FUNCTIONAL CHARACTERIZATION OF TRPM4 VARIANTS IDENTIFIED IN SUDDEN UNEXPLAINED DEATH

Ekaterina Subbotina, Nori Williams, Barbara Sampson, Yingying Tang, and William Coetzee

Supplemental Methods

Study population, genetic analysis and variant interpretation

Variants selected for functional analysis were identified through molecular analysis performed in sudden

cardiac death cases by the New York City Office of Chief Medical Examiner (NYC-OCME) by the in-house

Molecular Genetics Laboratory, accredited by the college of American Pathologists (CAP). At the NYC-

OCME, forensic investigations in sudden death include: scene investigation and family interview (by

certified physician assistants), complete gross autopsy, cardiac pathology and neuropathology

examinations, toxicological tests, microbiological tests (in infants), metabolic screen tests (in infants),

and medical record reviews. Cases defined as SUD had negative or unremarkable results from the

studies described above. A total of 296 SUD cases investigated between 2001 and 2014 met these

criteria and we have recently reported the results of genetic testing of 89 cardiac disease genes in this

cohort. 1 Between October 20th, 2015 and April 1st, 2016 we have performed prospective testing of an

additional 34 SUD cases using a panel that now comprised of 95 cardiac disease genes (Table S1), which

now includes additional genes, such as TRPM4, disease markers for Noonan syndrome (PTPN11),

cardiomyopathy (CALR3, JPH2, NPPA, PRDM16), and Tetralogy of Fallot (GJA5). The TRPM4 gene was

analyzed in both retrospective and prospective SUD cases (a total of 330). As in our prior study, variant

interpretation followed the 2015 ACMG-AMP guidelines and the maximum credible allele frequency for

a rare variant was set at 0.001%. 1 This study is exempt from human subject research approval

requirements because cadaver specimens were used for genetic analysis. NYC-OCME approved this

study for diagnosis of the underlying causes of SUD.

cDNAs and site-directed mutagenesis

We used a commercial service (GenScript, Piscataway, NJ) to a synthesize the human full-length coding

region (CDS) of TRPM4b (Genbank accession number NM_017636.3) in pcDNA3.1(+). Site directed

mutagenesis was also performed by GenScript. The constructs were verified by Sanger sequencing.

Cell culture and transfection

Human Embryonic Kidney (HEK) 293 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM),

supplemented with heat-inactivated 10% fetal bovine serum (FBS) and penicillin-streptomycin (Thermo

Fisher Scientific, Waltham, MA). Cells were grown in 35 mm culture dishes until 70-80% confluence was

reached and co-transfected using Lipofectamine 2000 (Life Technologies, Carlsbad, CA) with 1.8 µg of

TRPM4 and 0.2 µg of a GFP plasmid, to allow visualization of successfully transfected cells for patch

clamping. In case of heterozygous state simulation, HEK293 cells were co-transfected with 0.9 µg of WT

TRPM4, 0.9 µg of missense variant, and 0.2 µg of a GFP cDNAs. Cells were transferred to coverslips the

next day and used for experimentation at 48 h after transfection.

Semi-quantitative real-time polymerase chain reaction

Total RNA was isolated (RNaesy Mini kit, Qiagen, MD, USA) and first-strand cDNA synthesis was

performed (Superscript III, ThermoFisher Scientific, Waltham, MA) using 1 µg of total RNA and random

hexonucleotides following the manufacturer’s protocol. Semi-quantitative real-time RT-PCR (qRT-PCR)

was performed with 1 µL of reverse transcription reaction and 10 pmol of gene-specific primers using

PowerUP SYBR Green Master Mix (ThermoFisher Scientific). The PCR (StepOne™ Real-Time PCR System,

Applied Biosystems, Foster City, CA) Cycling conditions consisted of an initial denaturation 15 s step at

95 °C, followed by 40 cycles with a 60 s annealing/extension step at 60 °C. The amplicon quality was

verified by inspecting the thermal denaturation curve. The threshold cycle (CT) was determined by

StepOne Software v2.1 (Applied Biosystems) and semi-quantitative analysis was performed using the -

∆∆CT method, using GAPDH as a reference. For TRPM4 we used primers 5’-GATGCACACACCACGGAGAA-

3’ and 5’-AGAGCCGGAGGAAATTGCTG-3’, whereas primers 5’-CACTAGGCGCTCACTGTTCT-3’ and 5’-

GACCAAATCCGTTGACTCCG-3’ were used for GAPDH.

