Recessive CHRM5 variant as a potential cause of neurogenic bladder

Abstract

Neurogenic bladder is caused by disruption of neuronal pathways regulating bladder relaxation and contraction. In severe cases, neurogenic bladder can lead to vesicoureteral reflux, hydroureter, and chronic kidney disease. These complications overlap with manifestations of congenital anomalies of the kidney and urinary tract (CAKUT). To identify novel monogenic causes of neurogenic bladder, we applied exome sequencing (ES) to our cohort of families with CAKUT. By ES, we have identified a homozygous missense variant (p.Gln184Arg) in CHRM5 (cholinergic receptor, muscarinic, 5) in a patient with neurogenic bladder and secondary complications of CAKUT. CHRM5 codes for a seven transmembrane-spanning G-protein-coupled muscarinic acetylcholine receptor. CHRM5 is shown to be expressed in murine and human bladder walls and is reported to cause bladder overactivity in Chrm5 knockout mice. We investigated CHRM5 as a potential novel candidate gene for neurogenic bladder with secondary complications of CAKUT. CHRM5 is similar to the cholinergic bladder neuron receptor CHRNA3, which Mann et al. published as the first monogenic cause of neurogenic bladder. However, functional in vitro studies did not reveal evidence to strengthen the status as a candidate gene. Discovering additional families with CHRM5 variants could help to further assess the genes' candidate status.

Keywords: CAKUT; CHRM5; neurogenic bladder; whole-exome sequencing.

Figure 1.. Pedigree and clinical information of individual B2797–21.

A) Pedigree of individual B2797–21: affected boy with neurogenic bladder, small trabeculated urinary bladder, bilateral severe hydronephrosis, grade V VUR right, chronic kidney disease (stage 4) and unaffected parents (−11 and 12); red dot, individual included in ES. B-D) FLU-micturating cystourethrogram: lateral projection, right side (B), lateral projection, left side (C), and anterior-posterior projection (D) showing right-sided grade V vesicoureteric reflux (black arrow) with trabeculated urinary bladder (black arrowhead), likely caused by neurogenic bladder. E-G) Ultrasound of right kidney (E), left kidney (F), and urinary bladder (white triangle) (G), showing severe hydronephrosis (white asterisk) and hydroureter (black arrows).

Figure 2.. Tc99m-Mag3 Renal Scan at the age of 2 years and 10 months

A) Tc99m-Mag3 Renal Scan and B) Flow study of the Tc99m-Mag3 Renal Scan both show the left kidney is contributing to renal function by 84%. The right kidney is contributing to renal function by 16%. Red line, left kidney; blue line, right kidney; black line, aorta.

Figure 3.. Exon and protein structure of human CHRM5 cDNA.

A) Exon structure of human CHRM5. Exon numbers are denoted in black and white. B) Protein domain structure of the human CHRM5 protein with seven transmembrane regions (TM1-TM7). C) Position of the homozygous missense variant p.Gln184Arg identified in individual B2797–21. Sanger sequencing showing the homozygous variant in the affected individual (B2797–21) compared to heterozygous variants in both parents (father B2797–11 and mother B2797–12). D) Clustal alignment of amino acid sequences of CHRM5 to demonstrate evolutionary conservation from mammalia to insectae for each amino acid residue. Glossary: UTR, untranslated region; ATG, start codon; TGA, stop codon; TM, transmembrane region; HOM, homozygous; HET heterozygous; H.s., Homo sapiens; M.m., Mus musculus; G.g., Gallus gallus; X.t., Xenopus tropicalis; D.r., Danio rerio; C.i., Ciona intestinalis; C.e., Caenorhabditis elegans; D.m., Drosophila melanogaster.

Figure 4.. Structural model of the CHRM5 variant and functional studies.

A-B) Crystal structure of CHRM5. B) The p.Gln184Arg variant is located in the second extracellular loop (ECL2) of the receptor and replaces the polar amino acid glutamine with a positively charged amino acid arginine. C) Schema of seven transmembrane-spanning muscarinic acetylcholine (ACh) receptor. The red section marks the second extracellular loop (ECL2) where the variant p.Gln184Arg is located. D) Schema of M₅ mAChR activation. ACh activates the M₅ mAChR causing activation of the Gq signaling pathway leading to production of the second messenger IP1. Asterisks mark allosteric binding sites. E-G) Radioligand binding experiments using the antagonist [³H]-NMS at FlpIn CHO cells. E) The Gln184Arg variant does not affect receptor expression or binding affinity of the radioligand [³H]-NMS in comparison to the wild-type (WT) M₅ mAChR as determined through saturation binding experiments. F) A modest 2-fold difference in binding affinity of ACh at the Gln184Arg variant is observed in comparison to WT M₅ mAChR as determined through competition binding between a range of ACh concentrations and a K_D concentration of [³H]-NMS. G) IP1 accumulation following activation of the Gln184Arg mutant in response to ACh is indistinguishable from the response observed at the WT M₅ mAChR. For all experiments, data represent the mean ± S.E.M. of the three individual experiments performed in duplicate.