Calmodulin Mutations Associated With Recurrent Cardiac Arrest in

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)
“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.”
• Peak incidence at 2 to 4 months of age
• Presence of intrathoracic petechiae
• Death linked to a sleep period
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
Exposure to cigarette smoke, alcohol and illicit substances
Extrinsic factors: prone and side-sleeping
position, bed sharing, high ambient
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
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
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
• Synonymous and
intronic variants (except
for canonical splice
• Inherited variants
CALM1 (D130G)
CALM2 (D96V)
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
• No mutations found in the 1,800 exomes (Helmholtz Zentrum
• 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,
The novel mutation was absent in control subjects and in reference
De novo calmodulin gene mutations in infants with severe cardiac arrhythmias
Heterozygous missense
Predicted to be
damaging (SIFT, PolyPhen2)
EF-hand III
EF-hand IV
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
Ref gene: β-actin
Human heart samples:
fetal (n=4), infant (n=4), adult
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
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):
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
Alseikhan BA et al. Proc Natl Acad Sci U S A. 2002
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

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