Western blot

Transiently transfected HEK293 cells were harvested using RIPA buffer (Sigma-Aldrich, St. Louis, MO)

supplemented with HaltTM protease inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA). Cells

were homogenized on ice (3 bouts of 15 pulses at 50 % strength) using a Microson XL sonicator (Qsonic,

Newtown, CT) and centrifuged for 15 min (14,000 x g at 4 °C). The protein concentration of the

supernatant was measured with a BCA Protein Assay Kit (ThermoFisher Scientific), aliquoted and stored

at -80 °C. Before use, 5 µg protein was mixed with sample buffer (Thermo Fisher Scientific, Waltham,

MA) containing 5 % β-mercaptoethanol and kept at room temperature prior to resolving with a 4-20 %

polyacrylamide gradient gel. The fractionated proteins were transferred to polyvinylidene difluoride

(PVDF) membranes (Bio-Rad, Hercules, CA) using a semi-dry system Trans-Blot Turbo (Bio-Rad, Hercules,

CA). Blocking was performed with 5% fat free milk in TBS-Tween (0.01%) for 1 h, followed by incubation

with primary antibodies overnight at 4°C and secondary antibodies, dissolved in blocking buffer, for 1h

at room temperature. Detection was performed with chemiluminesence using SuperSignal West Dura

(Thermo Fisher Scientific, Waltham, MA) and photographic documentation. Antibodies used were anti-

TRPM4 (1:5,000 dilution, clone OTI10H5, OriGene, Rockville, MD), anti-GAPDH (1:5,000 dilution, clone

G8795) and horseradish peroxidase conjugated AffiniPure donkey anti-mouse IgG (Jackson Immuno

Research Labs, West Grove, PA).

Surface biotinylation assay

Transfected HEK293 cells were washed twice with ice-cold Hank's balanced salt solution and biotinylated

for 30 min using PBS containing 0.25 mg/mL of EZ Link Sulfo-NHS-SS-Biotin (Pierce Thermo Fisher

Scientific). Excess biotin was removed by washing twice with PBS and quenched by 5 min incubation

with 100 mM glycine in PBS. Cells were scraped from the dish and washed twice with TBS by

centrifugation (5 min at 1000 x g). The pelleted cells were lysed with 100 µl RIPA buffer (Sigma-Aldrich,

St. Louis, MO) supplemented with HALT Protease Inhibitor Cocktail (Thermo Fisher Scientific, Waltham,

MA). All steps were performed at 4°C. The biotinylated proteins were mixed with 2x Laemmli sample

buffer (Bio-Rad, Hercules, CA) with 5% β-Mercaptoethanol at room temperature for 1 h and analyzed by

Western blotting.

Electrophysiology

TRPM4 currents were recorded at room temperature using patch clamp techniques (Axopatch 200B;

Molecular Devices, Sunnyvale, CA). Patch electrodes were manufactured (Zeitz puller, Germany) using

borosilicate glass (1.5 mm OD; World Precision Instruments, Sarasota, FL). Cells were initially kept in

Tyrode’s solution, consisting of (in mmol/L): 135 NaCl, 5.4 KCl, 0.33 NaH2PO4, 1 MgCl2, 10 HEPES, 1.8

CaCl2, 10 glucose, and adjusted to pH 7.4 with NaOH. Pipettes were filled with (in mmol/L): 145 NaCl, 1

MgCl2, 10 glucose, 10 HEPES, 1 CaCl2 and pH adjusted to 7.4 with 2N NaOH. Excised patches were

analyzed with two ‘intracellular’ bath solutions that were applied using a rapid solution exchange system

(RCS-160, Bio-Logic Science Instruments, Seyssinet-Pariset, France). The nominally Ca2+-free solution

consisted of (in mmol/L): 145 NaCl, 1 MgCl2, 10 glucose, 10 HEPES, 1 EGTA. A Ca2+-containing solution

was prepared by addition of CaCl2 to the nominally Ca2+-free solution to obtain a calculated free Ca2+

activity of 1 mM, using Maxchelator.2 The pH of the bath solutions was adjusted to 7.2 with 2 M NaOH.

