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Targeted resequencing identifies TRPM4 as a major gene predisposing to progressive familial heart block type I

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Background: Progressive cardiac conduction disease (PCCD) is one of the most common cardiac conduction disturbances. It has been causally related to rare mutations in several genes including SCN5A, SCN1B, TRPM4, LMNA and GJA5. Methods and results: In this study, by applying targeted next-generation sequencing (NGS) in 95 unrelated patients with PCCD, we have identified 13 rare variants in the TRPM4 gene, two of which are currently absent from public databases. This gene encodes a cardiac calcium-activated cationic channel which precise role and importance in cardiac conduction and disease is still debated. One novel variant, TRPM4-p.I376T, is carried by the proband of a large French 4-generation pedigree. Systematic familial screening showed that a total of 13 family members carry the mutation, including 10 out of the 11 tested affected individuals versus only 1 out of the 21 unaffected ones. Functional and biochemical analyses were performed using HEK293 cells, in whole-cell patch-clamp configuration and Western blotting. TRPM4-p.I376T results in an increased current density concomitant to an augmented TRPM4 channel expression at the cell surface. Conclusions: This study is the first extensive NGS-based screening of TRPM4 coding variants in patients with PCCD. It reports the third largest pedigree diagnosed with isolated Progressive Familial Heart Block type I and confirms that this subtype of PCCD is caused by mutation-induced gain-of-expression and function of the TRPM4 ion channel.
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Targeted resequencing identies TRPM4 as a major gene predisposing to
progressive familial heart block type I
Xavier Daumy
a,b,c,1
, Mohamed-Yassine Amarouch
d,1,2
, Pierre Lindenbaum
a,b,c,e
, Stéphanie Bonnaud
a,b,c,e
,
Eric Charpentier
a,b,c
, Beatrice Bianchi
d
,SabineNafzger
d
, Estelle Baron
a,b,c
, Swanny Fouchard
d
,
Aurélie Thollet
d
, Florence Kyndt
a,b,c,e
, Julien Barc
a,b,c
, Solena Le Scouarnec
a,b,c
, Naomasa Makita
f
,
Hervé Le Marec
a,b,c,e
, Christian Dina
a,b,c,e
, Jean-Baptiste Gourraud
a,b,c,e
, Vincent Probst
a,b,c,e
, Hugues Abriel
d,
,1
,
Richard Redon
a,b,c,e,1
, Jean-Jacques Schott
a,b,c,e,
⁎⁎
,1
a
Institut National de la Santé et de la Recherche Médicale (INSERM) Unité Mixte de Recherche (UMR) 1087, l'institut du thorax, Nantes, France
b
Centre National de la Recherche Scientique (CNRS) UMR 6291, l'institut du thorax, Nantes, France
c
Université de Nantes, l'institutdu thorax, Nantes, France
d
Department of Clinical Research, and Swiss National Centre of Competence in Research (NCCR) TransCure, University of Bern, Switzerland
e
Centre Hospitalier Universitaire (CHU) de Nantes, l'institut du thorax, Service de Cardiologie, Nantes, France
f
Molecular Physiology, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan
abstractarticle info
Article history:
Received 15 July 2015
Received in revised form 5 November 2015
Accepted 1 January 2016
Available online 11 January 2016
Background: Progressive cardiac conduction disease (PCCD) is one of the most common cardiac conduction
disturbances. It has been causally related to rare mutations in several genes including SCN5A,SCN1B,TRPM4,
LMNA and GJA5.
Methods and results: In this study, by applying targeted next-generation sequencing (NGS) in 95 unrelated pa-
tients with PCCD, we have identied 13 rare variants in the TRPM4 gene, two of which are currently absent
from publicdatabases. This geneencodes a cardiac calcium-activated cationic channel which precise role andim-
portance in cardiac conduction and disease is still debated. One novel variant, TRPM4-p.I376T, is carried by the
proband of a large French 4-generation pedigree. Systematic familial screening showed that a total of 13 family
members carry the mutation, including 10 out of the 11 tested affected individuals versus only 1 out of the 21
unaffected ones. Functional and biochemical analyses were performed using HEK293 cells, in whole-cell
patch-clamp conguration and Western blotting.TRPM4-p.I376T results in an increased current density concom-
itant to an augmented TRPM4 channel expression at the cell surface.
Conclusions: This study is the rst extensive NGS-based screening of TRPM4 coding variants in patients with
PCCD. It reports the third largest pedigree diagnosed with isolated Progressive Familial Heart Block type I and
conrms that this subtype of PCCD is caused by mutation-induced gain-of-expression and function of the
TRPM4 ion channel.
© 2016 Published by Elsevier Ireland Ltd.
Keywords:
TRPM4
Atrio-ventricular block
PFHBI
Gain-of-function mutation
1. Introduction
Progressive cardiac conduction defect (PCCD) was rst described in
the sixties by Lenègre [1] and Lev [2] as a brosis process affecting the
conduction system. It is one of the most common cardiac conduction
disturbances characterized by a progressive alteration of cardiac con-
duction through the His-Purkinje system with right or left bundle
branch block (RBBB or LBBB) and widening of QRS complexes, leading
to complete atrioventricular block (AVB), syncope and sudden death.
Familial cases of PCCD have been reported with an autosomal
dominant inheritance and causally related to rare mutations in genes
involved in cardiac impulse propagation (SCN5A [3,4],SCN1B [5] and
TRPM4 [6,7]), in the structure of the nuclear lamina (LMNA [8,9]) and
in cell-to-cell communication (GJA5 [10]).
Among these genes, TRPM4, encoding a calcium-activated cationic
channel that is expressed in Purkinje bers and nodal tissue [6,7],has
rst been linked to progressive familial heart block type I (PFHBI) in
two large pedigrees, respectively from South Africa [6] and Lebanon
International Journal of Cardiology 207 (2016) 349358
Correspondence to: H. Abriel, University of Bern, Department of Clinical Research,
Murtenstrasse, 35, 3010 Bern, Switzerland.
⁎⁎ Correspondence to: J.-J. Schott, Inserm UMR 1087, l'institut du thorax IRT-UN, 8 quai
Moncousu, 44007 Nantes, France.
E-mail address: Hugues.Abriel@dkf.unibe.ch (H. Abriel), jjschott@univ-nantes.fr
(J.-J. Schott).
1
These authors contributed equally to this work.
2
Current afliation: Materials, Natural Substances, Environment & Modeling
Laboratory, University of Sidi Mohamed Ben Abdellah- Fes, Multidisciplinary Faculty of
Taza, Taza, Morocco.
http://dx.doi.org/10.1016/j.ijcard.2016.01.052
0167-5273/© 2016 Published by Elsevier Ireland Ltd.
