1. “Dominant-Negative ALK2 Allele Associates with Congenital Heart Defects” [Smith et. al, 2009] Presented by Arron Sikka & 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 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 Volume 1, Issue 2, http://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 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

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