Targeted resequencing identiﬁes TRPM4 as a major gene predisposing to
progressive familial heart block type I
, Mohamed-Yassine Amarouch
, Pierre Lindenbaum
, Stéphanie Bonnaud
, Beatrice Bianchi
, Estelle Baron
, Swanny Fouchard
, Florence Kyndt
, Julien Barc
, Solena Le Scouarnec
, Naomasa Makita
Hervé Le Marec
, Christian Dina
, Jean-Baptiste Gourraud
, Vincent Probst
, Hugues Abriel
, Jean-Jacques Schott
Institut National de la Santé et de la Recherche Médicale (INSERM) Unité Mixte de Recherche (UMR) 1087, l'institut du thorax, Nantes, France
Centre National de la Recherche Scientiﬁque (CNRS) UMR 6291, l'institut du thorax, Nantes, France
Université de Nantes, l'institutdu thorax, Nantes, France
Department of Clinical Research, and Swiss National Centre of Competence in Research (NCCR) TransCure, University of Bern, Switzerland
Centre Hospitalier Universitaire (CHU) de Nantes, l'institut du thorax, Service de Cardiologie, Nantes, France
Molecular Physiology, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan
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 identiﬁed 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 conﬁguration 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 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.
© 2016 Published by Elsevier Ireland Ltd.
Progressive cardiac conduction defect (PCCD) was ﬁrst described in
the sixties by Lenègre  and Lev  as a ﬁbrosis 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  and
TRPM4 [6,7]), in the structure of the nuclear lamina (LMNA [8,9]) and
in cell-to-cell communication (GJA5 ).
Among these genes, TRPM4, encoding a calcium-activated cationic
channel that is expressed in Purkinje ﬁbers and nodal tissue [6,7],has
ﬁrst been linked to progressive familial heart block type I (PFHBI) in
two large pedigrees, respectively from South Africa  and Lebanon
International Journal of Cardiology 207 (2016) 349–358
⁎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), firstname.lastname@example.org
These authors contributed equally to this work.
Current afﬁliation: Materials, Natural Substances, Environment & Modeling
Laboratory, University of Sidi Mohamed Ben Abdellah- Fes, Multidisciplinary Faculty of
Taza, Taza, Morocco.
0167-5273/© 2016 Published by Elsevier Ireland Ltd.
Contents lists available at ScienceDirect
International Journal of Cardiology
journal homepage: www.elsevier.com/locate/ijcard
. 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
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 identiﬁed
as causing diverse forms of cardiac conduction defects and/or Brugada
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 ﬁndings strongly support a central role of TRPM4 in car-
diac conduction and cardiac conduction disorders.
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 deﬁned using the conventional classiﬁcation [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 misclassiﬁcation, only the
most obviously affected patients were considered as affected. QRS axis
was classiﬁed 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) deﬁned 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-IT™dsDNA 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 Scientiﬁc). DNA integrity was assessed by separation in a E-Gel®
96 Agarose Gels, 1% (Life Technologies, G700801). For multiplex ampli-
ﬁcation, we used the HaloPlex™Target Enrichment System (Agilent
Technologies, 1–500 kb, ILMFST, 96 reactions, G9901B), Protocol
Version D.2 (November, 2012). We applied a custom HaloPlex™design
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
BMPR1A,GATA4,MSX2 and TNNI3K as likely candidate genes. The
targeted coding regions (exons) ± 10 bp correspond to141 kb of geno-
Target enrichment and sequencing were performed as previously
described . 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 ampliﬁcation. The ampliﬁed target DNA was puriﬁed
using AMPure XP bead (Beckman Coulter, A63881). To validate enrich-
ment of target DNA in each library sample by microﬂuidics analysis,
we used the 2200 TapeStation (Agilent Technologies, G2964AA), with
D1K ScreenTape (Agilent Technologies, 5067–5361), and D1K Reagents
(Agilent Technologies, 5067–5362). We ensured that the majority of
amplicons range from 175 to 625 bp. Finally we quantiﬁed each library
by qPCR using KAPA Library Quantiﬁcation 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 ﬁnal 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 UniﬁedGenotyper (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: 1—They 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_
(SO:0001889); 2—They 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_
In case of missense variants SIFT  and PolyPhen-2 (PPH-2) 
were used to predict the impact of the amino acid substitutions. Filter-
ing was performed using Knime4Bio .
350 X. Daumy et al. / International Journal of Cardiology 207 (2016) 349–358
2.4. Segregation analysis
Familial segregation analyses were c arried out by bidirectional direct
sequencing of ampliﬁed genomic DNA amplicons with variant-speciﬁc
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
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  calcu-
lation was performed with Superlink-Online SNP version 1.1 (http://
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 signiﬁcant 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 streptomycin–penicillin 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
2.6. Cell surface biotinylation assay
Following 48 h of incubation, transiently transfected HEK293 cells
were treated with EZlinkTM Sulfo-NHS-SS-Biotin (Thermo Scientiﬁc,
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
; 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 ﬁve 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 afﬁnity puriﬁed.
2.8. Cellular electrophysiology
For patch-clamp experiments in whole-cell conﬁguration, glass pi-
pettes (tip resistance, 1.5–3MΩ)wereﬁlled with an intracellular solu-
tion containing (in mM): 100 CsAsp, 20 CsCl, 4 Na
ATP, 1 MgCl
EGTA, and 10 HEPES. The pH was adjusted to 7.20 with CsOH, and the
concentration at 100 μM with CaCl
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
, 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 conﬁguration at room temperature
(23–25 °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 ﬁltered 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
2.9. Data analysis and statistical methods
Currents were analyzed with Clampﬁt 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 signiﬁcant.