Contents lists available at ScienceDirect
International Journal of Cardiology
journal homepage: www.elsevier.com/locate/ijcard
[7]. PFHBI is associated with a progressive impairment of the His bundle
branchesconduction, typically starting with RBBB and then left anterior
hemiblock (LAHB) and that may progress to a complete AVB. QRS dura-
tion increases with time while PR and QTc intervals remain constant
[1113].
Conduction defects in TRPM4-dependent familial cases were shown
to be related to gain-of-function mutations proposed to be caused by a
reduction of the deSUMOylation of TRPM4 channels and an impaired
endocytosis resulting in stabilization and overexpression of mutant
channels at the plasma membrane [6,7]. Since these two seminal re-
ports, eighteen gain- or loss-of-function variants have been identied
as causing diverse forms of cardiac conduction defects and/or Brugada
syndrome [1416].
Here, using next generation sequencing (NGS) technologies, we re-
port novel TRPM4 variants including one (c.T1127C; p.I376T) segregat-
ing with the third largest reported PCCD family. This missense
mutation segregates in 39 relatives of a 4-generation pedigree and
was observed to lead to gain-of-expression and function of the mutant
channel. These ndings strongly support a central role of TRPM4 in car-
diac conduction and cardiac conduction disorders.
2. Methods
2.1. Patient phenotyping
The study was conducted according to the French guidelines for
genetic research and approved by the ethic committee of the Nantes
University Hospital. A written consent was obtained for each family
member who accepted to participate in the study.
The investigation included a physical examination with particular
attention to the cardiovascular system and a 12-lead ECG. Heart rate,
PR interval, QRS, QTc duration, P and QRS axes were measured automat-
ically at rest (MacVu Marquette Inc., Milwaukee, Wisconsin, USA). Con-
duction defects were dened using the conventional classication [17,
18]. Two expert physicians, blinded to the clinical status, independently
and systematically reviewed the ECG parameters.
Because of the prevalence of minor conduction defects in the general
population and in order to decrease the riskof misclassication, only the
most obviously affected patients were considered as affected. QRS axis
was classied as normal when its value was between 30° and +90°.
PR duration shorter than 210 ms was considered as normal. Patients
were considered as affected if they have been implanted with a pace-
maker (PM) for PCCD or if they have an ECG showing a major conduc-
tion defect (complete AVB, complete RBBB, complete LBBB, parietal
block (PB) dened as a QRS wider than 115 ms without morphology
of RBBB or LBBB, LAHB or left posterior hemiblock (LPHB)). Given the
progressive nature of the disease, only patients older than 45 without
any conduction defect were considered as unaffected. All the other
patients were considered as undetermined and not included for the
evaluation of the ECG parameters. Cardiac morphological diseases
were excluded by echocardiography in all patients.
2.2. Targeted sequencing
Genomic DNA was extracted from peripheral blood lymphocytes by
standard protocols. The DNA yields were assessed by measurements
using Quant-ITdsDNA Assay Kit, Broad Range (Life Technologies,
Q33130). The purity of the DNA was assessed by spectrophotometry
(OD 260:280 and 260:230 ratios) using a Nanodrop instrument (Ther-
mo Scientic). DNA integrity was assessed by separation in a E-Gel®
96 Agarose Gels, 1% (Life Technologies, G700801). For multiplex ampli-
cation, we used the HaloPlexTarget Enrichment System (Agilent
Technologies, 1500 kb, ILMFST, 96 reactions, G9901B), Protocol
Version D.2 (November, 2012). We applied a custom HaloPlexdesign
enabling high-throughput sequencing of the coding regions of 45 genes
previously linked to cardiac arrhythmias or conduction defects and/or
sudden cardiac death, including 19 genes known or suspected to be in-
volved in PCCD. In this study we focused solely on the relevant 19 PCCD
genes including SCN5A,SCN1B,TRPM4,GJA5 and LMNA that havealready
been associated with isolated cardiac conduction defects together with
GJA1,GJC1,SCN10A,NKX2-5,TBX5,SNTA1,PRKAG2,RYR2,EMD,BMP2,
BMPR1A,GATA4,MSX2 and TNNI3K as likely candidate genes. The
targeted coding regions (exons) ± 10 bp correspond to141 kb of geno-
mic sequence.
Target enrichment and sequencing were performed as previously
described [19]. First, 200 ng of gDNA samples were digested in eight
differentrestriction reactions, eachcontainingtwo restriction enzymes,
to create a library of gDNA restriction fragments. These gDNA restriction
fragments were hybridized to the HaloPlex probe capture library.
Probes are designed to hybridize and circularize targeted DNA frag-
ments. During the hybridization process, Illumina sequencing motifs in-
cluding index sequences were incorporated into thetargeted fragments.
The circularized target DNA biotinylated HaloPlex probe complexes
were captured on magnetic streptavidin beads. We proceeded to a liga-
tion reaction of the circularized complexes followed by an elution reac-
tion before PCR amplication. The amplied target DNA was puried
using AMPure XP bead (Beckman Coulter, A63881). To validate enrich-
ment of target DNA in each library sample by microuidics analysis,
we used the 2200 TapeStation (Agilent Technologies, G2964AA), with
D1K ScreenTape (Agilent Technologies, 50675361), and D1K Reagents
(Agilent Technologies, 50675362). We ensured that the majority of
amplicons range from 175 to 625 bp. Finally we quantied each library
by qPCR using KAPA Library Quantication Kit (Clinisciences, KK4854).
Libraries were pooled to an equimolar concentration and DNA was
then denatured with NaOH. Finally libraries pool was diluted to a 4
pM nal concentration before proceeding to 100 bp paired-end Illumina
sequencing on HiSeq.
2.3. Detection of rare coding variation in TRPM4
Raw sequence reads were aligned to the human reference genome
(GRCh37) using BWAMEM (version 0.7.5a) after removing sequences
corresponding to Illumina adapters with Cutadapt v1.2. GATK was
used for indel realignment and base recalibration, following GATK
DNAseq Best Practices. Variants were called for each sample separately
using GATK UniedGenotyper (version 2.8) and Samtools mpileup
(version 0.1.19), and variants were considered for further analyses if
found by both GATK and Samtools with a minimum quality score of 25.
Variants were considered of interest if: 1They present a potential
pathogenicity as predicted by Variant Effect Predictor (Ensembl).
