Muscarinic Acetylcholine Receptor M3 Mutation Causes Urinary Bladder Disease and a Prune-Belly-like Syndrome
2011; Elsevier BV; Volume: 89; Issue: 5 Linguagem: Inglês
10.1016/j.ajhg.2011.10.007
ISSN1537-6605
AutoresStefanie Weber, Hölger Thiele, Sevgı Mır, Mohammad R. Toliat, Betül Sözeri, Heiko Reutter, Markus Draaken, Michael Ludwig, Janine Altmüller, Peter Frommolt, Helen M. Stuart, Parisa Ranjzad, Neil A. Hanley, Rachel Jennings, William G. Newman, Duncan T. Wilcox, Uwe Thiel, Karl P. Schlingmann, Rolf Beetz, Peter F. Hoyer, Martin Konrad, Franz Schaefer, Peter Nürnberg, Adrian S. Woolf,
Tópico(s)Ion channel regulation and function
ResumoUrinary bladder malformations associated with bladder outlet obstruction are a frequent cause of progressive renal failure in children. We here describe a muscarinic acetylcholine receptor M3 (CHRM3) (1q41-q44) homozygous frameshift mutation in familial congenital bladder malformation associated with a prune-belly-like syndrome, defining an isolated gene defect underlying this sometimes devastating disease. CHRM3 encodes the M3 muscarinic acetylcholine receptor, which we show is present in developing renal epithelia and bladder muscle. These observations may imply that M3 has a role beyond its known contribution to detrusor contractions. This Mendelian disease caused by a muscarinic acetylcholine receptor mutation strikingly phenocopies Chrm3 null mutant mice. Urinary bladder malformations associated with bladder outlet obstruction are a frequent cause of progressive renal failure in children. We here describe a muscarinic acetylcholine receptor M3 (CHRM3) (1q41-q44) homozygous frameshift mutation in familial congenital bladder malformation associated with a prune-belly-like syndrome, defining an isolated gene defect underlying this sometimes devastating disease. CHRM3 encodes the M3 muscarinic acetylcholine receptor, which we show is present in developing renal epithelia and bladder muscle. These observations may imply that M3 has a role beyond its known contribution to detrusor contractions. This Mendelian disease caused by a muscarinic acetylcholine receptor mutation strikingly phenocopies Chrm3 null mutant mice. Lower urinary tract and/or kidney malformations account for 40% of childhood end-stage renal failure.1North American Pediatric Renal Trials and Collaborative Studies (NAPRTCS) (2010). Annual Transplant Report. https://web.emmes.com/study/ped/annlrept/2010_Report.pdf.Google Scholar Advances have been made in unraveling the genetic pathogenesis of kidney anomalies,2Weber S. Moriniere V. Knüppel T. Charbit M. Dusek J. Ghiggeri G.M. Jankauskiené A. Mir S. Montini G. Peco-Antic A. et al.Prevalence of mutations in renal developmental genes in children with renal hypodysplasia: results of the ESCAPE study.J. Am. Soc. Nephrol. 2006; 17: 2864-2870Crossref PubMed Scopus (285) Google Scholar but less is known about the genetic origin of malformations of the lower tract. Congenital bladder outflow obstruction (BOO) has several causes, the commonest being posterior urethral valves (PUVs), with a high risk of developing chronic renal insufficiency.1North American Pediatric Renal Trials and Collaborative Studies (NAPRTCS) (2010). Annual Transplant Report. https://web.emmes.com/study/ped/annlrept/2010_Report.pdf.Google Scholar, 3Farrugia M.-K. Woolf A.S. Congenital urinary bladder outlet obstruction.Fetal Matern. Med. Rev. 2010; 21: 55-73Crossref Scopus (21) Google Scholar Recently, mutations of HPSE2 (MIM 613469; see Web Resources), encoding a heparanase inhibitor expressed in developing urinary tracts, were described in urofacial syndrome (UFS [MIM 236730]), which presents with a dysmorphic, poorly emptying bladder.4Daly 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 Also associated with congenital bladder dysfunction is prune belly syndrome (PBS [MIM 100100]). In its rare complete form, it comprises megacystis with disorganized detrusor muscle, cryptorchidism and thin abdominal musculature with overlying lax skin. PUV and PBS generally occur sporadically, although families have been reported with more than one affected member.3Farrugia M.