Clinical Trial Details
— Status: Completed
Administrative data
| NCT number |
NCT03387072 |
| Other study ID # |
RC17_0357 |
| Secondary ID |
|
| Status |
Completed |
| Phase |
|
| First received |
|
| Last updated |
|
| Start date |
March 14, 2018 |
| Est. completion date |
March 16, 2021 |
Study information
| Verified date |
March 2021 |
| Source |
Nantes University Hospital |
| Contact |
n/a |
| Is FDA regulated |
No |
| Health authority |
|
| Study type |
Observational
|
Clinical Trial Summary
Despite major progress in molecular and phenotypic characterization of primary electrical
disorders, many (aborted) sudden cardiac deaths (SCD) occur in young victims without
identifiable abnormalities. Investigator recently identified, in 4 families presenting
unexplained SCD, a new arrhythmia entity (catecholamine-induced QT prolongation; CIQTP)
characterized by normal QT duration at rest but major QT lengthening during mental stress
test (MST).
Investigators aim to determine the prevalence of this new phenotype in unexplained SCD and
identify its underlying pathophysiological mechanism.
More specifically, investigators aim to:
- determine the prevalence of CIQTP in unexplained SCD and identify new affected families;
- identify the role of mental stress in QT prolongation;
- identify the genetics basis underlying this life threatening disease;
- perform transcriptomic and electrophysiological profiling of induced pluripotent stem
cell-derived cardiomyocytes (iPSC-CM) from CIQTP patients to identify putative
biomarkers and pathophysiological mechanisms.
MST will be performed, additionally to the conventional screening, in families affected by
unexplained SCD or long QT syndrome (LQTS) referred to university hospitals of Nantes,
Rennes, Tours and Brest. Relevance of the MST on the different type of LQTS will be evaluated
and compared to conventional provocative tests (epinephrine, exercise).
Whole-genome sequencing will first be performed in 3 distantly affected relatives within each
of the 4 largest families identified. As previously performed in Nantes, analysis of the
shared rare variants will allow identifying gene(s) associated with the disease.
Transcriptomic (high-throughput 3' Digital Gene Expression mRNA sequencing) and
electrophysiological (96-well automated optical recordings of action potentials and
patch-clamp recordings of ionic currents, using specific ion channel activators and
inhibitors) profiling will be performed on iPSC-CMs from 2 affected and one unaffected
first-degree relatives of these 4 large families.
Description:
Sudden cardiac death (SCD) is a major cause of death in developed countries. An estimated
40,000 to 50,000 individuals die suddenly each year in France. Most of these deaths involve
elderly people and are related to coronary artery disease. However, an estimated 4000 to 5000
individuals between 1 and 45 years of age die suddenly each year in France. SCD often occurs
in young victims without identifiable ECG abnormalities and without structural defects. In
consequence, most SCD events in the young adult remain unexplained and are classified in an
elusive group known as idiopathic ventricular fibrillation (IVF).
Investigator recently identified a new arrhythmic entity (catecholamine-induced QT
prolongation; CIQTP) responsible for SCD and characterized by normal QT duration at rest but
major QT lengthening during mental stress test. The aim of this translational project is to
determine the prevalence of this new phenotype of rare disease in unexplained SCD and
identify its underlying pathophysiological mechanism.
The use of mental stress test could be extended to other inherited arrhythmic disease such as
long QT syndrome. Given the potential of identification of new affected relatives it may lead
to a significant reduction of the risk of sudden cardiac death recurrence in a family.
The main issue of this project is to describe the clinical implication and the
pathophysiological mechanism of this new syndrome leading to SCD.
Based on our recent description of this cathecholamine induced QT prolongation, the
investigators hypothesize that:
- This syndrome is partly involved in the 60% of undiagnosed familial IVF after complete
familial screening7. Indeed, as the baseline ECG is normal without MST, identification
of such clinical features may be performed by mental stress test in proband and
relatives previously considered as unaffected by inherited arrhythmic disease.
