SUPPLEMENTAL MATERIAL GENOTYPING WITH MULTIPLEXING TARGETED RESEQUENCING All of the patients and control subjects were sequenced and genotyped in the same way. Shotgun libraries of approximately 250 bp were prepared from genomic DNA of peripheral blood mononuclear cells with Illumina s Genomic DNA Sample Prep Kit (Illumina Inc, CA, USA). Each sample was barcoded at this step by using short index adaptors, instead of using the universal adaptors provided with the commercial kit. After fraction selection of 200-300 bp fragments with AMPureXP beads (Agencourt, Beckman Coulter, CA, USA) and PCR amplification for 8 cycles, the PCR products were purified and quantified. Every 12 individual genomic libraries with different barcode tags were pooled in equal amounts. All coding exonic regions plus their adjacent 5 bp intronic regions of 9 reported ARVC-related genes were enriched by using a custom designed in-solution probe library (Agilent Technologies, Santa Clara, CA, USA). After hybridization, the captured genomic DNA fragment was eluted and amplified for 10 cycles by using extending primers to make the final PCR products suitable for pair-end sequencing. The products from two captures were then subjected to a single lane of Illumina GAIIx to generate paired-end sequencing reads of 120 bp at each end. Illumina reads were sorted by and trimmed off the 6 bp barcoding sequences in the index adaptors and PCR duplications were removed by using PICARD. Sequence alignment and variant calling were performed with CLC Genomics Workbench (CLC-bio, Aarhus, Denmark). The sequence data of each individual were mapped to the human genome (GRCh37/hg19). The coverage of 9 ARVC-causing genes was analyzed and variants in these
genes were called using the following filter parameters: coverage 15 and variant frequency of at least 20%. All the variants, either affecting the obligatory splice sites or changing the amino acid sequence (missense, nonsense and indels), were considered potential ARVC-causing mutations and subjected to further analysis. In this study, the mean sequencing depth was above 250x, with above 99.6% of the targeted region in the 9 ARVC related genes covered and above 98.5% of the targeted regions sequenced 15 in all of the study patients and healthy controls.
Table S1. The Information of Primers Used in the Study. Gene Exon Name* Sequence Product Size (bp) Annealing Temperature ( ) PKP2 Exon2 PKP2-E2F 5' GTT CTT GGC CTT CAT TAC TTA 3' 525 56.4 (NM_004572) PKP2-E2R 5' TTA CCC AAC ATG TCC GTT TCT 3' Exon3 PKP2-E3F 5' ACT GCC ACT TAT GAA GGT CGC 3' 737 61 PKP2-E3R 5' CAG AAT GTG CTG GCA ATG ACT 3' Exon4 PKP2-E4F 5' TGC TTC CCA GTA TTC GCT GAG 3' 374 61 PKP2-E4R 5' GCT GGG AAT ATA GGC GTG AAC 3' Exon5 PKP2-E5F 5' GAC AGA TAC TGC CTT ATA GCC 3' 508 61 PKP2-E5R 5' CTC TAG CAT AAC AAT GAG CCC 3' Exon7 PKP2-E7F 5' GGC CTT TAT TTG TAT ATC TGA 3' 467 52.5 PKP2-E7R 5' AGG GCA GGG TAC AGG TAG CAT 3' Exon8 PKP2-E8F 5' ACC TGG AAG AAG CAT CAA GTA 3' 482 52.5
PKP2-E8R 5' TTG TAT TTT TAG TAG AGA CGG 3' Exon9 PKP2-E9F 5' TAA GAG CTA AAG CCT GTG TCC 3' 298 56.4 PKP2-E9R 5' CTC TCA TTC TCT CCC TTT CTC AT 3' Exon10 PKP2-E10F 5' TAT TTC TGG TCT CCT GGT TTG 3' 544 61 PKP2-E10R 5' GAG TTT TCA GTG CCA TAA GCC 3' Exon11 PKP2-E11F 5' AAA ACT TAA TGT TAT CGT GAA ATC 3' 310 52.5 PKP2-E11R 5' CAT CTT TGT GAA CGG GAG GTG 3' Exon12 PKP2-E12F 5' ACA GAG CAA GAT TCC GTC TCA 3' 449 61 PKP2-E12R 5' ATC TGG CCT GGC TTT ACA TCC 3' Exon13&14 PKP2-E13&14F 5' TAG CAG TTG AGG AGC GAA GAG 3' 658 56.4 PKP2-E13&14R 5' GTA CCA TAA GCC CTA ATA ACA 3' DSP Exon5 DSP-E5F 5' AAG CCG TGA TGG TGT GCG TAA 3' 389 52.5 (NM_004415) DSP-E5R 5' TAA TCT CTA ATA GGG CTA CTG 3' Exon23B DSP-E23BF 5' CAC TGA GCA GCG AAG GCG AGC 3' 590 56.4