-K. Woolf A.S. Congenital urinary bladder outlet obstruction.Fetal Matern. Med. Rev. 2010; 21: 55-73Crossref Scopus (21) Google Scholar, 5Weber S. Mir S. Schlingmann K.P. Nürnberg G. Becker C. Kara P.E. Ozkayin N. Konrad M. Nürnberg P. Schaefer F. Gene locus ambiguity in posterior urethral valves/prune-belly syndrome.Pediatr. Nephrol. 2005; 20: 1036-1042Crossref PubMed Scopus (28) Google Scholar, 6Ramasamy R. Haviland M. Woodard J.R. Barone J.G. Patterns of inheritance in familial prune belly syndrome.Urology. 2005; 65: 1227Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar We here describe a homozygous loss-of-function mutation of muscarinic acetylcholine receptor M3 (CHRM3) (1q41-q44) in five brothers with a PBS-like syndrome (Figure 1, Figure 2) . CHRM3 (MIM 118494) encodes the M3 subtype of muscarinic acetylcholine (ACh) receptors, the major receptor mediating urinary bladder contraction upon micturition.7Fowler C.J. Griffiths D. de Groat W.C. The neural control of micturition.Nat. Rev. Neurosci. 2008; 9: 453-466Crossref PubMed Scopus (951) Google Scholar We restudied a family reported in 20055Weber S. Mir S. Schlingmann K.P. Nürnberg G. Becker C. Kara P.E. Ozkayin N. Konrad M. Nürnberg P. Schaefer F. Gene locus ambiguity in posterior urethral valves/prune-belly syndrome.Pediatr. Nephrol. 2005; 20: 1036-1042Crossref PubMed Scopus (28) Google Scholar when there were four surviving boys with congenital BOO born to consanguineous Turkish parents. A fifth affected male sibling had died soon after birth as a result of renal failure and urosepsis (II-1). He and one affected surviving brother (II-4) displayed marked abdominal wall distension and were assigned as having PBS, whereas their three brothers were considered to have "PUV" because cystopscopy noted urethral valve-like structures. Recently, a sixth brother (II-7) was born, also with a malformed bladder. Evaluation of available cystograms showed massive and irregular-walled bladders with diverticulae (Figure 1). Vesicoureteric reflux was a variable feature. Urethral patency appeared normal in available cystograms. Cystometry performed in four affected boys in the first years of life revealed detrusor hyporeflexia with high residual volumes after micturition. Thus, the collective features in this kindred are most consistent with the PBS spectrum. Affected individuals also had bilaterally impaired pupillary constriction to light (Figure 1) and dry mouths. These examinations were performed after informed consent was obtained from the patients and their parents. Ethical approval for this study was obtained from the ethics review board at the Ruprecht-Karls-University Heidelberg and the collaborating institutions, according to the Declaration of Helsinki.Figure 2Deletion-Insertion Mutation of CHRM3Show full caption(A) Family pedigree. The index person II-4 and a deceased boy (II-1) presented with the full picture of PBS. Their male siblings, II-2, II-5, II-6, and II-7, had malformed bladders only. Chronic kidney disease (CKD) grade II was observed in affecteds II-5 and II-6, CKD grade IV in index person II-4.(B) p.Pro392Alafs∗43 introduces a premature termination codon in the third intracellular loop (M3R) resulting in a truncated protein that lacks the last two transmembrane domains (shown in red).(C) A homozygous CHRM3 frameshift mutation c.1173_1184 delinsT;p.Pro392Alafs∗43 was identified in index person II-4 by massively parallel sequencing; five forward (upper case) and three reverse reads (lower case) were recognized as an insertion-deletion mutation.