- Mental stress test will help to identify borderline cases of other inherited arrhythmia
such as long QT syndrome. Indeed, this inherited arrhythmia is characterised by a
genetic-based prolongation of the QT interval observed on the baseline ECG, however more
than a third of the patients present with only a slight QT prolongation at baseline.
Consequently, they require the use of provocation tests (e.g. exercise test and
adrenaline test) to unmask pathological QT prolongation18. The Investigator hypothesize
that MST may help to unmask this QT prolongation.
- Based on the autosomal inheritance mode investigator recently described for this
disease, investigator hypothesize that this syndrome belongs to the mendelian disorders.
Therefore, the investigators propose to apply a familial-based approach to identify the
major genes associated with the CIQTP syndrome.
The scope of this project is to describe the clinical implication and the pathophysiological
mechanism of this new syndrome leading to sudden cardiac death. More specifically, the
investigators aim to:
- determine the prevalence of CIQTP in unexplained SCD and identify new large affected
families
- identify the role of mental stress in QT prolongation
- identify associated genes with the disease combining whole genome sequencing and
Identity By Descent analysis in large families
- perform transcriptomic and electrophysiological profiling of induced pluripotent stem
cell-derived cardiomyocytes (iPSC-CM) from CIQTP patients to identify putative
biomarkers and pathophysiological mechanisms.
Based on this translational approach, the investigators aim to describe pathophysiology and
clinical implication of CIQTP in sudden cardiac death. The description of this new syndrome
using innovative approach such as whole genome sequencing and iPSC-CM profiling may provide a
comprehensive and major progress in the field of unexplained SCD. Beyond the identification
and the care of newly affected relatives, it may help defining a genetic/pathophysiology
based therapy.
The investigators already proved that MST is a highly efficient diagnostic tool to detect
this new syndrome, and the investigators believe that it may also help to unmask QT
prolongation in silent LQTS mutation carriers. MST has the advantage of being rapid and
simple to apply. It could thus be of great interest to uncover familial segregation for
unexplained cardiac electrical defects showing autosomal dominant transmission. It could
additionally decrease the apparent non-penetrance encountered in LQTS.
Tasks of the project
Task 1: determine the prevalence of CIQTP in unexplained SCD and identify new affected
families
Most cases of SCD before the age of 45 remain unexplained and are likely to be due to
inherited arrhythmic disease6. After unexplained SCD, all consecutive families referred to
Nantes, Rennes, Tours or Brest University Hospitals will be included in the study.
All available clinical proband data will be collected from the referring physicians and
relatives. When autopsy is performed, the probands whose diagnosis will be achievable will be
excluded from the study.
Familial screening After unexplained SCD before the age of 45, familial screening will be
performed in first-degree relatives and then extended to other relatives if positive. When
first-degree relatives are not available for screening, it will be performed in more distant
relatives.
Clinical screening will include a review of past medical history, clinical examination and
baseline ECG. In the absence of diagnosis after this first set of exams, stress test,
transthoracic echocardiogram (TTE), sodium channel blocker challenge (SCBC) either with
ajmaline (1 mg/kg) or flecainide (2 mg/kg) and an epinephrine test23,24 will be performed.
Mental stress test will be additionally performed to unmask QT prolongation with normal
baseline ECG 25.
Mental stress test protocol (appendix 2) MST will be performed as follows. Patients will be
placed in supine position with no explanation on the course of the test. ECG will be recorded
at baseline and permanently during the test. MST will be then performed abruptly according to
the patient's level of education (mental arithmetic, geography or names of Disney characters
for children) with a requirement for correct and rapid answers. MST will be considered as
relevant in the presence of at least a 20 beats-per-minute increase in heart rate. In case of
absence of a 20 beats-per-minute increase in heart rate, the test will be considered as
non-interpretable unless a significant prolongation of the QTc is identified. MST usually
lasts less than 2 minutes. A new ECG will be recorded 30 minutes after the end of the test in
an attempt to obtain an ECG trace with the lowest heart rate.
