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“Dominant-Negative ALK2 Allele Associates with Congenital Heart Defects” [Smith et. al, 2009] Presented by Arron Sikka & Anita Isama Highlights from Review Article • Genetics and embryological mechanisms of congenital heart diseases (Bajolle et. al, 2008) • Heart develops as different portions are added to the primitive linear cardiac tube in a sequential manner • Cardiac chamber maturation is distinguished by a ballooning process, atria maturing symmetrically and ventricles maturing sequentially • Genetic contribution • Deletion of chromosome 22q1.1 • One molecular abnormality that results in one group of heart diseases that are “heterogeneous anatomically but homogenous in terms of embryological mechanism” • One heart disease several mechanisms several genes • Pathology that is a result of mutations other than 22q1.1 • Heterogeneity introduces complexity What is congenital atrial septal heart defect? • Congenital means existing at birth • Atrial septum thin wall of tissue that separates the left and right atria • Abnormalities occur when an opening exists in this region Causes, Signs, and Symptoms • Occurs during fetal development • May be genetic, but for most children cause unclear • Severity of symptoms determined by the size and location of the atrial septum defect • Most children are symptomless • For severe cases symptoms include: • • • • • Poor appetite Poor growth Fatigue Shortness of breath Lung infections • Untreated condition leads to abnormal heart rhythm and potentially increased risk for stroke or pulmonary hypertension Diagnosis and Treatment • ASD caused heart murmur, identified during check-ups • Hard to hear, diagnosis can occur any time between infancy and adolescence • Confirmed via chest x-ray, EKG, or echocardiogram (primary) • Treatment depends on severity, ranges from: • Regular cardiologist visits • Cardiac catheterization Implant • Open heart surgery Introduction I • Primitive heart tube • Inner layer of endothelial cells • Endocardium • Outer layer of myocardial cells • After formation of the heart tube, endothelial cells migrate to the cardiac jelly, undergo endothelium-mesenchyme transition and give rise to endocardial cushions (ECs) • “ECs contribute to the valves and septa of the heart and disruptions in their formation result in valvular and septal defects.” • Signaling pathways implicated in atrioventricular septum development • • • • Vascular endothelial growth factor signaling Notch Wnt/β-catenin Bone morphogenetic protein (BMP)/transforming growth factor-β signaling (TGFβ) Introduction II • Identification of novel causative mutations in genes previously identified can be made through candidate screening approaches combined with kindred linkage and/or detailed functional analyses • Authors employed this approach • Sequenced the coding region of 32 genes from patients with endocardial cushion development abnormalities • Focused on several cSNPs in the ALK2 gene • ALK2 is a BMP receptor, evolutionarily conserved • Investigate aberrant BMP signaling in patients with atrial defects • Identify 11 genetic lesions in 10 different genes, characterizing 2 genetic lesions for the ALK2 receptor gene Signals controlling neural crest contributions to the heart Wiley Interdisciplinary Reviews: Systems Biology and Medicine Volume 1, Issue 2, pages 220-227, 29 APR 2009 DOI: 10.1002/wsbm.8 http://onlinelibrary.wiley.com/doi/10.1002/wsbm.8/full#fig3 Methods I • SNP/Mutation Discovery • Coding single-nucleotide polymorphisms in selected genes were determined by dideoxy resequencing of PCR-amplified exonic fragments • Clinical Evaluation, Sample Collection, and Genotyping • DNA material from the Netherlands national congenital heart disease registry • Controls- DNA material from patients with non-cardiac related diseases • Probands-ALK2 variants R307L and L343P • Patients and kindred were evaluated (history and physical examination) • Positive carriers for variant alleles were identified by transthoracic echocardiographic examination • Big Dye Terminator chemistry Methods II • Cell Lines and Transfections • Luciferase assay • Bovine aortic endothelial cells • Kinase assay • Cos7 cells • Constructs, Luciferase Assay, and Kinase Activity Assays • Constructed full length human ALK2 and mutant variants • Inserts sequenced and recloned into parental vector or pCS2+ vector • Luciferase assay performed in combination with a short hairpin RNA targeting bovine ALK2 construct • Kinase assay performed with γ-ATP Methods III • Protein Isolation and Western Blot Analysis • Protein isolated from transfected cells by direct lysis • Blotting performed with an HA antibody at [1:1000] and rabit anti-phospho-Smad 1, 5, 8 at [1:1000] • Fish Lines, mRNA synthesis, Injections, In Situ Hybridization, and Immunofluorescence • Wild type and Tg(Tie2:EGFP) fish lines • Statistical Analysis Figure 1 Figure 2 • In-vitro analysis • Luciferase acitivty significantly compromised by L343P variant, but not changed by A15G and R307L variants • Although ALK2 was still expressed, its kinase activity was inhibited Figure 3 • Zebrafish embryos injected with variant RNA at single-cell stage • L343P RNA injection embryos resulted in severe dorsalization • Phenotypic strength is defined as using a series from class 1 (C1) to class 5 (C5), C5 being the strongest phenotype of dorsalization Figure 3 • C – eye and boundary of brain is visible, but head is disorganized, fragmentation in various positions of yolk sac • a reduction in cell types of ventral origin in the blastoderm (blood and tail) and an expansion of dorsally derived tissues (anterior somites and notochord). • Coinjection with wtALK2 partially rescued dorsalization Figure 3 • SMAD is downstream of ALK2 • pSMAD activates downstream TGF-β gene transcription (transforming growth factor) • Although SMAD is present, its activation is inhibited by the L343P mutation Figure 4 • Cmlc2 disruption in overall morphology of the heart tube (cardiac myosin light chain 2) • Tbx2b expression lost or reduced (required for the specification of midline mesoderm) Figure 4 • ANF expression was consistent in mutant allele (atrial natriuretic factor = vasodilator hormone secreted by heart muscle cells) Figure 4 • Has2 expression is lost (enzyme that synthesizes hyaluronic acid = polysaccharide that extends through plasma membrane, serves to fill space, lubricate joints and create a matrix for cells to migrate Figure 4 • Reduced GFP expression reduced transcription/translation • Loss of endocardial cushion and atrioventricular canal lost or improperly formed Supplementary Figure 1 • No obvious inheritance pattern for either mutation • ALK2 L343P demonstrated in father and son, who both have a structural cardiac defect. Son has premium-type atrial septal defect, whereas father has anomaly that cant be unambiguously classified as CHD (non-penetrant) complex inheritance Supplementary Figure 2 • Eve1, gata2 dramatic reduction of ventral tissue • Chordin expansion of dorsal cell fate • myoD, krox20 severe dorsalisation (krox20 marks hindbrain rhombomeres) Discussion • ALK2 gene variations associated with Congenital Heart Defects • L343P varation inhibits kinase activity of ALK2 receptor interfering with BMP signaling hindered growth factor determining architecture of body • Overall morphology of heart was compromised in zebrafish embryos • ALK2-deficient mice die during gastrulation • Humans survive either wt ALK2 gets minimally expressed (L343P variant is less stable), or ALK3 helps to compensate for the deficiency • Still needs further studies