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• W2942705173 abstract "Congenital lower urinary-tract obstruction (LUTO) is caused by anatomical blockage of the bladder outflow tract or by functional impairment of urinary voiding. About three out of 10,000 pregnancies are affected. Although several monogenic causes of functional obstruction have been defined, it is unknown whether congenital LUTO caused by anatomical blockage has a monogenic cause. Exome sequencing in a family with four affected individuals with anatomical blockage of the urethra identified a rare nonsense variant (c.2557C>T [p.Arg853∗]) in BNC2, encoding basonuclin 2, tracking with LUTO over three generations. Re-sequencing BNC2 in 697 individuals with LUTO revealed three further independent missense variants in three unrelated families. In human and mouse embryogenesis, basonuclin 2 was detected in lower urinary-tract rudiments. In zebrafish embryos, bnc2 was expressed in the pronephric duct and cloaca, analogs of the mammalian lower urinary tract. Experimental knockdown of Bnc2 in zebrafish caused pronephric-outlet obstruction and cloacal dilatation, phenocopying human congenital LUTO. Collectively, these results support the conclusion that variants in BNC2 are strongly implicated in LUTO etiology as a result of anatomical blockage. Congenital lower urinary-tract obstruction (LUTO) is caused by anatomical blockage of the bladder outflow tract or by functional impairment of urinary voiding. About three out of 10,000 pregnancies are affected. Although several monogenic causes of functional obstruction have been defined, it is unknown whether congenital LUTO caused by anatomical blockage has a monogenic cause. Exome sequencing in a family with four affected individuals with anatomical blockage of the urethra identified a rare nonsense variant (c.2557C>T [p.Arg853∗]) in BNC2, encoding basonuclin 2, tracking with LUTO over three generations. Re-sequencing BNC2 in 697 individuals with LUTO revealed three further independent missense variants in three unrelated families. In human and mouse embryogenesis, basonuclin 2 was detected in lower urinary-tract rudiments. In zebrafish embryos, bnc2 was expressed in the pronephric duct and cloaca, analogs of the mammalian lower urinary tract. Experimental knockdown of Bnc2 in zebrafish caused pronephric-outlet obstruction and cloacal dilatation, phenocopying human congenital LUTO. Collectively, these results support the conclusion that variants in BNC2 are strongly implicated in LUTO etiology as a result of anatomical blockage. Congenital lower urinary-tract obstruction (LUTO) generally manifests as urinary bladder outflow obstruction, which can represent an anatomical blockage or a functional obstruction. LUTO of any kind has an estimated birth prevalence of three per 10,000 pregnancies.1Malin G. Tonks A.M. Morris R.K. Gardosi J. Kilby M.D. Congenital lower urinary tract obstruction: A population-based epidemiological study.BJOG. 2012; 119: 1455-1464Crossref PubMed Scopus (103) Google Scholar The largest population-based study from the West Midlands Congenital Anomaly Register in the UK suggests a significantly higher prevalence among black and minority ethnic groups than among white Europeans.1Malin G. Tonks A.M. Morris R.K. Gardosi J. Kilby M.D. Congenital lower urinary tract obstruction: A population-based epidemiological study.BJOG. 2012; 119: 1455-1464Crossref PubMed Scopus (103) Google Scholar Little is known about the genetic causes of anatomical LUTO. Single cases in the literature are associated with trisomy 21 or chromosomal aberrations, but for the majority of cases, the origin of isolated anatomical LUTO remains unknown. Functional LUTO has been extensively genetically defined, e.g., variants of CHRM3 (MIM: 118494) in prune-belly syndrome,2Weber S. Thiele H. Mir S. Toliat M.R. Sozeri B. Reutter H. Draaken M. Ludwig M. Altmüller J. Frommolt P. et al.Muscarinic acetylcholine receptor M3 mutation causes urinary bladder disease and a prune-belly-like syndrome.Am. J. Hum. Genet. 