The patch was excised at a membrane potential of +80 mV (pipette potential of -80 mV) and then set to

0 mV when steady state conditions were achieved (typically 30-60 s). Current-voltage relationships were

constructed with a voltage clamp protocol that consisted of 500 ms voltage steps between -80 mV and

+80 mV in 20 mV increments. Data were corrected to reflect the membrane potential and current by

inverting the pipette potential and direction of pipette currents. In all cases, current signals were low-

pass filtered with an 8-pole low-pass filter with a Bessel response at a -3 dB point of 1 kHz. Analog

signals were digitized at 3 kHz DigiData 1550A (Molecular Devices, Sunnyvale, CA) using pClamp v10.5

software (Molecular Devices, Sunnyvale, CA). Data were not corrected for liquid junction potentials,

which was calculated to be -0.4 mV.

RNA-seq analysis

Paired read RNA-Sequencing data from non-failing adult human heart were obtained from the Gene

Expression Omnibus (GEO accession number GSE71613; samples GSM1841251, GSM1841259,

GSM1841261 and GSM1841263). RNA-seq reads from human fetal heart were also obtained (GSE68279;

samples GSM1666976 and GSM1666977). Sequenced reads were trimmed for adaptor sequence and for

low-quality sequence, then mapped to the human hg19 whole genome using STAR on the NYU Phoenix

High Performance Cluster computing facility. Output BAM files were sorted by coordinates and gene

counts were produced. Index files were generated with Samtools and data were visualized with the

Integrated Genomics Viewer with splice junction tracks enabled.

Data analysis

Immunoblots were digitized by scanning and analyzed using ImageJ software.3 Patch clamp data were

analyzed using Clampfit 10.5. (Molecular Devices, Sunnyvale, CA) and statistical analysis was performed

with SigmaPlot 12 (Systat Software, San Jose, CA). For comparisons between two groups, we used the

Student’s t-test. Multiple group comparison was performed using a one-way analysis of variance

(ANOVA), followed by post hoc analysis using the Dunnett's test. Statistical significance was assumed at

p < 0.05.

Supplemental Figures

Figure S1: Amino acid sequence alignment of TRPM4 orthologs to demonstrate species conservation.

Shown are full-length proteins of human (Genbank accession number NP_060106.2), dog

(XP_541500.3), mouse (NP_780339) and rat (NP_001129701). Multiple sequence alignment was

performed using Clustal-Omega (https://www.ebi.ac.uk/services/teams/clustal-omega). The positions of

the variants are indicated by red boxes.

Figure S2: Amino acid sequence alignment of TRPM homologs to demonstrate conservation. Shown are

full-length proteins of human TRPM1 (Genbank accession number NP_001238949), TRPM2

(NP_003298), TRPM3 (NP_066003), TRPM4 (NP_060106.2), TRPM5 (NP_055370), TRPM6 (NP_060132),

TRPM7 (NP_060142), and TRPM8 (NP_076985). Displayed are only segments that contain the variants

under study (red boxes). Multiple sequence alignment was performed using Clustal-Omega

(https://www.ebi.ac.uk/services/teams/clustal-omega).

Figure S3: The position of the A380 variant in the TRPM4 protein. Shown is a portion of cryo-EM structure of mouse TRPM4 in ATP bound state at 2.9 angstrom resolution (PB: 6BCO). The three colors (pink, blue and green) indicate three adjacent TRPM4 subunits of the tetrametic channel protein. The display focuses on two of the four ATP binding regions and two bound ATP molecules are indicated. The A380 residue is within an α-helix between two ATP binding pockets. The coordinates were displayed using USCF Chimera (https://www.cgl.ucsf.edu/chimera/) using the command line, as follows:

open 6BCO;turn x 90;turn y 92;

color #ffc9c3,r :*.a;color #90e087,r :*.b;color #7567ff,r :*.c;color #d2d2d2,r :*.d;

sel :ATP.a;focus :ATP.a;~sel :ATP.a;

sel :381.*;display :381.*;~sel :381.*;

