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j.hrcr.2018.07.014

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Accepted Manuscript
A Unique Triadin Exon Deletion causing a Null Phenotype
Barry M. O’Callaghan, MRCPCH, Jules C. Hancox, FBPhS, Alan G. Stuart, FRCP,
Catherine Armstrong, MRCPCH, Maggie M. Williams, BSc FRCPath, Alison Hills,
PhD, Hazel Pearce, BSc, Carolyn L. Dent, PhD, Mary Gable, BSc, Mark A. Walsh,
MRCPCH
PII:
S2214-0271(18)30179-9
DOI:
10.1016/j.hrcr.2018.07.014
Reference:
HRCR 574
To appear in:
HeartRhythm Case Reports
Received Date: 26 May 2018
Revised Date:
12 July 2018
Accepted Date: 27 July 2018
Please cite this article as: O’Callaghan BM, Hancox JC, Stuart AG, Armstrong C, Williams MM, Hills A,
Pearce H, Dent CL, Gable M, Walsh MA, A Unique Triadin Exon Deletion causing a Null Phenotype,
HeartRhythm Case Reports (2018), doi: 10.1016/j.hrcr.2018.07.014.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
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ACCEPTED MANUSCRIPT
1Bristol
Royal Hospital for Children
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Barry M O’Callaghan MRCPCH 1, Jules C. Hancox FBPhS 2, Alan G. Stuart FRCP 1,
Catherine Armstrong MRCPCH 1, Maggie M. Williams BSc FRCPath 3, Alison Hills PhD 3,
Hazel Pearce BSc 3, Carolyn L. Dent PhD 3, Mary Gable BSc3, Mark A. Walsh 1, MRCPCH
School of Physiology and Pharmacology, Cardiovascular Research Laboratories,
University of Bristol
Diagnostics laboratory, University Hospital Bristol
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Correspondence: Mark Walsh, Bristol Royal Hospital for Children, Upper Maudlin
Street, Bristol, BS2 8BJ
Email: mark.walsh@UHBristol.nhs.uk
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Phone: 0117 3428852
Running title : A unique triadin deletion
None of the authors have any conflict of interest to declare.
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A Unique Triadin Exon Deletion causing a Null Phenotype
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Figures – 3
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Keywords
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Triadin, cardiac arrest, pediatrics, CPVT, ventricular fibrillation
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Introduction
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Triadin is a transmembrane protein located in the sarcoplasmic reticulum; it
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interacts with both ryanodine (RYR2) and calsequestrin (CASQ2) to facilitate
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calcium homeostasis in the human cardiac and skeletal muscle cells.1 Pathogenic
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variants in the RYR2 and CASQ2 genes are more commonly associated with
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catacholaminergic polymorphic ventricular tachycardia (CPVT). Triadin is a more
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recently acknowledged protein, wherein genetic aberrations in triadin are
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responsible for a number of malignant arrhythmic syndromes, particularly in
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younger children.2, 3 These triadin mutations are recessively inherited, they are
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associated with a prolonged QT interval, and T-wave abnormalities; the term
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“triadin knockout syndrome” has been suggested by Altmann et. al.2
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The TRDN gene localizes to chromosome 6, and is seen in a variety of isoforms
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including trisk 32, which is expressed predominantly in cardiac muscle. These
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isoforms are generated through alternative splicing of the triadin gene. The protein
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is comprised of a transmembrane domain with both cytosolic and luminal
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components and is 286 amino acids long. Knockout of this gene in murine cardiac
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muscle causes a loss of calcium regulation, impaired excitation contraction coupling
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and cardiac arrhythmia, particularly during beta-adrenergic stimulation.3-5
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Case Presentation
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We present the case of an infant born to non-consanguineous parents originating
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from Oman. His antenatal and postnatal course was uneventful. He had a history of
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G6PD, was not on any medications and was developmentally normal. He presented
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with an out-of-hospital cardiac arrest at 16 months of age, without any preceding
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warning. He received 18 minutes of CPR and following stabilization, he was
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transferred to a tertiary paediatric center for further management. His ECG
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demonstrated a prolonged rate-corrected QT (QTc: Bazett’s correction) of 490 msec;
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it also demonstrated T-wave inversion in the anterior precordial leads (figure 1(a)).
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A head MRI demonstrated possible hypoxic changes in keeping with a cardiac arrest.