2011; 89: 668-674Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar variants of HPSE2 (MIM: 613469) and LRIG2 (MIM: 608869) in urofacial syndrome,3Daly S.B. Urquhart J.E. Hilton E. McKenzie E.A. Kammerer R.A. Lewis M. Kerr B. Stuart H. Donnai D. Long D.A. et al.Mutations in HPSE2 cause urofacial syndrome.Am. J. Hum. Genet. 2010; 86: 963-969Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 4Stuart H.M. Roberts N.A. Hilton E.N. McKenzie E.A. Daly S.B. Hadfield K.D. Rahal J.S. Gardiner N.J. Tanley S.W. Lewis M.A. et al.UK VUR Study Group4C Study GroupUrinary tract effects of HPSE2 mutations.J. Am. Soc. Nephrol. 2015; 26: 797-804Crossref PubMed Scopus (25) Google Scholar and variants of MYH11 (MIM: 160745) and ACTG2 (MIM: 102545) in microcolon megabladder hypoperistalsis syndrome.5Gauthier J. Ouled Amar Bencheikh B. Hamdan F.F. Harrison S.M. Baker L.A. Couture F. Thiffault I. Ouazzani R. Samuels M.E. Mitchell G.A. et al.A homozygous loss-of-function variant in MYH11 in a case with megacystis-microcolon-intestinal hypoperistalsis syndrome.Eur. J. Hum. Genet. 2015; 23: 1266-1268Crossref PubMed Scopus (57) Google Scholar, 6Wangler M.F. Gonzaga-Jauregui C. Gambin T. Penney S. Moss T. Chopra A. Probst F.J. Xia F. Yang Y. Werlin S. et al.Baylor-Hopkins Center for Mendelian GenomicsHeterozygous de novo and inherited mutations in the smooth muscle actin (ACTG2) gene underlie megacystis-microcolon-intestinal hypoperistalsis syndrome.PLoS Genet. 2014; 10: e1004258Crossref PubMed Scopus (91) Google Scholar Anatomical congenital LUTO can also be familial, albeit with interfamilial phenotypic variability, even between identical twins.7Schreuder M.F. van der Horst H.J.R. Bökenkamp A. Beckers G.M.A. van Wijk J.A.E. Posterior urethral valves in three siblings: a case report and review of the literature.Birth Defects Res. A Clin. Mol. Teratol. 2008; 82: 232-235Crossref PubMed Scopus (18) Google Scholar, 8Hanlon-Lundberg K.M. Verp M.S. Loy G. Posterior urethral valves in successive generations.Am. J. Perinatol. 1994; 11: 37-39Crossref PubMed Scopus (8) Google Scholar, 9Frese S. Weigert A. Hoppe B. Feldkötter M. Ludwig M. Weber S. Kiliś-Pstrusińska K. Zaniew M. Reutter H. Hilger A.C. A classic twin study of lower urinary tract obstruction: Report of 3 cases and literature review.Low. Urin. Tract Symptoms. 2018; 2018: 17Google Scholar Severe forms of LUTO are usually diagnosed prenatally on the basis of a distended bladder that fails to empty with dilatation of the upper urinary tract. However, milder forms manifest postnatally, often with recurrent urinary tract infections (UTI).10Scott J.E. Management of congenital posterior urethral valves.Br. J. Urol. 1985; 57: 71-77Crossref PubMed Scopus (57) Google Scholar Severe forms cause oligohydramnios and are associated with dysplastic kidney malformations that can be secondary to LUTO.11Dinneen M.D. Dhillon H.K. Ward H.C. Duffy P.G. Ransley P.G. Antenatal diagnosis of posterior urethral valves.Br. J. Urol. 1993; 72: 364-369Crossref PubMed Scopus (89) Google Scholar This can be deduced because similar kidney disease occurs in ovine fetuses with surgically generated LUTO.12Farrugia M.