1

ONLINE MATERIALS

I. Online Methods

II. Online Results

III. Online Tables

Table 1A Clinical features of 15 French probands with congenital AV block and their parents

Table 1B Clinical features and ECG profiles of 31 Japanese AV block and SSS cases

Table 2 Candidate arrhythmia susceptibility 457 genes for targeted exon sequencing

Table 3 ECG parameters of cardiac-specific conditional Gjc1 knockout mice

Table 4 Transesophageal pacing study of cardiac-specific conditional Gjc1 knockout mice

Table 5 Nucleotide sequences of the oligonucleotide primers

IV. Online Figures

Figure 1 Lateral cephalometric angular and linear measurement

Figure 2 Study protocol of conditional knockout and immunohistological demonstration of

Gjc1 depletion in SA node

Figure 3 Current ECGs of the affected members of family A and family B

Figure 4 Extracardiac abnormalities of family B members

Figure 5 Efficacy of gap junction plaque formation and the voltage-dependency of WT- and

WT/R75H-Cx45

Figure 6. Histological evaluation of nodal areas of control and Gjc1-CKO mouse

V. References

2

I. ONLINE METHODS

Trio whole exome sequencing

Library preparation and sequencing

Genomic DNA was extracted from peripheral blood lymphocytes by standard protocols. For the trio whole exome

sequencing, we recruited a total of 15 European de novo AV block cases and their parents. Clinical history and

characterization are summarized in Online Table 1A. For the trio whole exome sequencing of 15 de novo cases and their

parents, DNA concentrations of 45 samples were assessed by using Quant-IT™ dsDNA Assay Kit, Broad Range (Life

Technologies). The purity of the DNA was assessed by spectrophotometry (OD 260:280 and 260:230 ratios) using

NanoDrop (Thermo Scientific). DNA integrity was assessed on E-Gel® 96 Agarose Gels, 1% (Life Technologies). After

these assessments, each 3 µg of genomic DNA was sheared by Bioruptor sonicator (Diagenode) during about 100 min

to get 150-200 bp fragments. Ends of DNA fragments were repaired, and 3’ ends were adenylated and indexing-specific

paired-end adaptors were ligated using SureSelect XT kit (Agilent). Exome capture was performed with SureSelect

Human All Exon kit V4 and V5 (Agilent). The captured fragments were then selected using streptavidin-coated magnetic

beads, and amplified by on-beads post-capture PCR during which Illumina sequencing motifs including index sequences

were incorporated into the targeted fragments. After the purification of the enriched DNA fragments using AMPure XP

beads (Beckman Coulter), each library sample was quantified by qPCR using KAPA Library Quantification kit

(CliniSciences). DNA libraries were pooled to an equimolar concentration, and DNA was then denatured with NaOH.

Finally, it was diluted, and then proceeded to 75 or 100 bp paired-end Illumina sequencing on HiSeq1500 (Illumina).

Detection of de novo variants

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 HaplotypeCaller (version 3.5) and Samtools mpileup (version 1.0.29). Variants were considered of interest if: 1-

They present a potential pathogenicity as predicted by Variant Effect Predictor on Ensembl(1). Variants were considered

as having a potential functional consequence if they were annotated with one or more of the following Sequence

Ontology (SO) terms for at least one RefSeq transcript: “transcript_ablation” (SO:0001893), “splice_donor_variant”

(SO:0001575), “splice_acceptor_variant” (SO:0001574), “splice_region_variant” (SO:0001630), “stop_gained”

(SO:0001587), “frameshift_variant” (SO:0001589), “stop_lost” (SO:0001578), “inframe_insertion” (SO:0001821),

“inframe_deletion” (SO:0001822), “missense_variant” (SO:0001583), “transcript_amplification” (SO:0001889),

“coding sequence variant” (SO:0001580), “protein_altering_variant” (SO:0001818), “start lost” (SO:0002012); 2- They

have been called by both algorithms GATK HaplotypeCaller (with a Quality score >100) and Samtools mpileup (with a

Quality score >20); 3- They were not found within the proband’s parents; 4- They were not found within any healthy

(other) parents (n=28) to prevent recurrent technical sequencing error or frequent variants within a low covered region;

5- They were rare: minor allele frequency (MAF) was <0.1% within the 1000 genomes database(2), the Exome

3

Aggregation Consortium (ExAC) database(3), the FREX database(4), and the Genome Aggregation Database

(gnomAD)(5) (assess date: September 2016). Variants within repeat or segmental duplication regions were filtered out.

Filtering was performed using Knime4Bio(6). Variant validations were carried out in patients and in parents to filter out

false positive (de novo) variants. We used bidirectional direct sequencing of amplified genomic DNA amplicons with

variant-specific primers using Big Dye Terminator v3.1 (Life Technologies) and Applied Biosystems 3730 DNA

Analyzer (Life Technologies) following the manufacturer’s instructions. In case of missense variants, SIFT(7) and

PolyPhen-2(8) were used to predict the impact of the amino acid substitutions.

Targeted exon sequencing

For the targeted exon sequencing of 457 conduction susceptibility genes, a total of 31 Japanese patients with familial

AV block and/or SSS were recruited, which included AV block (n=6), SSS (n=22), and combination of AV block and

SSS (n=3) (Online Table 1B) Mutations in SCN5A, KCNQ1, KCNH2, HCN4, GJA5, MYH6, and LMNA were previously

excluded by Sanger sequencing. We applied targeted exon sequencing strategy for 457 arrhythmia susceptibility genes

which were selected based on the following criteria (Online Table 2); (1) Genes associated with heart rate, PR interval,

QT interval, or QRS duration by genome-wide association studies (GWAS), (2) Cardiovascular gene ontology

annotation initiative; http://www.ebi.ac.uk/GOA/CVI, (3) plausible genes based on protein function and association

with other arrhythmias and cardiac diseases. These include the relevant genes SCN5A, SCN1B, TRPM4, GJA5, MYH6

and LMNA that have already been associated with SSS and AV block, together with GJA1, GJC1, SCN10A, NKX2-5,

TBX5, PRKAG2, and GATA4 as likely candidate genes. Targeted regions of DNA were captured by a custom exon

capture probe panel (SeqCap EZ Choice Library, Roche) and KAPA HTP Library Preparation kit (Kapa Biosystems)

according to the manufacturer’s instructions, and sequenced on the Hiseq2500 platform (Illumina) with paired-end reads

of 101 bp for insert libraries consisting of 150 to 200 bp fragments. Acquired sequence data were mapped to the human

reference genome (GRCh37). The candidate variations were determined causing nonsynonymous, stop-gain, stop-loss

or splice site substitutions and InDels occurring frame-shift, in-frame, or splice sites substitution by using GATK v3.3-

0 and annotated by using the ANNOVAR software package (version 2014-07-14). Variations with MAF>0.1% in any

of the following four public databases were filtered out; HGVD(9), Integrative Japanese Genome Database(10), the

1000 Genomes(2), and ExAC(3) (assess date: September 2016). Candidate variations after the filtration were confirmed

by Sanger sequencing using an automated capillary electrophoresis DNA sequencing platform Applied Biosystems 3130

DNA Analyzer (Life Technologies). Family cosegregation of the candidate variations were confirmed by the Sanger

sequencing. The impact on protein function was further predicted by SIFT(7) and Polyphen-2(8) programs.

Dental, cephalometric, and orthopedic analyses

Dental casts were obtained from dental impression in each patient. Mesiodistal diameter of crown (crest of curvature on

the mesial surface to crest of curvature on the distal surface) was measured with Boley gauge in the each dental cast(11).

Tooth numbers are presented by the standard tooth numbering systems (http://www.stedmansonline.com/webFiles/Dict-

Dental2/14_med_dent_tooth_numbering.pdf).

