ABSTRACT
BACKGROUND: Mutations in the genes encoding sarcomere proteins have been associated with both hypertrophic and dilated cardiomyopathy. Recently, mutations in myosin heavy chain (MYH7), cardiac actin (ACTC), and troponin T (TNNT2) were associated with left ventricular noncompaction, a form of cardiomyopathy characterized with hypertrabeculation that may also include reduced function of the left ventricle. METHODS AND RESULTS: We used clinically available genetic testing on 3 cases referred for evaluation of left ventricular dysfunction and noncompaction of the left ventricle and found that all 3 individuals carried sarcomere mutations. The first patient presented with neonatal heart failure and was referred for left ventricular noncompaction cardiomyopathy. Genetic testing found 2 different mutations in MYBPC3 in trans. The first mutation, 3776delA, Q1259fs, rendered a frame shift at 1259 of cardiac myosin-binding protein C and the second mutation was L1200P. The frameshift mutation was also found in this mother who displayed mild echocardiographic features of cardiomyopathy, with only subtle increase in trabeculation and an absence of hypertrophy. A second pediatric patient presented with heart failure and was found to carry a de novo MYH7 R369Q mutation. The third case was an adult patient with dilated cardiomyopathy referred for ventricular hypertrabeculation. This patient had a family history of congestive heart failure, including pediatric onset cardiomyopathy where 3 individuals in the family were found to have the MYH7 mutation R1250W. CONCLUSIONS: Genetic testing should be considered for cardiomyopathy with hypertrabeculation.
Subject(s)
Hypertrophy, Left Ventricular/genetics , Mutation , Sarcomeres/genetics , Cardiac Myosins/genetics , Carrier Proteins/genetics , Child , Child, Preschool , Female , Humans , Hypertrophy, Left Ventricular/diagnostic imaging , Infant , Infant, Newborn , Male , Middle Aged , Myosin Heavy Chains/genetics , Pedigree , UltrasonographyABSTRACT
Deletions on chromosome 22q11.21 disrupt pharyngeal and cardiac development and cause DiGeorge and related human syndromes. CRKL (CRK-Like) lies within 22q11.21, and Crkl-/- mice have phenotypic features of 22q11 deletion (del22q11) syndromes. While human FGF8 does not localize to 22q11, deficiency of Fgf8 also generates many features of del22q11 syndrome in mice. Since Fgf8 signals via receptor-type tyrosine kinases, and Crk family adaptor proteins transduce intracellular signals downstream of tyrosine kinases, we investigated whether Crkl mediates Fgf8 signaling. In addition to discovering genetic interactions between Crkl and Fgf8 during morphogenesis of structures affected in del22q11 syndrome, we found that Fgf8 induces tyrosine phosphorylation of FgfRs 1 and 2 and their binding to Crkl. Crkl is required for normal cellular responses to Fgf8, including survival and migration, Erk activation, and target gene expression. These findings provide mechanistic insight into disrupted intercellular interactions in the pathogenesis of malformations seen in del22q11 syndrome.
Subject(s)
Chromosomes, Human, Pair 22 , DiGeorge Syndrome/metabolism , Fibroblast Growth Factor 8/metabolism , Gene Deletion , Proto-Oncogene Proteins c-crk/deficiency , Signal Transduction/physiology , Animals , Apoptosis , Blotting, Western/methods , Bone and Bones/embryology , Bone and Bones/metabolism , Cardiovascular System/embryology , Cardiovascular System/metabolism , Cell Count/methods , Cells, Cultured , Chemotactic Factors/metabolism , DiGeorge Syndrome/genetics , Disease Models, Animal , Embryo, Mammalian , Enzyme Activation , Fluorescent Antibody Technique/methods , Gene Expression Regulation, Developmental/genetics , Genotype , Humans , Mice , Mice, Knockout , Models, Biological , Neural Crest/metabolism , Pharynx/embryology , Pharynx/metabolism , Phenotype , Receptors, Fibroblast Growth Factor/metabolism , Time FactorsABSTRACT
22q11 deletion (del22q11) syndrome is characterized genetically by heterozygous deletions within chromosome 22q11 and clinically by a constellation of congenital malformations of the aortic arch, heart, thymus, and parathyroid glands described as DiGeorge syndrome (DGS). Here, we report that compound heterozygosity of mouse homologs of two 22q11 genes, CRKL and TBX1, results in a striking increase in the penetrance and expressivity of a DGS-like phenotype compared to heterozygosity at either locus. Furthermore, we show that these two genes have critical dose-dependent functions in pharyngeal segmentation, patterning of the pharyngeal apparatus along the anteroposterior axis, and local regulation of retinoic acid (RA) metabolism and signaling. We can partially rescue one salient feature of DGS in Crkl+/-;Tbx1+/- embryos by genetically reducing the amount of RA produced in the embryo. Thus, we suggest that del22q11 is a contiguous gene syndrome involving dose-sensitive interaction of CRKL and TBX1 and locally aberrant RA signaling.