-K. Woolf A.S. Fry C.H. Peebles D.M. Cuckow P.M. Godley M.L. Radiotelemetered urodynamics of obstructed ovine fetal bladders: correlations with ex vivo cystometry and renal histopathology.BJU Int. 2007; 99: 1517-1522Crossref PubMed Scopus (13) Google Scholar Indeed, LUTOs are the leading cause for end-stage renal disease (ESRD) in children.13Parkhouse H.F. Woodhouse C.R. Long-term status of patients with posterior urethral valves.Urol. Clin. North Am. 1990; 17: 373-378Abstract Full Text PDF PubMed Google Scholar The most common anatomical causes of LUTO are posterior urethral valves (PUVs) at the level of the prostatic urethra, a lesion unique to males.14Robertson W.B. Hayes J.A. Congenital diaphragmatic obstruction of the male posterior urethra.Br. J. Urol. 1969; 41: 592-598Crossref PubMed Scopus (31) Google Scholar Less common are anterior urethral valves, also called urethral atresia, that can occur in either sex.11Dinneen M.D. Dhillon H.K. Ward H.C. Duffy P.G. Ransley P.G. Antenatal diagnosis of posterior urethral valves.Br. J. Urol. 1993; 72: 364-369Crossref PubMed Scopus (89) Google Scholar, 15Morris R.K. Kilby M.D. Long-term renal and neurodevelopmental outcome in infants with LUTO, with and without fetal intervention.Early Hum. Dev. 2011; 87: 607-610Crossref PubMed Scopus (38) Google Scholar Here, we present evidence that monoallelic variants in BNC2, coding for basonuclin 2, are strongly implicated in isolated anatomical LUTO. The study was conducted in adherence to the Declaration of Helsinki. The respective informed consent was obtained from the affected individuals or by proxies in the case of minors. The study was approved by the ethics committee of the medical faculty of the University of Bonn (No. 146/12) as well as by the respective ethics committees of the collaborating centers in Boston, Manchester, and Nijmegen (AGORA data and biobank). Human embryonic material, collected with maternal consent and ethical approval (REC 08/H0906/21+5), was sourced from the MRC-Wellcome Trust Human Developmental Biology Resource. Zebrafish and mice were kept according to national law and to general recommendations in our fish facility in Bonn, Germany and the mouse facility in Frankfurt, Germany, respectively. We used exome sequencing in a previously unreported family (family 1; for a detailed description, see Supplemental Data and Table 1) whose affected members had autosomal-dominant LUTO of variable phenotypic expression (Figure 1C). The male index individual (IV-2) received vesicoamniotic shunting at 13 weeks of gestation as a result of early diagnosis of severe LUTO. Postnatally he was diagnosed with high-grade urethral stenosis. His mother (III-4) was diagnosed with urethral stenosis at the age of 16. Prior to her pregnancy with the index individual, she had two spontaneous abortions of unknown cause (Figure 1C). The maternal grandmother (II-4) was diagnosed with distal urethral stenosis (meatal stenosis) at the age of 47. Her sister (II-2) denied having voiding dysfunction but did not consent to any urological or genetic assessment. In accordance with the previously described, well-noted, high within-family phenotypic variability, we considered her to be healthy. The female maternal cousin of the index individuals’ mother (III-2) was diagnosed with high-grade urethral stenosis at the age of 42.Table 1Heterozygous Variants of BNC2 in Families With LUTOFamilyFamily 1Family 2Family 3Family 4Family 5IndividualIV-2III-4III-2II-4III-1II-1I-1II-1I-1II-1II-1SexmalefemalefemalefemalemalemalefemalemalemalemalemalePhenotypedistal urethral stenosis, BL VUR grade 5urethral stenosis, urinary tract infections, pollakisuria with nycturiameatal urethral stenosis, bladder descensus, pollakisuria with nycturia, urinary incontinenceurethral stenosisPUV, Rt VUR grade 5, BL renal hypodysplasiapathological voiding on uroflowmetrynormal RUS and renal functionPUV, VUR grade 1-2.5, bladder diverticlesNAPUV, right hydonephrosis,left VUR grade 1, incontinence