(D and E) Three-dimensional protein structure modeling of wild-type (D) and mutant (E) receptor M3. Transmembrane helices (I-VII) and the amino- (N) and carboxy- (C) termini are indicated. Structure predictions of mutant M3 suggest complete deterioration of folding of the third intracellular loop (M3R) in addition to loss of transmembrane helices VI and VII in mutant M3. Three-dimensional protein structure modeling was obtained as a homology (comparative) protein structure with (PS)2-v2 using Protein Data Bank (PDB) entries 2rh1, 2rh1A and 1r5sA as template.31Chen C.C. Hwang J.K. Yang J.M. (PS)2-v2: template-based protein structure prediction server.BMC Bioinformatics. 2009; 10: 366Crossref PubMed Scopus (92) Google Scholar Structures were processed with Jmol. Model reliability is ≥75% for wild-type and mutant (excluding the mutant C terminus) and 55%–75% for the mutant C terminus.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Family pedigree. The index person II-4 and a deceased boy (II-1) presented with the full picture of PBS. Their male siblings, II-2, II-5, II-6, and II-7, had malformed bladders only. Chronic kidney disease (CKD) grade II was observed in affecteds II-5 and II-6, CKD grade IV in index person II-4. (B) p.Pro392Alafs∗43 introduces a premature termination codon in the third intracellular loop (M3R) resulting in a truncated protein that lacks the last two transmembrane domains (shown in red). (C) A homozygous CHRM3 frameshift mutation c.1173_1184 delinsT;p.Pro392Alafs∗43 was identified in index person II-4 by massively parallel sequencing; five forward (upper case) and three reverse reads (lower case) were recognized as an insertion-deletion mutation. (D and E) Three-dimensional protein structure modeling of wild-type (D) and mutant (E) receptor M3. Transmembrane helices (I-VII) and the amino- (N) and carboxy- (C) termini are indicated. Structure predictions of mutant M3 suggest complete deterioration of folding of the third intracellular loop (M3R) in addition to loss of transmembrane helices VI and VII in mutant M3. Three-dimensional protein structure modeling was obtained as a homology (comparative) protein structure with (PS)2-v2 using Protein Data Bank (PDB) entries 2rh1, 2rh1A and 1r5sA as template.31Chen C.C. Hwang J.K. Yang J.M. (PS)2-v2: template-based protein structure prediction server.BMC Bioinformatics. 2009; 10: 366Crossref PubMed Scopus (92) Google Scholar Structures were processed with Jmol. Model reliability is ≥75% for wild-type and mutant (excluding the mutant C terminus) and 55%–75% for the mutant C terminus. Using a SNP chip-based genome-wide linkage approach, we previously identified two regions of homozygosity on chromosomes 1 (35 cM) and 11 (9 cM) in four affected surviving males of this family (Figure 2).5Weber S. Mir S. Schlingmann K.P. Nürnberg G. Becker C. Kara P.E. Ozkayin N. Konrad M. Nürnberg P. Schaefer F. Gene locus ambiguity in posterior urethral valves/prune-belly syndrome.Pediatr. Nephrol. 2005; 20: 1036-1042Crossref PubMed Scopus (28) Google Scholar Exon capture and massively parallel sequencing now lead to the identification of the causative genetic defect in CHRM3, located in the chromosome 1 critical interval (Figure 2). DNA of index patient II-4 was enriched for protein-coding genes with the Agilent SureSelect Human All Exon 38 Mb kit and ran on two lanes of the Illumina GAIIx Sequencer with the paired-end protocol and a read length of 95 bp at each end, generating ∼202 × 106 raw reads of which ∼193 × 106 could be mapped to the human reference sequence. Alignment and variant calling was performed with MAQ for SNP detection (version 0.7.1)8Li H. Ruan J. Durbin R. Mapping short DNA sequencing reads and calling variants using mapping quality scores.Genome Res. 2008; 18: 1851-1858Crossref PubMed Scopus (2019) Google Scholar and BWA-short (version 0.5.7)9Li H. Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform.Bioinformatics. 2009; 25: 1754-1760Crossref PubMed Scopus (26826) Google Scholar in combination with SAMTOOLS (version 0.1.7)10Li H. Handsaker B. Wysoker A. Fennell T. Ruan J. Homer N. Marth G. Abecasis G. Durbin R. 1000 Genome Project Data Processing SubgroupThe Sequence Alignment/Map format and SAMtools.Bioinformatics. 