Diagnosis
ECG performed at baseline, during the stress test and during the pharmacological test will be
reviewed by two physicians blinded to the clinical and genetic status. All the data will be
collected in a core database.
The diagnosis criteria adhered to the guidelines of the latest consensus conference24,26. The
investigators will additionally consider a delta QTm > 30 ms during epinephrine or the mental
stress test and a delta QTc > 30 ms at 3 min after the stress test, as sufficient for
diagnosis of LQTS 27,28.
A minimum interval of 30 minutes between each test will be observed to enable catecholamine
clearance. All tests will be performed at least 5 days after interrupting each treatment that
could interfere with the tests.
This study is conducted according to European guidelines for clinical and genetic research.
Protocol approval will be obtained from institutional ethical committees. Informed written
consent will be obtained from each patient who agreed to participate to the clinical and
genetic study.
Statistical analysis Statistical analyses will be performed with Social Science® software
(IBM Corp., Armonk, New York). Categorical variables will be presented with their numbers and
percentages. The chi-square or Fisher exact test (based on expected frequency) will be used
to compare categorical variables between groups.
Quantitative variables will be presented as mean ± standard deviation or median (25th; 75th).
Analysis of continuous variables will be performed using a t-test or Mann Whitney test based
on distribution. Comparisons among families will be performed using intra-familial mean value
or median of continuous variable in order to limit the bias toward family size. A two-sided
P-value of less than 0.05 will be considered to indicate statistical significance in all
tests.
Task 2: identify the role of mental stress in QT prolongation
To identify the role of mental stress in QT prolongation the investigators will additionally
enroll positive and negative control groups in Nantes, Rennes, Tours or Brest University
Hospitals.
This will consist in the enrolment of consecutive relatives of families affected by long QT
syndrome without QT prolongation at baseline. The result of this test will be compared to
conventional screening using exercise test and epinephrine test. Thereby, the investigators
aim to enroll patient further defined as carriers of the main genetic-based forms of long QT
syndrome (LQT1, LQT2 and LQT3, positive control group).
The investigators will additionally enrol a negative control group of healthy patients with
normal baseline ECG, no cardiac structural abnormalities and without any medication that
could interfere with the MST. This population will consist in relatives of families negative
for unexplained SCD screening.
Diagnosis criteria and test protocol will be identical to those defined in task 1.
Three consecutive beats will be used for each measurement and the average value obtained by
the two investigators will be taken into account. QT interval will be measured in DII or V5,
from the beginning of the QRS complex to the end of the T-wave (defined as the intersection
of the tangent of the T-wave and the isoelectric line). U-wave will be excluded from the
measurement of the QT interval.
This study is conducted according to European guidelines for clinical and genetic research.
Protocol approval will be obtained from institutional ethical committees. Informed written
consent will be obtained from each patient who agreed to participate in the clinical and
genetic study.
Statistical analyses will be performed with Social Science® software (IBM Corp., Armonk, New
York). Comparisons between groups will be performed for each clinical and ECG parameter at
baseline and during both tests (mental stress test, exercise test, epinephrine test).
Categorical variables will be presented with their numbers and percentages. The chi-square or
Fisher exact test (based on expected frequency) will be used to compare categorical variables
between groups. Quantitative variables will be presented as mean ± standard deviation or
median (25th; 75th). Analysis of continuous variables will be performed using a t-test or
Mann Whitney test based on distribution. A two-sided P-value of less than 0.05 will be
considered to indicate statistical significance in all tests.
Investigator have already enrolled in Nantes hospital a population of 30 healthy controls and
25 subjects affected with LQTS (LQT1=9 or LQT2=16) but with a resting QTc<480 ms. First
results have identified a sensitivity of exercise test, epinephrine test and MST respectively
of 60%, 95% and 94% within the families, and 76%, 93% and 92% within the positive controls,
for a specificity of 100% reached for every test. This confirms that MST test may be of
interest in long QT patients with an absence of clear QT prolongation.