until 6 yearsPUV, VUR, ESRDAge of clinical onset13 weeks of gestation12 years47 years42 years at surgical correctionprenatal38 years at finding of pathological uroflowmetryNA7 yearsNA8 years∼11 yearsNucleotide changec.2557C>Tc.2663A>Gc.473C>Tc.1036G>Cc.13G>ATranscriptENST00000418777ENST00000380672ENST00000380672ENST00000380672ENST00000545497RefSeqGenBank: NM_001317939.1GenBank: NM_017637.6GenBank: NM_017637.6GenBank: NM_017637.6NAStateheterozygousheterozygousheterozygousheterozygousheterozygousChr. Pos. (GRCh37/hg19)chr9: 16435054chr9: 16419624chr9: 16552724chr9: 16437156chr9: 16832274Aminoacid changep.Arg853∗p.His888Argp.Thr158Ilep.Glu346Glnp.Val5IleConserved toNAdrosophila melanogasterdanio reriodanio reriopongo albeliiProtein domainnot knownc2h2 zinc finger domainnot knownnot knownnot knownGERP scoreNA5.595.845.96-0.728PPH (score)NAprobably damaging (0.981)possibly damaging (0.878)benign (0.037)benign (0.015)SIFT (score)NAdeleterious (0)deleterious (0)deleterious (0)tolerated (0.59)CADD score19.0925.726.622.11.466ACMGpathogenicpathogenicuncertain significanceuncertain significancebenignrsNumberrs1350162888novelrs144242525rs945575406rs750936655gnomAD Allele Frequencies (hom/het/WT)0.000028 (0/5/245862)novel0.000012 (0/3/245862)0.000004 (0/1/245862)0.000048 (0/6/124816)ethnicity, gender3 female AFR, 2 female NFEnovel1 NFE male, 2 NFE female1 NFE male4 SAS, 1AMR, 1 ASJ, 4 female, 2 maleComplete pedigrees of identified families in which affected members have both the LUTO phenotype and rare variants in BNC2 can be found in Figure S2.Abbreviations are as follows: het, heterozygous; PPH, PolyPhen prediction score; BL, bilateral; ESRD, end-stage renal disease; PUV, posterior urethral valve; Rt, right; VUR, vesico ureteral reflux; RUS, renal ultrasound; NA, not available; ACMG, American College of Human Genetics Standards and Guidelines Classification as pathogenic, likely pathogenic, uncertain significance, likely benign or benign; NFE, non-Finnish European; SAS, south Asian; AMR, American; and ASJ, Ashkenazi Jewish.Variants that were rated as pathogenic by the ACMG guidelines were annotated with a ClinVar Accession Number (ENST00000418777, c.2557C>T [p.Arg853∗], ClinVar: SCV000891778; ENST00000380672, c.2663A>G [p.His888Arg], ClinVar: SCV000854630). Open table in a new tab Complete pedigrees of identified families in which affected members have both the LUTO phenotype and rare variants in BNC2 can be found in Figure S2. Abbreviations are as follows: het, heterozygous; PPH, PolyPhen prediction score; BL, bilateral; ESRD, end-stage renal disease; PUV, posterior urethral valve; Rt, right; VUR, vesico ureteral reflux; RUS, renal ultrasound; NA, not available; ACMG, American College of Human Genetics Standards and Guidelines Classification as pathogenic, likely pathogenic, uncertain significance, likely benign or benign; NFE, non-Finnish European; SAS, south Asian; AMR, American; and ASJ, Ashkenazi Jewish. Variants that were rated as pathogenic by the ACMG guidelines were annotated with a ClinVar Accession Number (ENST00000418777, c.2557C>T [p.Arg853∗], ClinVar: SCV000891778; ENST00000380672, c.2663A>G [p.His888Arg], ClinVar: SCV000854630). Exome sequencing was performed on the affected individuals, II-4, III-2, and IV-2 from family 1 (Figure 1C). According to recent reports on rare dominantly inherited congenital anomalies of the kidney and urinary tract (CAKUT), we considered only variants with an allele frequency <0.0001.16Vivante A. Kleppa M.-J. Schulz J. Kohl S. Sharma A. Chen J. Shril S. Hwang D.-Y. Weiss A.-C. Kaminski M.M. et al.Mutations in TBX18 cause dominant urinary tract malformations via transcriptional dysregulation of ureter development.Am. J. Hum. Genet. 