Muscarinic Acetylcholine Receptor M3 Mutation Causes Urinary Bladder Disease and a Prune-Belly-like Syndrome

Abstract

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.

Figure 1

Clinical Features in Family with CHRM3 Mutation (A) Cystogram of individual II-7 demonstrating a massively enlarged bladder. (B) Cystogram of index person II-4 showing an irregular-walled bladder with diverticula (black arrows) and unilateral high-grade vesicoureteral reflux into a distorted renal pelvis (white arrow). (C) Abdominal wall laxicity of index person II-4. (D) Impaired pupillary constriction to bright light in index person II-4.

Figure 2

Deletion-Insertion Mutation of CHRM3 (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. 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.

Figure 3

M3 Localization in the Developing Renal Tract Immunohistochemistry of M3 on sections of normal mouse (A–E) and human (F–I) embryos by respectively applying M3 antibodies Sigma M0194 and Abcam 60981. (A and B) Embryonic day 13 sagittal sections. (A) Localization of M3 in epithelia of the urogenital sinus, which has separated from the hindgut (hg); the upper (cranial) part of the sinus is forming the bladder (bl) and the lower part of the sinus is forming the urethra (u) extending into the genital tubercule (gt). M3 is also detected in hindgut epithelia. The spine (sp) is dorsal to the hindgut. (B) Adjacent section with primary antibody omitted. (C) Embryonic day 15 bladder. M3 localization in detrusor smooth muscle (dsm) as well as urothelia; the asterisk indicates lumen. (D) Longitudinal section through embryonic day 14 ureter, when urothelium is surrounded by mesenchyme which has yet to form muscle; M3 is localized in epithelia. (E) Forming nephron in embryonic day 13 metanephric kidney. The upper part is the precursor of the glomerulus (g) while the lower section will form the tubule (t); in both M3 is detectable. (F) Sagittal section at 51 days post conception when the urogenital sinus (ugs) and hindgut (hg) terminate in a common cloaca (c). M3 is localized to both urogenital sinus and hindgut epithelia. (G) Transverse section through the urethra at the bladder outlet (10 weeks after conception); M3 localization to urothelia while the surrounding mesenchyme (m) is negative is shown. (H) Bladder wall 12 weeks after conception; M3 detection in human detrusor smooth muscle (dsm; indicated by arrows) and urothelia; the asterisk indicates lumen. (I) Adjacent section with primary antibody omitted. Scale bars represent 500 μm (A and B), 100 μm (C), 125 μm (D), 50 μm (E), 200 μm (F), 100 μm (G), and 400 μm (H and I).