Variants were considered as having a potential functional consequence
if they were annotatedwith one or more of the following SO terms for at
least oneRefSeq transcript: transcript_ablation(SO:0001893), splice_
donor_variant(SO:0001575), splice_acceptor_variant(SO:0001574),
stop_gained(SO:0001587), frameshift_variant(SO:0001589),
stop_lost(SO:0001578), initiator_codon_variant(SO:0001582),
inframe_insertion(SO:0001821), inframe_deletion(SO:0001822),
missense_variant(SO:0001583), transcript_amplication
(SO:0001889); 2They were rare that is if the minor allele frequency
(MAF) was b1% compared to the 1000 genomes phase 1 data (379
individuals of European origin, integrated release v3, downloaded
from ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/release/20110521), to
the NHLBI GO Exome Sequencing Project (ESP) data Exome Variant
Server (EVS) (4300 individuals of European origin, ESP6500SI-V2 re-
lease, downloaded from http://evs.gs.washington.edu/EVS), and to the
Exome Aggregation Consortium (ExAC) data (60,706 unrelated individ-
uals including more than 33,300 non-Finnish European individuals,
release v1, downloaded from ftp://ftp.broadinstitute.org/pub/ExAC_
release).
In case of missense variants SIFT [20] and PolyPhen-2 (PPH-2) [21]
were used to predict the impact of the amino acid substitutions. Filter-
ing was performed using Knime4Bio [22].
350 X. Daumy et al. / International Journal of Cardiology 207 (2016) 349358
2.4. Segregation analysis
Familial segregation analyses were c arried out by bidirectional direct
sequencing of amplied genomic DNA amplicons with variant-specic
primers (forward: CCTCCATCCCTTTGGACAG; reverse: CAGGCCAGGA
AAGGTGTCTA) using the Big Dye Terminator v3.1 Cycle Sequencing
Kit(Applied Biosystems - Life Technologies) following the
manufacturer's recommendations. The capillary sequencing was per-
formed on Applied Biosystems 3730 DNA Analyzer using standard pro-
cedures provided by Applied Biosystems (Life Technologies).
The RefSeq NM_017636.3 transcript has been used to compare our
sequencing data.
Linkage was assessed between the variant I376K and the disease
using standard method comparing likelihood under a recombination
fraction of 50% (no linkage) and 0% (full linkage). LOD score [23] calcu-
lation was performed with Superlink-Online SNP version 1.1 (http://
cbl-hap.cs.technion.ac.il/superlink-snp/main.php)[24].
We postulated a rare causal variant (frequency set at 1/10,000) and
dominant model with high penetrance (80%). The prevalence is 5% and
therefore, the phenocopy rate is 0.0499.
A LOD score higher than 3 is considered as signicant for linkage.
2.5. Cell culture and transfection
Human embryonic kidney (HEK293) cells were cultured with
DMEM medium supplemented with 4 mM Glutamine, 10% FBS and a
cocktail of streptomycinpenicillin antibiotics. For the electrophysiolog-
ical studies, the cells were transiently transfected with 80 ng of HA-
TRPM4 WT or HA-TRPM4 p.I376T in a 35 mm dish mixed with 4 µl of
JetPEI (Polyplus transfection, Illkirch, France) and 46 μl of 150 mM
NaCl. The cells were incubated for 24 h at 37 °C with 5% CO2. All trans-
fections included 400 ng of cDNA encoding CD8 antigen as a reporter
gene. Anti-CD8 beads (Dynal®, Oslo, Norway) were used to identify
transfected cells, and only CD8-displaying cells were analyzed. Cells
were used 24 h after transfection.
For the biochemical studies, HEK 293-cells were transiently
transfected with 960 ng of either HA-TRPM4 WT, HA-TRPM4 p.I376T
variants or empty vector (pcDNA4TO) in a P100 dish (BD Falcon, Dur-
ham, North Carolina, USA) mixed with 30 μl of JetPEI (Polyplus transfec-
tion, Illkirch, France) and 250 μl of 150 mM NaCl. The cells were
incubatedfor48hat3Cwith5%CO2.
2.6. Cell surface biotinylation assay
Following 48 h of incubation, transiently transfected HEK293 cells
were treated with EZlinkTM Sulfo-NHS-SS-Biotin (Thermo Scientic,
Rockford, Illinois, USA) 0.5 mg/ml in cold 1X PBS for 15 min at 4 °C. Sub-
sequently, the cells were washed twice with 200 mM glycine in cold 1X
PBS and twice with cold 1X PBS to inactivate and remove the excess bi-
otin, respectively. The cells were then lysed with 1X lysis buffer (50 mM
HEPES pH 7.4; 150 mM NaCl; 1.5 mM MgCl
2
; 1 mM EGTA pH 8.0; 10%
Glycerol; 1% Triton X-100; 1X Complete Protease Inhibitor Cocktail
(Roche, Mannheim, Germany)) for 1 h at 4 °C. Cell lysates were centri-
fuged at 16,000 g; 4 °C for 15 min. Two milligram of the supernatant
was incubated with 50 μl Streptavidin Sepharose High Performance
beads (GE Healthcare, Uppsala, Sweden) for 2 h at 4 °C, and the remain-
ing supernatant was kept as the input. The beads were subsequently
washed ve times with 1X lysis buffer before elution with 50 μlof2X
NuPAGE sample buffer (Invitrogen, Carlsbad, California, USA) plus
100 mM DTT at 37 °C for 30 min. These biotinylated fractions were an-
alyzed as TRPM4 expressed at the cell surface. The input fractions, ana-
lyzed as total expression of TRPM4, were resuspended with 4X NuPAGE
Sample Buffer plus 100 mM DTT to give a concentration of 1 mg/mland
incubated at 37 °C for 30 min.
2.7. Western blot experiments
Protein samples were analyzed on 9% polyacrylamide gels, trans-
ferred with the TurboBlot dry blot system (Biorad, Hercules, CA, USA)
and detected with anti-TRPM4 (generated by Pineda, Berlin,
Germany), anti α-actin A2066 (Sigma-Aldrich, St. Louis, Missouri,
USA) antibodies using SNAP i.d. (Millipore, Billerica, MA, USA). The
anti-TRPM4 antibody was generated by Pineda (Berlin, Germany)
using the following peptide sequence: NH2-CRDKRESDSERLKRTSQKV-
CONH2. A fraction of the antisera, which was subsequently used in
this study, was then afnity puried.