2009; 25: 2078-2079Crossref PubMed Scopus (31781) Google Scholar for indel detection. On average, 90% of the exome was covered at least 30-fold and 97% ten-fold. Scripts developed in-house were applied to detect protein changes, affected splice sites, and overlaps with known variants (Table S1 available online). In detail, using MAQ and SAMtools, we identified a total of 672,588 variations. After filtering for a minimal phred-like consensus quality score > 15, 517303 variations remained. A stringent filter step discarding known variations with dbSNP, 1,000 genomes data, and the CCG in-house database with ∼100 exomes reduced the list to 48811. Further filtering for homozygosity (allele frequency > 75%) together with changes in protein sequence or location ±25 nts inside 3′/5′ splice sites reduced the list to 38 candidate variations. Finally, only two variations were found to overlap with the previously identified regions of significant linkage—a missense change in HEATR1:c.6037A > G;p.Lys2013Glu (RefSeq accession number NM_018072.5) and an insertion-deletion frameshift mutation in CHRM3: c.1173_1184 delinsT; p.Pro392Alafs∗43 (RefSeq accession number NM_000740.2). Both variations were not present at the Exome Variant Server, NHLBI Exome Sequencing Project (ESP, Seattle, WA; see Web Resources). HEATR1 encodes BAP28, a protein involved in rRNA transcription and processing, and knockdown of Heatr1 in zebrafish causes embryonic death due to CNS degeneration,11Azuma M. Toyama R. Laver E. Dawid I.B. Perturbation of rRNA synthesis in the bap28 mutation leads to apoptosis mediated by p53 in the zebrafish central nervous system.J. Biol. Chem. 2006; 281: 13309-13316Crossref PubMed Scopus (70) Google Scholar a phenotype considerably different from the one observed in our index patient. CHRM3, however, encodes the human muscarinic acetylcholine receptor M3 (hereafter simply called M3), a G protein-coupled receptor with seven transmembrane domains and the major receptor involved in mediating urinary bladder contraction upon micturition.7Fowler C.J. Griffiths D. de Groat W.C. The neural control of micturition.Nat. Rev. Neurosci. 2008; 9: 453-466Crossref PubMed Scopus (951) Google Scholar Both the obvious loss-of-function nature of the CHRM3 mutation and the striking similarity of the human phenotype to Chrm3 null mutant mice (discussed below) underscored the likelihood of this mutation being the causal genetic lesion in our family. The p.Pro392Alafs∗43 mutation induces a premature termination codon (PTC) at position 435. If mutant RNA transcripts were stable and translated, a truncated M3 protein would be generated lacking part of the third cytosolic loop, transmembrane domains VI and VII and the C terminus of the receptor. Three-dimensional structure predictions of mutant M3 suggest perturbed folding of the remaining third intracellular loop (Figure 2), known to be essential for signal transduction through G protein activation and scaffold protein interaction.12Borroto-Escuela D.O. Correia P.A. Perez Alea M. Narvaez M. Garriga P. Fuxe K. Ciruela F. Impaired M(3) muscarinic acetylcholine receptor signal transduction through blockade of binding of multiple proteins to its third intracellular loop.Cell. Physiol. Biochem. 2010; 25: 397-408Crossref PubMed Scopus (19) Google Scholar Sanger sequencing was performed for individuals II-2, II-3, II-4, II-5, II-6, II-7, I-1, and I-2 of the family. The c.1173_1184 delinsT; p.Pro392Alafs∗43 mutation of CHRM3 cosegregated consistently with the disease phenotype according to a recessive mode of inheritance5Weber S. Mir S. Schlingmann K.P. Nürnberg G. Becker C. Kara P.E. Ozkayin N. Konrad M. Nürnberg P. Schaefer F. Gene locus ambiguity in posterior urethral valves/prune-belly syndrome.Pediatr. Nephrol. 