DSP-E23BR 5' CTG TTC GCA TCT GAG TGA CTT 3' Exon24 DSP-E24F1 5' ACC CAA AGG AGA GCC ATC GTT 3' 636 56.4 DSP-E24R1 5' TTA CTG ATT CAT GTC GGG AGC 3' Exon24 DSP-E24F2 5' TCC ACC ATA TCC AGC GTC AGG 3' 476 56.4 DSP-E24R2 5' CGG ATG GCT TCT TCG GTG CTT 3' DSG2 Exon3 DSG2-E3F 5' GGC CCT ATG CAG TTT GCT AGA 3' 588 56.4 (NM_001943) DSG2-E3R 5' CAG TGC CAA ATC CCT TTA CTC 3' Exon6 DSG2-E6F 5' TAT CCC ATT CAC GCT TAT GTC 3' 482 52.5 DSG2-E6R 5' TCC CTA AAA GAG AGT AAT TCC 3' Exon8 DSG2-E8F 5' ATG GCA ATG GAG AAG TTA CAG 3' 563 56.4 DSG2-E8R 5' ACA GAA CTT GGG CAA ATA GGG 3' Exon10 DSG2-E10F 5' AGC TGT AGA TGT TAG AGG TTT 3' 507 61 DSG2-E10R 5' ATT CTC CTG CCT CAG TCT CTT 3' Exon11 DSG2-E11F 5' TAA GAT GAA ATA AGC ACC TAA 3' 668 52.5
DSG2-E11R 5' ATA CCC AGC TAC CTT CAA GAC 3' Exon15 DSG2-E15F 5' AGC CAA CTA TCA CAC TAT CTT 3' 838 56.4 DSG2-E15R 5' GTG GTT GGT GGC ATA GTG GAC 3' DSC2 Exon3 DSC2-E3F 5' TCC CCA CGT GCA TAC ATT ACT 3' 383 56.4 (NM_024422) DSC2-E3R 5' AAG CCA AAC TAT ACC ATA TCC 3' Exon5 DSC2-E5F 5' TTT ACT CAT GCC TTG CTC TTG 3' 443 51.2 DSC2-E5R 5' ACT CAG AAA GCA GAA AAG GTT 3' JUP Exon8 JUP-E8F 5' CAG GGT GTG GCG GGT CAT AGG 3' 576 61 (NM_002230) JUP-E8R 5' CTT CTT TGC TAC AGC GGC TTT 3' Exon13 JUP-E13F 5' CTC AAG TGA TCC GCC TGC TGT 3' 415 61 JUP-E13R 5' GCT GCC TCC TCT ACC CAT GCC 3' TMEM43 Exon4 TMEM43-E4F 5' TTC CTC TTC CAG TCC AGC TTC C 3' 261 56.4 (NM_024334) TMEM43-E4R 5' TCT GCA TGT TAT CCT GAC ACC T 3' Exon12 TMEM43-E12F 5' CCA ACC ACG CAG CTC ATA GCA 3' 441 56.4
TMEM43-E12R 5' TAG GGC TCA CGC AGG TGT CGG 3' TGFβ3 Exon1 TGFB3-E1F 5' CTG CTG AAC TTT GCC ACG GTC 3' 463 61 (NM_003239) TGFB3-E1R 5' CAA CGG AGG AGC ATC GCA CAT 3' *F indicates upper primer; R, reverse primer. The amplicon of primer pair of DSP-E24F1 and DSP-E24R1 covers partial 24 exon of DSP gene from chr.6:7,584,631 to chr.6:7,585,266 (human genome GRCh37/hg19). The amplicon of primer pair of DSP-E24F2 and DSP-E24R2 covers partial 24 exon of DSP gene from chr.6:7,585,296 to chr.6:7,585,729 (human genome GRCh37/hg19). Table S2. Common Polymorphisms and Neutral Rare Variants Excluded in the Study. Gene Exon/Intron cdna Change Protein Change Patient (180 chromosomes) Control (600 chromosomes) PolyPhen* SIFT PKP2 (NM_004572) Exon4 c.1097t>c p.leu366pro 3 22
DSP Exon10 c.1197c>g p.ile399met 3 0 -, (0.13) -, (0.10) (NM_004415) Exon12 c.1481a>t p.tyr494phe 14 42 Exon23B c.3601g>a p.glu1201lys 1 0 -, (0.276) -, (0.36) Exon23B c.3923g>a p.arg1308gln 2 3 Exon23B c.4528g>a p.val1510ile 1 1 Exon23B c.4535a>g p.tyr1512cys 57 158 Exon23B c.4943a>g p.gln1648arg 4 2 Exon23B c.5213g>a p.arg1738gln 35 87 Exon24 c.6799a>t p.thr2267ser 2 3 Exon24 c.7916g>a p.arg2639gln 4 8 Exon24 c.8455a>c p.met2819leu 2 3 Exon24 c.8605a>g p.ile2869val 2 17 DSG2 Exon14 c.2036g>t p.gly679val 1 0 -, (0) -, (0.28) (NM_001943) Exon14 c.2318g>a p.arg773lys 80 203
Exon15 c.2568a>c p.lys856asn 2 37 Exon15 c.3209c>t p.thr1070met 1 8 JUP Exon4 c.702g>t p.met234ile 1 1 (NM_002230) Exon10 c.1714c>t p.arg572trp 1 1 Exon14B c.2089a>t p.met697leu 52 147 TMEM43 Exon6 c.504a>t p.lys168asn 68 161 (NM_024334) Exon7 c.536t>c p.met179thr 115 259 Exon12 c.1111t>c p.tyr371his 1 3 TGFB3 (NM_003239) DES (NM_001927) Exon1 c.188c>a p.thr63asn 2 0 -, (0.009) -, (0.18) Exon7 c.1265c>a p.thr422asn 1 0 -, (0.004) -, (0.48) *PolyPhen prediction: -, benign. SIFT prediction: -, tolerated.