4

Lateral and frontal X-ray cephalograms were analyzed using the cephalometric program Quick Ceph Studio (v3.9.1,

Quick Ceph Systems). Cephalometric parameters including facial width (FW), maxillary width (MxW), mandibular

width (MdW), mandibular length, midfacial length, facial axis angle (angle between the basion-nasion plane (BA-NAP)

and the facial axis (FX)), facial depth angle (angle between the Frankfort horizontal plane (FHP) and the facial plane

(FP)), and mandibular plane angle (angle between the FHP and the mandibular plane (MdP)) were statistically analyzed

by Ricketts’ method with the ethnic-, age- and sex-matched normal values(12-16) (Online Figure 1). Facial pattern was

diagnosed based on the ratio of width and height, i.e. dolichofacial (long and narrow face), brachyfacial (short and wide

face), or mesofacial (average facial proportion) patterns. The values are expressed in standard deviations (SD) according

to the control data.

Diagnosis of digital abnormalities including clinodactyly (radial angulation at an interphalangeal joint in the radio-

ulnar or palmar planes),(17) brachydactyly (hypoplasia or aplasia of middle phalanges of 2-5 digit in hands and feet and

proximal phalanges of the thumbs and great toe)(18), camptodactyly (congenital contracture or flexion at proximal

interphalangeal (PIP) joint and /or distal interphalangeal (DIP) joint)(19,20) and syndactyly (congenital fusion of two

or more digits)(21) were made by visual inspection and/or X-ray imaging.

Plasmid construction

A 1.3 kb coding region of GJC1 (exon 3) was amplified by polymerase chain reaction (PCR) from human genomic

DNA, and was cloned into pEGFP-N1 (Takara Bio) to concatenate the enhanced green fluorescence protein (EGFP) at

the C-terminus of Cx45, and the R75H mutation was introduced by QuikChange II XL Site-Directed Mutagenesis Kits

(Agilent) (WT-Cx45-pEGFP, R75H-Cx45-pEGFP). WT and R75H cDNAs were cloned into bicistronic expression

vectors pIRES2-EGFP and pIRES2-DsRed (Takara Bio) (WT-Cx45-pIRES2-EGFP and R75H-Cx45-pIRES2-DsRed),

respectively. For tagging polypeptide proteins to Cx45, WT and R75H cDNAs were cloned into c-myc-tag plasmid

pcDNA3.1 myc-His A (Thermo Fisher Scientific) (myc-tagged Cx45). Flag-tag was introduced at the C-terminal end of

Cx45 by PCR, and cloned into pcDNA3.1 (Thermo Fisher Scientific) (Flag-tagged Cx45). DNA sequences of the

plasmids were verified by Sanger method. Primer information is listed in Online Table 5.

Co-immunoprecipitation to evaluate the hemichannel assembly

To evaluate the functional ability of Cx45 mutant molecules to assemble each other to form a hemichannel, the binding

affinity between Flag-tagged Cx45 and myc-tagged Cx45 were investigated by co-immunoprecipitation assay. HeLa

cells (1.0x107 on a 10cm dish) were transiently transfected with 3 µg myc-tagged Cx45 (either WT or R75H) and 3 μg

Flag-tagged Cx45 (either WT or R75H) using Transfectin lipid reagent (BioRad). After 48 hrs later, cells were lysed in

TNE buffer (1% Nonidet P-40, 1 mM EDTA, 150 mM NaCl, and 10 mM Tris-HCl, pH 7.8) containing 1% Protease

Inhibitor Cocktail (Sigma-Aldrich). Total cellular lysate was obtained by centrifugation at 13,000 x rpm for 5 min, and

its protein concentration was measured by BCA protein assay kit (Thermo Fisher Scientific). For co-

immunoprecipitation assay, equal amounts of cellular lysate were incubated with goat anti-myc polyclonal antibody

(Sigma-Aldrich) and the protein G sepharose 4 fast flow (GE Healthcare) overnight at 4ºC. Immunoprecipitates were

5

separated on a 9% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane (Invitrogen). After blocking

with 5% skim milk in phosphate-buffered saline (PBS), membranes were incubated with either primary anti-Flag

monoclonal antibody (1:100, MBL) or anti-myc polyclonal antibody (1:100, Santa Cruz Biotechnology) overnight at

4ºC. Membranes were incubated with rabbit-anti mouse and goat-anti rabbit IgG HRP-conjugated antibody (1:1000, GE

Healthcare), respectively for 1 hr at room temperature (RT), and the signals were visualized by Immobilon Western

Chemiluminescent HRP Substrate (Millipore) and Fluor Chem FC2 (R&D systems).

Immunocytochemistry

Murine neuroblastoma cell Neuro-2a (N2a), 1x105 on an 8-well culture slide (Thermo Fisher Scientific), were

transfected with the myc-tagged Cx45 plasmid of WT or R75H with Lipofectamine LTX (Invitrogen). Twenty-four hrs

later, the cells were washed with PBS, fixed in 4% paraformaldehyde for 15 min at RT, and permeabilized by 0.15%

Triton X-100 in PBS with 3% bovine serum albumin for 20 min at RT. The cells were then stained by primary anti-myc

polyclonal antibody (1:100, Santa Cruz Biotechnology) overnight at 4ºC, and incubated with secondary Alexa Fluor 488

goat anti-rabbit IgG antibody (1:500, Thermo Fisher Scientific). Cells were mounted on a slide glass using Slow Fade

Gold antifade reagent with DAPI (Invitrogen). The fluorescent images were analyzed using LSM780 laser-scanning

confocal microscopy equipped with ELYRA superresolution microscopy PS.1 and a 100x oil immersion objective lens

(Zeiss).

Dye transfer experiments

To test the formation of functional Cx45 gap junction (GJ) channels between the adjacent cells, dye transfer experiments

were conducted as previously described(22). Briefly, N2a cells (2x106 on a 6 cm dish) were transfected with WT-Cx45-

pEGFP or R75H-Cx45-pEGFP plasmids using Transfectin reagent, and the medium was changed 48 hrs after

transfection with normal Tyrode’s solution (in mmol/L: 145 NaCl, 4 KCl, 1.8 CaCl2, 1 MgCl2, 10 HEPES and 10 glucose,

with a pH of 7.35 adjusted with NaOH). A micropipette filled with an intracellular solution containing 1 mg/ml Lucifer

yellow carbohydrazide dilithium (Sigma-Aldrich) was attached to the surface of the cells showing GFP-positive GJ

plaques at border of adjacent cells (Figure 2c, arrows) to form a cell-attached patch. Then, the dye was injected by

rupturing the patch with suction to form a whole-cell patch, and the images were obtained by using fluorescence

microscopy IX70 and DP controller (Olympus). The fluorescence intensity of dye in the manipulated and the adjoining

cells were respectively quantified by using ImageJ software(23), and then the arbitrary units of dye intensities in the

adjoining cells were calculated by the adjustment of the fluorescence at 10 min after the dye diffusion in the manipulated

cell to 1. The number of the adjoining cell whose arbitrary units of dye intensity were over 0.4 of the dye-injected cell

at 10 min after the dye diffusion was compared between WT, R75H, and the combination of WT and R75H.

Patch-clamp recordings

Gap junction currents were recorded from heterologously expressed N2a cell pairs using whole-cell double patch clamp

techniques as previously described.(24,25) N2a cells with 20-30% confluence on a 35-mm culture dish were transiently

6

transfected with either WT-Cx45-pIRES2-EGFP (0.5 μg), R75H-Cx45-pIRES2-DsRed (0.5 μg), or a combination of

both (0.25 µg each) using Effectene (Qiagen). Twenty-four hrs later, gap junction channel conductance (Gj) was

measured by dual whole-cell patch-clamp methods. The external solution contained (in mmol/L) 160 NaCl, 10 CsCl2, 2

CaCl2, 0.6 MgCl2, and 10 HEPES, at pH 7.4, and the intracellular solution contained (in mmol/L) 130 CsCl2, 0.5 CaCl2,

10 HEPES, 10 EGTA, 2 Na2ATP, and 3 MgATP (prepared at time of use). Both cells in cell pairs (Cell 1 and Cell 2)

were independently voltage-clamped using the same holding potential (-40 mV). Cell 1 was then stepped up to a new

voltage of -140 to +60 mV at 10 mV increments, thus creating a potential difference of -100 to +100 mV across the

junction (Vj). The current in Cell 2 corresponds to the junctional current (Ij) with opposite polarity, and the Gj was

calculated as Ij/Vj (Figure 4E). When filled with internal solution, the pipettes had a DC resistance of 5-10 MΩ.