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He suffered two subsequent episodes of arrhythmia, one episode of ventricular
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fibrillation requiring CPR for 11 minutes and torsades de point lasting 1 minute. His
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echocardiogram demonstrated a structurally normal heart. He had a cardiac MRI
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that demonstrated no clear evidence of cardiomyopathy.
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He was subsequently fitted with an epicardial defibrillator (weight 9.5kg).
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Medtronic epicardial leads (4968 – 25 cm) were placed on the atrium and ventricle,
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and a Medtronic endocardial shock lead (6935 – 65 cm) was placed around the back
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of the heart and sutured into the pericardium. A Medtronic EveraTM XTDR was
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placed in an abdominal pocket in the right upper quadrant. The post-operative
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course was complicated by renal impairment, liver dysfunction, and electrolyte
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imbalance. The defibrillator administered 4 shocks in the immediate postoperative
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period for ventricular fibrillation. A neurological assessment prior to discharge
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demonstrated limb hypokinesia. He continues on nadolol 10mg BD (1 mg/kg BD)
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and Flecainide 10mg BD (1 mg/kg BD). He had a single shock for ventricular
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fibrillation 7 months after implantation (figure 1(b)). This episode seemed to have
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been related to non-adherence with medications. The flecainide was increased (2
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mg/kg BD) after this episode of ventricular fibrillation.
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Genetic analysis was performed using next generation sequencing targeting 54
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genes associated with cardiac arrhythmia using Agilent SureSelect, Custom Design
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sequenced on the Next Seq 2500 (Illumina). There was a change seen in the RyR2
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gene (Glu3783) that was of uncertain significance. It was a missense mutation,
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which was present in the asymptomatic father; predicted pathogenicity revealed
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mixed results. There was also a missense mutation in the KCNE2 gene (c.170T>C, p.
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(Ile57Thr)), which has been previously described in long QT type 6. Copy number
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analysis revealed an apparently homozygous deletion of exon 2 of the TRDN gene.
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This was confirmed using multiplex ligation dependent probe amplification (MLPA)
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analysis (figure 2). Normal copy number was confirmed for flanking exons. Both
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parents were subsequently confirmed to be carriers of this deletion; both had a
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normal ECG and both had a normal stress test.
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Discussion
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We have described a case of a homozygous deletion of the TRDN gene, which has
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resulted in a more severe phenotype than those described previously.2 The patient
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presented with ventricular fibrillation at a young age and recurrence of these
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arrhythmias despite beta-blockade and flecainide. This deletion is predicted to have
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a profound effect on protein function, resulting in a triadin null phenotype. The
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classic features of ventricular fibrillation, T-wave inversion, and muscle weakness
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are consistent with previous reports of triadin knockout syndrome.6
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This deletion of exon 2 of the TRDN gene is predicted to remove amino acids 8-78.
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This section composes of the N-terminus and all of the transmembrane components
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(residues 48 – 68). The protein likely does not function normally in the membrane
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of the sarcoplasmic reticulum, and as a consequence of this, the C terminus region
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(KEKE repeat region) would be unavailable to interact with RYR2 and CASQ2 to
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form the calcium-regulating unit. (figure 3) This means that the protein is
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essentially non-functional, resulting in a triadin null phenotype.4, 7 It is the large
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deletion of the triadin protein that makes this particular mutation novel. It is
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possible that deletions and copy number variations make up some inherited
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arrhythmias that remain genetically elusive.8
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The term “triadin knockout syndrome”, was used by Altmann et al, in a series of
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genotypically negative long QT patients.2 They described a group of 5 patients that
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all presented with a cardiac arrest less than 3 years of age. The salient features of
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the syndrome were: T-wave inversion in the anterior precordial leads, QT
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prolongation and ventricular fibrillation at a young age. Muscle weakness was also
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described in some patients. The presence of T-wave inversion in the anterior
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precordial leads in our patient is consistent with this part of the syndrome. Some of
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the patients were also described as having skeletal muscle weakness: this was seen
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in our patient. Other families with triadin mutations have been described by Roux-
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Buisson: a homozygous mutation (TRDN c.53_56delACAG) who died at 3 years of
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age following a cardiac arrest.3 The other family had a compound heterozygous
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mutation, (c.176C.G: a missense mutation), a substitution mutation (59 (p.T59R)),
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and a nonsense mutation (c.613C.T). We have also reported siblings previously with
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compound hetrozygous triadin mutations, one novel and one as described above
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(TRDN c.53_56delACAG).9
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The deletion described by Roux-Buisson, and Walsh et al (TRDN c.53_56delACAG),
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also on exon 2, is a frameshift mutation (p.D18Afs*13).3, 9 It results in a premature
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stop codon at residue 31; this is before the bridging section of the transmembrane
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protein. Altman et al saw this particular mutation in homozygous form in one of
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their patients. In the 2 previously described homozygous premature stop codon
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mutations, cardiac arrests occurred at 2 in the case described by Roux-Buisson, and
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3 in the case described by Altman et al.2 Given the homozygous status of the
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premature stop codon on residue 31 these specific mutations, they would also be
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predicted to function as a null phenotype.
