medical Center, Tel Hashomer, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; §§Centre for Nephrology, Institute of Child Health, University College London, London, United Kingdom; ||Division of Pediatric Nephrology, Sheba Medical Center, Tel Hashomer, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; and ¶¶Metabolic Disease Unit, Edmond and Lily Safra Children’s Hospital, Sheba Medical Center, Tel Hashomer, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel ABSTRACT Abnormal differentiation of the renal stem/progenitor pool into kidney tissue can The pathologic basis of RHD is the lead to renal hypodysplasia (RHD), but the underlying causes of RHD are not well disturbance of normal nephrogenesis, understood. In this multicenter study, we identified 20 Israeli pedigrees with isola- possibly due to mutations in genes that ted familial, nonsyndromic RHD and screened for mutations in candidate genes in- direct the process.1,8 Most of the genes volved in kidney development, including PAX2, HNF1B, EYA1, SIX1, SIX2, SALL1, known to be involved are transcriptional GDNF, WNT4,andWT1. In addition to previously reported RHD-causing genes, we factors and genes that encode for pro- found that two affected brothers were heterozygous for a missense variant in the teins involved in the mesenchymal to ep- WNT4 gene. Functional analysis of this variant revealed both antagonistic and ag- ithelial transition.2,8 To date, most forms onistic canonical WNT stimuli, dependent on cell type. In HEK293 cells, WNT4 inhibi- of RHD have been found negative for tedWNT3Ainducedcanonicalactivation,andtheWNT4 variant significantly abnormalities in recognized renal devel- enhanced this inhibition of the canonical WNT pathway. In contrast, in primary cul- opmental genes,3,9,10 and it is highly tures of human fetal kidney cells, which maintain WNT activation and more closely likely that other, still unreported, genes represent WNT signaling in renal progenitors during nephrogenesis, this mutation will be identified, especially genes for caused significant loss of function, resulting in diminished canonical WNT/b-catenin which renal maldevelopment has been signaling. In conclusion, heterozygous WNT4 variants are likely to play a causative role in renal hypodysplasia. Received January 28, 2012. Accepted December 6, J Am Soc Nephrol 24: 550–558, 2013. doi: 10.1681/ASN.2012010097 2012. Published online ahead of print. Publication date available at www.jasn.org. ESRD in children most commonly results diagnosed sporadically or with familial Correspondence: Dr. Benjamin Dekel, Pediatric from congenital anomalies of the kidney aggregation.1,3–6 For familial cases, the sug- Stem Cell Research Institute, Edmond and Lily Safra Children’s Hospital, Sheba Medical Center, Tel 1 and urinary tract. The most common gested mode of inheritance in most pedi- Hashomer, 52621, Israel. Email: binyamin.dekel@ congenital anomalies of the kidney and grees is autosomal dominant with variable sheba.health.gov.il or [email protected] urinary tract clinic-pathologic type is re- expression and reduced penetrance, esti- Copyright © 2013 by the American Society of nal hypodysplasia (RHD).2 RHD can be mated to range between 50% and 90%.7 Nephrology 550 ISSN : 1046-6673/2404-550 J Am Soc Nephrol 24: 550–558, 2013 www.jasn.org BRIEF COMMUNICATION fi demonstrated in genetically modi ed A B models.11 C T GG A A G T C A T G G A C T C G G Weinvestigatedtheprevalenceof WT mutations in 9 kidney developmental genes among a selected group of 20 families with isolated nonsyndromic fa- milial RHD. We showed that mutations are present in a significant part of this C T GG A A G T C A T G G A C T C G G >C group, and we outlined the possible role M64T of mutated WNT4 as a cause for dis- turbed early kidney development lead- ing to the RHD phenotype. Viewing RHD as a disease of the renal stem/pro- genitor cell pool, we hypothesized that C D human developing kidney cell–based systems would be extremely useful for disease modeling. Our study group comprised 51 RHD- affected individuals and 91 unaffected family members from 20 unrelated fam- ilies. The families’ clinical characteristics are shown in Supplemental Figure 1 and Supplemental Table 1. We identified 13 Figure 1. Mutation analysis of the p.M64T, WNT4 variant shows high degree of conser- vation. (A) Sequence analysis reveals the WNT4 variant caused by heterozygous transition mutation-carrying participants from (c.t191c) (lower panel, blue arrow) resulting in amino acid substitution M64T. The wild-type four unrelated families harboring muta- (WT) sequence is given for comparison (upper panel). (B) WNT4 variant is found in patients tions in three different genes: one family 1, 3, 4, and 5. Whereas patients 3 and 4 have a clear isolated renal phenotype of severe left with a PAX2 mutation, two families with renal hypodysplasia, patient 1 has an isolated left renal cyst (sized 12.837 mm) in addition HNF1B mutations (for full genetic and to normal BP and normal GFR. Patient 5, age 3 years, has normal pelvic and renal ultra- clinical characterization see Supplemen- sound results, normal BP, normal GFR, and low morning serum cortisol levels. Incomplete tal Appendix), and one family (family 4) penetrance or variable expression can be considered in this family. Squares indicate male demonstrating a novel heterozygous family members and circles female family members; black filled squares indicate that the missense variant in the human WNT4 patients are affected with RHD. Gray filled squares and circles indicate subtle clinical signs. gene. The latter family includes two af- (C) cDNA sequences of human WNT4 are compared with WNT4 orthologs in other species. fected brothers with severe left RHD (D) Conservation scale of WNT4 protein shows the amino acid methionine in position 64 (blue arrow) to be highly conserved (8 of 9), with “b” indicating a buried residue and “e” who were found to harbor a novel mis- indicating an exposed residue. Note the high degree of conservation. sense WNT4 variant, c.t191c– p.M64T. This is the first description of an associ- ation between a WNT4 heterozygous variant and isolated human RHD. Geno- [http://www.hgmd.cf.ac.uk/ac/index. is known to cause renal malformation as type-phenotype correlation in this fam- php]). Automatic computer prediction part of the general Mayer–Rokitansky- ily revealed an autosomal dominant for possible effects of amino acid substi- like syndrome that was first described pattern of inheritance with incomplete tution on the structure and function of the in an 18-year-old female presenting penetrance, similar to other renal hypo- WNT4 protein using PolyPhen software with primary amenorrhea and a single dysplasia-causing genes (Figure 1). predictions (http://genetics.bwh.harvard. kidney.13 This is strikingly similar to The WNT4 p.M64T variant affects a edu/pph)12 showed that this variant may the WNT4 knockout mouse model that highly conserved methionin residue be possibly damaging (position-specific showed defective mesenchymal-epithe- found in all living organisms for which independent count [PSIC] score, 1.79). lial transition (MET), lack of nephron the WNT4 sequence is known (Figure 1), Finally, screening of 280 ethnically formation, and development of renal hy- suggesting possible functional impor- matched control participants (560 alleles) podysplasia,14,15 in addition to defective tance. The variant was not found in a for presence of the WNT4 variant was sexual differentiation exclusive to the fe- search of the single nucleotide polymor- negative, suggesting that this is not a com- male model. Supporting our findings, phism (SNPs) or mutation databases mon polymorphism. the male mouse model showed only iso- (dbSNP, 1000 genomes [http://brows- Further evidence supporting the cau- lated RHD, similar to both affected er.1000genomes.org/index.html]; Hu- sality of this rare WNT4 variant include males described herein.15 The interde- man Gene Mutation Database HGMD that heterozygous mutation in this gene pendence
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