2005; 20: 1036-1042Crossref PubMed Scopus (28) Google Scholar and was absent from 374 Turkish control chromosomes (power >95% to distinguish a normal sequence variant given an allele frequency of 0.0113Collins J.S. Schwartz C.E. Detecting polymorphisms and mutations in candidate genes.Am. J. Hum. Genet. 2002; 71: 1251-1252Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). The mutation was also not annotated in the Exome Variant Server SNP database. All affected males were shown to be homozygous for the CHRM3 mutation whereas both parents and the unaffected sister (II-3) were heterozygous carriers. CHRM3 mutations and homozygous deletions were not identified by Sanger sequencing in five multiplex PUV families, 38 simplex PUV cases, 19 simplex PBS cases, four simplex megacystis cases, three unrelated individuals affected by UFS lacking HPSE2 mutations,4Daly 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 and a cohort of 86 individuals of the 4C-Kidney Disease Study with bladder and/or urethral anomalies (see Web Resources). Because of the recessive nature of the disease, we did not look for heterozygous deletions in this cohort. Notably, Pomper et al.14Pomper J.K. Wilhelm H. Tayebati S.K. Asmus F. Schüle R. Sievert K.D. Haensch C.A. Melms A. Haarmeier T. A novel clinical syndrome revealing a deficiency of the muscarinic M3 receptor.Neurology. 2011; 76: 451-455Crossref PubMed Scopus (8) Google Scholar recently reported an adult with micturition failure since childhood and with pupils that failed to constrict upon muscarinic stimulation. This phenotype is strikingly similar to that affecting the family in our current report. An absence of M3 protein was found in bladder tissue and a CHRM3 mutation was postulated. However, sequencing and copy number analyses were normal in the individual presented by Pomper et al., consistent with a yet-to-be-defined genetic or acquired disorder affecting generation of M3 protein and/or its stability. To date, human mutations of nicotinic acetylcholine receptor subunits have been described, e.g., in the fetal akinesia deformation sequence (MIM 208150) where they seem to be involved in morphogenetic signal transduction in addition to skeletal muscle contraction.15Michalk A. Stricker S. Becker J. Rupps R. Pantzar T. Miertus J. Botta G. Naretto V.G. Janetzki C. Yaqoob N. et al.Acetylcholine receptor pathway mutations explain various fetal akinesia deformation sequence disorders.Am. J. Hum. Genet. 2008; 82: 464-476Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar Our report describes a Mendelian human disease caused by a muscarinic acetylcholine receptor mutation, linking a developmental disorder of the renal tract to a component of the parasympathetic nervous system. Key functions of the bladder are to act as a low-pressure reservoir for urine and to expel urine per urethra. The latter is driven by detrusor contractions stimulated by parasympathetic nerves. Although male and female adult human bladders express mRNA of two muscarinic acetylcholine receptor subtypes (M2 and M3),16Mansfield K.J. Liu L. Moore K.H. Vaux K.J. Millard R.J. Burcher E. Molecular characterization of M2 and M3 muscarinic receptor expression in bladder from women with refractory idiopathic detrusor overactivity.BJU Int. 2007; 99: 1433-1438Crossref PubMed Scopus (23) Google Scholar, 17Shakirova Y. Mori M. Ekman M. Erjefält J. Uvelius B. Swärd K. Human urinary bladder smooth muscle is dependent on membrane cholesterol for cholinergic activation.Eur. J. Pharmacol. 2010; 634: 142-148Crossref PubMed Scopus (13) Google Scholar only M3 is the critical receptor for micturition,7Fowler C.J. Griffiths D. de Groat W.C. The neural control of micturition.Nat. Rev. Neurosci. 