Supplemental Tables

Table S1. List of genes in the prospective testing panel

Gene Protein Description Ref SEQ (HGMD) Associated SUD-related Diseases

ABCC9 a member of the superfamily of ATP-binding cassette (ABC) transporters NM_005691.2 AF, Brs, DCM

ACTC1 Actin, alpha, cardiac muscle NM_005159.4 HCM, RCM, LVNC, DCMACTN2 Actinin, alpha 2 NM_001103.2 HCM, DCM, LVNC

AKAP10 a member of the A-kinase anchor protein family NM_007202.3 conduction

AKAP9 a member of the AKAP family NM_005751.4 LQT11

ANK2 a member of the ankyrin family of proteins NM_001148.4 LQT4

ANKRD1 Ankyrin repeat domain 1 (cardiac muscle) NM_014391.2 HCM,DCM

ARHGAP24 Rho GTPase activating protein 24 NM_001025616.2 conduction

BAG3 BCL2-associated athanogene 3 NM_004281.3 RCM, HCM, DCM

CACNA1C alpha-1 subunit of a voltage-dependent calcium channel. NM_000719.6 LQT, Brs, Timothy

CACNA2D1 Calcium channel, voltage-dependent, alpha 2/delta subunit 1 NM_000722.2 heart block, Brs, SQT

CACNB2 Calcium channel, voltage-dependent, beta 2 subunit NM_201590.2 Brs4

CALM1 Calmodulin 1 (phosphorylase kinase, delta) NM_006888.4 LQT14, CPVTCALM2 Calmodulin 2 (phosphorylase kinase, delta) NM_001743.4 LQT15CALR3 Calreticulin 3 NM_145046.4 HCMCASQ2 Calsequestrin 2 (cardiac muscle) NM_001232.3 CPVT, LVNC (AR)CAV1 Caveolin 1, caveolae protein, 22kDa NM_001753.4 SIDSCAV3 Caveolin 3 NM_033337.2 LQT9, HCM, DCMCRYAB Crystallin, alpha B NM_001885.1 DCM

CSRP3 Cysteine and glycine-rich protein 3 (cardiac LIM protein) NM_003476.4 HCM,DCM

CTF1 Cardiotrophin 1 NM_001330.3 HCM,DCMDES Desmin NM_001927.3 ARVC, RCM, DCMDPP6 Dipeptidyl-peptidase 6 NM_001936.3 VFDSC2 Desmocollin 2 NM_024422.3 ARVC, DCMDSG2 Desmoglein 2 NM_001943.3 ARVC, DCMDSP Desmoplakin (DPI,DPII) NM_004415.2 ARVC, DCMDTNA Dystrobrevin, alpha NM_001390.4 LVNCEMD Emery-Dreyfuss muscular dystrophy (Emerin) NM_000117.2 DCM, Emery-Dreifuss

muscular dystrophyFHL2 Four and a half LIM domains 2 NM_201555.1 DCMGATAD1 GATA zinc finger domain containing 1 NM_021167.4 DCMGJA1 Gap junction protein alpha 1 (connexin 43) NM_000165.3 SIDS

GJA5 Gap junction protein, alpha 5, 40kDa (connexin 40) NM_005266.5 AF, Atrial standstill,

digenic (GJA5/SCN5A)GLA Galactosidase alpha NM_000169.2 FabryGPD1L Glycerol-3-phosphate dehydrogenase 1-like NM_015141.3 Brs, SIDS

HCN4 Hyperpolarization activated cyclic nucleotide-gated potassium channel 4 NM_005477.2 Brs, SSS

JPH2 Junctophilin 2 NM_020433.4 ARVC, HCM, DCmJUP Junction plakoglobin NM_002230.2 ARVC, Naxos disease

KCNA5 Potassium voltage-gated channel, shaker-related subfamily, member 5 NM_002234.3 AF

KCND2 Potassium voltage-gated channel, Shal-related subfamily, member 2 NM_012281.2 J-wave syndrome,