2015; 97: 291-301Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 17Sanna-Cherchi S. Westland R. Ghiggeri G.M. Gharavi A.G. Genetic basis of human congenital anomalies of the kidney and urinary tract.J. Clin. Invest. 2018; 128: 4-15Crossref PubMed Scopus (53) Google Scholar According to these filter criteria, we detected 11 missense and two nonsense variants that segregated in the family (Table S1). All variants were confirmed by Sanger sequencing. Among these variants, we prioritized the two nonsense variants as the most likely to be disease causing. One variant was found to be a novel p.Trp3∗ in ALB (MIM: 103600), encoding albumin. Recessive truncating variants in ALB have been described to cause analbuminaemia (MIM: 616000), whereas heterozygous variants in ALB can cause dysalbuminemic hyperthyroxinemia (MIM: 615999). Neither condition is associated with LUTO, and therefore the variant was not considered to be causative for the LUTO phenotype in the family. The second truncating variant, p.Arg853∗ rs1350162888 (ClinVar: SCV000891778) (total 860 amino acids) (Table 1), was found in BNC2 (MIM: 608669, GenBank: NM_001317939, ENST00000418777) (Figure 1A), which is a candidate gene for human hypospadias and encodes basonuclin 2, a zinc-finger-containing protein.18Bhoj E.J. Ramos P. Baker L.A. Garg V. Cost N. Nordenskjöld A. Elder F.F. Bleyl S.B. Bowles N.E. Arrington C.B. et al.Human balanced translocation and mouse gene inactivation implicate Basonuclin 2 in distal urethral development.Eur. J. Hum. Genet. 2011; 19: 540-546Crossref PubMed Scopus (18) Google Scholar, 19Kon M. Suzuki E. Dung V.C. Hasegawa Y. Mitsui T. Muroya K. Ueoka K. Igarashi N. Nagasaki K. Oto Y. et al.Molecular basis of non-syndromic hypospadias: systematic mutation screening and genome-wide copy-number analysis of 62 patients.Hum. Reprod. 2015; 30: 499-506Crossref PubMed Scopus (26) Google Scholar Little is known about the function of BNC2. Previously, Vanhoutteghem et al. reported on the localization of BNC2 in nuclear speckles and its potential involvement in nuclear processing of mRNA.20Vanhoutteghem A. Djian P. Basonuclins 1 and 2, whose genes share a common origin, are proteins with widely different properties and functions.Proc. Natl. Acad. Sci. USA. 2006; 103: 12423-12428Crossref PubMed Scopus (33) Google Scholar Bhoj et al. analyzed Bnc2 expression in the penis and urethra of Bnc2+/− and Bnc2−/− newborn mice and determined that Bnc2 expression is highest in the phallic periurethral tissue,18Bhoj E.J. Ramos P. Baker L.A. Garg V. Cost N. Nordenskjöld A. Elder F.F. Bleyl S.B. Bowles N.E. Arrington C.B. et al.Human balanced translocation and mouse gene inactivation implicate Basonuclin 2 in distal urethral development.Eur. J. Hum. Genet. 2011; 19: 540-546Crossref PubMed Scopus (18) Google Scholar consistent with BNC2 having an important role in urethral development. They further suggested that BNC2 acts locally in urethral development in both sexes. The variant in BNC2 has been reported five times heterozygously in 175,684 alleles in the gnomAD database (version 2.0.2, date of inquiry 2018/09/20 for all mentioned gnomAD inquiries) (allele frequency 0.000028, rs1350162888). Notably, all of the heterozygous carriers were females. We next re-sequenced all 14 BNC2 transcripts in 697 individuals with LUTO from the AGORA study in the Netherlands and from pediatric nephrology departments in Germany, Poland, the UK, and the US. 13 of the 14 transcripts are predicted to be protein coding. The protein product for the two transcripts has been verified in humans so far (ENST00000380672, GenBank: NM_017637.6, canonical transcript; and ENST00000418777, GenBank: NM_001317939.1). In total, all rare missense or nonsense variants identified in