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

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.


Whole-exome sequencing family B
Target DNA enrichment was designed to capture all exons and 40 base pair (bp) of the intron/exon boundaries and was achieved using the Agilent SureSelectXT custom Kit. NGS was performed on the Illumina HiSeq® 2500 platform according to manufacturer's protocol.
Identified variant has been submitted to Clinvar.

Array CGH family C
Array-CGH analysis was performed on proband and parents using Agilent 180K oligo-array (Amadid 023363, Agilent, Santa Clara, CA), with 13-kb overall median probe spacing and a GRCh37/hg19 browser. Standard methods were used for labeling and hybridization. Samples were hybridized against a pool of 40 healthy sex-matched human reference samples. Data were analyzed with Genomic Workbench 6.5 (Agilent) and Cartagenia [BENCHlab CNV v5.0 (r6643); Agilent]. Identified variant has been submitted to Clinvar. Mutagenesis primers are shown in supplemental Table 3.

Expression vectors, cell culture, tranfection
To activate endogenous smooth muscle gene expression by MYOCD and the mutants, MYOCD constructs were transfected in mouse fibroblasts (CH3-10T1/2 cells, ATCC), which were cultured in DMEM supplemented with 10% FBS, 1% penicillin-streptomycin and 200 mM L-glutamine (all from Gibco, Life Technologies) in a humidified incubator at 37 °C with 5% CO2.
1 µg pcDNA-MYOCD plasmid was transfected per well of a 6-well plate using Genejammer (Stratagene) according to manufacturer's instruction at 50-75% confluence. 48 hours after transfection, cells were harvested for RNA isolation using TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.

Quantitative PCR
For gene expression analysis in the 10T1/2 cells and P1 mouse bladders, heart and aorta, ~1 μg RNA was treated with DNAse I (Invitrogen, Carlsbad, CA, USA) and cDNA was synthesized using Superscript II RT (Invitrogen, Carlsbad, CA, USA). Quantitative PCR (qPCR) was performed on a Lightcycler 480 (Roche Diagnostics, Risch-Rotkreuz, Switzerland) using SYBR green (Roche Diagnostics, Risch-Rotkreuz, Switzerland). Gene expression was normalized to Gapdh expression. Analysis of qPCR data was performed using LinRegPCR analysis software (2).
For gene expression analysis in E15 mouse bladders, cDNA was synthesized from ~300ng RNA using the QuantiTect Reverse Transcription kit (Qiagen, Cat. No. 205313). Quantitative PCR (qPCR) was performed on a QuantStudio 5 (ThermoFisher Scientific) using PowerUp Sybr Green Master Mix (ThermoFisher Scientific, Cat. No. A25741). Gene expression was normalized to Gapdh expression. Analysis of qPCR data was performed using LinRegPCR analysis software (2). Primers used are included in Supplemental Table 3.

Semi-quantitative RT-PCR
Neonatal mouse bladders were frozen in liquid nitrogen and the frozen tissue was crushed with a pestle. The resulting powder was resuspended in Buffer RLT from the RNeasy Micro Kit (Qiagen, Cat. No. 74004), passed through a QiaShredder column (Qiagen, Cat. No. 79654), and RNA was isolated according to the RNeasy Micro Kit protocol. One microgram total RNA was used as input for reverse transcription using the QuantiTect Reverse Transcription Kit (Qiagen, Cat. No. 205313) according to the manufacturer's protocol. To control for genomic DNA contamination, one reaction was set up using 1µg RNA from Myocd∆LZ/+ bladder with all reaction components except the reverse transcriptase enzyme (-RT). One microliter from each RT reaction was used as template for PCR using the primers LZ-fwd (5'-acggacgagagtctgctgag-3'), LZ-rev (5'-ctgcagctgctcttctgcttc-3'), and GAPDH fwd (5'-ggtggacctcatggcctaca-3'), GAPDH rev (5'-ctctcttgctcagtgtccttgct-3') as an internal control in each reaction. Samples were taken from each reaction after the indicated number of cycles and run on a 2% agarose/TBE gel for 80 minutes at 85V.

