A homozygous missense variant in CHRM3 associated with familial urinary bladder disease

Abstract CHRM3 codes for the M3 muscarinic acetylcholine receptor that is located on the surface of smooth muscle cells of the detrusor, the muscle that effects urinary voiding. Previously, we reported brothers in a family affected by a congenital prune belly‐like syndrome with mydriasis due to homozygous CHRM3 frameshift variants. In this study, we describe two sisters with bladders that failed to empty completely and pupils that failed to constrict fully in response to light, who are homozygous for the missense CHRM3 variant c.352G > A; p.(Gly118Arg). Samples were not available for genotyping from their brother, who had a history of multiple urinary tract infections and underwent surgical bladder draining in the first year of life. He died at the age of 6 years. This is the first independent report of biallelic variants in CHRM3 in a family with a rare serious bladder disorder associated with mydriasis and provides important evidence of this association.


| INTRODUCTION
Urinary bladder abnormalities not only devastate children's health but also cause major disruption to the lives of families. The last decade has witnessed discoveries of gene variants in specific classes of congenital urinary bladder abnormalities, including prune belly, 1,2 urofacial 3-6 and visceral myopathy (megacystis-microcolon-intestinalhypoperistalsis) [7][8][9][10][11] syndromes. In some of these cases, the associated genes encode proteins involved in the intracellular contractile apparatus of bladder smooth muscle. These genes comprise MYH11 encoding myosin light chain kinase, 8 ACTA2 encoding α-smooth muscle actin, 2 and ACTG2 encoding γ2-smooth muscle actin, 7 all muscle cytoskeletal proteins; and MYLK that codes for myosin light chain kinase, required for myosin activation. 9 In other cases, genes that have been associated with bladder malformations code for proteins implicated in the neuro-muscular circuits required for bladder voiding. 12,13 As examples, HPSE2 codes for heparanase 2 and LRIG2 codes for leucine rich repeats and immunoglobulin-like domains 2, proteins detected in foetal bladder nerves; variants of these genes are implicated in urofacial syndrome. 5,6,14 Also in the neuro-muscular category is CHRM3 that encodes M3, the key acetylcholine receptor expressed by detrusor smooth muscle cells that is required for parasympathetic-driven detrusor contraction and bladder emptying. 2 In this report, we present a second family carrying a homozygous variant in CHRM3 associated with familial urinary bladder disease.

| METHODS
The family ( Figure 1A) was recruited as part of a nationally and internationally sourced cohort of 20 unrelated families with phenotypes overlapping urinary bladder voiding dysfunction of unknown cause. HEK293 cells were cultured overnight to 40% to 50% confluency in Dulbecco's modified Eagle's medium high-glucose, DMEM (Sigma), supplemented with 10% foetal bovine serum (Sigma) in six well tissue culture-treated plates at 37 C with 5% CO 2 . Cells were transiently transfected with 1 μg of mini-gene vector using Lipofectamine LTX (Thermofisher Scientific) and the manufacturer's recommended protocol. Following 20 hours incubation at 37 C with 5% CO 2 , RNA was   likely also their brother who died in childhood. In contrast, in mice carrying a homozygous targeted Chrm3 mutation, the overdistended bladder phenotype only manifests in males. 17 This observation might be in part explained by the fact that the male urethra is longer than the female counterpart, so higher intra-vesical pressures are needed to expel urine. In fact, the male phenotype may be more severe in humans, with early deaths only observed in males in this report and that of Weber et al. 1 The precise mechanism whereby males are more severely affected is as yet unclear but may be related to the anatomical differences, including urethral length. In both the current family CHRM3 sequencing and copy number analyses were normal. We speculate that this patient may have harboured non-coding variants that affect transcription of CHRM3.
CHRM3 comprises five exons although only one of these is coding.
Therefore, the frameshift variant identified in the original report 1 is likely to escape nonsense-mediated decay and a truncated protein will likely be formed. The missense variant reported here has no effect on splicing in an in vitro system and although it is predicted to result in reduced function, the specific mechanism requires further elucidation.
Unfortunately, CHRM3 genotyping data representing a healthy Malaysian population is not available either publically or could be CHRM3 is overexpressed in bladders of adults with benign prostatic hypertrophy, 20 most likely a compensatory response to anatomic bladder outflow obstruction. The M3 acetylcholine receptor is in fact more widely expressed than in the detrusor and ciliary muscles.
Although dilated pupils are present in all affected individuals reported to date with biallelic CHRM3 variants indicating that this is a core feature of the phenotype, the association of bladder dysfunction with mydriasis is not exclusive to individuals with CHRM3 variants. Of note individuals with biallelic pathogenic variants in MYL9 have mydriasis as part of megacystis microcolon intestinal hypoperistalsis phenotype. 11 The similarity in the bladder-eye phenotype in the two families would indicate that the missense variant results in a loss of function comparable to that predicted by the frameshift variant identified by Weber et al. 1 It will be important through murine and cellular studies to undertake functional studies to determine the mechanism of action of the reported disease-associated variants. The report of this second family provides independent evidence that biallelic variants in CHRM3 result in severe bladder voiding dysfunction which on the basis of the families described to date is associated with mydriasis and is more severe, resulting in early lethality, in males.