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , DiGeorge Syndrome/metabolism , Gene Deletion , Nuclear Proteins/metabolism , Signal Transduction/physiology , T-Box Domain Proteins/metabolism , Tretinoin/metabolism , Adaptor Proteins, Signal Transducing/deficiency , Animals , Aorta/embryology , Aorta/metabolism , Aorta/pathology , Branchial Region/embryology , Branchial Region/metabolism , Branchial Region/pathology , Chromosomes, Human, Pair 22 , Cytochrome P-450 Enzyme System/genetics , Cytochrome P-450 Enzyme System/metabolism , Disease Models, Animal , Embryo, Mammalian , Gene Expression/genetics , Gene Expression Regulation, Developmental/genetics , Genotype , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Humans , In Situ Hybridization/methods , Mice , Mice, Inbred C57BL , Mice, Knockout , Nuclear Proteins/deficiency , Retinoic Acid 4-Hydroxylase , T-Box Domain Proteins/deficiency , Thymus Gland/embryology , Thymus Gland/metabolism , Thymus Gland/pathologyABSTRACT
During brain development, many neurons migrate long distances before settling and differentiating. These migrations are coordinated to ensure normal development. The secreted protein Reelin controls the locations of many types of neurons, and its absence causes the classic "Reeler" phenotype. Reelin action requires tyrosine phosphorylation of the intracellular protein Dab1 by Src-family kinases. However, little is known about signaling pathways downstream of Dab1. Here, we identify several proteins in embryonic brain extract that bind to tyrosine-phosphorylated, but not non-phosphorylated, Dab1. Of these, the Crk-family proteins (CrkL, CrkI, and CrkII ), bind significant quantities of Dab1 when embryonic cortical neurons are exposed to Reelin. CrkL binding to Dab1 involves two tyrosine phosphorylation sites, Y220 and 232, that are critical for proper positioning of migrating cortical plate neurons. CrkL also binds C3G, an exchange factor (GEF) for the small GTPase Rap1 that is activated in other systems by tyrosine phosphorylation. We report that Reelin stimulates tyrosine phosphorylation of C3G and activates Rap1. C3G and Rap1 regulate adhesion of fibroblasts and other cell types. Regulation of Crk/CrkL, C3G, and Rap1 by Reelin may be involved in coordinating neuron migrations during brain development.
Subject(s)
Adaptor Proteins, Signal Transducing , Cell Adhesion Molecules, Neuronal/metabolism , Extracellular Matrix Proteins/metabolism , Guanine Nucleotide-Releasing Factor 2/metabolism , Nerve Tissue Proteins/metabolism , Neurons/metabolism , Nuclear Proteins/metabolism , rap1 GTP-Binding Proteins/metabolism , Animals , Baculoviridae , Blotting, Western , Cell Adhesion Molecules, Neuronal/pharmacology , Cells, Cultured , Chromatography, Affinity , Electrophoresis, Polyacrylamide Gel , Enzyme Activation/drug effects , Extracellular Matrix Proteins/pharmacology , Genetic Vectors , Mass Spectrometry , Mice , Nuclear Proteins/isolation & purification , Phosphorylation/drug effects , Precipitin Tests , Reelin Protein , Serine Endopeptidases , Signal Transduction/drug effects , Signal Transduction/physiology , TransfectionABSTRACT
The adapter protein Crk-Like (CrkL) can associate with the Src substrate p130(Cas) (Cas). The biological role of CrkL downstream of Cas, however, has been largely obscure. Consistent with the ability of CrkL to biochemically associate with Cas, we found that Src triggers translocation of CrkL to focal adhesions (FAs) in a manner dependent on Cas. Forced localization of CRKL to FAs (FA-CRKL) by itself was sufficient to induce activation of Rac1 and Cdc42 and rescued haptotaxis defects of mouse embryonic fibroblasts (MEFs) lacking Src, Yes, and Fyn, three broadly expressed Src family members required for integrin-induced migration. Consistent with Rac1 activation, FA-CRKL induced cotranslocation of a Rac1 activator, Dock1, to focal adhesions. These results therefore indicate a role for CrkL in mediating Src signaling by activating small G proteins at focal adhesions. Furthermore, MEFs lacking CrkL show impaired integrin-induced migration despite expression of a closely related protein, Crk-II, in these cells. These results therefore provide formal evidence that CrkL plays a specific role in integrin-induced migration as a downstream mediator of Src.
