“ Dominant-Negative ALK2 Allele Associates with Congenital Heart Defects ” [Smith et. al, 2009]

1. “Dominant-Negative ALK2 Allele Associates with Congenital Heart Defects”[Smith et. al, 2009] Presented by ArronSikka & Anita Isama

2. 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

3. 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

5. 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

6. 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

7. 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)

8. 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

9. Signals controlling neural crest contributions to the heart Wiley Interdisciplinary Reviews: Systems Biology and MedicineVolume 1, Issue 2, pages 220-227, 29 APR 2009 DOI: 10.1002/wsbm.8http://onlinelibrary.wiley.com/doi/10.1002/wsbm.8/full#fig3

10. Methods I • SNP/Mutation Discovery • Coding single-nucleotide polymorphisms in selected genes were determined by dideoxyresequencing 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

11. 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

12. 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

13. Figure 1

14. 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

15. 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

16. 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

17. 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

18. 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)

19. Figure 4 • ANF  expression was consistent in mutant allele (atrial natriuretic factor = vasodilator hormone secreted by heart muscle cells)

20. 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

21. Figure 4 • Reduced GFP expression  reduced transcription/translation • Loss of endocardial cushion and atrioventricular canal lost or improperly formed

22. 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

23. Supplementary Figure 2 • Eve1, gata2  dramatic reduction of ventral tissue • Chordin  expansion of dorsal cell fate • myoD, krox20  severe dorsalisation (krox20 marks hindbrain rhombomeres)

24. 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