this study were found in one of these two verified proteins except that one variant was identified in ENST00000545497. All variants identified in the canonical transcript ENST00000380672 are also contained by the alternatively spliced transcript ENST00000418777, in which the variant of family 1 was identified. By re-sequencing, we detected one novel (c.2663A>G [p.His888Arg], ENST00000380672, ClinVar: SCV000854630) and three rare missense variants (c.13G>A [p.Val5Ile], ENST00000545497, rs750936655, allele frequency 0.000048) (c.473C>T [p.Thr158Ile], ENST00000380672, rs144242525, allele frequency 0.000012) (c.1036G>C [p.Glu346Gln], ENST00000380672, rs945575406, allele frequency 0.000004) in four independent individuals with LUTO (Table 1, Figure S2). The novel variant c.2663A>G, detected in an individual with PUV and severe secondary dilatation of his upper urinary tract (III-1, family 2, Figure 1E), was inherited from an affected father (II-1) who had a pathological uroflowmetry (Figure S1). His renal ultrasound and renal function were normal. Investigation of the paternal grandparents (I-1 and I-2) showed that the variant was inherited from the healthy grandmother who did not have any history of UTI, pollakisuria, or nycturia and who had normal kidneys on renal ultrasound and normal uroflowmetry. The amino acid change p.His888Arg is highly conserved to D. melanogaster (GERP 5.59). Three in silico prediction programs classify the amino acid change as potentially disease causing and (according to the American College of Medical Genetics [ACMG] guidelines) as pathogenic (Table 1). The variant (c.2663A>G [p.His888Arg], ENST00000380672) is located in the fourth C2H2 zinc finger domain (Figure 1B). According to SwissProt entry Q6ZN30 and PROSITE, this variant is located near the second (residues 833–856) of three C2H2 zinc finger domains in BNC2 protein. However, InterPro and SMART include residue His888 in their signature matches (residues 833–856 and 861–888) for zinc-finger domains of the C2H2-type. These predictions suggest that this zinc finger's very last histidine, which is replaced by arginine, might interfere directly with the zinc finger domain's ability to bind to specific DNA sequences. Moreover, PROFAcc and ISIS, implemented in the PredictProtein server,21Rost B. Yachdav G. Liu J. The PredictProtein server.Nucleic Acids Res. 2004; 32: W321-W326Crossref PubMed Scopus (1180) Google Scholar, 22Ofran Y. Rost B. ISIS: interaction sites identified from sequence.Bioinformatics. 2007; 23: e13-e16Crossref PubMed Scopus (218) Google Scholar characterize His888 as an exposed residue, located in a protein-binding region. Because some C2H2 zinc-finger domains were also shown to facilitate binding to proteins or RNA,23Brayer K.J. Kulshreshtha S. Segal D.J. The protein-binding potential of C2H2 zinc finger domains.Cell Biochem. Biophys. 2008; 51: 9-19Crossref PubMed Scopus (63) Google Scholar the substitution p.His888Arg in BNC2 might affect not only DNA, but also RNA, and/or might target protein binding. Because this novel variant has been transmitted from an affected father to his affected son and because this variant resides in a highly conserved functional domain of BNC2, we believe this variant to be possibly disease causing (Table 1). Of the remaining three rare missense variants detected in BNC2, the first variant, c.13G>A, detected in a boy with PUV and severe dilatation of his upper urinary tract (vesicoureteral reflux [VUR]) and secondary ESRD, has been reported in a heterozygous context six times (in two male and four female carriers) in 124,816 alleles according to gnomAD (allele frequency 0.000048). The GERP conservation score, the benign classification of three in silico prediction