Recordings were carried out independently using two Axopatch 200B amplifiers (Molecular Devices). All the signals

were sampled at 2 kHz and digitally filtered at 200 Hz using the pClamp10 suite (Molecular Devices).

Establishment of conditional Cx45 knockout mice

All mice were bred and utilized according to the procedures approved by the Animal Care and Ethics Committee at

Tokyo Women’s Medical University. Cx45flox/flox mice we previously created(26) were crossed with the tamoxifen-

inducible α-myosin heavy chain (αMHC)-MerCreMer transgenic mice(27) to establish time-specific conditional

knockout of Cx45 gene Gjc1 (Gjc1-CKO) in the heart on demand. Tamoxifen (Sigma) was dissolved in sesame oil (10

mg/ml), and administered via intraperitoneal injections (1 mg/mouse) once a week started at 5-9 weeks after birth

(Online Figure 2A). Cx45flox/flox mice were used as the control.

To define the spacial and temporal extent of Cre-mediated recombination, we further crossed the Gjc1-CKO mice

with the double-fluorescent Cre-recombinase reporter mice (mT/mG)(28). Gjc1 and Cre genotypes were determined by

multiplex genomic PCR as previously reported.(26) The mT/mG allele represents 634 bp GFP fragment (primer pair;

GFPF1 and GFPR1) and control showed 413 bp Dad1 genomic region (Online Fig 2B) (primer pair; DadF1 and DadR1;

Online Table 5).(29) Alternatively, fluorescence detection using the Twin LED Light system (RelyOn) was used.

Cryosections of the heart tissue including SA node and AV node were stained with primary anti-mouse HCN4 antibody

(#APC-052, Alomone Labs, 1:20), and detected by biotinylated horse polyclonal anti-goat IgG (Vetcor Laboratories,

1:200), and Alexa Fluor594 (or 488)-conjugated Streptavidin (Jackson ImmunoResearch), using a fluorescent

microscope (Leica DMI6000B/CTR6000).

Transesophageal electrophysiological studies in Gjc1-CKO mice

Gjc1-CKO mice (n=9) and control mice (n=14) were used for the control electrophysiological evaluations at the age of

5-9 weeks without tamoxifen. After 4-times of tamoxifen injection, the same mice were subjected to the 2nd round of

body surface ECG and electrophysiological study at the age of 16.7 ± 8.6 weeks in Gjc1-CKO and 10.9 ± 6.6 weeks in

control (p>0.05). Transesophageal electrophysiological studies were performed as follows; after the induction of

inhalation anesthesia with 2-2.5% Isofluran, mice were placed in the supine position on a custom-designed ECG

recording board and maintained anesthetized under 1-1.2% Isofluran. The body temperature was strictly maintained

7

between 37.5°C and 38.5°C with a heating light during recordings. To avoid inter-individual variability, we recoded

ECGs of the identical mice before and after tamoxifen administration, and compared the ECG parameters including

heart rate, PR interval, P wave duration, QRS interval, QTc interval (corrected by Bazett equation), P wave amplitude,

R wave amplitude, and T wave amplitude.

Electrophysiological properties of the SA node, AV node, and the atrium were evaluated by the transesophageal

pacing strategy as previously described(30) before and after the tamoxifen administration using the identical mice, rather

than more invasive transjugular vein procedures. Transesophageal atrial pacing was performed using a 1.1-french pacing

catheters with 8 ring electrodes ERP-800 (Millar) and a digital stimulator PG4000A (Cygnus Technology) as previously

described(30),(31), and the data were recorded and analyzed using LabChart 8 (AD Instruments). To evaluate the SA

node function, repetitive atrial pacing was applied for 30 s with a cycle length progressively decremented in 10 ms

starting from the value 10 ms shorter than the intrinsic cycle length. Sinus node recovery time (SNRT) was determined

as the longest pause from the last pacing spike to the first P wave on the lead I or II, or the first A wave on the esophageal

lead. Corrected SNRT (cSNRT) was calculated by subtracting the intrinsic cycle length from the SNRT. SA conduction

time (SACT) was determined by Narula method.(32) Eight-stimulus drive train at a cycle length 10 ms less than intrinsic

cycle length was delivered and SACT was determined as the coupling interval between last pacing spike and first P

wave after pacing. AV conduction parameter AVw is defined as the minimum cycle length inducing Wenckebach type

AV block by rapid atrial pacing. Atrial effective refractory periods (AERP) were determined by delivering an 8-stimulus

drive train (S1) at a cycle length of 100 ms followed by a premature stimulus (S2) progressively decremented in 10 ms

intervals. AERP is defined as the shortest S1-S2 interval before failure of S1-S2 to capture the atrium.

8

II. ONLINE RESULTS

Trio whole exome sequencing

Trio of 15 families affected by congenital AV block (45 individuals) have been exome sequenced resulting in an average

depth of 84 and a mean coverage of 96.5% at 10-fold. Variants of potential pathogenicity predicted by Variant Effect

Predictor and de novo (absent from parents) were studied further. Remaining variants were compared to public databases

and filtered with MAF<0.1%. Forty-two variants followed these criteria. Three of them were contained within segmental

duplication region and were filtered out. After Sanger sequencing validation, 2 were false positive signals and 19 were

found in parents. We identified 18 de novo variants including 13 missense variants, 3 splice region variants, 1 nonsense

variant and an 8-base frameshift deletion resulting in a premature stop codon.

In the proband of family 12 (Online Table 1A) (Family A), we identified 13,035 coding variants (Table 2). Trio

exome sequencing revealed 7 de novo variants, and filtering MAF<0.1% and Sanger sequencing validation confirmed

only 4 candidate rare variations: 2 truncation mutations: ZNF683-c.1219_1226delCTGCACTG (p.Q407CfsX461),

ZNF22-c.C85T (p.Q29X), and 2 missense mutations: GJC1-c.G224A (p.R75H), ATAD2B-c.C4212G (p.L1404F). These

variations were not found in databases of 1000 Genomes, ToMMo-2KJPN, ExAC, gnomAD, and HGVD. GJC1-

c.G224A, ATAD2B-c.C4212G were assumed to be deleterious by in-silico prediction programs (Table 3).

Targeted exon sequencing

Targeted exon sequencing of 457 conduction susceptibility genes using HiSeq2500 platform yielded a total of 6.9 giga

bp data with 99.1% mapped and 239-fold coverages, and identified 1,040 coding variations in the proband of family 10

of Online Table 1B (Family B, Table 2). After filtering variations with MAF<0.1% using public variation databases, 8

variations remained. Sanger validation and the family cosegregation further narrowed down them to three candidate

variations; CACNB2-c.A305G (p.Q102R), TTN-c.G6984T (p.Q2328H), and GJC1-p.R75H (Table 3). These variations

were not found in databases of 1000 Genomes, ToMMo-2KJPN, ExAC, and HGVD, but CACNB2-c.A305G and TTN-

c.G6984T were found in gnomAD with MAF of 4.0x10-5. Only GJC1-c.G224A was assumed to be deleterious by in-

silico prediction programs. This mutation was found in both family A and B.

