Novel frameshift variant in MYL2 reveals molecular differences between dominant and recessive forms of hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is characterized by thickening of the ventricular muscle without dilation and is often associated with dominant pathogenic variants in cardiac sarcomeric protein genes. Here, we report a family with two infants diagnosed with infantile-onset HCM and mitral valve dysplasia that led to death before one year of age. Using exome sequencing, we discovered that one of the affected children had a homozygous frameshift variant in Myosin light chain 2 (MYL2:NM_000432.3:c.431_432delCT: p.Pro144Argfs*57;MYL2-fs), which alters the last 20 amino acids of the protein and is predicted to impact the most C-terminal of the three EF-hand domains in MYL2. The parents are unaffected heterozygous carriers of the variant and the variant is absent in control cohorts from gnomAD. The absence of the phenotype in carriers and the infantile presentation of severe HCM is in contrast to HCM associated with dominant MYL2 variants. Immunohistochemical analysis of the ventricular muscle of the deceased patient with the MYL2-fs variant showed a marked reduction of MYL2 expression compared to an unaffected control. In vitro overexpression studies further indicate that the MYL2-fs variant is actively degraded. In contrast, an HCM-associated missense variant (MYL2:p.Gly162Arg) and three other MYL2 stop-gain variants (p.E22*, p.K62*, p.E97*) that result in loss of the EF domains are stably expressed but show impaired localization. The degradation of the MYL2-fs can be rescued by inhibiting the cell's proteasome function supporting a post-translational effect of the variant. In vivo rescue experiments with a Drosophila MYL2-homolog (Mlc2) knockdown model indicate that neither the MYL2-fs nor the MYL2:p.Gly162Arg variant supports normal cardiac function. The tools that we have generated provide a rapid screening platform for functional assessment of variants of unknown significance in MYL2. Our study supports an autosomal recessive model of inheritance for MYL2 loss-of-function variants in infantile HCM and highlights the variant-specific molecular differences found in MYL2-associated cardiomyopathy.

TGF-α ligands can substitute for the neuregulin Vein in Drosophila development.

ErbB receptors, including the epidermal growth factor receptor (Egfr), are activated by EGF ligands to govern cell proliferation, survival, migration and differentiation. The different EGF-induced cell responses in development are regulated by deployment of multiple ligands. These inputs, however, engage only a limited number of intracellular pathways and are thought to elicit specific responses by regulating the amplitude or duration of the intracellular signal. The single Drosophila Egfr has four ligands: three of the TGF-α-type and a single neuregulin-like called vein (vn). Here, we used mutant combinations and gene replacement to determine the constraints of ligand specificity in development. Mutant analysis revealed extensive ligand redundancy in embryogenesis and wing development. Surprisingly, we found that the essential role of vn in development could be largely replaced by expression of any TGF-α ligand, including spitz (spi), in the endogenous vn pattern. vn mutants die as white undifferentiated pupae, but the rescued individuals showed global differentiation of adult body parts. Spi is more potent than Vn, and the best morphological rescue occurred when Spi expression was reduced to achieve an intracellular signaling level comparable to that produced by Vn. Our results show that the developmental repertoire of a strong ligand like Spi is flexible and at the appropriate level can emulate the activity of a weak ligand like Vn. These findings align with a model whereby cells respond similarly to an equivalent quantitative level of an intracellular signal generated by two distinct ligands regardless of ligand identity.

Dpp-induced Egfr signaling triggers postembryonic wing development in Drosophila.

The acquisition of flight contributed to the success of insects and winged forms are present in most orders. Key to understanding the origin of wings will be knowledge of the earliest postembryonic events promoting wing outgrowth. The Drosophila melanogaster wing is intensely studied as a model appendage, and yet little is known about the beginning of wing outgrowth. Vein (Vn) is a neuregulin-like ligand for the EGF receptor (Egfr), which is necessary for global development of the early Drosophila wing disc. vn is not expressed in the embryonic wing primordium and thus has to be induced de novo in the nascent larval wing disc. We find that Decapentaplegic (Dpp), a Bone Morphogenetic Protein (BMP) family member, provides the instructive signal for initiating vn expression. The signaling involves paracrine communication between two epithelia in the early disc. Once initiated, vn expression is amplified and maintained by autocrine signaling mediated by the E-twenty six (ETS)-factor PointedP2 (PntP2). This interplay of paracrine and autocrine signaling underlies the spatial and temporal pattern of induction of Vn/Egfr target genes and explains both body wall development and wing outgrowth. It is possible this gene regulatory network governing expression of an EGF ligand is conserved and reflects a common origin of insect wings.

New negative feedback regulators of Egfr signaling in Drosophila.

The highly conserved epidermal growth factor receptor (Egfr) pathway is required in all animals for normal development and homeostasis; consequently, aberrant Egfr signaling is implicated in a number of diseases. Genetic analysis of Drosophila melanogaster Egfr has contributed significantly to understanding this conserved pathway and led to the discovery of new components and targets. Here we used microarray analysis of third instar wing discs, in which Egfr signaling was perturbed, to identify new Egfr-responsive genes. Upregulated transcripts included five known targets, suggesting the approach was valid. We investigated the function of 29 previously uncharacterized genes, which had pronounced responses. The Egfr pathway is important for wing-vein patterning and using reverse genetic analysis we identified five genes that showed venation defects. Three of these genes are expressed in vein primordia and all showed transcriptional changes in response to altered Egfr activity consistent with being targets of the pathway. Genetic interactions with Egfr further linked two of the genes, Sulfated (Sulf1), an endosulfatase gene, and CG4096, an A Disintegrin And Metalloproteinase with ThromboSpondin motifs (ADAMTS) gene, to the pathway. Sulf1 showed a strong genetic interaction with the neuregulin-like ligand vein (vn) and may influence binding of Vn to heparan-sulfated proteoglycans (HSPGs). How Drosophila Egfr activity is modulated by CG4096 is unknown, but interestingly vertebrate EGF ligands are regulated by a related ADAMTS protein. We suggest Sulf1 and CG4096 are negative feedback regulators of Egfr signaling that function in the extracellular space to influence ligand activity.

Loss of the tumor suppressor Pten promotes proliferation of Drosophila melanogaster cells in vitro and gives rise to continuous cell lines.

In vivo analysis of Drosophila melanogaster has enhanced our understanding of many biological processes, notably the mechanisms of heredity and development. While in vivo analysis of mutants has been a strength of the field, analyzing fly cells in culture is valuable for cell biological, biochemical and whole genome approaches in which large numbers of homogeneous cells are required. An efficient genetic method to derive Drosophila cell lines using expression of an oncogenic form of Ras (Ras(V12)) has been developed. Mutations in tumor suppressors, which are known to cause cell hyperproliferation in vivo, could provide another method for generating Drosophila cell lines. Here we screened Drosophila tumor suppressor mutations to test if they promoted cell proliferation in vitro. We generated primary cultures and determined when patches of proliferating cells first emerged. These cells emerged on average at 37 days in wild-type cultures. Using this assay we found that a Pten mutation had a strong effect. Patches of proliferating cells appeared on average at 11 days and the cultures became confluent in about 3 weeks, which is similar to the timeframe for cultures expressing Ras(V12). Three Pten mutant cell lines were generated and these have now been cultured for between 250 and 630 cell doublings suggesting the life of the mutant cells is likely to be indefinite. We conclude that the use of Pten mutants is a powerful means to derive new Drosophila cell lines.