Calmodulin Mutations Associated With Recurrent Cardiac Arrest in Infants

1. Calmodulin Mutations Associated With Recurrent CardiacArrest in Infants Scuola di Dottorato in Scienze Biomediche e Oncologia Umana Indirizzo Genetica Umana Russo Alessia Ciclo: XXVI Tutor: Prof. Matullo

2. The sudden Infant Death Syndrome (SIDS) • Definition: • “Sudden death of an infant or young child, which is unexpected by history, and in which a thorough post mortem examination fails to demonstrate an adequate cause of death.” • Features: • Peak incidence at 2 to 4 months of age • Presence of intrathoracicpetechiae • Death linked to a sleep period Causes: • 20% of cases: • Infection • Inherited disorders of fatty acid oxidation (MCAD gene) • Genetic cardiac channelopathies • 80% of cases: unexpected deaths Kinney et al. N Engl J Med, 2009

3. The sudden Infant Death Syndrome (SIDS) Risk factors: Male predominance (2:1) Race/ethnic background Prematurity Exposure to cigarette smoke, alcohol and illicit substances Extrinsic factors: prone and side-sleeping position, bed sharing, high ambient temperature Kinney et al. N Engl J Med, 2009

4. The sudden Infant Death Syndrome (SIDS) Genetic risk factors: Autonomicnervoussystemdevelopment • Paired-likehomeobox 2a (Phox2a) • Rearrangedduringtransfection (RET) • Endothelin-converting enzyme-1 (ECE1) • T-cellleukemiahomeobox (TLX3) • Engrailed-1 (EN1) Cardiacionchannelopathies • Sodiumchannel (SCN5A) • Potassiumchannel Serotinintransporter gene (5-HTT) Infection and inflammation • Complement C4A and C4B • Interleukin-10 Hunt, Hauck. CMAJ, 2006; Kinney et al. N Engl J Med, 2009

5. The sudden Infant Death Syndrome (SIDS) Highly malignant arrhythmias may predispose to the sudden death Arrhythmia: group of conditions in which the electrical activity of the heart is irregular or is faster (tachycardia) or slower (bradycardia) than normal. Ventricular fibrillation: uncoordinated contraction of the cardiac muscle of the ventricles in the heart. The most commonly identified arrhythmia in cardiac arrest patients.

6. The identification of novel arrhythmia susceptibility genes has great value for understanding the molecular basis of sudden cardiac death, including unexplained infant mortality, and has the potential to inspire new therapeutic approaches.

7. Study subjects • Hispanic female infant • Cardiac arrest and multiple episodes of ventricular fibrillation • Prolonged long QTc interval • No family history for sudden cardiac arrest • Parents and the older sister with normal ECGs • Normal cardiac anatomy and function • No mutations in KCNQ1, KCNH2, SCN5A, KCNE1, and KCNE2 genes (LQTS) • Seizures and developmental delay • White girl from Italy • Cardiac arrest caused by ventricular fibrillation at 6 months of age • Prolonged long QTc interval • No family history for sudden cardiac arrest • Asymptomatic parents with normal ECGs • Normal cardiac anatomy and function • No mutations in KCNQ1, KCNH2, SCN5A, KCNE1, and KCNE2 genes (LQTS) • Mild delay in language development

8. Study subjects Cases: Controls: Two probands Unaffected parents 82 unrelated patients with LQTS without an identified genetic cause 92 Hispanic Americans (CoriellInstitute for Medical Research) 1,800 Europeans (Helmholtz ZentrumMünchen) Exome sequencing analysis (Illumina HiSeq2000) on the two probands and their parents Peripheral blood leukocytes

9. Exome sequencing results • Filtering criteria: • Variants identified in dbSNP, 1000Genomes, Exome Variant Server, and Helmholtz exome databases • Synonymous and intronic variants (except for canonical splice sites) • Inherited variants C6orf108 Not expressed in cardiac tissue No known function Nucleotide position with poor evolutionary conservation CALM1 (D130G) CALM2 (D96V)

10. Extreme rarity of Calmodulin gene mutations: • Neither mutations found in the DNA of ethically matched control subjects • No mutations found in the 1,800 exomes (Helmholtz ZentrumMünchen) • Only 2 nonsynonymouscoding variants in CALM1 (T10I and L143V) and none in CALM2 were called in 8,599 alleles of European ancestry by the ExomeSequencing Project • Mutations analysis in CALM1, CALM2 and CALM3 in 82 LQTS patients without an identified genetic cause: • 2 mutations (one novel) identified in 2 patients in CALM1 gene (D130G, F142L) • The novel mutation was absent in control subjects and in reference databases

11. De novo calmodulin gene mutationsin infants with severe cardiac arrhythmias Heterozygous missense mutations Predicted to be damaging (SIFT, PolyPhen2)

12. CALMODULIN EF-hand III Ca2+ N2H COOH EF-hand IV Ca2+ Residues involved are highly conserved across species • Three different genes encode an identical calcium binding protein, the phosphorylase kinase delta

13. mRNAs for CALM1, CALM2, and CALM3are expressed in normal human heart (left ventricle) throughout development Rank order of expression: CALM3>CALM2>CALM1 qRT-PCR Ref gene: β-actin Human heartsamples: fetal(n=4), infant (n=4), adult(n=8)

14. CALM1-D130G and CALM2-D96V were predicted to reduce Ca2+ affinity CALM1-F142L was predicted to alterate the energetic coupling of Ca2+ binding and the conformational change associated with calmodulin activation In vitro Ca2+ bindingstudies: All 3 mutations reduce Ca2+ affinity in the C-domain

15. Discussion Discovery of de novo calmodulin mutations in a severe, early-onset cardiac arrhythmia syndrome with features of LQTS In electricallyexcitabletissues (i.e. heart and brain): Ca2+ Calcium channels Potassium channels Sodium channels Calmodulin-dependent Kinase II Calmodulin Calciumsensor Signaltransducer Dramatic prolonged QT interval Predisposition to ventricular arrhythmia Impaired Ca2+-dependent inactivation of L-type voltage-gated Ca2+ channels AlseikhanBA et al. ProcNatlAcad Sci U S A. 2002

16. Discussion CALM1-N53I (EF-hand domain II) and CALM1-N97S (EF-hand domain III) mutations: no evidence of prolonged QT intervals in any of these mutations carriers NyegaardM et al. Am J HumGenet. 2012 • Genotype-phenotype correlation among calmodulin mutations • Ubiquitously expressed calmodulin genes present predominantly with a cardiac phenotype Less physiological reserve of Ca2+ in heart • Calmodulin mutations could also confer increased susceptibility to neurodevelopmental phenotypes and epilepsy • Possibility of a dominant-negative mechanism of actions

17. Thank you!