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It is possible that our more severe phenotype is due to chance variation, however it
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is also possible that parentally inherited novel RYR2 missense variant, c.11347G>C,
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p.(Glu3783Gln), has caused an increased vulnerability to arrhythmia, causing the
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patient to present at a younger age (i.e. digenic compound heterozygous). The
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Ile57Thr KCNE2 mutation, reduces repolarizing potassium current (Ikr) in vitro, and
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has been reported to be associated with long QT in the absence of VF or torsade de
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pointes.10, 11 This makes it unlikely to be the cause of the severe phenotype seen in
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our patient, however it may have contributed towards the QT prolongation. Given
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the rarity of this TRDN gene deletion, it is likely that the parents shared a common
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ancestor; we were not however able to confirm this with the current genetic
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information.
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We did deliberate extensively about placing a defibrillator in our patient as he was
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returning to Oman, where they would reside permanently. After consulting with
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cardiologists in the patient’s country of origin, we were reassured that adequate
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follow and support was available. The subsequent cardiac arrests underlie the
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importance of placing defibrillators even in smaller patients who have clinically
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apparent triadin mutations.
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The effectiveness of flecainide has been described previously in both CPVT and
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triadin mutations.7 There is debate as to the mechanism of action of flecainide, with
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some reports suggesting it works via a direct effect on the ryanodine channel.
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Recent reports have suggested that flecainide works by altering Na+-dependent
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modulation of intracellular Ca
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cytosolic proteins that modulate RYR2 such as calmodulin. Further work is needed
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to determine the precise mechanism of action for flecainide.13
handling.12 Flecainide may also act by binding
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Conclusion
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We have described a homozygous intragenic TRDN gene deletion that is predicted to
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produce a non-functioning triadin protein. The case demonstrated all of the salient
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features of the “triadin knockout syndrome”, which reinforces the phenotype. The
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infant presented at a very young age, and an epicardial defibrillator was placed
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which delivered appropriate shocks on 2 separate occasions. Patients who are
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genetically confirmed to have biallelic pathogenic triadin triadin variants should be
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treated aggressively with anti-arrhythmic medications and strong consideration
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given to defibrillator placement.
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Chopra N, Yang T, Asghari P, et al. Ablation of triadin causes loss of cardiac
Ca2+ release units, impaired excitation-contraction coupling, and cardiac
arrhythmias. Proc Natl Acad Sci U S A. 2009;106:7636-7641.
Altmann HM, Tester DJ, Will ML, Middha S, Evans JM, Eckloff BW, Ackerman
MJ. Homozygous/Compound Heterozygous Triadin Mutations Associated
With Autosomal-Recessive Long-QT Syndrome and Pediatric Sudden Cardiac
Arrest: Elucidation of the Triadin Knockout Syndrome. Circulation.
2015;131:2051-2060.
Roux-Buisson N, Cacheux M, Fourest-Lieuvin A, et al. Absence of triadin, a
protein of the calcium release complex, is responsible for cardiac arrhythmia
with sudden death in human. Hum Mol Genet. 2012;21:2759-2767.
Rooryck C, Kyndt F, Bozon D, Roux-Buisson N, Sacher F, Probst V, Thambo JB.
New family with catecholaminergic polymorphic ventricular tachycardia
linked to the Triadin gene. J Cardiovasc Electrophysiol 2015.