Sudden death

KCND3 Potassium voltage-gated channel, Shal-related subfamily, member 3 NM_004980.4 Brs, SIDS

KCNE1 Potassium voltage gated channel, Isk related family, member 1 NM_000219.4 LQT5

KCNE1L KCNE1-like NM_012282.2 VF, AF

KCNE2 Potassium voltage-gated channel, Isk-related family, member 2 NM_172201.1 LQT6, AF

KCNE3 Potassium voltage-gated channel, Isk-related family, member 3 NM_005472.4 Brs6

KCNE4 Potassium voltage-gated channel, Isk-related family, member 4 NM_080671.2 AF

KCNH2 Potassium voltage-gated channel, subfamily H, member 2 (HERG) NM_000238.3 LQT2, SQT1

KCNJ2 Potassium inwardly-rectifying channel, subfamily J, member 2 NM_000891.2 LQT,SQT, CPVT, AF,

Andersen syndrome

KCNJ5 Potassium inwardly-rectifying channel, subfamily J, member 5 NM_000890.3 LQT13

KCNJ8 Potassium inwardly-rectifying channel, subfamily J, member 8 NM_004982.3 Brs, Cantú syndrome,

SIDS, VF

KCNQ1 Potassium voltage gated channel, KQT-like subfamily, member 1 (KVLQT1) NM_000218.2 LQT, SQT, AF

LAMA4 Laminin, alpha 4 NM_002290.4 DCM

LAMP2 Lysosomal-associated membrane protein 2 NM_002294.2 HCM, DCM, Danon disease

LDB3 LIM domain binding 3 (Z1 isoform) NM_001080116.1 ARVC, LVNC, HCM, DCM

LMNA Lamin A/C NM_170707.3 LVNC, DCMMYBPC3 Myosin binding protein C, cardiac NM_000256.3 LVNC, HCM, DCMMYH6 Myosin, heavy polypeptide 6, cardiac muscle, NM_002471.3 SSS, HCM, DCM

alpha (cardiomyopathy, hypertrophic 1)

MYH7 Myosin, heavy polypeptide 7, cardiac muscle, beta NM_000257.2 LVNC, RCM, HCM, DCM

MYL2 Myosin, light chain 2, regulatory ventricular NM_000432.3 HCM10

MYL3 Myosin, light chain, alkali; ventricular and skeletal slow NM_000258.2 HCM8

MYLK2 Myosin light chain kinase 2, skeletal muscle NM_033118.3 HCM1MYOZ2 Myozenin 2 NM_016599.4 HCM16MYPN Myopalladin NM_032578.3 HCM,DCM, RCMNEBL Nebulette NM_006393.2 DCMNEXN Nexilin (F actin binding protein) NM_144573.3 HCM20,DCMNPPA Natriuretic peptide precursor A NM_006172.3 AF, Atrial standstillPKP2 Plakophilin 2 NM_004572.3 ARVC9, Brs, DCMPLN Phospholamban NM_002667.3 HCM18, DCMPRDM16 PR domain containing 16 NM_022114.3 DCM, LVNC

PRKAG2 Protein kinase, AMP-activated, gamma 2 non-catalytic subunit NM_016203.3 WPW, HCM6

PTPN11 Protein tyrosine phosphatase, non-receptor type 11 NM_002834.3 Noonan, HCM

RANGRF RAN guanine nucleotide release factor NM_016492.4 Brs

RBM20 RNA binding motif protein 20 NM_001134363.1 DCM

RyR2 Ryanodine receptor 2 (cardiac) NM_001035.2 CPVT, ARVC

SCN10A Sodium channel, voltage-gated, type X, alpha subunit NM_006514.2 Brs, AF

SCN1B Sodium channel, voltage-gated, type 1, beta polypeptide NM_001037.4 Brs, AF

SCN2B Sodium channel, voltage-gated, type II, beta NM_004588.4 LQT, AF, BrsSCN3B Sodium channel, voltage-gated, type III, beta NM_018400.3 Brs, AFSCN4B Sodium channel, voltage-gated, type IV, beta NM_174934.3 LQT10, AF

SCN5A Sodium channel, voltage gated, type V, alpha polypeptide NM_198056.2 LQT, Brs, AF, DCM,