Detailed clinical information of affected individuals with the GJC1 mutation c.G224A (Cx45-p.R75H)

Family A proband (II:1)

A 2 yo boy was diagnosed with 1st degree AV block during a physical checkup, which turned out to be complete AV

block with very similar rates of P and QRS (~100 bpm), although asymptomatic (Figure 1A). He showed all degrees of

AV blocks of 1st through 3rd degrees and the P waves were progressively diminished, when he was diagnosed as SSS

at 7 yo. Holter ECG showed alternans of 2nd and 3rd degree AV blocks with mean heart rate of 37 bpm, maximum heart

rate of 155 bpm while riding a bicycle, there was no pauses longer than 2 sec (Figure 1A). Echocardiography was normal

except for a minor hypoplasia of the aorta. Cephalometric analysis, using midfacial length, mandibular length, facial

axis angle, facial depth angle, and mandibular plane angle, revealed moderate brachyfacial pattern (Figure 2A, Table 1).

9

He had a mild clinodactyly at the 5th fingers (Figure 2B). Dental investigation revealed mandibular permanent incisor

agenesis (n=5), and microdontia of bilateral maxillary lateral incisor (n=2; 12 and 22; 5.76 mm and 5.12 mm), where

average size of upper lateral incisors in French is 6.72 mm (Figure 2C)(33). Both parents showed normal ECG, and

there is no family history of arrhythmias or sudden death. He is now 9 yo, asymptomatic, and shows junctional rhythm

with narrow QRS complex. (Online Figure 3A)

Family B proband (II:2).

A 9 yo girl undertook further examinations for evaluating the bradycardia associated with family history of SSS, atrial

standstill, and permanent pacemaker implantation observed in her father (I:1). Wenckebach and Mobitz type-II 2nd

degree AV block were identified by Holter ECG. In the next four years, she experienced repetitive episodes of

presyncope associated with exercise and P wave diminished on ECG. Treadmill exercise ECG exhibited 1st and 3rd

degree AV block, and the atropine stimulation test indicated impaired exercise intolerance. His bundle electrocardiogram

showed complete AH block and the right atrium pacing failed to capture, indicating a condition partial atrial standstill.

A permanent pacemaker was implanted at the age of 13. Current ECG took at the time of the generator exchange in the

absence of pacing showed junctional rhythm without involving ventricular conduction abnormalities (Online Figure 3B).

As extra-cardiac findings, she had severe brachyfacial appearance in comparison to the age- and sex-matched

control Japanese value (http://www.e-stat.go.jp/SG1/estat/List.do?bid=000001054955&cycode=0, from the database of

Japanese Statistic Bureau). Among the cephalometric parameters for the diagnosis of facial patterns, midfacial length,

mandibular length, and mandibular plane angle were markedly smaller than control normal values (-1.0 to -6.0 SD).

Furthermore, facial axis angle and facial depth angle were also substantially larger than control (+0.9 to +2.0 SD) (Figure

2D, Table 1). Based on this cephalometric analysis, she was diagnosed with the typical brachyfacial pattern. She had

bilateral camptodactyly on DIP joint of 3rd to 5th fingers and clinodactyly on the 5th fingers (Fig 2E). X-ray showed

brachymesophalangy and radial curvature of 5th fingers (Bell type A). As dental abnormalities, she had mandibular

permanent incisor agenesis (n=3; 31, 41 and 42) and microdontia of upper lateral incisors (n=2; 12 and 22; 5.2 mm and

6.6 mm). (Figure 2F, Tables 1).

Family B daughter (III:1)

First degree AV block and Wenckebach type 2nd degree AV block were identified during the regular school checkup at

6 yo. Holter ECG showed 1st degree AV block at 8 yo, AV dissociation with nearly identical atrial and ventricular rates

associated with progressively diminished P wave amplitude (12 yo), and total loss of P waves (14 yo) (Figure 1B). She

was admitted to the hospital because she had the first episode of syncope after taking bath at 14 yo. Electroanatomical

mapping using CARTO system virtually failed to detect voltage activities larger than 0.05 mV in the almost entire right

atrium, which is defined as scar(34,35) and compatible with the status of atrial standstill (Figure 1C). A permanent

pacemaker of VVI-mode was implanted. Other cardiac abnormalities have not been identified. Current ECG took at the

time of the generator exchange in the absence of pacing showed junctional rhythm without involving ventricular

conduction abnormalities (Online Fig 3C)

10

She had camptodactyly (DIP joint) and clinodactyly (radial curvature) on 5th fingers of the hands. X-ray further

revealed brachymesophalangy (Bell type A)(36). Her facial pattern was severe brachyfacial. Dental investigations

revealed mandibular permanent incisor agenesis (n=2; 31 and 41), microdontia of upper lateral incisors (n=2; 12 and

22; 5 mm), and prolonged retention of deciduous lower frontal incisors (n=2; 31 and 41) (Tables 1,4, Online Figure 4).

Average size of upper lateral incisors in Japanese is 6.9 mm(12).

Family B son (III:2)

The proband’s brother also developed sinus bradycardia and junctional escape beats at the age of 12. Holter ECG

recorded at the age of 15 showed junctional bradycardia and suppressed diurnal heart rate variability, but he has been

asymptomatic. Current ECG showed junctional rhythm without involving ventricular conduction abnormalities (Online

Fig 3D). As extra-cardiac features, he had bilateral camptodactyly on DIP joint of 3rd through 5th fingers of both hands

and clinodactyly on 5th fingers. X-ray showed brachymesophalangy and radial curvature of the 5th fingers (Bell type

A). He had severe brachyfacial pattern associated with mandibular incisor agenesis (n=2, 31 and 41) and prolonged

retention of deciduous lower incisor (n=1, 31or 41) (Tables 1,4, Online Figure 4).

Family B father (I:1)

The proband’s father was diagnosed with AV block and atrial standstill. He had exertional palpitations and repetitive

episodes of presyncope at the age of 36 (Figure 1B). ECG and transesophageal pacing demonstrated atrial standstill,

and he received a permanent pacemaker implanted. He also had bilateral camptodactyly and clinodactyly (not shown).

Dental records are not available. He died of a non-cardiac cause during the 7th decade of his life. Genomic DNA and

X-ray imaging are unavailable, and he is an obligate mutation carrier.

Intermolecular binding affinity of Cx45

The R75 residue in Cx45 was assumed to be essential for the inter-monomer interactions based on the crystal structure

analysis(37). We tested if the mutation R75H of Cx45 disrupt the hemi-channel assembly property using co-IP assay.

The binding affinity of R75H-Cx45 was comparable to that of WT-Cx45 (Figure 4A).

Gap junction plaque formation

To investigate the localization of the mutant Cx45 channel molecules, neuroblastoma N2a cells were transfected with

myc-Cx45 plasmids of either WT, R75H, or in combination, and were labelled with the GFP-conjugated anti-myc

antibody. In N2a cells overexpressing WT-Cx45, the GFP signals were mainly localized on the borderline between the

adjoining cells as well as the cells homozygously or heterozygously overexpressing R75H (Figure 4B). Fluorescence-

positive and gap junction plaque-positive cell pairs were counted in 5 different views for each group, and the efficacy

of gap junction plaque formation were statistically analyzed by calculating the ratio of cell pairs with gap junction

plaques to the number of fluorescence-positive cell pairs. The efficacy of gap junction plaque formation was not

significantly different in three groups (WT: 64.3 ± 6.7%, n=171; WT/R75H: 59.4 ± 11.0%, n=165; R75H: 60.4 ± 8.4%,

11

n=150; NS, Online Figure 5), suggesting that the mutant Cx45 does not have defect of membrane trafficking or

localization on the border line between the adjoining cells. Combined with the result of the binding assay by co-IP

(Figure 4A), it is indicated that R75H-Cx45 can assemble each other and be transported to cell surface to form GJ

channels between the adjoining cells as well as WT-Cx45.