Schwartz PJ, Ackerman MJ, George AL, Jr., Wilde AA. Impact of genetics on the
clinical management of channelopathies. J Am Coll Cardiol. 2013;62:169-180.
Oddoux S, Brocard J, Schweitzer A, Szentesi P, Giannesini B, Brocard J, Faure
J, Pernet-Gallay K, Bendahan D, Lunardi J, Csernoch L, Marty I. Triadin
deletion induces impaired skeletal muscle function. J Biol Chem.
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Khoury A, Marai I, Suleiman M, Blich M, Lorber A, Gepstein L, Boulos M.
Flecainide therapy suppresses exercise-induced ventricular arrhythmias in
patients with CASQ2-associated catecholaminergic polymorphic ventricular
tachycardia. Heart Rhythm 2010:1671-1675.
Pilichou K, Lazzarini E, Rigato I, et al. Large Genomic Rearrangements of
Desmosomal Genes in Italian Arrhythmogenic Cardiomyopathy Patients. Circ
Arrhythm Electrophysiol. 2017;10.
Walsh MA, Stuart AG, Schlecht HB, James AF, Hancox JC, Newbury-Ecob RA.
Compound Heterozygous Triadin Mutation Causing Cardiac Arrest in Two
Siblings. Pacing Clin Electrophysiol. 2016;39:497-501.
Abbott GW, Sesti F, Splawski I, Buck ME, Lehmann MH, Timothy KW, Keating
MT, Goldstein SA. MiRP1 forms IKr potassium channels with HERG and is
associated with cardiac arrhythmia. Cell. 1999;97:175-187.
Sesti F, Abbott GW, Wei J, Murray KT, Saksena S, Schwartz PJ, Priori SG,
Roden DM, George AL, Jr., Goldstein SA. A common polymorphism associated
with antibiotic-induced cardiac arrhythmia. Proc Natl Acad Sci U S A.
2000;97:10613-10618.
Bannister ML, Thomas NL, Sikkel MB, Mukherjee S, Maxwell C, MacLeod KT,
George CH, Williams AJ. The mechanism of flecainide action in CPVT does not
involve a direct effect on RyR2. Circ Res. 2015;116:1324-1335.
Mehra D, Imtiaz MS, van Helden DF, Knollmann BC, Laver DR. Multiple modes
of ryanodine receptor 2 inhibition by flecainide. Mol Pharmacol.
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References
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Hancox JCJ, Andrew F.; Walsh M.A.; and Stuart A.G. . Triadin mutations - a
cause of ventricular arrhythmias in children and young adults. Journal of
Congenital Cardiology 20171: 9. 2017.
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Figure 1(a): ECG 6 days post cardiac arrest. There was a prolonged QTc interval of 490
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msec and T-wave inversion in the anterior precordial leads from V1-5. (b) Episode of
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ventricular fibrillation that was effectively converted by a single shock, 7 months post
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implantation.
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Figure 2
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Quantitative analysis data for showing the TRDN homozygous deletion of exon 2
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in (1) the index case and heterozygous deletion in (2) both parents. A: Multiplex
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Ligation-dependent Probe Amplification (MLPA®) analysis using control probes
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targeting exon 1 and 3 in addition to standard control probes included within MLPA
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kit (P200). (1) The index case shows complete absence of two probes targeting
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different regions of TRDN exon 2. (2) Both parents have patient/reference ratios <0.61
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for both exon 2 probes, indicating a heterozygous deletion. B: Droplet Digital PCR
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(ddPCR) analysis indicating absence of TRDN exon 2 in the index case and reduced
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signal in the maternal sample confirming heterozygous state.
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Figure 3
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(A and Bi) Normal functioning of the TRDN gene with the KEKE component close to
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the C-terminus, that interacts with CASQ2 and RYR2. (Bii) Dashed lines show that
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components of TRDN missing in our patient which will result in the inability of the
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protein to interact with CASQ2 and RYR2, and inability to anchor in the membrane of
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the sarcoplasmic reticulum.14
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Key teaching points
Homozygous pathogenic TRDN gene mutations result in a severe phenotype
with cardiac arrest often seen at a young age
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Invariably patients presenting with these mutations will require defibrillator
placement
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Medical therapy with B-blockade and flecainide is also required due to the
high risk of appropriate shocks
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Large deletions and duplications may be responsible for some of the cause
genetically elusive arrhythmia syndromes
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