Task 3: identify specific genes associated with the disease
Objective: To identify rare variants in new genes contributing strongly the risk of VF within
pedigrees.
Four large IVF families presenting 6 SCD (with 2 resuscitated) and 7 unexplained syncope
episodes occurring at young age have been characterized in Nantes. Among them, respectively
9, 10, 11, and 4 family members show a prolonged QT interval after MST and/or another
provocative test while all LQTS-associated genes were negative. The description of this new
clinical entity has been recently accepted for publication in the Journal of the American
College of Cardiology17. The occurrence of VF/SCD or ECG abnormalities in genetically distant
individuals within families makes pedigrees highly powerful for genetic studies, since the
greater the genetic distance, the less variants the patients will share. Of note additional
relatives will be clinically investigated (individual with question mark symbols on pedigrees
below) and may increase the power of the genetics study. Previous works of J. Barc and Nantes
group demonstrated our capability to lead such ambitious projects and specifically on gene
discovery based on familial approach coupled to next generation sequencing.
task3.1: Whole genome genotyping and analysis of the affected family members Aim: Identify a
genomic region shared by the affected members.
Genome-wide genotyping on all affected family members will be performed using Axiom
Genome-Wide Human Precision Medicine Research Array (PMRA) Plate on Affymetrix GeneTitan®
Multi-Channel, instrument available through the genomic core facility of Nantes of our lab.
Genomic regions shared by affected family members will be assessed by Identity By Descent
(IBD) analysis as previously described. This step is crucial to refine the genome region
where the causal variant is located. According the large size of the families the
investigators can expect that a relatively small genomic region will come up from this
analysis. Attention will be dedicated to potential shared region among families that would
indicate a potential common gene.
task3.2: Whole genome sequencing and analysis Aim: identify rare variants among the genomic
regions shared by affected family members.
Here the investigators shall carry out genome sequencing in 3 distantly related individuals
affected by CIQTP syndrome from each of the 4 pedigrees. Advantages with genome sequencing
reside on the absence of capturing and PCR steps, limiting strongly the capture artefacts,
the non-covered regions and offering the possibility of uncovering a mutation within an
essential regulatory element belonging to the non-coding region. Sequencing will be performed
in collaboration with the National Genotyping Center (CEA-CNG). The NGS pipeline follows the
recommendations of the Broad Institute GATK "Best Practices"
broadinstitute.org/gatk/guide/best-practices.php. Basically, reads are mapped using bwa-mem,
Duplicates are removed using picard, GATK is used to realign and recalibrate the reads. The
variants are called with HaplotypeCaller and are recalibrated with GATK. The generated VCFs
are then annotated with Ensembl Variant Effect predictor. From VCF files and within each
pedigree, the investigators shall prioritize variants shared by the 3 affected individuals
and contained within shared genomic regions (IBD). From the shared variants the investigators
shall focus on rare variants and then filter out variants that are common in the general
population (minor allele frequency >0.1%). For this the investigators will use public and
in-house exome/genome databases gathering in total about 130 000 exomes and 20 000 genomes
such as The gnomAD consortium, 1000Genomes, the Genome of the Netherlands and demographic
matched controls specifically from Nantes area (PREGO study-www.vacarme-project.org). This is
expected to result in a short-list of few "candidate" variants/genes per family. A
bioinformatics prediction of their functional effect will be assessed by the programs SIFT
and Polyphen2 to highlight putative malignant variants among those. The investigators will
validate all variants identified in such way by Sanger sequencing and investigate their
co-segregation with other affected family members in the respective pedigrees. Experience of
the genomics and bioinformatics core facility of Nantes of our lab, which are recognized as
French national infrastructures in genomics (IBiSA) and bioinformatics (Institut Français de
Bioinformatique) coupled to our experience in NGS data managing will assure the feasibility
of this Task. Furthermore a large genome sequencing project gathering more than 1000 whole
genome (including cardiac patients and individuals from the general population) is on going
in our group. Experience from this project will reinforce the efficiency of the task#3
analysis and chance of success.