Mutant R75H-Cx45 exhibited severely reduced dye transfer

To evaluate the GJ permeability of the mutant Cx45 channels, Lucifer yellow dye transfer was performed in N2a cell

pairs overexpressing Cx45 of either WT, R75H or both. Immediately after rupturing the membrane to make a whole-

cell patch, Lucifer yellow in the pipette solution was rapidly diffused into the manipulated cells (donor cells, Figure 4C).

Fluorescent intensity of the transferred dye from the donor cells (open symbols) to the adjoining cells (closed symbols)

was time-dependent, and was nearly saturated in 5 min in almost all the cells expressing WT-Cx45 (Figure 4D). In

contrast, dye transfer to the adjoining cells was severely suppressed in the cells expressing homomeric (R75H) or

heteromeric (WT/R75) Cx45. These data show that the mutant Cx45 protein dominant-negatively suppresses permeation

property of WT-Cx45 protein without affecting plaque formation.

Electrophysiological properties of the mutant Cx45 channels

The electrophysiological properties of R75H-Cx45 were determined using conventional dual whole-cell patch-clamp

techniques. The probability of observing electrical coupling in homomeric mutant R75H channel (R75H-Cx45-pIRES2-

EGFP) was 0% (0/8), whereas those of WT (WT-Cx45-pIRES2-EGFP) and WT (WT-Cx45-pIRES2-DsRed) were

respectively 91.7% (11/12) and 100% (5/5). Heteromeric channels (WT/R75H) expressing both WT-Cx45-pIRES2-

DsRed and R75H-Cx45-pIRES2-EGFP showed electrical coupling of 72.7% (8/11). In contrast, macroscopic

conductance (Gj) measured at a transjunctional voltage of +60 mV was significantly suppressed in the heteromeric

channel WT/R75H and homomeric channel R75H than WT (WT: 24.2 ± 7.9 nS, n=5; WT/R75H: 4.9 ± 5.3 nS, n=11;

R75H: 0 nS, n=8; p=1.00x10-7) (Figure 4E). These data show that R75H exhibits the dominant-negative suppression

effects on the electrophysiological properties of WT-Cx45 channel.

Immunohistological evaluation of Gjc1 depletion at the SA node area

Gjc1-CKO mice after tamoxifen administration showed red fluorescence in all organs, while green fluorescence was

observed only in the heart. Furthermore, immunohistological evaluation with cryosections of the hearts in these mice

showed colocalization of HCN4 (red) and Cre recombination (green) in the SA node areas, confirming that tamoxifen

successfully depleted Gjc1 in the heart (Online Figure 2C).

Histological evaluation of nodal areas and time course of nodal functions in Gjc1-CKO mice

Paraffin-embedded sections of SA node and AV node area were histologically examined using Masson Trichrome

staining in Gjc1-CKO and control mice. There was no apparent difference in fibrosis of between Gjc1-CKO and control

mouse (Online Figure 6).

12

We repeated EPS several times for an extended period of time (up to 32 weeks) in some mice, but the SA node

and AV node functions were largely unchanged after tamoxifen injection (data not shown).

13

III. ONLINE TABLES

Table 1A. Clinical features of 15 French probands with congenital AV block and their parents

a. Probands

Family Sex Age at last clinicalexamination (y.o.)

HR (bpm) PR (ms)* QRS(ms)

QTc (ms) QRSwaveform

AVConduction

Age at PMI

1 female 12 47 NA 78 389 Normal 3rd AVB 22 female 22 60 NA 158 494 RBBB 3rd AVB 223 male 0 75 NA 70 498 Normal 3rd AVB 34 female 0 39 NA 125 755 LBBB 3rd AVB 05 male 0 56 NA 122 325 LBBB 3rd AVB 06 female 5 49 NA 60 495 Normal 3rd AVB 57 male 2 60 NA 63 442 Normal 3rd AVB8 female NA NA NA NA NA NA NA 09 male 4 50 NA 55 385 Normal 3rd AVB10 male NA NA NA NA NA NA NA 9

11 male 18 51 237 73 483 Normal 1st andMobitz AVB

12 male 3 100 392 64 415 Normal 1st-3rd AVB13 female 5 50 NA 65 443 Normal 3rd AVB 514 male 2 60 NA 75 397 Normal 3rd AVB 215 male 4 43 NA 70 433 Normal 3rd AVB 4

mean M:F=9:6 6 ± 7 57 ± 16 83 ± 31 458 ± 103 5 ± 6

Family Age at last clinicalexamination (y.o.)

HR PR (ms) QRS(ms)

QTc (ms) QRSwaveform

AVConduction

Status

mother 43 53 170 88 412 Normal Normal Unaffectedfather 46 74 184 94 418 Normal Normal Unaffectedmother 49 75 121 101 404 Normal Normal Unaffectedfather 50 67 173 91 398 Normal Normal Unaffectedmother 36 75 157 95 426 Normal Normal Unaffectedfather 37 60 145 83 389 Normal Normal Unaffectedfather 43 61 160 100 400 Normal Normal Unaffectedmother 43 59 140 80 360 Normal Normal Unaffectedfather 51 85 120 80 405 Normal Normal Unaffectedmother 30 73 150 80 397 Normal Normal Unaffectedmother 37 80 132 90 418 Normal Normal Unaffectedfather 36 90 194 101 473 Normal Normal Unaffectedmother 33 80 97 55 341 Normal Normal Unaffectedfather 37 75 102 79 318 Normal Normal Unaffectedfather 43 65 126 100 445 Normal Normal Unaffectedmother 40 60 123 85 390 Normal Normal Unaffectedfather 45 75 137 93 421 Normal Normal Unaffectedmother 41 78 127 103 446 Normal Normal Unaffectedmother 41 79 160 90 413 Normal Normal Unaffected

father 40 61 140 110 380 IncompleteRBBB

Normal Probablyunaffected

father 46 70 175 99 412 Normal Normal Unaffectedmother 44 100 154 98 403 Normal Normal Unaffectedmother 39 69 122 88 437 Normal Normal Unaffectedfather 42 43 188 88 403 Normal Normal Unaffectedfather 39 48 159 88 353 Normal Normal Unaffectedmother 39 75 140 66 391 Normal Normal Unaffectedmother 38 60 116 69 351 Normal Normal Unaffectedfather 39 80 170 70 388 Normal Normal Unaffectedmother 37 60 130 92 409 Normal Normal Unaffectedfather 43 78 143 104 420 Normal Normal Unaffected

mean 41 ± 5 70 ± 12 145 ± 25 89 ± 12 401 ± 33

15

QTc : Bazett's corrected QT interval. HR, heart rate; NA, data not available; PMI, pacemaker implantation; LBBB, left bundlebranch block; RBBB, right bundle branch block; AVB, atrioventricular block. *; PR duration was rarely recorded due to a largenumber of cases with complete atrioventricular dissociation. Family 12 is the Family A of this work.

b. Parents

1

2

3

4

5

6

7

8

9

10

11

12

13

14

14

Table 1B. Clinical features and ECG profiles of 31 Japanese AV block and SSS cases

Proband Sex diagnosis Age at diagnosis(y.o.)