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The Myocd ∆LZ allele was generated by CRISPR-Cas9 genome editing at the Cardiovascular Research Institute, University of California, San Francisco (UCSF), CA as previously described (4). Briefly, candidate spCas9 guide sequences were identified within a 200bp region of the mouse genome encompassing the coding sequence of the Myocd leucine zipper (LZ) domain. The guide sequence 5'-aagtgatcaaccagctcacc(tgg)-3' (PAM sequence indicated in parentheses) was selected based on predicted high on-target and low off-target effects.
Annealed oligonucleotides were filled in using Klenow fragment (NEB, M0210) and PCR amplified. sgRNA was transcribed using the MEGAshortscript T7 kit (Life Technologies, AM1354) and then purified using the MEGAclear kit (Life Technologies, AM1908). Purified sgRNA and humanized Streptococcus pyogenes Cas9 (hspCas9) mRNA (SBI, CAS500A-1) were co-injected into the cytoplasm of fertilized mouse oocytes using standard techniques as described previously (5). F0 founders were screened for editing events by PCR amplification and BclI digestion of an 839bp fragment encompassing the targeted genomic region using the following primers: Myocd LZ genotyping F, 5'-tgctgctgggtgaagagac-3' and myocd LZ genotyping R, 5'-agtgtcaggcaccaagacag-3'. BclI digestion of this fragment from a wild type allele results in two fragments of ~400bp, whereas the edited allele was predicted to be resistant to BclI digestion owing to the coincidence of the BclI site with the guide sequence.
One F0 male founder carried a 24bp deletion, leading to loss of 9 amino acids (INQLTWKLR) and the creation of one amino acid (Met) from the encoded protein (p.I531_R539delinsM in NP_666498.2), including one isoleucine and two leucine residues previously shown to be critical for myocardin dimerization (6). This founder was outcrossed to wild type females to establish the F1 generation. A single F1 male carrying the 24bp deletion was used to establish the Myocd ∆LZ line, and a colony was generated by outcrossing to wild type animals on a mixed 6 genetic background. Animals were maintained according to the NIH Guide for the Care and Use of Laboratory Animals.

Histology of Myocd ∆LZ mice and Myocd knockout mice
Myocd ∆LZ/+ males were crossed to female mice harboring a Myocd gene knock out allele (Myocd +/-)(7). Neonates were collected shortly after birth and euthanized by decapitation with tail tissue collected for PCR genotyping. Whole neonates were fixed in 4% formaldehyde, embedded in paraffin, and 7µm transverse sections were obtained using a microtome and affixed to glass slides. Serial sections were stained with haemotoxylin and eosin or processed for immunohistochemistry as follows: Following deparaffinization and rehydration, heatmediated antigen retrieval was performed in citrate buffer. Staining was performed using the Vector ImmPRESS Excel Staining Kit (Vector Labs, MP-7601) according to the manufacturer's instructions using alpha smooth muscle actin antibody (Abcam, ab5694) or Rabbit IgG isotype control (Cell Signaling Technology, #3900) diluted 1:100 in PBS + 0.1% Tween-20 with 2.5% Horse serum. Following 3,3'-Diaminobenzidine (DAB) staining, sections were counterstained with haemotoxylin and mounted using VectaMount Medium (Vector Laboratories). The megabladder phenotype was defined by megacystis at autopsy, and on histology, finding both a lack of SM-like cells and immunostaining for αSMA.

Genomic organization of the megabladder (mgb) mouse model
Generation of the Mgb mouse model has been described (9). In short, the MLR19 transgene was randomly inserted into chromosome 16 between 26.6 and 27.5 Mb, and in conjunction with a portion of chromosome 16, the transgene then translocated into a second domain on chromosome 11, resulting in the development of megabladder in homozygous mice (9). Triple fluorescence in situ hybridization (FISH) analysis was used to narrow the translocation breakpoint on chromosome 11 (Suppl. Figure 5). FISH using 8 BACs from chromosome 11 (Supplemental Table 4) and an Mgb transgene positive BAC clone was performed to screen and map the Mgb interchromosomal insertional translocation site under guidance of the Molecular Cytogenetics core at The Ohio State University as described (9). Briefly, bone marrow was aspirated from the femur of transgenic and wild-type mice and grown overnight in RPMI medium supplemented with 15% FBS. Cells were harvested using standard

Myocd In Situ Hybridization.
In situ hybridization for Myocd expression was performed as previously described (10). Briefly, E15 embryos from timed matings of Myocd mgb/+ mice were collected and specimens were fixed and processed using standard procedures. Serial paraffin-embedded transverse sections (10 uM) of the bladder were affixed to slides. In situ hybridization using 35 S-UTP radiolabeled riboprobes was performed by prehybridizing sections with hybridization solution for 90 minutes while riboprobes were labeled with 35 S-UTP. The riboprobes were purified with a NucAway kit (Amersham) and the slides were hybridized with 70,000 DPM counts of riboprobe in hybridization solution. Slides were incubated O/N at 50 o C in a hybridization oven. Following incubation, the slides were washed in high stringency washes (FSM, STE x2, STE + yeast-tRNA and RNase, STE + BME, FSM x2, 2x SSC, 0.1x SSC) and dehydrated. Slides were airdried, placed on film, and exposed for 3 days. Following exposure, slides were emulsion coated (Kodak), incubated at 4 o C for 10-14 days, and developed. Lastly, slides were cover slipped with permaslip and observed under dark field and fluorescence. The myocardin in situ probe used was derived from published exon sequences as previously described and annotated(10).

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Data are presented as the mean value ± standard deviation. Measurements were taken from distinct samples. Statistical tests used to compare between conditions are indicated in the figure legends.

Data availability
All data generated or analysed during this study are included in this published article (and its supplementary information files).