Heart block, SSS,VFSGCD Sarcoglycan, delta NM_000337.5 DCMSLMAP Sarcolemma associated protein NM_007159.2 Brs

SNTA1 Syntrophin, alpha 1 (dystrophin-associated protein A1) NM_003098.2 LQT12

TAZ Tafazzin NM_000116.3 LVNC, DCM, Barth syndrome

TCAP Titin-cap (telethonin) NM_003673.3 HCM, DCM

TGFB3 Transforming growth factor, beta 3 NM_003239.2 ARVC, Rienhoff syndrome

TMEM43 Transmembrane protein 43 NM_024334.2 ARVC, Emery-Dreifuss muscular dystrophy

TMPO Thymopoietin NM_003276.2 DCM

TNNC1 Troponin C, slow NM_003280.2 HCM, DCMTNNI3 Troponin I, cardiac muscle isoform NM_000363.4 HCM, RCM, DCM

TNNT2 Troponin T2, cardiac NM_001001430.1 LVNC, RCM, HCM, DCM

TPM1 Tropomyosin 1 alpha NM_001018005.1 LVNC, HCM, DCM

TRDN Triadin NM_006073.3 CPVT

TRPM4 transient receptor potential cation channel, subfamily M, member 4 NM_017636.3 heart block, Brs

TTN Titin NM_133378.4 ARVC, HCM, DCMVCL Vinculin NM_014000.2 LVNC, HCM, DCMLQT: long QT syndrome; SQT: short QT syndrome; JLNS: Jervell and Lange-Nielsen syndrome; AF: atrial fibrillation; Brs: Brugada Syndrome; SSS: sick sinus syndrome; VF; ventricular fibrillation; PCCD: progressive cardiac conduction defect; CPVT: Catecholaminergic Polymorphic Ventricular Tachycardia; HCM: hypertrophic cardiomyopathy; ARVC: arrhythmogenic right ventricular cardiomyopathy; DCM: idiopathic dilated cardiomyopathy; LVNC: left ventricular noncompaction cardiomyopathy; RCM: restrictive cardiomyopathy; SIDS: sudden infant death syndrome.

Table S2: Re-analysis of allele frequency of published TRPM4 variants related to cardiac arrhythmias.

Variant Disease Function MAF* ClinVar References

p.E7K PFHB1B GOF N.A. Pathogenic 5

p.C20S SUD LOF 0.006% VUS This studyp.M49V SUD N.C. N.A. N.A. This studyp.A101T CHB, cRBBB LOF 0.113% Benign 6

p.Q131H PFHB1B; incomplete RBBB N.C. 0.0045% N.A. 7

p.R144W Brugada N.C. 0.00001% N.A. 8

p.R164W PFHB1B GOF 0.00002% Pathogenic 9

p.R195Q SUD N.C. 0.00002% N.A. This studyp.Q293R PFHB1B; AV block N.C. 0.00017% N.A. 10

p.I376T PFHB1B GOF N.A. N.A. 11

p.A380V SUD LOF 0.00001% N.A. This studyp.A432T PFHB1B; AV block, Brugada Mixed 0.00045% C.I.P. 8-10, 12

p.G582S/p.A432T

AV block GOF 0.00048% C.I.P. (6)

p.V441M Brugada LOF 0.00004% VUS 13

p.K487_L498del

Brugada N.C. 0.00775% C.I.P. 8

p.E497G SUD N.C. 0.00002% N.A. This studyp.R499W p.R499P

Brugada LOF 0.00006% N.A. 13

p.G534R SUD N.C. N.A. N.A. This studyp.G555R Brugada N.C. 0.00008% N.A. 13

p.G582S First-degree AV block and atypical RBBB, Brugada

GOF N.A. C.I.P. 8, 10, 12

p.V586L SUD N.C. 0.00048% VUS This studyp.L595V SUD LOF 0.000105% N.A. This studyp.R706C SUD N.C. 0.000036% N.A. This studyp.F773I Brugada N.C. 0.00004% N.A. 8