SSS HR (bpm) AV Conduction QRSwaveform

Age atPMI

family history

1 female SSS 62 + 48 Normal Normal 62 mother (SSS, PMI)

2 female SSS, AFL, WPW,AVB

56 + 55 Wenckebach LBBB 66 father, sister, son(SSS, AVB, PMI)

3 male SSS 19 + 54 Normal Normal 19 grandfather, uncles(syncope, PMI)

4 female AVB 6 - 58 3rd AVB LBBB 11 father (arrhythmia)5 male SSS NA + NA NA NA NA NA6 male SSS NA + NA NA NA NA NA7 male SSS NA + NA NA NA NA NA8 female SSS NA + NA NA NA NA NA9 male AVB 42 - NA NA NA NA NA

10 female AVB, AS 7 + 54 3rd AVB Normal 14grandfather, mother,brother (AVB, AS,PMI)

11 female SSS 52 + NA NA NA 52 son, daughter (SSS)

12 female SSS, AFL 49 + 48 Normal Normal 49 father, sister, son(SSS, AFL)

13 male AVB 51 - NA NA NA 51 brother, mother(AVB, PMI)

14 female AVB 52 - NA 3rd AVB NA 52 mother (AVB)

15 male SSS, LVNC, AS 16 + 32 Normal Normal 17 grandfather, great-grandmother (PMI)

16 female SSS NA + NA NA NA NA father (SSS, PMI)17 male SSS NA + NA NA NA NA NA18 female SSS + 54 Normal Normal daughter (SSS)19 male SSS NA + NA NA NA NA NA20 male SSS, Af 50 + NA NA NA 52 mother (SSS, PMI)21 female SSS NA + NA NA NA NA NA22 female SSS NA + NA NA NA NA NA23 female AVB NA + NA NA NA NA NA24 male SSS 54 + 64 NA Normal none25 female SSS 15 + 33 Normal Normal 15 none26 male SSS NA - NA NA NA NA NA27 female AVB 33 - 50 3rd AVB Normal 33 brorther (SSS)28 male AVB 12 - 71 3rd AVB Normal NA29 male SSS, WPW 7 + 67 Normal Normal mother (SSS)

30 male SSS, AS 0 + 49 NA NA mother, brothers(SSS)

31 male SSS, AVB 7 + 48 1st AVB Normal 15 noneMean 31 ± 22 52 ± 11 36 ± 20 48.4% (15/31)

SSS: sick sinus syndrome, AS: Atrial standstill, AFL:atrial flutter, Af: atrial fibrillation, LVNC: Left ventricular non-compaction. Family 10 is the Family B ofthis work.

15

Table 2. Candidate arrhythmia susceptibility 457 genes for targeted exon sequencing

ABCC8 CACNA1G DENND2D GJC1 LAMA2 PLEKHG5 SLN VCLABCC9 CACNA1H DEPDC5 GLA LAMP2 PLN SMAD1 VCPABI1 CACNB1 DES GMPPB LARGE PMVK SMAD2 VTI1AACADVL CACNB2 DHRS9 GNB4 LDB3 POMGNT1 SMAD3 WASLACE CACNB3 DHX8 GNE LIG3 POMT1 SMAD4 WNT11ACHE CACNB4 DKK1 GNG11 LITAF POMT2 SMCHD1 XIRP1ACIN1 CALM1 DLG1 GOT2 LMNA POU1F1 SNN XIRP2ACOT7 CALM2 DLG2 GPD1L MAML2 POU2F1 SNTA1 ZC3H7AACTA1 CALM3 DLG4 GPR133 MAPK1 POU4F1 SOS1 ZFHX3ACTC1 CALR DMD GSK3B MAPK14 PPP2R3A SOX5ACTN2 CAMK2A DMPK GSN MATR3 PPP3CC SP1ACTN3 CAMK2B DNAH1 HAND1 MCU PPP3R1 STARD9ACTN4 CAMK2D DNAH11 HCN1 MEIS1 PRAM1 STIM1ACVR1 CAMK2G DNAH2 HCN2 MICAL1 PRKAG2 STK3ACVRL1 CAMK2N1 DNM2 HCN4 MIS12 PRKCE STRNADRA1A CAMK2N2 DPM1 HEY2 MSN PRRX1 STRN3ADRA1B CAND1 DPP6 HIP1 MSTN PSEN1 SYNE1ADRA2A CAND2 DPP8 HIPK1 MSX1 PSEN2 SYNE2ADRA2B CANX DSC2 HNRPDL MTM1 PTPN11 SYT10ADRA2C CAPG DSG2 HSPG2 MTMR2 PTRF TAZADRB1 CAPN3 DSP HTR4 MTND6 RAF1 TBX2ADRB2 CAPN7 DTNA IRX3 MTTG RASGRF1 TBX20ADRB3 CASQ1 DUX4 IRX4 MTTK RASGRF2 TBX3ADRBK1 CASQ2 DYSF IRX5 MYBPC3 RBM20 TBX5AGT CAV1 EDN1 ITGA7 MYH10 RDX TCAPAGTR1 CAV2 EDNRA ITPR1 MYH6 RETN TFPIAHNAK CAV3 EDNRB ITPR2 MYH7 RFFL TGFB1AKAP6 CD34 EMD ITPR3 MYL2 RFX4 TGFB2AKAP7 CD46 EME1 JAG1 MYL3 RNF19A TGFB3AKAP9 CDH3 ENG JPH2 MYL4 RNF207 TGFBR1AKT3 CDHR3 EPAS1 JUP MYL7 RP1L1 TGFBR2ALAS1 CDK5RAP1 EPB41 KCNA4 MYLK2 RYR1 TLN1AMIGO3 CDKN1A EYA4 KCNA5 MYO3B RYR2 TLN2AMPD1 CFL2 EZR KCNAB1 MYO7A RYR3 TMEM38AANK1 CH25H FADS1 KCNAB2 MYOT S100A1 TMEM38BANK2 CHRM2 FAM191B KCNB1 MYOZ2 SCN10A TMEM43ANKRD1 CHRM3 FAM5C KCNB2 MYPN SCN11A TMOD1ANKRD23 CHRM4 FAS KCND1 NCOA7 SCN1B TMOD4ANKRD46 CLASP2 FGF23 KCND2 NDRG4 SCN2B TMPOANO5 CLEC16A FHL1 KCND3 NEB SCN3B TNFRSF17APC CMYA5 FHL2 KCNE1 NEIL3 SCN4A TNNC1ARHGAP24 CNBP FHOD3 KCNE2 NEURL SCN4B TNNI3ARIH2 CNOT1 FKBP1A KCNE3 NF1 SCN5A TNNT1ASF1A CNOT2 FKBP1B KCNE4 NFKB1 SCN7A TNNT2ASPH CNOT3 FKRP KCNH2 NKX2-5 SCN8A TNPO3ASTN2 COL15A1 FKTN KCNIP1 NLRP10 SCN9A TOR1AIP1ATP1A1 COL17A1 FLNA KCNIP2 NLRP14 SDHA TPM1ATP1A2 COL1A1 FLNB KCNIP3 NOS1AP SEPN1 TPM2ATP1A3 COL1A2 FLNC KCNIP4 NOTCH1 SGCA TPM3ATP1B1 COL4A1 FLRT2 KCNJ11 NPHP4 SGCB TRDNATP2A1 COL4A2 FNDC3B KCNJ12 NPPA SGCD TRIM32ATP2A2 COL4A3 FRG1 KCNJ2 NPPB SGCG TRIM55ATP2A3 COL4A4 FRG2 KCNJ3 NPR3 SLC12A9 TRIM63ATP2B1 COL5A2 FRMD4A KCNJ4 OBSCN SLC22A5 TRPC1ATP2B2 COL6A1 FXR1 KCNJ5 ORAI1 SLC24A6 TRPC2ATP2B4 COL6A2 FXR2 KCNJ8 PABPN1 SLC25A25 TRPC3ATXN1 COL6A3 GAA KCNN2 PCDHGA7 SLC25A4 TRPC6B3GNT7 COL7A1 GATA4 KCNN3 PCOLCE SLC30A1 TRPM4BAG3 CPNE8 GFPT1 KCNQ1 PDE4B SLC35F1 TTNBIN1 CPT2 GINS3 KCNV1 PFN1 SLC38A7 TTRBMP2 CRYAB GJA1 KIAA1755 PITX2 SLC6A2 UBCC6orf204 CSRP3 GJA10 KLF12 PKP2 SLC8A1 UFSP1CACNA1C DBC1 GJA4 KLHL40 PLA2G6 SLMAP USP15CACNA1D DCAMKL1 GJA5 KRAS PLEC SLMO1 USP25

16

Table 3. ECG parameters of cardiac-specific conditional Gjc1 knockout mice

Table 4. Transesophageal pacing study of cardiac-specific conditional Gjc1 knockout mice