programs, and the ACMG guidelines suggest this variant to be benign. Parental DNA was not available for testing of inheritance (Table 1, family 5). The second rare missense variant, c.473C<T, detected in a male individual with PUV, has been mentioned in a heterozygous context three times (in one male and two female carriers) in 245,826 alleles according to gnomAD (allele frequency 0.000012). This variant is highly conserved and predicted by three in silico prediction programs to be disease causing. Because the variant was transmitted from a father with unknown affection status, we considered this variant to be a variant of unknown clinical significance (Table 1, family 3). The third rare missense variant, c.1036G>C, was detected in a male individual with PUV. It has been mentioned only once in a heterozygous context (in one male carrier) in 245,816 alleles according to gnomAD (allele frequency 0.000004). Although the amino acid at this position is highly conserved, only two out of three in silico prediction programs classified the variant to be disease causing. Parental DNA was not available for testing of inheritance (Table 1, family 4). Hence, we classified the latter two missense variants, c.473C>T and c.1036G>C, each residing at a highly conserved amino acid position and predicted to be disease causing by at least two out of three in silico prediction programs according to the ACMG guidelines, as variants of unknown clinical significance. In addition to these four missense variants, we detected four rare (minor-allele frequency <0.0001) and two novel synonymous variants and one rare intronic variant (minor-allele frequency <0.0001) within the direct vicinity to the respective splice site (Table S2). The novel synonymous variant c.1132A>C (p.Ser358Ser) was predicted by four splice-site analysis programs to have an impact on splicing at this side (Table S2). Nevertheless, it remains speculative whether splicing is affected, and further assessment of this variant is warranted. Thereafter we sought to characterize the embryonic effects on the contribution of BNC2 to the formation of LUTO. BNC2 encodes a highly conserved zinc-finger protein of poorly understood function. The Human Protein Atlas reports expression of BNC2 in the cytoplasm of urothelial cells of the bladder. To study the expression of BNC2 in the human urethra, we performed immunohistochemistry staining with an anti-BNC2 antibody targeting a region in the N-terminal-vicinity third zinc finger of the canonical transcript ENST00000380672 (amino acids (aa) 661-787) and ENST00000418777 (aa 618–743). In a 7-week human embryo (Figure 2), BNC2 expression was detected in the urogenital sinus, the precursor of the bladder, and its outflow tract. The most prominent signal was in the primitive urothelium, and there was weaker immunoreactivity in the surrounding mesenchyme (Figure 2 A–D). Expression was also detected in the urothelium of the adult human male urethra (Figure S3). We performed expression analysis by in situ hybridization (ISH) in mouse embryos by using a pan-Bnc2 probe that showed Bnc2 expression in the developing lower urinary-tract structures, with emphasis on the genital tubercle (gt) above the phallic urethra and below the pelvic urethra at embryonic day (E) 13.5 (Figures 2E–2G), the critical time point for urethral development. Expression of Bnc2 was also visible in the brain, in the mandibular region, and in dorsal parts above the spinal cord. The presence of the different Bnc2 mRNA (Bnc2-201 and Bnc2-214) in the urogenital region of developing mouse embryos was verified by Sanger sequencing. These mouse mRNA represent the two different homologous human transcripts that were found to be affected by the variants identified in family 1 (ENST00000418777) and family 2 (ENST00000380672). Our expression data for Bnc2 during murine development of the urinary tract and the existence of a heterozygous Bnc2 knockout mouse, which was reported by Bhoij et al.18Bhoj E.J. Ramos P. Baker L.A. Garg V. Cost N. Nordenskjöld A. Elder F.F. Bleyl S.B. Bowles N.E. Arrington C.B. et al.Human balanced translocation and mouse gene inactivation implicate Basonuclin 2 in distal urethral development.Eur. J. Hum. Genet. 