2008; 9: 453-466Crossref PubMed Scopus (951) Google Scholar mediating 95% of cholinergic contraction of the detrusor muscle.18Matsui M. Motomura D. Karasawa H. Fujikawa T. Jiang J. Komiya Y. Takahashi S. Taketo M.M. Multiple functional defects in peripheral autonomic organs in mice lacking muscarinic acetylcholine receptor gene for the M3 subtype.Proc. Natl. Acad. Sci. USA. 2000; 97: 9579-9584Crossref PubMed Scopus (384) Google Scholar In mice and rats, M3 is observed in bladders of both young animals and adults.19Zarghooni S. Wunsch J. Bodenbenner M. Brüggmann D. Grando S.A. Schwantes U. Wess J. Kummer W. Lips K.S. Expression of muscarinic and nicotinic acetylcholine receptors in the mouse urothelium.Life Sci. 2007; 80: 2308-2313Crossref PubMed Scopus (82) Google Scholar, 20Schneider T. Hein P. Michel-Reher M.B. Michel M.C. Effects of ageing on muscarinic receptor subtypes and function in rat urinary bladder.Naunyn Schmiedebergs Arch. Pharmacol. 2005; 372: 71-78Crossref PubMed Scopus (47) Google Scholar For determining the localization of M3 in embryonic and fetal tissue, immunohistochemistry studies were performed with M3 antibodies in wild-type mice and human embryos (Figure 3).21Jenkins D. Winyard P.J.D. Woolf A.S. Immunohistochemical analysis of Sonic hedgehog signalling in normal human urinary tract development.J. Anat. 2007; 211: 620-629Crossref PubMed Scopus (43) Google Scholar The latter were collected with informed consent under Polkinghorne Committee guidelines and staged by the Carnegie classification or foot length. In embryonic day 13 mice, M3 was present in nascent bladder and urethral epithelia. Later in mouse gestation, when the bladder becomes muscularized, M3 was immunodetected in bladder urothelial and detrusor muscle layers. M3 was also localized in ureteric and metanephric kidney nephron precursors. In normal human embryos, M3 protein was detected in urogenital sinus and urethral epithelia and in the late first trimester bladder in urothelia and muscle cells. M3 protein is therefore not only present in fetal bladder muscle but also in the urogenital sinus and fetal urethral epithelia, concurring with sites of embryonic Chrm3 mRNA expression according to Mouse Genome Informatics and GUDMAP databases (see Web Resources). GUDMAP also reports Chrm3 transcripts in the metanephros, and we detected M3 protein in nephron precursors. Collectively, these observations may imply that M3 has a role beyond its known contribution to detrusor contractions. Preliminary GUDMAP data exists that ChAT transcripts are expressed in embryonic kidneys as well as in embryonic bladder and urethral mesenchyme. If the encoded protein, choline acetyltransferase (ACh), is also detectable, non-neuronal ACh may be synthesized in the nascent renal tract. In future studies, it will be interesting to address the possibility that ACh is also expressed in non-neuronal tissues. In homozygous Chrm3 mutant mice lacking M3, adult detrusor contractility to carbachol ex vivo is impaired and (only) male mutant adult bladders are grossly distended in vivo.18Matsui M. Motomura D. Karasawa H. Fujikawa T. Jiang J. Komiya Y. Takahashi S. Taketo M.M. Multiple functional defects in peripheral autonomic organs in mice lacking muscarinic acetylcholine receptor gene for the M3 subtype.Proc. Natl. Acad. Sci. USA. 2000; 97: 9579-9584Crossref PubMed Scopus (384) Google Scholar Although it is unknown whether megacystis is present in younger Chrm3 mutant mice, the hypocontractile bladder phenotype is very similar to humans with CHRM3 mutations, described above. Of note, only male Chrm3 mice will develop clinical disease. This is, at least in mice most probably, related to differences between male and female urethral anatomy with male bladders being more contractile to force the urine through the longer urethra. Alternatively, female mice (but not males) might have other compensatory physiological mechanisms allowing them to empty their bladders independent of M3 action. Expression and function of M3 in rat and guinea pig urinary bladders do not seem to differ considerably between male and female animals22Creed K.E. Loxley R.A. Phillips J.K. Functional expression of muscarinic and purinoceptors in the urinary bladder of male and female rats and guinea pigs.J. Smooth Muscle Res. 2010; 46: 201-215Crossref PubMed Scopus (11) Google Scholar and with regard to overall muscarinic responsiveness this situation appears to be similar in humans.23Kories C. Czyborra C. Fetscher C. Schneider T. Krege S. Michel M.C. Gender comparison of muscarinic receptor expression and function in rat and human urinary bladder: differential regulation of M2 and M3 receptors?.Naunyn Schmiedebergs Arch. Pharmacol. 