SUPPLEMENTAL MATERIAL GENOTYPING WITH MULTIPLEXING TARGETED RESEQUENCING
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1 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 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
2 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.
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4 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' (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' PKP2-E3R 5' CAG AAT GTG CTG GCA ATG ACT 3' Exon4 PKP2-E4F 5' TGC TTC CCA GTA TTC GCT GAG 3' PKP2-E4R 5' GCT GGG AAT ATA GGC GTG AAC 3' Exon5 PKP2-E5F 5' GAC AGA TAC TGC CTT ATA GCC 3' PKP2-E5R 5' CTC TAG CAT AAC AAT GAG CCC 3' Exon7 PKP2-E7F 5' GGC CTT TAT TTG TAT ATC TGA 3' PKP2-E7R 5' AGG GCA GGG TAC AGG TAG CAT 3' Exon8 PKP2-E8F 5' ACC TGG AAG AAG CAT CAA GTA 3'
5 PKP2-E8R 5' TTG TAT TTT TAG TAG AGA CGG 3' Exon9 PKP2-E9F 5' TAA GAG CTA AAG CCT GTG TCC 3' PKP2-E9R 5' CTC TCA TTC TCT CCC TTT CTC AT 3' Exon10 PKP2-E10F 5' TAT TTC TGG TCT CCT GGT TTG 3' PKP2-E10R 5' GAG TTT TCA GTG CCA TAA GCC 3' Exon11 PKP2-E11F 5' AAA ACT TAA TGT TAT CGT GAA ATC 3' PKP2-E11R 5' CAT CTT TGT GAA CGG GAG GTG 3' Exon12 PKP2-E12F 5' ACA GAG CAA GAT TCC GTC TCA 3' 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' 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' (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'
6 DSP-E23BR 5' CTG TTC GCA TCT GAG TGA CTT 3' Exon24 DSP-E24F1 5' ACC CAA AGG AGA GCC ATC GTT 3' DSP-E24R1 5' TTA CTG ATT CAT GTC GGG AGC 3' Exon24 DSP-E24F2 5' TCC ACC ATA TCC AGC GTC AGG 3' DSP-E24R2 5' CGG ATG GCT TCT TCG GTG CTT 3' DSG2 Exon3 DSG2-E3F 5' GGC CCT ATG CAG TTT GCT AGA 3' (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' DSG2-E6R 5' TCC CTA AAA GAG AGT AAT TCC 3' Exon8 DSG2-E8F 5' ATG GCA ATG GAG AAG TTA CAG 3' DSG2-E8R 5' ACA GAA CTT GGG CAA ATA GGG 3' Exon10 DSG2-E10F 5' AGC TGT AGA TGT TAG AGG TTT 3' DSG2-E10R 5' ATT CTC CTG CCT CAG TCT CTT 3' Exon11 DSG2-E11F 5' TAA GAT GAA ATA AGC ACC TAA 3'
7 DSG2-E11R 5' ATA CCC AGC TAC CTT CAA GAC 3' Exon15 DSG2-E15F 5' AGC CAA CTA TCA CAC TAT CTT 3' DSG2-E15R 5' GTG GTT GGT GGC ATA GTG GAC 3' DSC2 Exon3 DSC2-E3F 5' TCC CCA CGT GCA TAC ATT ACT 3' (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' DSC2-E5R 5' ACT CAG AAA GCA GAA AAG GTT 3' JUP Exon8 JUP-E8F 5' CAG GGT GTG GCG GGT CAT AGG 3' (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' 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' (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'
8 TMEM43-E12R 5' TAG GGC TCA CGC AGG TGT CGG 3' TGFβ3 Exon1 TGFB3-E1F 5' CTG CTG AAC TTT GCC ACG GTC 3' (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
9 DSP Exon10 c.1197c>g p.ile399met 3 0 -, (0.13) -, (0.10) (NM_004415) Exon12 c.1481a>t p.tyr494phe 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 Exon23B c.4943a>g p.gln1648arg 4 2 Exon23B c.5213g>a p.arg1738gln 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
10 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 TMEM43 Exon6 c.504a>t p.lys168asn (NM_024334) Exon7 c.536t>c p.met179thr 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.
11 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 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
12 Exon3 c.746g>c p.ser249thr Missense Novel 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 Exon4 c.1063c>t p.arg355* Nonsense Novel 1 Intron4 c g>a splice Splice Known 1 Intron4 c a>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 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
13 Exon11 c _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 g>a splice Splice Known 4 Exon13 c.2554delg p.glu852fs Frameshift Known 3 DSP Exon5 c.621g>t p.trp207cys Missense Novel (NM_004415) Exon23B c.3862a>c p.lys1288gln Missense Novel Exon23B c.4043t>g p.leu1348arg Missense Novel Exon23B c.4198c>t p.arg1400* Nonsense Novel 1 Exon24 c.7623g>t p.arg2541ser Missense Novel Exon24 c.8066a>c p.lys2689thr Missense Novel DSG2 Exon3 c.105t>a p.asn35lys Missense Novel - + 2
14 (NM_001943) Exon3 c.146g>a p.arg49his Missense Known Exon6 c.593a>g p.tyr198cys Missense Novel Exon6 c.685a>g p.arg229gly Missense Novel Exon8 c.877a>g p.ile293val Missense Known Exon10 c.1334c>t p.ser445phe Missense Novel Exon11 c.1592t>g p.phe531cys Missense Known Exon15 c.2470c>t p.arg824cys Missense Novel DSC2 Exon3 c.229g>a p.gly77ser Missense Novel (NM_024422) Exon3 c.287t>c p.ile96thr Missense Novel Exon5 c.565g>c p.glu189gln Missense Novel JUP Exon8 c.1280c>t p.thr427met Missense Known (NM_002230) Exon13 c.2078a>g p.tyr693cys Missense Novel TMEM43 Exon4 c.332c>t p.pro111leu Missense Novel (NM_024334) Exon12 c.1073c>t p.ser358leu Missense Known
15 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.
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