GJC1-CKO (n=9)§ Before tamoxifen After tamoxifen P value*Heart Rate (bpm) 456.8 ± 64.6 477.9 ± 74.6 0.268PR Interval (ms) 35.7 ± 2.4 38.0 ± 4.6 0.126P wave duration (ms) 15.6 ± 4.1 16.5 ± 3.0 0.320QRS Interval (ms) 8.4 ± 1.0 8.2 ± 0.7 0.278QTc (ms) 59.6 ± 11.4 60.2 ± 10.7 0.457P wave amplitude (µV) 22.9 ± 13.9 25.8 ± 12.1 0.346R wave amplitude (µV) 398.4 ± 163.6 409.3 ± 209.9 0.409T wave amplitude (µV) 205.6 ± 87.5 237.1 ± 114.5 0.090

Control (n=14) Before tamoxifen After tamoxifen P value*Heart Rate (bpm) 457.4 ± 39.1 488.9 ± 49.9 0.031PR Interval (ms) 37.2 ± 5.8 38.3 ± 3.4 0.271P wave duration (ms) 17.0 ± 0.3 17.7 ± 0.3 0.309QRS Interval (ms) 8.0 ± 1.1 8.0 ± 0.6 0.484QTc (ms) 59.9 ± 7.0 56.1 ± 7.6 0.094P wave amplitude (µV) 20.4 ± 6.7 21.9 ± 3.9 0.296R wave amplitude (µV) 393.3 ± 160.3 356.8 ± 114.2 0.143T wave amplitude (µV) 193.1 ± 83.3 184.7 ± 83.0 0.318

§: Selected ECG traces showing appearent P waves were analyzed.*: Paired Student's t-test

Gjc1-CKO (n=9) Before tamoxifen After tamoxifen P value*SNRT (ms) 195.3 ± 28.4 226.1 ± 32.7 0.030cSNRT (ms) 67.2 ± 26.2 95.4 ± 31.7 0.040SACT (ms) 171.4 ± 19.8 174.4 ± 23.7 0.400AVw (ms) 73.3 ± 5.0 75.6 ± 7.3 0.080AERP (ms) 57.2 ± 7.5 57.8 ± 14.8 0.440

Control (n=14) Before tamoxifen After tamoxifen P value*SNRT (ms) 190.9 ± 23.3 185.8 ± 19.9 0.308cSNRT (ms) 64.6 ± 20.7 58.8 ± 19.1 0.250SACT (ms) 159.0 ± 12.3 158.7 ± 16.4 0.477AVw (ms) 74.2 ± 5.1 70.0 ± 6.0 0.048AERP (ms) 58.6 ± 3.2 55.5 ± 6.1 0.076

SNRT: sinus node recovery time from overdrive pacingcSNRT: corrected SNRTSACT: sinoatrial conduction timeAVw: minimum cycle length resulting in Wenckebach type AV blockAERP: atrial effective refractory period*: Paired Student's t-test

17

Table 5. Nucleotide sequences of the oligonucleotide primers

1. Sanger sequencingName sequence (5' to 3')GJC1-F1 GAGCCACCCTACCCAACTGAGJC1-R1 ACCAGAGCCAAATGTTTACTCAAGJC1-NC1 CTTCCGGATCGTCCTTACAGGJC1-NC2 ACCGGGCTCTGGAAGAAACGGJC1-NC3 TATGGTGTTACAGGCCTTTG

2. Plasmid cloning and mutagenesisName sequence (5' to 3')GJC1-EcoR1-F GCATACGAATTCCGCCACCATGAGTTGGAGCTTCCGJC1-R-Flag-Xho1 TTTCTCGAGCTACTTGTCGTCATCGTCTTTGTAGTCAATCCAGACGGAGGTCTTCCCGJC1-Xho1-R TAAGCCCTCGAGGACCCAAATCCAGACGGAGGTCGJC1-Xho1-F CCACTCGAGTCACCATGAGTTGGAGCTTCGJC1-EcoR1-R AAGAATTCGAATCCAGACGGAGGTCTTCCGJC1-R75H-QCM-F CCTCTCTCCCATGTACACTTCTGGGTGTTCGJC1-R75H-QCM-R GAACACCCAGAAGTGTACATGGGAGAGAGG

3. Genotyping of mT/mG mice Name sequence (5' to 3')GFPF1 CGGCCACAAGTTCAGCGTGTCGFPR1 GTCCATGCCGAGAGTGATCCCDadF1 CCGGTATCCGAAGTCCCCGTGTTCDadR1 CAGTTTCAACTCCTGTTAGGCATTAGAA

18

IV. ONLINE FIGURES

Figure 1. Lateral cephalometric angular and linear measurement. Facial axis angle is defined as an angle between the basion-nasion plane (BA-NAP) and the facial axis (FX). Facial depth angle is an angle between the Frankfort horizontal plane (FHP) and the facial plane (FP). Mandibular plane angle is an angle between FHP and the Mandibular plane (MdP). MfL and MdL stand for midfacial length (Condylion-A point) and mandibular length (Condylion-Gnathion), respectively.

Online Fig. 1

19

Figure 2. Study protocol of conditional knockout and immunohistological demonstration of Gjc1 depletion in SA node (A) Experimental protocol of in vivo assays of Gjc1-CKO mice. ECG parameters, transesophageal pacing study (EPS) were performed before and after tamoxifen administration (four times, weekly) in each mouse. (B) Gjc1-CKO crossed with mT/mG mice were genotyped by multiplex PCR. The 634 bp and 413 bp bands correspond to mT/mG and control Dad1 genes, respectively. (C) Efficiency of Cre-loxP recombinase activity to knockout Gjc1 gene in SA node after tamoxifen administration. GFP fluorescence (green signal) indicating successful Cre-LoxP recombination was colocalized with immunofluorescent Hcn4 (red signal) at the SA node area. RAA: right atrial appendage, CT: crista terminalis, SAN: sinoatrial node, IAS: interatrial septum. White scale bars: 200 μm

20

21

Figure 3. Current ECGs of the affected members of family A and family B. Current ECGs of (A) family A proband (II:1), (B) family B proband (II:2), (C) family B daughter (III:1), and (D) family B son (III:2). ECGs of (B) and (C) were taken in the presence of pacing during the generator exchange. All four individuals show junctional rhythm without ventricular conduction disturbance, despite exhibiting progressive conduction abnormalities in the AV node and atrium.

22

Figure 4. Extracardiac abnormalities of the family B members. (A)-(C); proband (III:1), and (D)-(F); brother of the proband (III:2). (A)(D) Front and lateral views and cephalograms of the patient. Cephalometric analysis showed significant brachyfacal pattern (see Table 1). (B) (E) Clinodactyly on the 5th fingers and camptodactyly on 3rd through 5th fingers of hand. X-ray showed shorter middle phalanx and radial curvature of the 5th fingers of hands (arrows). (C)(F) Intra-oral view and pantomography of the patient. Bilateral small maxillary lateral incisors (microdontia, asterisks), and defect of bilateral mandibular central incisors and right mandibular lateral incisor (arrows) were observed in III:1. Remnant of mandibular deciduous central incisor, and defect of bilateral mandibular central incisors (agenesis, arrows) were observed in III:2. No microdontia was observed. All phonographs are reproduced with the written permission of the patient or the guardian.