2003; 367: 524-531Crossref PubMed Scopus (56) Google Scholar In humans, we can neither rule out nor confirm a male-limited phenotype: the pedigree of the presented index family is not helpful to delineate the phenotype of female homozygous CHRM3 mutation carriers given that no female individual is affected by such a mutation. Mother and sister of the index person are both heterozygous mutation carriers with apparently normal bladder anatomy and function. With respect to PBS, the full clinical picture, including BOO, cryptorchidism, and abdominal wall distension, is limited to males; however, incomplete forms of PBS have also been described in females.24Gaboardi F. Sterpa A. Thiebat E. Cornali R. Manfredi M. Bianchi C. Giacomoni M.A. Bertagnoli L. Prune-belly syndrome: report of three siblings.Helv. Paediatr. Acta. 1982; 37: 283-288PubMed Google Scholar, 25Giuliani S. Vendryes C. Malhotra A. Shaul D.B. Anselmo D.M. Prune belly syndrome associated with cloacal anomaly, patent urachal remnant, and omphalocele in a female infant.J. Pediatr. Surg. 2010; 45: e39-e42Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar The etiology of abdominal muscle deficiency in individuals with PBS has been a matter of controversy. Either a common underlying defect of mesodermal development is proposed25Giuliani S. Vendryes C. Malhotra A. Shaul D.B. Anselmo D.M. Prune belly syndrome associated with cloacal anomaly, patent urachal remnant, and omphalocele in a female infant.J. Pediatr. Surg. 2010; 45: e39-e42Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar or, as other authors suggest, abdominal muscle deficiency seems to be a nonspecific anatomic defect that is secondary to fetal abdominal wall distention (caused by megacystis, hydronephrosis or constipation).26King C.R. Prescott G. Pathogenesis of the prune-belly anomalad.J. Pediatr. 1978; 93: 273-274Abstract Full Text PDF PubMed Scopus (37) Google Scholar In the present study, RT-PCR experiments were performed on RNA extracted from human mature abdominal wall musculature, but no M3 expression was apparent (Figure S1). These data are in favor of abdominal wall distension being a secondary event after megacystis and/or urinary stasis due to impaired urinary bladder contraction early in fetal life. Failure of bladder contractility before birth in humans with homozygous (or compound heterozygous) CHRM3 mutation might contribute to the observed bladder dysmorphology postpartum. Bladder dysfunction is likely to persist in individuals with CHRM3 mutation thoughout life, constituting an important risk factor for the progression of renal insufficiency.3Farrugia M.-K. Woolf A.S. Congenital urinary bladder outlet obstruction.Fetal Matern. Med. Rev. 2010; 21: 55-73Crossref Scopus (21) Google Scholar Chrm3 mutant mice also display impaired salivation and dilated pupils, consistent with a role for muscarinic ACh actions in serous salivary secretion and pupillary constriction,18Matsui M. Motomura D. Karasawa H. Fujikawa T. Jiang J. Komiya Y. Takahashi S. Taketo M.M. Multiple functional defects in peripheral autonomic organs in mice lacking muscarinic acetylcholine receptor gene for the M3 subtype.Proc. Natl. Acad. Sci. USA. 2000; 97: 9579-9584Crossref PubMed Scopus (384) Google Scholar and a similar phenotype is present when human CHRM3 is mutated. Deletions within 17q12 encompassing hepatocyte nuclear factor 1B (HNF1B [MIM 189907]) have been reported in sporadic PBS,27Murray P.J. Thomas K. Mulgrew C.J. Ellard S. Edghill E.L. Bingham C. Whole gene deletion of the hepatocyte nuclear factor-1beta gene in a patient with the prune-belly syndrome.Nephrol. Dial. Transplant. 