Calmodulin Mutations Associated With Recurrent Cardiac Arrest in Infants Scuola di Dottorato in Scienze Biomediche e Oncologia Umana Indirizzo Genetica Umana Russo Alessia Ciclo: XXVI Tutor: Prof. Matullo 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 intrathoracic petechiae • 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 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 The sudden Infant Death Syndrome (SIDS) Genetic risk factors: Autonomic nervous system development • Paired-like homeobox 2a (Phox2a) • Rearranged during transfection (RET) • Endothelin-converting enzyme-1 (ECE1) • T-cell leukemia homeobox (TLX3) • Engrailed-1 (EN1) Cardiac ion channelopathies • Sodium channel (SCN5A) • Potassium channel Serotinin transporter gene (5-HTT) Infection and inflammation • Complement C4A and C4B • Interleukin-10 Hunt, Hauck. CMAJ, 2006; Kinney et al. N Engl J Med, 2009 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. 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. Study subjects • White girl from Italy • Hispanic female infant • Cardiac arrest caused by ventricular • Cardiac arrest and multiple episodes of ventricular fibrillation fibrillation at 6 months of age • Prolonged long QTc interval • Prolonged long QTc interval • No family history for sudden cardiac arrest • No family history for sudden cardiac arrest • Asymptomatic parents with normal ECGs • Parents and the older sister with normal ECGs • Normal cardiac anatomy and function • Normal cardiac anatomy and function • No mutations in KCNQ1, KCNH2, SCN5A, • No mutations in KCNQ1, KCNH2, SCN5A, KCNE1, and KCNE2 genes (LQTS) KCNE1, and KCNE2 genes (LQTS) • Mild delay in language development • Seizures and developmental delay Study subjects Cases: Controls: Two probands 92 Hispanic Americans (Coriell Unaffected parents Institute for Medical Research) 82 unrelated patients with LQTS 1,800 Europeans (Helmholtz Zentrum without an identified genetic cause München) Exome sequencing analysis (Illumina HiSeq2000) on the two probands and their parents Peripheral blood leukocytes 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 CALM1 (D130G) CALM2 (D96V) C6orf108 Not expressed in cardiac tissue No known function Nucleotide position with poor evolutionary conservation 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 Zentrum München) • Only 2 nonsynonymous coding variants in CALM1 (T10I and L143V) and none in CALM2 were called in 8,599 alleles of European ancestry by the Exome Sequencing 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 De novo calmodulin gene mutations in infants with severe cardiac arrhythmias Heterozygous missense mutations Predicted to be damaging (SIFT, PolyPhen2) 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 mRNAs for CALM1, CALM2, and CALM3 are expressed in normal human heart (left ventricle) throughout development Rank order of expression: CALM3>CALM2>CALM1 qRT-PCR Ref gene: β-actin Human heart samples: fetal (n=4), infant (n=4), adult (n=8) 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+ binding studies: All 3 mutations reduce Ca2+ affinity in the C-domain Discussion Discovery of de novo calmodulin mutations in a severe, early-onset cardiac arrhythmia syndrome with features of LQTS In electrically excitable tissues (i.e. heart and brain): Ca2+ Calmodulin Calcium sensor Signal transducer Impaired Ca2+-dependent inactivation of Ltype voltage-gated Ca2+ channels Calcium channels Potassium channels Sodium channels Calmodulin-dependent Kinase II Dramatic prolonged QT interval Predisposition to ventricular arrhythmia Alseikhan BA et al. Proc Natl Acad Sci U S A. 2002 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 Nyegaard M et al. Am J Hum Genet. 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