Loss-of-function variants in myocardin cause congenital megabladder in humans and mice

Abstract

Myocardin (MYOCD) is the founding member of a class of transcriptional coactivators that bind the serum-response factor to activate gene expression programs critical in smooth muscle (SM) and cardiac muscle development. Insights into the molecular functions of MYOCD have been obtained from cell culture studies, and to date, knowledge about in vivo roles of MYOCD comes exclusively from experimental animals. Here, we defined an often lethal congenital human disease associated with inheritance of pathogenic MYOCD variants. This disease manifested as a massively dilated urinary bladder, or megabladder, with disrupted SM in its wall. We provided evidence that monoallelic loss-of-function variants in MYOCD caused congenital megabladder in males only, whereas biallelic variants were associated with disease in both sexes, with a phenotype additionally involving the cardiovascular system. These results were supported by cosegregation of MYOCD variants with the phenotype in 4 unrelated families by in vitro transactivation studies in which pathogenic variants resulted in abrogated SM gene expression and by the finding of megabladder in 2 distinct mouse models with reduced Myocd activity. In conclusion, we have demonstrated that variants in MYOCD result in human disease, and the collective findings highlight a vital role for MYOCD in mammalian organogenesis.

Keywords: Genetic diseases; Molecular genetics; Muscle Biology; Urology.

Figure 1. Identification of MYOCD variants in 4 families with congenital megabladder.

(A) Pedigrees of 4 families presenting with congenital megabladder. Affected individuals are marked with black symbols. Available genotypes are shown beneath symbols. Slashed symbols denote deceased individuals. Gestational age is indicated above the symbol. Gray symbols denote stillbirths with external features consistent with PBS. N, normal bladder ultrasound; P with arrow, proband of the family; npe, normal prenatal echo. (B) Schematic diagram showing functional domains within MYOCD and location of the identified mutations (7). Conservation of respective amino acid positions with the mutated residues are highlighted. (C) Ultrasound images showing enlarged bladder of indicated fetuses of families B and C; asterisks denote bladder.

Figure 2. Bladder and kidney abnormalities in family A.

(A and C) From healthy midgestation fetuses. (B and D) From affected fetus from family A. H&E staining from urinary bladders shows transverse sections of muscle bundles (TSM) and longitudinal sections of muscle bundles (LSM) in the healthy and affected fetuses. Note, however, that the bundles in the affected fetus appear disorganized and less compact compared with the well-defined muscle fibers in the control. (C) In a control fetal kidney, glomeruli (G) and tubules (T) are evident. (D) In the kidney from the affected fetus, glomeruli are cystic, with dilated Bowman’s spaces (asterisks), a characteristic of fetal urinary flow obstruction. Scale bars: 20 μm.

Figure 3. MYOCD mutations abrogate activation of SM cell gene expression in vitro.

(A) Mouse fibroblasts were transiently transfected for 48 hours with expression vectors for MYOCD or the indicated MYOCD mutants (mutation A1: p.S229Qfs*17; mutation A2: p.E530G; mutation B: p.R115*) and a luciferase reporter linked to the Transgelin (Sm22) promoter (n = 3/group). (B) Mouse fibroblasts were transfected with expression plasmids encoding MYOCD or the indicated mutants (n = 4/group). An empty expression plasmid served as a control. RNA was isolated, and SM gene expression was measured by qPCR. GAPDH was used to normalize expression. Overexpression levels of MYOCD were comparable between conditions (Supplemental Figure 3). *P < 0.01 compared with WT MYOCD according to 1-way ANOVA with Dunnett’s multiple comparison test. Shown are representative experiments of 2 independent repeats.

Figure 4. Myocd loss of function in mice causes the megabladder phenotype.

(A) Schematic representation of the Myocd^ΔLZ allele, in which 24 nucleotides are deleted in the LZ domain (p.I531_R539delinsM in NP_666498.2). (B–E) αSMA immunohistochemistry in 1-day-old neonates from Myocd^+/– and Myocd^ΔLZ/+ crosses. (C) Compound heterozygosity (Myocd^ΔLZ/–; reminiscent of the alleles present in the affected individuals in family A) results in wall thinning of the bladder and lack of SM cells compared with the WT bladder wall (B). (D and E) Higher magnifications of WT and Myocd^ΔLZ/– bladder walls showing lack of αSMA-expressing muscle bundles in the putative detrusor layer, although expression appeared retained in myofibroblast-like cells in the lamina propria directly below the urothelium and in the rectum. (F) Schematic representation of the Myocd^Mgb allele. (G–J) Representative αSMA immunohistochemistry in P1 bladder of WT (G and H) and Myocd^mgb/– compound heterozygote (I and J) (from a cross of Myocd^mgb/+ and Myocd^+/– mice). Note the severe bladder distention and absent detrusor muscle in the Myocd^mgb/– bladder. Bl, bladder; In, intestine; Re, rectum; U, urethelium; S, submucosa; DM, detrusor muscle. Scale bars: 500 μm (B, C, G, and I); 100 μm (D, E, H, and J). (K) Myocd mRNA levels were quantified by qPCR using E15 bladders of WT, Myocd^+/–, Myocd^mgb/+, Myocd^mgb/mgb, and Myocd^mgb/– mice. The absolute numbers of embryos developing megabladder as a fraction of the total number of embryos analyzed are indicated above the graph and reveals a highly penetrant phenotype in the Myocd^mgb/mgb and Myocd^mgb/– mice.