Table S3. Pathogenic Mutations Identified in the ARVC Subjects. Gene Exon/ Intron cdna Change Protein Change Type Comments PolyPhen* SIFT Patients, n PKP2 Intron1 c.224-3c>g splice Splice Novel 1 (NM_004572) Exon2 c.235c>t p.arg79* Nonsense Known 1 Exon2 c.253_256delgagt p.glu85fs Frameshift Known 1 Intron2 c.336+1g>a splice Splice Novel 1 Exon3 c.427c>t p.his143tyr Missense Novel - + 1 Exon3 c.456delt p.pro152fs Frameshift Novel 1 Exon3 c.498c>g p.tyr166* Nonsense Novel 1 Exon3 c.508c>t p.gln170* Nonsense Novel 1 Exon3 c.568delg p.val190fs Frameshift Novel 1 Exon3 c.729c>g p.tyr243* Nonsense Novel 1
Exon3 c.746g>c p.ser249thr Missense Novel ++ + 1 Exon3 c.801delt p.thr267fs Frameshift Novel 1 Exon3 c.870g>a p.trp290* Nonsense Novel 2 Exon3 c.895c>t p.arg299cys Missense Novel ++ + 1 Exon4 c.1063c>t p.arg355* Nonsense Novel 1 Intron4 c.1170+1g>a splice Splice Known 1 Intron4 c.1171-2a>g splice Splice Known 1 Exon5 c.1375dela p.thr459fs Frameshift Novel 1 Exon7 c.1597dela p.ile533fs Frameshift Novel 1 Exon7 c.1669_1671delaac p.asn513del Deletion Novel 1 Exon8 c.1749t>g p.ile583met Missense Novel ++ + 1 Exon9 c.1951c>t p.arg651* Nonsense Known 1 Exon10 c.1978c>t p.gln660* Nonsense Known 3 Exon10 c.1994c>g p.pro665arg Missense Novel ++ - 1
Exon11 c.2146-1_2146delga p.met716fs Frameshift Known 1 Exon11 c.2179a>t p.lys727* Nonsense Novel 1 Exon11 c.2203c>t p.arg735* Nonsense Known 3 Exon11 c.2240dela p.lys747fs Frameshift Novel 1 Exon12 c.2421c>a p.tyr807* Nonsense Known 2 Intron12 c.2489+1g>a splice Splice Known 4 Exon13 c.2554delg p.glu852fs Frameshift Known 3 DSP Exon5 c.621g>t p.trp207cys Missense Novel ++ + 1 (NM_004415) Exon23B c.3862a>c p.lys1288gln Missense Novel ++ - 1 Exon23B c.4043t>g p.leu1348arg Missense Novel ++ - 1 Exon23B c.4198c>t p.arg1400* Nonsense Novel 1 Exon24 c.7623g>t p.arg2541ser Missense Novel - + 1 Exon24 c.8066a>c p.lys2689thr Missense Novel ++ - 1 DSG2 Exon3 c.105t>a p.asn35lys Missense Novel - + 2
(NM_001943) Exon3 c.146g>a p.arg49his Missense Known ++ + 3 Exon6 c.593a>g p.tyr198cys Missense Novel ++ + 2 Exon6 c.685a>g p.arg229gly Missense Novel ++ + 1 Exon8 c.877a>g p.ile293val Missense Known ++ - 1 Exon10 c.1334c>t p.ser445phe Missense Novel ++ + 1 Exon11 c.1592t>g p.phe531cys Missense Known ++ + 5 Exon15 c.2470c>t p.arg824cys Missense Novel - + 2 DSC2 Exon3 c.229g>a p.gly77ser Missense Novel ++ + 1 (NM_024422) Exon3 c.287t>c p.ile96thr Missense Novel ++ + 1 Exon5 c.565g>c p.glu189gln Missense Novel ++ - 1 JUP Exon8 c.1280c>t p.thr427met Missense Known + - 1 (NM_002230) Exon13 c.2078a>g p.tyr693cys Missense Novel ++ - 1 TMEM43 Exon4 c.332c>t p.pro111leu Missense Novel + - 1 (NM_024334) Exon12 c.1073c>t p.ser358leu Missense Known ++ - 1
TGFβ3 (NM_003239) Exon1 c.113_115delaga p.lys38_arg39delinsarg Deletion Novel 1 *PolyPhen prediction: ++, probably damaging; +, possibly damaging; -, benign. SIFT prediction: +, not tolerated; -, tolerated.