2.8. Cellular electrophysiology
For patch-clamp experiments in whole-cell conguration, glass pi-
pettes (tip resistance, 1.53MΩ)werelled with an intracellular solu-
tion containing (in mM): 100 CsAsp, 20 CsCl, 4 Na
2
ATP, 1 MgCl
2
,10
EGTA, and 10 HEPES. The pH was adjusted to 7.20 with CsOH, and the
free Ca
2+
concentration at 100 μM with CaCl
2
using WEBMAXCLITEpro-
gram (http://www.stanford.edu/~cpatton/downloads.htm). Access re-
sistance ranges was from 3 to 5 MΩ. Extracellular solution contained
(in mM): 156 NaCl, 1.5 CaCl
2
,1MgCl
2
, 6 CsCl, 10 glucose and 10
HEPES. The pH was adjusted to 7.40 with NaOH. Patch-clamp recordings
were carried-out in the whole-cell conguration at room temperature
(2325 °C). TRPM4 currents were investigated using a ramp protocol.
The holding potential was 60 mV. The 400 ms increasing ramp from
100 to + 100 mV ends with a 300 ms step at + 100 mV then
300 ms at 100 mV. A new ramp was performed every 2 s. Before
seal formation, liquid junction potential was compensated to keep
the baseline at 0 mV. Using a Digidata 1440 A analog-digital inter-
face (Axon Instruments,Inc.), currents were ltered at 5 kHz and the
sampling frequency was at 50 kHz. Current densities were obtained by
dividing the peak current recorded at 100 mV by the cell capacitance
(17 ± 2 pF and 16 ± 1 pF, respectively transfected with WT and I376T-
TRPM4 channels). Of note, capacitances and series resistances were not
compensated.
2.9. Data analysis and statistical methods
Currents were analyzed with Clampt software (Axon Instruments,
Inc). Data were analyzed using a combination of pClamp10, Excel
(Microsoft) and Prism (Graphpad).
Comparisons between groups were performed with impaired two-
tailed Student's t test. Data are expressed as mean + SEM. A p-value
b0.05 was considered signicant.
3. Results
3.1. Mutational screening
Ninety-ve patients with PCCD were recruited through the French
National Referral Center for Sudden Cardiac Death as previously de-
scribed [25]. Nineteen genes known or suspected to be involved in con-
duction defects were sequenced in these patients using the HaloPlex
System, resulting in a mean coverage depth of 578 × per sample:
SCN5A,SCN1B,TRPM4,GJA5 and LMNA that have already been associated
with isolated cardiac conduction defects together with GJA1,GJC1,
SCN10A,NKX2-5,TBX5,SNTA1,PRKAG2,RYR2,EMD,BMP2,BMPR1A,
GATA4,MSX2 and TNNI3K.A graphical representationof the mean cover-
age obtained for the 5 major genes is provided in Supplemental Fig. 1.
When selecting only genetic variants with a potential pathogenicity
as predicted by Variant Effect Predictor (see methods) and an MAF
below 1% in public databases, we identied a total of 45 variants in 43
patients: 11 novel variants and 34 rare variants (see Supplemental
Table 1). Among these variants, 13 have already been associated with
cardiac pathologies such as the Brugada syndrome and cardiac
351X. Daumy et al. / International Journal of Cardiology 207 (2016) 349358
Table 1
Characteristics of identied amino acid variants in TRPM4.*
No. Patient Exon Nucleotide Amino
Acid
Effect Genotype Other
variant(s) in
susceptibility
genes
SIFT | PPH-2 dbSNP141
database id
EUR_AF
(1000
genomes)
(%)
EVS_UAMAF
(%)
AllelicFreq_NFE
(ExAC) (%)
ECG
morphology
Phenotype
1.1 35 6 c.755 GNA R252H [16] missense_variant Heterozygous 0 deleterious(0.01) |
possibly_damaging(0.772)
rs146564314 0 0.63 0.818 RBBB type 2 AVB 2°
1.2 36 6 c.755 GNA R252H [16] missense_variant Heterozygous 0 deleterious(0.01) |
possibly_damaging(0.772)
rs146564314 0 0.63 0.818 LBBB type 2 AVB 2°
1.3 37 6 c.755 GNA R252H [16] missense_variant Heterozygous 0 deleterious(0.01) |
possibly_damaging(0.772)
rs146564314 0 0.63 0.818 Normal type 2 AVB 2°
2 13 7 c.858 GNA T286T splice_region_variant &
synonymous_variant
Heterozygous 1 (GJA5) | 0 0.00 0.001 Normal AVB 3°
3 9 9 c.1127 TNC I376T missense_variant Heterozygous 0 deleterious(0.02) | benign(0.323) 0 0.00 0 RBBB + LAHB Normal
4 4 11 c.1294 GNA A432T [7,15,16] missense_variant Heterozygous 2 (TRPM4
and RYR2)
deleterious(0) |
probably_damaging(0.97)
rs201907325 0.13 0.10 0.056 LBBB AVB 3°
5 24 11 c.1324 CNT R442C missense_variant Heterozygous 1 (SCN5A) deleterious(0) |
probably_damaging(0.996)
rs148867331 0 0.02 0.018 RBBB + LAHB Normal
6.1 21 12 c.1682 ANC D561A [16] missense_variant Heterozygous 1 (SCN1B) tolerated(0.22) | benign(0.086) rs56355369 0.13 0.55 0.618 RBBB AVB 3°
6.2 28 12 c.1682 ANC D561A [16] missense_variant Heterozygous 1 (SCN5A) tolerated(0.22) | benign(0.086) rs56355369 0.13 0.55 0.618 RBBB AVB 3°
7 4 13 c.1744 GNA G582S [15,16] missense_variant &
splice_region_variant
Heterozygous 2 (TRPM4
and RYR2)
tolerated(0.34) | benign(0.037) rs172149856 0.13 0.10 0.060 LBBB AVB 3°
8.1 38 16 c.2209 GNA G737R [15] missense_variant &
splice_region_variant
Heterozygous 0 tolerated(0.59) | benign(0.007) rs145847114 0.4 0.17 0.180 LBBB AVB 1° + type 2
AVB 2°
8.2 39 16 c.2209 GNA G737R [15] missense_variant &
splice_region_variant
Heterozygous 0 tolerated(0.59) | benign(0.007) rs145847114 0.4 0.17 0.180 RBBB + LPHB AVB 1°
9 40 17 c.2531 GNA G844D[7,16] missense_variant Homozygous 0 tolerated(0.2) | probably_damaging(0.945) rs200038418 0.13 0.16 0.431 RBBB + LAHB AVB 2/1, 3/1
10 41 17 c.2561 ANG Q854R [15,16] missense_variant Heterozygous 0 tolerated(0.29) | benign(0.029) rs172155862 0.26 0.12 0.289 LAD type 2 AVB 2°
11 3 18 c.2674 CNT R892C missense_variant Heterozygous 3 (TNNI3K,
SCN1B and
RYR2)
deleterious(0) |
probably_damaging(0.985)
rs147854826 0 0.10 0.081 Normal AVB 3°
12 10 18 c.2675 GNA R892H missense_variant Heterozygous 1 (SCN5A) deleterious(0.02) | benign(0.252) 0 0.00 0 RBBB + LAHB AVB 3°
13.1 42 24 c.3611 CNT P1204L [15,16] missense_variant Heterozygous 0 tolerated(0.21) | unknown(0) rs150391806 0.13 0.33 0.505 Normal AVB 3°
13.2 43 24 c.3611 CNT P1204L [15,16] missense_variant Heterozygous 0 tolerated(0.21) | unknown(0) rs150391806 0.13 0.33 0.505 RBBB + LAHB AVB 1°
RBBB: Right Bundle Branch Block; LBBB: Left Bundle Branch Block; LAHB: Left Anterior HemiBlock; LPHB: Left Posterior HemiBlock; LAD: Left Axis Deviation; AVB: AtrioVentricular Block
Variants already described in some articles are noted: [7] Liu et al., [16] Stallmeyer et al., [15] Liu et al.