Task 4: perform transcriptomic and electrophysiological profiling of induced pluripotent stem
cell-derived cardiomyocytes from CIQTP patients to identify putative biomarkers and
pathophysiological mechanisms
The investigators make the hypothesis that induced pluripotent stem cell-derived
cardiomyocytes (iPSC-CMs) generated from CIQPT patients will allow us to identify the global
gene expression changes and electrophysiological alterations associated with the disease. For
that, the investigators aim at comparing iPSC-CMs generated from members of the 4 families
described in task 3. Within each pedigree the investigators will select one "control" healthy
subject (not presenting the familial QT prolongation), and 2 CIPQT patients.
Positive LQT control and negative healthy control iPS cell lines have already been generated
and studied in Nantes in a context of a previous study on LQT2 demonstrating the feasibility
of this task. This will also offer the opportunity to compare CIPQT transcriptomic and
electrophysiological signature to LTQ2 ones. For each line, 3 iPS cell clones have been
validated: all lines have normal karyotype and meet the current standards for pluripotency.
The investigators successfully differentiated the iPS cells into the cardiac lineage, using a
recently described method based on temporal modulation of Wnt/ß-catenin signaling using small
molecules. Preliminary data suggest that the investigators can generate high purity and
efficiency of cardiac differentiation. Regarding the iPS cells from the LQT2 patient, the
investigators showed that hERG A561P mutation leads to a trafficking defect that results in
reduced IKr current, and consequently e to action potential prolongation and cellular
arrhythmias(early afterdepolarizations) .
Upon obtainment and validation of all iPS cell lines, the first step of the project will
consist in the identification of the cardiac gene expression profile associated with CIQTP.
For that, the investigators will use cardiomyocytes differentiated from control and
patient-derived iPS cells (day 28) to perform RNA seq analyses, in collaboration with the
Genomics core facility of our lab. The investigators will also perform high-throughput action
potential and ionic current analyses using automated patch clamp and optical based
(CellOptiq®) techniques under baseline conditions and after drug-induced adrenergic
stimulation. All combined, these analyses will provide us with the first identification of
the electrophysiological profile associated with CIQTP in human cardiomyocytes. With this
analysis, the investigators will be able to link CIQTP-associated altered gene expression
program to defective cardiac physiology.
iPSC cardiac differentiation protocols: cells will be cultured on a Matrigel-coated plate and
exposed to cardiogenic cytokines: CHIR and IWR-1. Beating colonies appear after about 10
days. After 20 days, cells will be dissociated and reseeded for electrophysiological
analyses, 10 days later. Finally, after 28 days of differentiation, beating cells will be
collected for RNA seq analysis.
Whole transcriptome sequencing (RNAseq): beating iPSC-CMs at 28 days of 3 differentiation
from 2 clones of each iPS cell line will be collected and RNA will be extracted using
Macherey-Nagel kit. Presence and quantity of each RNA of the samples will then be assessed
using next generation sequencing (Illumina HiSeq technology). Bioinformatics analysis will be
performed by the BIRD core facility of our lab.
Ionic current recordings by automated patch clamp: CIQTP's repolarization defects likely
reflect dysfunction of specific ionic currents that will be recorded using whole cell
patch-clamp: sodium current, INa, calcium current, ICa,L, and potassium currents, IKs and
IKr.
Action potential recording: This recording will be performed using 2 techniques.
1. Isolated beating cells will be analyzed using perforated patch-clamp technique with
amphotericin B at 37oC. Patch pipettes will be filled with a solution that has an ionic
composition similar to the intracellular fluid.
2. Action potential optical recordings will be performed on cardiomyocytes after 28 days of
differentiation, with 5 µmol/L di-8-ANEPPS as the voltage-sensitive dye using the
CellOptiq® technology (Clyde Biosciences). Cells will be incubated at 37°C, 5% CO2 and
20% O2 during the 2 h-incubation of the dye and during the recording.
