The work by Newman and collaborators is an interesting case report of a second family with a very rare congenital urinary bladder disease. Using a small targeted NGS gene panel the authors identified a novel homozygous missense variant in the CHRM3 gene assumed to be associated with this phenotype. The manuscript is much around the clinical description of two female patients with urinary and bladder dysfunction. There was an additional patient, an affected male that died at 6 years of age. So little clinical information is known and no genetic studies have been conducted in this patient.

A limitation of this work is that no functional data were obtained to demonstrate the pathogenic effect of this missense substitution, which would be also useful to determine the underlying pathophysiological mechanism causing disease in this family.

My overall impression is that this manuscript could be presented in a more condensed format, such reduction would not compromise its content or scope.

Some aspects should be also addressed and improved as follows.

1. Major remarks:

1.1 The variant identified in the first case of prune belly-like syndrome by Weber et al. (2011) is truncating, thus predictable to cause a frameshift of the reading frame and the M3 acetylcholine receptor loss-of-function. In a subsequent report by Pomper and collaborators (2011), there was also a reduction in M3 protein levels in a patient with similar features, but the genetic defect remained elusive.

Thus, and concerning the missense variant presented here, the authors stated that it affects a “residue within the second transmembrane domain" of this receptor. The effect of this variant should be better explored in the manuscript. Also, further experimental data demonstrating its impact at the protein level (resorting to patients’ samples or a knock-in animal model) would certainly enrich this work. At least a more detailed bioinformatic analysis should be performed, for instance, showing a change in the transmembrane domain. Could this variant have an additional effect over splicing (gene expression) besides the foreseeable substitution of an aminoacid?

1.2 One major evidence towards the pathogenicity of the c.352G>A variant was its absence over 120,000 healthy individuals in gnomAD database. Is the Malaysian population well represented in this database? Should ethnical-matched controls more suitable to determine its frequency? Nonetheless, based on the information available in gnomAD, most of the variants in the CHRM3 gene (244 out of 255) were found in heterozygosity in only one or two individuals. CHRM3 homozygous missense variants are rare in healthy individuals.

1.3 The strategy chosen to conduct genetic analysis was an NGS gene panel of only 13 genes, previously known to be linked to urinary bladder voiding problems. As there was parental consanguinity reported could runs of homozygosity (using the genome/exome data) point towards possible additional genetic causes for the different clinical aspects found in these patients (i.e. intellectual disability)?

1. Minor remarks:

2.1 Concerning the international cohort with urinary bladder disturbance, could the authors mention the total number of patients enrolled/studied in this project, and how many were successfully characterized?

2.2 Please complete the version of the cDNA sequence accession number for CHRM3 used (probably NM_000740.3?).

2.3 The legend of figure 1 F) should be corrected to “no change” instead of “homozygous for variant”.

All my remarks have been considered or answered by the authors.