The patient 40 has been identied as homozygous for this variant (G844D).
352 X. Daumy et al. / International Journal of Cardiology 207 (2016) 349358
conduction defects (and one with Small Fiber Neuropathy; see Supple-
mental Table 1).
3.2. TRPM4 is the most frequently affected gene
The most frequently affected gene is TRPM4, with a total of 13 rare
variants identied and then validated by capillary sequencing (Table 1).
Seven of these variants (54%) are located in the intracellular N-terminal
region (Fig. 1a). Two of them p.I376T and p.R892H are absent from
public databases and thus considered as novel.
The TRPM4-p.I376T missense variant, which resides in the intracel-
lular N-terminal domain (Fig. 1a), was identied in the male patient 9
(Fig. 1b). No other rare variant altering any other known PCCD-
susceptibility genes could be identied in this patient. The affected
Fig. 1. TheTRPM4-p.I376Tvariant is responsible for PFHBI. (A) Distributionof rare coding variation detected among 95patients with PCCDin the TRPM4 channel.Novel variants areshown
in red, low-frequency ones inblue. The two rare variants previously reported as causing PFHBI[6,7] are indicatedin green. (B) Capillarysequencing of the exon 9 of TRPM4 for the patient9
conrms the presence of a novel variant resulting in the p.I376T substitution. (C) Family tree of patient 9 (the proband, IV-5). Plus symbols (+) denotes p.I376T mutation carriers and
minus symbol ()non-carriers.PMindicates patients implantedwith a pacemaker, LVNCstands for Left Ventricular Non-Compaction and Cindicatescongenital forms of conduction
defects.
Fig. 2. The ECG prole of the proband IV-5. This patient presented with a heart rate of 69 bpm, a complete right bundle branch block and a left anterior hemiblock enlarging the QRS
complex to 170 ms. ECG was recorded at a 25 mm/s paper speed and 0,1 mV/mm signal amplitude. A premature ventricular beat can also be observed in the rst QRS complex of the
precordial lead.
353X. Daumy et al. / International Journal of Cardiology 207 (2016) 349358
amino acid is located in a highly conserved region across vertebrates as
indicated by its Genomic Evolutionary Rate Proling score [26] of 4.24
(Supplemental Fig. 2). It is predicted as deleterious (0.02) by SIFT [20]
but benign (0.323) by PolyPhen-2 (PPH-2) [21].
The TRPM4-p.R892H variant has been identied in the patient 10,
who presents with a complete AVB. We found that the same patient
also carries a rare missense variant in SCN5A (p.A572D), suggesting
that the TRPM4-p.R892H variant alone may not be responsible for the
observed cardiac conduction defects. Another substitution affecting
the same amino acid - TRPM4-p.R892C - was detected in a second pa-
tient (patient 3), but was also reported atan MAF below 1% in public da-
tabases (Table 1).
3.3. Familial recruitment
Patient nine carrying the TRPM4-p.I376T variant (patient IV-5 in the
pedigree) was diagnosed with complete RBBB and LAHB (Fig. 2)and
was implanted with a PM for conduction disorders at the age of 32. Fa-
milial investigation has been undertaken for this patient, indicated as
the proband IV-5 on Fig. 1c.
A total of 96 family members could be identied, among which 57
have been recruited (Fig. 1c). Twelve patients werediagnosed with con-
duction defects, of which six (50%) were implanted with a PM (Table 2).
Ten of the 12 patients presented with RBBB, among which 8 showed
LAHB. The eleventh patient (V-18) exhibited an isolated LBBB; the last
one (III-2) PB (Table 2).
Two patients (V-16 and VI-1) exhibiting at birth 2:1 AVB with RBBB
and LAHB QRSmorphology alternant with complete AVB were classied
as patients with a congenital AVB, as well as the patient V-18 who ex-
hibited a permanent complete AVB with a 30 bpm ventricular escape
rhythm with a complete LBBB QRS morphology. This patient also met
the magnetic resonance image diagnostic criteria for a left ventricular
non compaction whileechocardiography had failed to identify this phe-
notype (Fig. 1c). Note that the patient IV-6 presented with minor con-
duction defects (QRS duration of 118 ms) and a slight left axis
deviation (14°), but was not considered as affected following our
criteria and thus was classied as unknown.
Age at last clinical evaluation (34 ± 25 vs 22 ± 16, ns), PR (169 ±
24 ms vs 138 ± 20 ms, P b0.001), QRS (138 ± 26 ms vs 88 ± 13 ms,
Pb10
10
) and QTc (438 ± 42 ms vs 417 ± 22 ms, P b0.05) durations
were higher in the affected members compared to non-affected
members while the heart rate was lower in the affected group (61 ±
16 bpm vs 77 ± 16 bpm, P b0.01) (Table 3).
The novel TRPM4-c.T1127C variant (TRPM4-p.I376T) was systemat-
ically assessed among family members (Fig. 1c). We were able to test 39
family members: 10 out of the 11 affected patients that we tested
(90.9%) carried the TRPM4 variant versus only 1 out of 21 unaffected
family members (4.8%). The twelfth patient suffering from cardiac con-
duction disease (patient VI-1) was born in 2012 andthus was not geno-
typed given his young age. The two-point logarithm of the odds ratio
(LOD) score was estimated at 4.1182 for this locus - assuming a disease
allele frequency of 0.01%, a disease penetrance of 80% and arecombina-
tion fraction of 0%. These ndings indicate a genotypephenotype co-
segregation in an autosomal dominant manner in this large French fam-
ily affected by PFHBI.The patient III-2, while appearing as a phenocopy,
may show conductiondefects caused by a previous anterior myocardial
infarct while the patient V-17 (born in 1994) was still youngat recruit-
ment time (17 years old), which may explain the absence of conduc-
tance disturbance for this variant carrier. This patient will be subjected
to regular clinical follow-up since carrying the putative causal variant
may confer higher risk to develop PCCD with aging.