2008; 23: 2412-2415Crossref PubMed Scopus (32) Google Scholar but whether HNF1B itself or a nearby disrupted gene is causative remains unclear, and HNF1B mutations have not yet been implicated in familial PBS. Furthermore, although HNF1B mutations are a not uncommon cause of dysplastic kidneys,2Weber S. Moriniere V. Knüppel T. Charbit M. Dusek J. Ghiggeri G.M. Jankauskiené A. Mir S. Montini G. Peco-Antic A. et al.Prevalence of mutations in renal developmental genes in children with renal hypodysplasia: results of the ESCAPE study.J. Am. Soc. Nephrol. 2006; 17: 2864-2870Crossref PubMed Scopus (285) Google Scholar, 28Adalat S. Woolf A.S. Johnstone K.A. Wirsing A. Harries L.W. Long D.A. Hennekam R.C. Ledermann S.E. Rees L. van't Hoff W. et al.HNF1B mutations associate with hypomagnesemia and renal magnesium wasting.J. Am. Soc. Nephrol. 2009; 20: 1123-1131Crossref PubMed Scopus (198) Google Scholar such patients rarely have overt bladder lesions. Importantly, in our CHRM3 mutation family, HNF1B was normal by sequencing and seeking copy number variants (data not shown). Mice mutant for β2 and β4 or α3 subunits of the nicotinic ACh receptor29Xu W. Orr-Urtreger A. Nigro F. Gelber S. Sutcliffe C.B. Armstrong D. Patrick J.W. Role L.W. Beaudet A.L. De Biasi M. Multiorgan autonomic dysfunction in mice lacking the beta2 and the beta4 subunits of neuronal nicotinic acetylcholine receptors.J. Neurosci. 1999; 19: 9298-9305Crossref PubMed Google Scholar, 30Xu W. Gelber S. Orr-Urtreger A. Armstrong D. Lewis R.A. Ou C.N. Patrick J. Role L. De Biasi M. Beaudet A.L. Megacystis, mydriasis, and ion channel defect in mice lacking the alpha3 neuronal nicotinic acetylcholine receptor.Proc. Natl. Acad. Sci. USA. 1999; 96: 5746-5751Crossref PubMed Scopus (246) Google Scholar display enlarged, hypocontractile bladders, as a result of compromised neural relaying via autonomic ganglia, and may serve as models for humans with congenital BOO who do not have CHRM3, HPSE2, or HNF1B mutations. We suggest that the next decade will witness the unraveling of genetic causes of bladder malformations, thus providing insights into the mechanisms of both normal development and explaining how bladder dysmorphology arises in the context of PBS, PUV, UFS and other related syndromes.3Farrugia M.-K. Woolf A.S. Congenital urinary bladder outlet obstruction.Fetal Matern. Med. Rev. 2010; 21: 55-73Crossref Scopus (21) Google Scholar We thank the patients and their family for participating in this study, Bettina Cirkel and Christian Becker for excellent technical support and Anne Deix for critical advice. Financial grant support was received from the Kidney Research UK (W.G.N. and A.S.W.), Kids Kidney Research and Kidneys for Life (A.S.W.), Manchester NIHR Biomedical Research Centre (A.S.W., R.J., N.A.H., H.S., and W.G.N.), and The Wellcome Trust (A.S.W. and N.A.H.). S.W. and F.S. received financial support from the Fritz Thyssen grant. H.R. and M.D. are members of the German network for congenital uro-rectal malformations (CURE-Net), which is supported by a research grant (01GM08107) from the German Federal Ministry of Education and Research. The 4C-Kidney Disease Study is supported by research grants of the KfH Foundation for Preventive Medicine, ERA-EDTA and IFB Transplantation. Download .pdf (.13 MB) Help with pdf files Document S1. One Table and One Figure The URLs for data presented herein are as follows:The Cardiovascular Comorbidity in Children with Chronic Kidney Disease Study, http://www.4c-study.orgExome Variant Server, http://snp.gs.washington.edu/EVSGUDMAP, http://www.gudmap.orgMouse Genome Informatics, http://www.informatics.jax.orgOnline Mendelian Inheritance in Man (OMIM), http://www.omim.org
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