p.P779R Brugada LOF N.A. N.A. 8

p.Y790H PFHB1B; AV block N.C. N.A. N.A. 10

p.S834R SUD N.C. N.A. N.A. This studyp.G844D PFHB1B; RBBB, Brugada GOF 0.000108% N.A. 8-10, 14

p.Q854R Brugada, CHB GOF 0.000978% Likely benign 6, 8

p.T873I Brugada N.C. 0.000762% N.A. 8

p.K914R PFHB1B; AV block, RBBB N.C. 0.0000098% Pathogenic 10

p.K914X Brugada LOF N.A. C.I.P. 8

p.R965H SUD N.C. 0.00121% N.A. This studyp.P970S PFHB1B; incomplete RBBB N.C. 0.0000722% VUS 10

p.L1075P Brugada N.C. 0.000004% N.A. 8

p.S1044C CHB LOF 0.000123% N.A. 6

p.I1082S SUD LOF 0.000004% N.A. This studyp.R1086G SUD N.C. N.A. N.A. This study

p.P1204L Brugada N.C. N.A. Benign/Likely benign

8

p.A101T/P1204L

IVF LOF0.00347%

Benign/ Benign/Likely benign

6

The Table includes the novel TRPM4 variants described in this study. *Allele frequency was determined

by gnomAD. CHB = Complete heart block; RBBB = right bundle-branch block; cRBBB = complete right

bundle branch block; PFHB1B = Progressive familial heart block type IB (OMIM #604559); AV block =

atrioventricular block; IVF, idiopathic ventricular fibrillation; SUD = Sudden Unexpected Death GOF =

Gain-of-function, LOF = Loss-of-function; N.C. = Not characterized; N.A. = Not available; C.I.P. =

Conflicting interpretations of pathogenicity; VUS = variant of uncertain significance.

References

1. Lin Y, et al. Applying High-Resolution Variant Classification to Cardiac Arrhythmogenic Gene Testing in a Demographically Diverse Cohort of Sudden Unexplained Deaths. Circ Cardiovasc Genet. 2017;10.2. Bers DM, et al. A practical guide to the preparation of Ca2+ buffers. Methods Cell Biol. 1994;40:3-29.3. Schneider CA, et al. NIH Image to ImageJ: 25 years of image analysis. Nat Methods. 2012;9:671-5.4. Adzhubei IA, et al. A method and server for predicting damaging missense mutations. Nat Methods. 2010;7:248-9.5. Kruse M, et al. Impaired endocytosis of the ion channel TRPM4 is associated with human progressive familial heart block type I. J Clin Invest. 2009;119:2737-44.6. Bianchi B, et al. Four TRPM4 Cation Channel Mutations Found in Cardiac Conduction Diseases Lead to Altered Protein Stability. Frontiers in physiology. 2018;9:177.7. Duthoit G, et al. Brugada ECG pattern: a physiopathological prospective study based on clinical, electrophysiological, angiographic, and genetic findings. Frontiers in physiology. 2012;3:474.8. Liu H, et al. Molecular genetics and functional anomalies in a series of 248 Brugada cases with 11 mutations in the TRPM4 channel. PLoS ONE. 2013;8:e54131.9. Liu H, et al. Gain-of-function mutations in TRPM4 cause autosomal dominant isolated cardiac conduction disease. Circ Cardiovasc Genet. 2010;3:374-85.10. Stallmeyer B, et al. Mutational spectrum in the Ca(2+)--activated cation channel gene TRPM4 in patients with cardiac conductance disturbances. Hum Mutat. 2012;33:109-17.11. Daumy X, et al. Targeted resequencing identifies TRPM4 as a major gene predisposing to progressive familial heart block type I. Int J Cardiol. 2016;207:349-58.12. Syam N, et al. Variants of Transient Receptor Potential Melastatin Member 4 in Childhood Atrioventricular Block. Journal of the American Heart Association. 2016;5.13. Hof T, et al. TRPM4 non-selective cation channel variants in long QT syndrome. BMC medical genetics. 2017;18:31.14. Celestino-Soper PB, et al. Evaluation of the Genetic Basis of Familial Aggregation of Pacemaker Implantation by a Large Next Generation Sequencing Panel. PLoS ONE. 2015;10:e0143588.