3.4. The p.I376T variant induces a gain-of-function of TRPM4 channel
To investigate the effect of the p.I376T variant on TRPM4 expression
levels, we performed Western blot and cell surface biotinylation exper-
iments. As previously published [27], we observed that the TRPM4
channel is expressed in fully and core glycosylated forms (Fig. 3). In
the presence of the p.I376T variant, we observed an increased expres-
sion of these two forms at the cell membrane (Fig. 3). The functional
consequences of the p.I376T variant were investigated using the
whole-cell conguration of the patch-clamp technique. As reported by
our group [28], TRPM4 currents recorded over time show two distinct
phases (Fig. 4a). After the membrane rupture, a fast transient phase is
observed; it is followed by a plateau phase in which the current ampli-
tude is stable (Fig. 4a). The functional characterization of the p.I376T
variant shows in this condition an increase of TRPM4 current densities
in both transient and plateau phases, (Table 4,Fig. 4b, c and d).
Table 2
Clinical data of the affected family members.
Patient no. Age at last clinical
examination
Heart rate (bpm) PR (ms) QRS (ms) QTc (ms) ECG morphology Conduction PM (age)
IV-5 (proband) 32 69 160 170 474 RBBB + LAHB Normal 32 y.o.
II-1 85 63 138 126 439 RBBB Normal
III-2 60 81 170 128 439 PB Normal
III-3 50 35 200 120 336 RBBB + LAHB type 2 AVB 2° 50 y.o.
III-4 51 55 188 158 439 RBBB + LAHB Normal
IV-8 33 59 180 168 436 RBBB + LAHB Normal 31 y.o.
IV-9 25 76 170 166 504 RBBB + LAHB Normal
IV-25 40 74 200 130 434 RBBB + LAHB AVB 1°
V-16 8 45 148 398 RBBB + LAHB type 2 AVB 2°
and AVB 3°
at birth
V-1812 35 LBBB AVB 3° at birth
V-21 11 73 136 123 441 RBBB Normal
VI-1 0 62 144 90 439 RBBB + LAHB type 2 AVB 2° 8-month-old
RBBB: Right Bundle Branch Block; LAHB: Left Anterior HemiBlock; PB: Parietal Block; LBBB: Left Bundle Branch Block; AVB: AtrioVentricular Block.
This patientpresented with a left ventricular non-compaction phenotype.
Table 3
Comparison of age at last clinical evaluation, heart rate, PR, QRS and QTc durations
between affected and unaffected members of the family.
Affected Unaffected p value (affected
vs unaffected)
Patients (N) 12 45
Age (years) 34 ± 25 24 ± 16 NS
Heart rate (bpm) 61 ± 16 75 ± 17 b0.01
PR (ms) 169 ± 24 140 ± 21 b0.001
QRS (ms) 138 ± 26 90 ± 13 b10
10
QTc (ms) 438 ± 42 415 ± 24 b0.05
354 X. Daumy et al. / International Journal of Cardiology 207 (2016) 349358
4. Discussion
In the present study, thirteen variants in the TRPM4 gene were iden-
tied using NGS technologies upon screening of a cohort of 95 patients
with PCCD. Eleven of these variants were previously listed in at least one
of the used public databases. Two of them (p.A432T and p.G844D)
were previously reported in familial autosomal conduction block and
were shown as deleterious [7]. Five other variants (p.R252H, p.D561A,
Fig. 3. Expression of the WTand p.I376T TRPM4 channels. (A) Western blots showing the expression of TRPM4at the total (left panel)and surface levels(right panel) with whiteand black
arrowheads representing fullyglycosylated and core-glycosylated forms of TRPM4, respectively. (B) Quantication ofthe Western blots is shown as relative intensity of proteinbands for
both fully- and core-glycosylated forms of TRPM4 in each fraction. *P b0.05, **P b0.01.
355X. Daumy et al. / International Journal of Cardiology 207 (2016) 349358
p.G582S, p.Q854R and p.P1204L) have been reported in sporadic cases
presenting with conduction disorders and/or Brugada syndrome [15,
16]. Of interest p.R252H was identied in 3 unrelated patients all of
which exhibiting a type 2 second-degree AVB (patients 35, 36 and
37). The four other variants (three missense variants p.R442C,
p.G737R and p.R892C and one synonymous variant predicted to affect
splicing of the seventh intron p.T286T) have not been causally related
to conduction disorders and/or arrhythmia so far.
Another variant, TRPM4-p.R892H, is novel since absent from public
databases. However, as the patient carrying this variant also carries an
SCN5A-p.A572D variant, no conclusion could be drawn on the relative
pathogenicity of each of these two variants.
The last variant identied in TRPM4 (p.I376T) is also novel. Familial
investigations led to the identication of 96 members including 12 pa-
tients with conduction disorders. This is the third largest pedigree diag-
nosed withPFHBI in which a TRPM4 mutation signicantly segregates in
an autosomal dominant manner with the pathology. Thus, this study
represents the rst NGS-based detection of a TRPM4 variant that has
led to the recruitment of a large 4-generation pedigree from the pro-
band (patient IV-5 on Fig. 1c) carrying the mutation p.I376T. Of interest,
the novel variant p.I376T is located in the same intracellular N-terminal
domain as the 2 causal variants previously identied in large pedigrees
Fig. 4. Whole cell patch clamp recording for the WT and p.I376T TRPM4channels. (A) Time course recording of the TRPM4 current. (B) Individual current traces of the WT and p.I376T
TRPM4 channels recorded as transient and plateau phases. (C) Quantication of current density of the WT and p.I376T TRPM4 channels for both phases. The current densities are
measured at the pic current at 100 mV. (D) Currentvoltage relationships of the WT and p.I376T TRPM4 channels. *P b0.05, **P b0.01, ***P b0.001.
Table 4
The functional characterization of the I376T variant.
TRPM4 WT TRPM4 p.I376T
Current density
of Transient Phase (pA/pF)
161 ± 31
n=12
678 ± 113
n=9
Current density
of plateau phase (pA/pF)
772 ± 138
n=8
1390 ± 134
n=7
356 X. Daumy et al. / International Journal of Cardiology 207 (2016) 349358
(Fig. 1a) [11,18]. Noteworthy, 6 out of the 11 low-frequency variants
identied in this study also reside in the same intracellular N-terminal
domain, thus suggesting that this domain could be a preferential site
for PFHBI causing mutations.
