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.

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 six 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.

Purpose: To develop a comprehensive analysis framework to identify pre-messenger RNA splicing mutations in the context of rare disease. Methods: We assessed ‘variants of uncertain significance’ through six in-silico prioritization strategies. Firstly, through comparison to functional analyses, we determined the precise effect on splicing of variants identified through clinical multi-disciplinary meetings. Next, we calculated the sensitivity of in-silico prioritization strategies to distinguish known splicing mutations from common variation (>2% in allele frequency in gnomAD) within relevant disease genes. These approaches defined an accurate in-silico strategy for variant prioritization, which we retrospectively applied to a large cohort of 2783 individuals who had previously received genomic testing for rare genomic disorders. We assessed the clinical impact of such prioritization strategies alongside routine diagnostic testing strategies. Results: We identified 21 variants that potentially impacted splicing, and used cell based splicing assays to identify those variants which disrupted normal splicing. These findings underpinned new molecular diagnoses for 14 individuals. This process established that the use of pre-defined thresholds from a machine learning splice prediction algorithm, SpliceAI, was the most efficient method for variant prioritization, with a positive predictive value of 86%. We analysed 1,346,744 variants identified through diagnostic testing for 2783 individuals and observed that splicing variant prioritization strategies would improve clarity in clinical analysis for 15% of the individuals surveyed. Prioritized variants could provide new molecular diagnoses or provide additional support for molecular diagnosis for up to 81 individuals within our cohort. Conclusion: We present an in-silico and functional analysis framework for the assessment of variants impacting pre-messenger RNA splicing which is applicable across monogenic disorders. Incorporation of these strategies improves clarity in diagnostic reporting, increases diagnostic yield and, with the advent of targeted treatment strategies, can directly alter patient clinical management. Key Highlights: We establish an in-silico and functional analysis framework for the incorporation of splice variant assessment into diagnostic testing that is applicable across monogenic disorders. After assessment of six distinct variant prioritization strategies, we concluded that SpliceAI was the best method to accurately identify genomic variation disrupting normal pre-mRNA splicing. We determined this through (i) functional assessment of novel ‘variants of uncertain significance’ described in this study, and (ii) calculation of sensitivity and specificity for prioritization strategies to distinguish known splicing mutations from common variants in the general population. We describe novel disease-causing variants with support from cell based functional assays which underpin autosomal recessive, autosomal dominant and X-linked Mendelian disorders. This includes variants which are deeply intronic, within the nearby splice region of canonical splice sites and variants which activate cryptic splice sites within the protein-coding regions of genes. We integrated the best performing variant prioritization strategy alongside clinical diagnostic testing for 2783 individuals referred to a well-established targeted gene panel test available through the UK National Health Service. We show that integration of such strategies will increase accuracy and clarity of diagnostic reporting, including the identification of variants which could provide new diagnoses and new carrier findings for referred individuals. Functional assessment is essential for accurate clinical assessment of variants disrupting pre-mRNA splicing. We show through cell based functional assessments that variants impacting splicing may have complex impacts on pre-mRNA splicing, which may cause multiple interpretable consequences according to ACMG guidelines.

The bladder exstrophy–epispadias complex (BEEC) comprises of a spectrum of anterior midline defects, all affecting the lower urinary tract, the external genitalia, and the bony pelvis. In extreme cases, the gastrointestinal tract is also affected. The pathogenesis of BEEC is unclear but chromosomal aberrations have been reported. In particular, duplications of 22q11.2 have been identified in eight unrelated individuals with BEEC. The current study aimed to identify chromosomal copy number variants in BEEC. Analyses was performed using the Affymetrix Genome‐wide SNP6.0 assay in 92 unrelated patients cared for by two UK pediatric urology centers. Three individuals had a 22q11.2 duplication, a significantly higher number than that found in a control group of 12,500 individuals with developmental delay who had undergone microarray testing (p < .0001). Sequencing of CRKL, implicated in renal tract malformations in DiGeorge syndrome critical region at 22q11, in 89 individuals with BEEC lacking 22q11 duplications revealed no pathogenic variants. To date, 22q11.2 duplication is the genetic variant most commonly associated with BEEC. This is consistent with the hypothesis that altered expression of a single, yet to be defined, gene therein is critical to the pathogenesis of this potentially devastating congenital disorder.

The bladder exstrophy–epispadias complex (BEEC) is an abdominal midline malformation comprising a spectrum of congenital genitourinary abnormalities of the abdominal wall, pelvis, urinary tract, genitalia, anus, and spine. The vast majority of BEEC cases are classified as non-syndromic and the etiology of this malformation is still unknown. This review presents the current knowledge on this multifactorial disorder, including phenotypic and anatomical characterization, epidemiology, proposed developmental mechanisms, existing animal models, and implicated genetic and environmental components.

Understanding the genomic basis of inherited respiratory disorders can assist in the clinical management of individuals with these rare disorders. We apply whole genome sequencing for the discovery of disease-causing variants in the non-coding regions of known disease genes for two individuals with inherited respiratory disorders. We describe analysis strategies to pinpoint candidate non-coding variants within the non-coding genome and demonstrate aberrant RNA splicing as a result of deep intronic variants in DNAH11 and CFTR . These findings confirm clinical diagnoses of primary ciliary dyskinesia and cystic fibrosis, respectively.