2011; 19: 540-546Crossref PubMed Scopus (18) Google Scholar and presents with a cleft in the ventral part of the external genitalia, support the impact of BNC2 in urethral development. Furthermore, Bhoij et al. describe homozygous knockout mice that have the same defect but in a more penetrant and severe form. These mice die soon after birth. Corresponding to that, the Bnc2−/− knockout mice, as described by Vanhoutteghem et al., die soon after birth. Because of their cleft palates and ingestion of air, these mice display aerial distention of their digestive tracts and therefore have distended abdomens.24Vanhoutteghem A. Maciejewski-Duval A. Bouche C. Delhomme B. Hervé F. Daubigney F. Soubigou G. Araki M. Araki K. Yamamura K. Djian P. Basonuclin 2 has a function in the multiplication of embryonic craniofacial mesenchymal cells and is orthologous to disco proteins.Proc. Natl. Acad. Sci. USA. 2009; 106: 14432-14437Crossref PubMed Scopus (25) Google Scholar The urinary tract was not examined in these mice, although a distended abdomen is also observed in human newborns with LUTO.25Docimo S.G. Canning D. Khoury A. Salle J.L.P. The Kelalis–King–Belman Textbook of Clinical Pediatric Urology. CRC Press, 2018Crossref Google Scholar In order to further investigate the function of BNC2 during urinary tract development, we used zebrafish as a model organism. Their embryonic kidney, the pronephros, consists of two nephrons, with a fused glomerulus, two tubules, and ducts. The ducts terminate in a cloaca, altogether reflecting a simplified model of the human nephron and urinary tract.26Drummond I.A. Majumdar A. Hentschel H. Elger M. Solnica-Krezel L. Schier A.F. Neuhauss S.C. Stemple D.L. Zwartkruis F. Rangini Z. et al.Early development of the zebrafish pronephros and analysis of mutations affecting pronephric function.Development. 1998; 125: 4655-4667Crossref PubMed Google Scholar BLAST analysis with human BNC2 identified a single zebrafish bnc2 ortholog encoding six different transcripts. To study the expression of bnc2 in zebrafish larvae (zfl), we generated a labeled RNA probe for ISH. We detected strong expression of bnc2 in the brain as described before27Lang M.R. Patterson L.B. Gordon T.N. Johnson S.L. Parichy D.M. Basonuclin-2 requirements for zebrafish adult pigment pattern development and female fertility.PLoS Genet. 2009; 5: e1000744Crossref PubMed Scopus (64) Google Scholar but also in the pronephric ducts and the developing cloaca region at 33 hours post-fertilization (hpf) (Figure 2H). Expression of Bnc2 in the pronephric ducts was confirmed by immunohistochemistry analysis in developing zfl at 100 hpf (Figure 2I, 2J). Next, we performed phenotypic analysis after a Bnc2 knockdown in developing zfl. We designed one specific antisense Morpholino (MO), targeting the translation initiation site of transcript bnc2_202 (ENSDART00000128671.4). The canonical transcript in zebrafish bnc2_202 (ZFIN and Ensembl genome browser) is the most similar to the two human BNC2 transcripts that bear the discovered variants (ENST00000380672 and ENST00000418777). The zebrafish protein shows an amino acid similarity of 85%, similar protein structure, and a c" @default.
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