In the present family, a large majority of affected members present
with RBBB and anterior hemiblocks, without any LBBB. This pattern is
similar to the clinical descriptions of the families previously linked to
mutationsin TRPM4 [6,7], which corresponds to the PFHB type IB deni-
tion. Our study, in combination with previously published works [6,7,
16] strongly support the prominent role of this cardiac TRP channel in
this subtype of conduction disease. The clinical onset of conduction
disturbances tends to occur at an early age among affected patients. In
particular, the presence of three cases of congenital AVB implanted
with a PM during the rst year of life also suggests an important role
of heritability in disease severity. Furthermore, the observation that
these three congenital AVB patients are rst- or second-degree relatives
suggests that additional genetic factors are strengthening the disease
susceptibility in these patients.
Expression and functional analyses were performed using
HEK293 cells, in whole-cell patch-clamp conguration and Western
blotting. The TRPM4 p.I376Tresultsinanincreasedcurrentdensity
that may be caused by an augmented TRPM4 channel expression at
the cell surface as previously described [6,7]. The underlying mecha-
nisms leading to conduction blockcausedbyTRPM4dysfunction
are not yet understood. It has been proposed [15,29] that gain-of-
function mutations may depolarize the cells of the conduction sys-
tem, reduce the availability of the cardiac sodium channels and cur-
rent and thereby alter the normal impulse propagation in Purkinje
bers. This model is consistent with the large QRS complexes ob-
served in PFHBI patients. Conversely, loss-of-function mutations of
TRPM4 may lead to a hyperpolarization of the membrane potential,
and so reduce cellular excitability and conduction. A detailed analy-
sis of the molecular mechanisms leading to the mutation-induced
gain of expression and function was out of the scope of the present
work. These ndings, however, strongly support the role of TRPM4
gain-of-function in slowed cardiac impulse propagation.
5. Limitations
Next generation-based targeted resequencing such as HaloPlex
System allows high-throughput genetic screening in a large number of
individuals but some target sequences may be uncovered due to biases
in DNA digestion by restriction enzymes. Thus some relevant variations
may be missed in small subsets of coding regions: this problem is inher-
ent to sequencing strategies based on DNA enrichment.
Furthermore this high-throughput candidate-gene approach was
used to screen 19 candidate genes in 95 unrelated patients. Except for
the patient carrying the TRPM4-p.I376T variant (patient 9) for which a
familial recruitment, segregation tests and a LOD score calculation
strongly suggest an association between this variant and the phenotype,
the implication of variants in other unknown involved genes cannot be
excluded in isolated PCCD cases.
6. Conclusion
In this study we identied one large family with 10 members diag-
nosed with PFHBI and carrying a TRPM4 gain-of-expression and func-
tion mutation. This represents the rst NGS-based detection of a
TRPM4 variant that has led to the recruitment of a large PCCD pedigree.
This work conrms that gain-of-function mutations in the intracellular
N-terminal region of TRPM4 are responsible for PFHBI and further
underline the crucial role of TRPM4 channel in cardiac conduction
disorders.
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.ijcard.2016.01.052.
Authors' contributions
XD, MYA, HA, RR and JJS conceived the study, wrote the manuscript
and are the guarantors of the project; PL, EC, JB and SLS contributed to
the data processing; SB and EB performed the sequencing; BB and SN
contributed to the functional and biochemical analyses; SF, AT and FK
recruited the patients; HLM, NM, JBG and VP provided expert clinical
advice; CD led the statistical analysis. All authors interpreted the data,
contributed and commented on drafts of the article, and approved the
nal version.
Fundings
This work was supported by the Fondation pour la Recherche
Médicale (FRM grant DEQ20140329545) to Jean-Jacques Schott; by
the Institut National de la Santé et de la Recherche Médicale (INSERM,
ATIP-Avenir program), the ANR-14-CE10-0001-01 (GenSuD) and the
French Regional Council of Pays-de-la-Loire to Richard Redon; by the
Centre National de la Recherche et de la Santé (CNRS grant PRC
CNRS/JSPS) to Jean-Jacques Schott and Naomasa Makita; by the French
Ministry of Health (grant from the Clinical Research Hospital Program
PHRC-I PROG11/33 in 2011) and the Fédération Française de Cardiologie
(grant no. RC13_0012 in 2012) to Vincent Probst; and by the Swiss Na-
tional Science Foundation to Hugues Abriel (310030B_14706035693),
and the TransCure NCCR network, the Berne University Research
Foundation.
Conict of interest
The authors declare no conict of interest.
Acknowledgments
We would like to thank the French clinical network against inherited
cardiac arrhythmias as well as the patients who participated to this
study for participation. We are also grateful to the members of the
Genomics and Bioinformatics Core Facility of Nantes (Biogenouest) for
their technical expertise.
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358 X. Daumy et al. / International Journal of Cardiology 207 (2016) 349358
... In 2009, Kruse et al. identified the first TRPM4 gene mutation in PFHBI patients [11]. Very recently, Dong et al. reported a novel TRPM4 mutation in a Chinese Family with atrioventricular block [26], supporting the idea that TRPM4 may act as a major gene predisposing to progressive familial heart block type I [27]. ...
... Twenty-four of them are missense/nonsense mutations and one, a small insertion. Of these 25 mutations, 9 were associated to isolated cardiac conduction disease [11,[27][28][29], 8 + 1 (?) to Brugada syndrome [30,31], 3 to long QT syndrome [32], 2 to type 1 AVB [11,27] and 1 to unexpected sudden death in infancy [33] (Table 1). The Gly844Asp variant found in our family had already been reported in multiple individuals, in association with cardiac conduction disorders [27][28][29][30][31]. ...
... Twenty-four of them are missense/nonsense mutations and one, a small insertion. Of these 25 mutations, 9 were associated to isolated cardiac conduction disease [11,[27][28][29], 8 + 1 (?) to Brugada syndrome [30,31], 3 to long QT syndrome [32], 2 to type 1 AVB [11,27] and 1 to unexpected sudden death in infancy [33] (Table 1). The Gly844Asp variant found in our family had already been reported in multiple individuals, in association with cardiac conduction disorders [27][28][29][30][31]. ...
