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

Cardiac ion channelopathies Sodium channel (SCN5A) Potassium channel Serotinin transporter gene (5-HTT) Genetic risk factors: Infection and inflammation Complement C4A and C4B Interleukin-10 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) The sudden Infant Death Syndrome (SIDS) Hunt, Hauck. CMAJ, 2006; Kinney et al. N Engl J Med, 2009

The sudden Infant Death Syndrome (SIDS) 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. Highly malignant arrhythmias may predispose to the sudden death

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 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 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

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

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)

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)

EF-hand III EF-hand IV CALMODULIN COOHN2HN2H Three different genes encode an identical calcium binding protein, the phosphorylase kinase delta Residues involved are highly conserved across species Ca 2+

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) mRNAs for CALM1, CALM2, and CALM3 are expressed in normal human heart (left ventricle) throughout development

CALM1-D130G and CALM2-D96V were predicted to reduce Ca 2+ affinity CALM1-F142L was predicted to alterate the energetic coupling of Ca 2+ binding and the conformational change associated with calmodulin activation In vitro Ca 2+ binding studies: All 3 mutations reduce Ca 2+ affinity in the C-domain

Discussion Discovery of de novo calmodulin mutations in a severe, early-onset cardiac arrhythmia syndrome with features of LQTS Calcium sensor Signal transducer Calcium channels Potassium channels Sodium channels Calmodulin-dependent Kinase II In electrically excitable tissues (i.e. heart and brain): Impaired Ca 2+ -dependent inactivation of L- type voltage-gated Ca 2+ channels Dramatic prolonged QT interval Predisposition to ventricular arrhythmia Alseikhan BA et al. Proc Natl Acad Sci U S A Ca 2+ Calmodulin

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 Genotype-phenotype correlation among calmodulin mutations Calmodulin mutations could also confer increased susceptibility to neurodevelopmental phenotypes and epilepsy Ubiquitously expressed calmodulin genes present predominantly with a cardiac phenotype Less physiological reserve of Ca 2+ in heart Possibility of a dominant-negative mechanism of actions Discussion Nyegaard M et al. Am J Hum Genet. 2012