23

Online Fig. 5

Figure 5. Efficacy of gap junction plaque formation and the voltage-dependency of WT- and WT/R75H-Cx45. (A) Fluorescence-positive and gap junction plaque-positive cell pairs were counted in 5 different views for each group. The efficacy of gap junction plaque formation, the ratio of cell pairs with gap junction plaques to the number of fluorescence-positive cell pairs, was not significantly different among WT, heteromeric channel (WT/R75), and homomeric mutant channel (R75H). (WT: 64.3 ± 6.7%, n=171; WT/R75H: 59.4 ± 11.0%, n=165; R75H: 60.4 ± 8.4%, n=150; NS.) (B) Steady-state Gj/Vj relationships of gap junction conductance were determined by plotting normalized steady-state conductance with respect to peak conductance (Gjss/Gjpeak) versus Vj during the long voltage pulses. The normalized junctional conductance (Gj) values were fitted with Boltzmann equation: Gj= (Gjmax-Gjmin)/(1+exp(A(Vj-V0))) + Gjmin where Gj is macroscopic junctional conductance, Gjmax is theoretical maximum conductance, V0 is voltage at which voltage-sensitive conductance is reduced by 50%; A is the slope factor. Parameters of gap junction properties were comparable between WT/R75H heteromeric channels (n=3) and WT (n=1); Parameters of WT/R75H and WT on negative side were; V0: -34.1 mV, -36.5 mV; Gjmin: 0.11, 0.07; A: 0.11 mV-1, 0.18 mV-1, respectively. Parameters of WT/R75H and WT on positive side were; V0: +34.0 mV, +33.8 mV; Gjmin : 0.07, 0.04; A: 0.11 mV-1, 0.10 mV-1, respectively.

24

Figure 6. Histological evaluation of nodal areas of control and Gjc1-CKO mouse Masson-Trichrome staining showing fibrotic changes (blue) in the SA node (A, C) and AV node (B, D) areas in a Gjc1-CKO mouse (22-week-old; A, B) and a control mouse (24-week-old; C, D). Boxed area in each inset is magnified as A-D. No obvious differences of fibrosis levels at the nodal areas between Gjc1-CKO and control.

25

V. REFERENCES

1. Variant Effect Predictor on Ensembl. http://grch37.ensembl.org/Homo_sapiens/Tools/VEP. 2. 1000 Genomes project database (phase 3, 2504 individuals).

ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/release/20130502. 3. Exome Aggregation Consortium database (ExAC; release v0.3, 60,706 unrelated individuals including

more than 33,300 non-Finnish European individuals). ftp://ftp.broadinstitute.org/pub/ExAC_release. 4. FREX database (573 exomes from French unrelated individuals). www.france-

genomique.org/spip/spip.php?article158. 5. Genome Aggregation Database (gnomAD; 126,216 exome and 15,136 whole-genome sequences)

http://gnomad.broadinstitute.org. 6. Lindenbaum P, Scouarnec SL, Portero V, Redon R. Knime4Bio: a set of custom nodes for the

interpretation of next-generation sequencing data with KNIME. Bioinformatics 2011;27:3200-3201. 7. SIFT. http://sift.jcvi.org/www/SIFT_enst_submit.html. 8. PolyPhen-2. http://genetics.bwh.harvard.edu/pph2/. 9. Human Genetic Variation Database (HGVD; Japanese 1,200 exomes) http://www.genome.med.kyoto-

u.ac.jp/SnpDB/. 10. Integrative Japanese Genome Database (ToMMo-2KJPN; whole-genome sequence database of 2,049

healthy Japanese). https://ijgvd.megabank.tohoku.ac.jp/. 11. Ash MM, Nelson SL. Introduction to dental anatomy. 8th ed. Philadelphia: Saunders, 2003. 12. Fujita T, Kirino, T., Yamashita, Y. Upper lateral incisor (second incisors). 22nd ed. Tokyo: Kanahara,

2012. 13. Duminache M, Gabison K, Atrtchine A, Chabre C. Trois analyses céphalométriques usuelles. EMC-

Odontologie/Orthopédie Dentofaciale 2013;8:1-14. 14. Ursi WJ, Trotman CA, McNamara JA, Jr., Behrents RG. Sexual dimorphism in normal craniofacial

growth. Angle Orthod 1993;63:47-56. 15. Lux CJ, Conradt C, Burden D, Komposch G. Transverse development of the craniofacial skeleton and

dentition between 7 and 15 years of age--a longitudinal postero-anterior cephalometric study. Eur J Orthod 2004;26:31-42.

16. Ricketts RM, Bench RW, Gugino CF, Hilgers JJ, Schulhof RJ. Bioprogressive therapy. Denver: Rocky Mountain Orthodontics, 1979:59.

17. Flatt AE. The troubles with pinkies. Proc (Bayl Univ Med Cent) 2005;18:341-4. 18. Temtamy SA, Aglan MS. Brachydactyly. Orphanet J Rare Dis 2008;3:15. 19. Welch JP, Temtamy SA. Hereditary contractures of the fingers (camptodactyly). J Med Genet

1966;3:104-13. 20. Hamilton KL, Netscher DT. Multidigit camptodactyly of the hands and feet: A case study. Hand (N Y)

2013;8:324-9. 21. Chong AK. Common congenital hand conditions. Singapore Med J 2010;51:965-71. 22. Piazza V, Beltramello M, Menniti M et al. Functional analysis of R75Q mutation in the gene coding for

Connexin 26 identified in a family with nonsyndromic hearing loss. Clin Genet 2005;68:161-6. 23. Schneider CA, Rasband WS, Eliceiri KW. NIH Image to ImageJ: 25 years of image analysis. Nat

Methods 2012;9:671-5. 24. Seki A, Coombs W, Taffet SM, Delmar M. Loss of electrical communication, but not plaque formation,

after mutations in the cytoplasmic loop of connexin43. Heart Rhythm 2004;1:227-33. 25. Makita N, Seki A, Sumitomo N et al. A connexin40 mutation associated with a malignant variant of

progressive familial heart block type I. Circ Arrhythm Electrophysiol 2012;5:163-72. 26. Nishii K, Seki A, Kumai M et al. Connexin45 contributes to global cardiovascular development by

establishing myocardial impulse propagation. Mech Dev 2016;140:41-52. 27. Sohal DS, Nghiem M, Crackower MA et al. Temporally regulated and tissue-specific gene

manipulations in the adult and embryonic heart using a tamoxifen-inducible Cre protein. Circ Res 2001;89:20-5.

28. Muzumdar MD, Tasic B, Miyamichi K, Li L, Luo L. A global double-fluorescent Cre reporter mouse. Genesis 2007;45:593-605.

26

29. Nishii K, Tsuzuki T, Kumai M et al. Abnormalities of developmental cell death in Dad1-deficient mice. Genes Cells 1999;4:243-52.

30. Hagendorff A, Schumacher B, Kirchhoff S, Luderitz B, Willecke K. Conduction disturbances and increased atrial vulnerability in Connexin40-deficient mice analyzed by transesophageal stimulation. Circulation 1999;99:1508-15.

31. Appleton GO, Li Y, Taffet GE et al. Determinants of cardiac electrophysiological properties in mice. J Interv Card Electrophysiol 2004;11:5-14.

32. Narula OS, Shantha N, Vasquez M, Towne WD, Linhart JW. A new method for measurement of sinoatrial conduction time. Circulation 1978;58:706-14.

33. Lavergne J. Dimensions mésio-distales et vestibulo-linguales des dents humaines permanentes. Bulletins et Mémoires de la Société d'anthropologie de Paris, XIII° Série, 1974:351-355.

34. de Groot NM, Schalij MJ, Zeppenfeld K, Blom NA, Van der Velde ET, Van der Wall EE. Voltage and activation mapping: how the recording technique affects the outcome of catheter ablation procedures in patients with congenital heart disease. Circulation 2003;108:2099-106.

35. Nakagawa H, Shah N, Matsudaira K et al. Characterization of reentrant circuit in macroreentrant right atrial tachycardia after surgical repair of congenital heart disease: isolated channels between scars allow "focal" ablation. Circulation 2001;103:699-709.

36. Bell J. Brachydactyly and symphalangism. London: Cambridge University Press, 1951. 37. Maeda S, Nakagawa S, Suga M et al. Structure of the connexin 26 gap junction channel at 3.5 A

resolution. Nature 2009;458:597-602.