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Expert Consensus Statement on the state of genetic testing for cardiac diseases
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The transient receptor potential channel, TRPM4, and its closest homolog, TRPM5, are non-selective cation channels that are activated by an increase in intracellular calcium. They are expressed in many cell types, including neurons and myocytes. Although the electrophysiological and pharmacological properties of these two channels have been previously studied, less is known about their regulation, in particular their post-translational modifications. We, and others, have reported that wild-type (WT) TRPM4 channels expressed in HEK293 cells, migrated on SDS-PAGE gel as doublets, similar to other ion channels and membrane proteins. In the present study, we provide evidence that TRPM4 and TRPM5 are each N-linked glycosylated at a unique residue, Asn(992) and Asn(932), respectively. N-linked glycosylated TRPM4 is also found in native cardiac cells. Biochemical experiments using HEK293 cells over-expressing WT TRPM4/5 or N992Q/N932Q mutants demonstrated that the abolishment of N-linked glycosylation did not alter the number of channels at the plasma membrane. In parallel, electrophysiological experiments demonstrated a decrease in the current density of both mutant channels, as compared to their respective controls, either due to the Asn to Gln mutations themselves or abolition of glycosylation. To discriminate between these possibilities, HEK293 cells expressing TRPM4 WT were treated with tunicamycin, an inhibitor of glycosylation. In contrast to N-glycosylation signal abolishment by mutagenesis, tunicamycin treatment led to an increase in the TRPM4-mediated current. Altogether, these results demonstrate that TRPM4 and TRPM5 are both N-linked glycosylated at a unique site and also suggest that TRPM4/5 glycosylation seems not to be involved in channel trafficking, but mainly in their functional regulation.
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Brugada syndrome (BrS) is a condition defined by ST-segment alteration in right precordial leads and a risk of sudden death. Because BrS is often associated with right bundle branch block and the TRPM4 gene is involved in conduction blocks, we screened TRPM4 for anomalies in BrS cases. The DNA of 248 BrS cases with no SCN5A mutations were screened for TRPM4 mutations. Among this cohort, 20 patients had 11 TRPM4 mutations. Two mutations were previously associated with cardiac conduction blocks and 9 were new mutations (5 absent from ∼14'000 control alleles and 4 statistically more prevalent in this BrS cohort than in control alleles). In addition to Brugada, three patients had a bifascicular block and 2 had a complete right bundle branch block. Functional and biochemical studies of 4 selected mutants revealed that these mutations resulted in either a decreased expression (p.Pro779Arg and p.Lys914X) or an increased expression (p.Thr873Ile and p.Leu1075Pro) of TRPM4 channel. TRPM4 mutations account for about 6% of BrS. Consequences of these mutations are diverse on channel electrophysiological and cellular expression. Because of its effect on the resting membrane potential, reduction or increase of TRPM4 channel function may both reduce the availability of sodium channel and thus lead to BrS.
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The Brugada syndrome (BrS) is a rare heritable cardiac arrhythmia disorder associated with ventricular fibrillation and sudden cardiac death. Mutations in the SCN5A gene have been causally related to BrS in 20-30% of cases. Twenty other genes have been described as involved in BrS, but their overall contribution to disease prevalence is still unclear. This study aims to estimate the burden of rare coding variation in arrhythmia-susceptibility genes among a large group of patients with BrS. We have developed a custom kit to capture and sequence the coding regions of 45 previously reported arrhythmia-susceptibility genes and applied this kit to 167 index cases presenting with a Brugada pattern on the electrocardiogram as well as 167 individuals aged over 65 year-old and showing no history of cardiac arrhythmia. By applying burden tests, a significant enrichment in rare coding variation (with a minor allele frequency below 0.1%) was observed only for SCN5A, with rare coding variants carried by 20.4% of cases with BrS versus 2.4% of control individuals (p=1.4 x 10(-7)). No significant enrichment was observed for any other arrhythmia-susceptibility gene, including SCN10A and CACNA1C. These results indicate that, except for SCN5A, rare coding variation in previously reported arrhythmia-susceptibility genes do not contribute significantly to the occurrence of BrS in a population with European ancestry. Extreme caution should thus be taken when interpreting genetic variation in molecular diagnostic setting, since rare coding variants were observed in a similar extent among cases versus controls, for most previously reported BrS-susceptibility genes. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.
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Human embryonic kidney cells 293 (HEK293) are widely used as cellular heterologous expression systems to study transfected ion channels. This work characterizes the endogenous expression of TRPM4 channels in HEK293 cells. TRPM4 is an intracellular Ca2+-activated non-selective cationic channel expressed in many cell types. Western blot analyses have revealed the endogenous expression of TRPM4. Single channel 22pS conductance with a linear current-voltage relationship was observed using the inside-out patch clamp configuration in the presence of intracellular Ca2+. The channels were permeable to the monovalent cations Na+ and K+, but not to Ca2+. The open probability was voltage-dependent, being higher at positive potentials. Using the whole-cell patch clamp "ruptured patch" configuration, the amplitude of the intracellular Ca2+-activated macroscopic current was dependent on time after patch rupture. Initial transient activation followed by a steady-increase reaching a plateau phase was observed. Biophysical analyses of the macroscopic current showed common properties with those from HEK293 cells stably transfected with human TRPM4b, with the exception of current time course and Ca2+ sensitivity. The endogenous macroscopic current reached the plateau faster and required 61.9±3.5μM Ca2+ to be half-maximally activated versus 84.2±1.5μM for the transfected current. The pharmacological properties, however, were similar in both conditions. One hundred μM of flufenamic acid and 9-phenanthrol strongly inhibited the endogenous current. Altogether, the data demonstrate the expression of endogenous TRMP4 channels in HEK293 cells. This observation should be taken into account when using this cell line to study TRPM4 or other types of Ca2+-activated channels.
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The transient receptor potential channel (TRP) family comprises at least 28 genes in the human genome. These channels are widely expressed in many different tissues, including those of the cardiovascular system. The transient receptor potential channel melastatin 4 (TRPM4) is a Ca(2+)-activated non-specific cationic channel, which is impermeable to Ca(2+). TRPM4 is expressed in many cells of the cardiovascular system, such as cardiac cells of the conduction pathway and arterial and venous smooth muscle cells. This review article summarizes the recently described roles of TRPM4 in normal physiology and in various disease states. Genetic variants in the human gene TRPM4 have been linked to several cardiac conduction disorders. TRPM4 has also been proposed to play a crucial role in secondary hemorrhage following spinal cord injuries. Spontaneously hypertensive rats with cardiac hypertrophy were shown to over-express the cardiac TRPM4 channel. Recent studies suggest that TRPM4 plays an important role in cardiovascular physiology and disease, even if most of the molecular and cellular mechanisms have yet to be elucidated. We conclude this review article with a brief overview of the compounds that have been shown to either inhibit or activate TRPM4 under experimental conditions. Based on recent findings, the TRPM4 channel can be proposed as a future target for the pharmacological treatment of cardiovascular disorders, such as hypertension and cardiac arrhythmias.