3.1. Mutational screening
Ninety-ﬁve patients with PCCD were recruited through the French
National Referral Center for Sudden Cardiac Death as previously de-
scribed . 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,
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 identiﬁed 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) 349–358
Characteristics of identiﬁed amino acid variants in TRPM4.*
No. Patient Exon Nucleotide Amino
Effect Genotype Other
SIFT | PPH-2 dbSNP141
1.1 35 6 c.755 GNA R252H  missense_variant Heterozygous 0 deleterious(0.01) |
rs146564314 0 0.63 0.818 RBBB type 2 AVB 2°
1.2 36 6 c.755 GNA R252H  missense_variant Heterozygous 0 deleterious(0.01) |
rs146564314 0 0.63 0.818 LBBB type 2 AVB 2°
1.3 37 6 c.755 GNA R252H  missense_variant Heterozygous 0 deleterious(0.01) |
rs146564314 0 0.63 0.818 Normal type 2 AVB 2°
2 13 7 c.858 GNA T286T splice_region_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
rs201907325 0.13 0.10 0.056 LBBB AVB 3°
5 24 11 c.1324 CNT R442C missense_variant Heterozygous 1 (SCN5A) deleterious(0) |
rs148867331 0 0.02 0.018 RBBB + LAHB Normal
6.1 21 12 c.1682 ANC D561A  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  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 &
Heterozygous 2 (TRPM4
tolerated(0.34) | benign(0.037) rs172149856 0.13 0.10 0.060 LBBB AVB 3°
8.1 38 16 c.2209 GNA G737R  missense_variant &
Heterozygous 0 tolerated(0.59) | benign(0.007) rs145847114 0.4 0.17 0.180 LBBB AVB 1° + type 2
8.2 39 16 c.2209 GNA G737R  missense_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,
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:  Liu et al.,  Stallmeyer et al.,  Liu et al.
⁎The patient 40 has been identiﬁed as homozygous for this variant (G844D).
352 X. Daumy et al. / International Journal of Cardiology 207 (2016) 349–358
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 identiﬁed 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 identiﬁed in the male patient 9
(Fig. 1b). No other rare variant altering any other known PCCD-
susceptibility genes could be identiﬁed 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
conﬁrms 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.‘PM’indicates patients implantedwith a pacemaker, ‘LVNC’stands for Left Ventricular Non-Compaction and ‘C′indicatescongenital forms of conduction
Fig. 2. The ECG proﬁle 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 ﬁrst QRS complex of the
353X. Daumy et al. / International Journal of Cardiology 207 (2016) 349–358
amino acid is located in a highly conserved region across vertebrates as
indicated by its Genomic Evolutionary Rate Proﬁling score  of 4.24
(Supplemental Fig. 2). It is predicted as deleterious (0.02) by SIFT 
but benign (0.323) by PolyPhen-2 (PPH-2) .
The TRPM4-p.R892H variant has been identiﬁed 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 identiﬁed, 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 classiﬁed
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 classiﬁed 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,
) 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 ﬁndings indicate a genotype–phenotype 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 , 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 conﬁguration of the patch-clamp technique. As reported by
our group , 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).
Clinical data of the affected family members.
Patient no. Age at last clinical
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°
V-18⁎12 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.
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
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
QTc (ms) 438 ± 42 415 ± 24 b0.05
354 X. Daumy et al. / International Journal of Cardiology 207 (2016) 349–358
In the present study, thirteen variants in the TRPM4 gene were iden-
tiﬁed 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 . 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) Quantiﬁcation 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) 349–358
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 identiﬁed 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 identiﬁed in TRPM4 (p.I376T) is also novel. Familial
investigations led to the identiﬁcation of 96 members including 12 pa-
tients with conduction disorders. This is the third largest pedigree diag-
nosed withPFHBI in which a TRPM4 mutation signiﬁcantly segregates in
an autosomal dominant manner with the pathology. Thus, this study
represents the ﬁrst 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 identiﬁed 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) Quantiﬁcation 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) Current–voltage relationships of the WT and p.I376T TRPM4 channels. *P b0.05, **P b0.01, ***P b0.001.
The functional characterization of the I376T variant.
TRPM4 WT TRPM4 p.I376T
of Transient Phase (pA/pF)
−161 ± 31
−678 ± 113
of plateau phase (pA/pF)
−772 ± 138
−1390 ± 134
356 X. Daumy et al. / International Journal of Cardiology 207 (2016) 349–358
(Fig. 1a) [11,18]. Noteworthy, 6 out of the 11 low-frequency variants
identiﬁed 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 deﬁni-
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 ﬁrst year of life also suggests an important role
of heritability in disease severity. Furthermore, the observation that
these three congenital AVB patients are ﬁrst- 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 conﬁguration 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
ﬁbers. 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 ﬁndings, however, strongly support the role of TRPM4
gain-of-function in slowed cardiac impulse propagation.
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.
In this study we identiﬁed one large family with 10 members diag-
nosed with PFHBI and carrying a TRPM4 gain-of-expression and func-
tion mutation. This represents the ﬁrst NGS-based detection of a
TRPM4 variant that has led to the recruitment of a large PCCD pedigree.
This work conﬁrms 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
Supplementary data to this article can be found online at http://dx.
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
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
Conﬂict of interest
The authors declare no conﬂict of interest.
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
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