Supplemental Figure 1: Nisoldipine Concentration-Response Relationship on ipsc- CMs. Supplemental Figure 2: Exome Sequencing Prioritization Strategy

Transcription

1 Supplementary Appendix Table of Contents Figures: Supplemental Figure 1: Nisoldipine Concentration-Response Relationship on ipsc- CMs Supplemental Figure : Exome Sequencing Prioritization Strategy Supplemental Figure : sirna mediated knockdown of KCNK1 and REM Transcript Supplemental Figure : Effect of REM sirna on APD and ICaL in patient derived ipsc-cm. Supplemental Figure : Immunohistochemistry of canonical cardiac markers in patient derived ipsc-cm Tables: Supplemental Table 1: Cleveland LQT Family Summary Statistics Supplemental Table : Phenotype Details from All herg RW Mutation-Positive Individuals from Cleveland LQT Family Supplemental Table : Comprehensive Action Potential Characteristics from Patient Specific ipsc-cms Supplemental Table : Summary data of action potential and ICaL characteristics from patient specific ipsc-cms - and + Nisoldipine treatment. Supplemental Table : Exome Sequencing Gene Candidates Supplemental Table : Additional Action Potential Characteristics from Effects of KCNK1 sirna on icells Supplemental Table : Summary data of action potential and ICaL characteristics from patient specific ipsc-cms - and + REM sirna. Supplemental Table : Summary data of action potential and ICaL characteristics from IV- patient ipsc-cm - and + CRISPR-Cas9 genome editing. 1

2 Supplemental Figure 1. Concentration-Response Relationship for Nisoldipine on ipsc-cms. Concentration-response relationship for the effect of nisoldipine (Cav1. blocker) on ICaL (L-type Ca + channel) was measured at -10 mv (between - cells, mean±sem). EC0 value yielded 10 nm. Inset: structural schematic of nisoldipine.

3 Supplemental Figure. Exome Sequencing Prioritization Strategy. Four patients were exome sequenced (two in each group: IV-1 and IV- in the mildly affected and III- and IV- in the severely affected). The severely affected herg RW mutation-positive and mildly affected mutation-positive individuals were filtered looking for variants in genes exclusive to each cohort. Total coding variants describes all synonymous, nonsynonymous, and frameshift-inducing insertions or deletions as well as canonical splice site variants within captured exons (but does not include or untranslated regions and introns). From this group we filtered first for only nonsynonymous coding variants (i.e. variants resulting in an amino acid substitution) or for Insertion/Deletion. Next, we filtered for genes specific to cardiac tissue. Then, we filtered for cardiac ion channels or genes that were cross listed in the LQTS Neighborhood() or linked by GWAS to QT interval modulation(1, 19, 1). 1

4 A B 1 Supplemental Figure. sirna knockdown of KCNK1 and REM. Panel A shows qpcr data of icell cardiomyocytes transfected with either scrambled control sirna or KCNK1 sirna revealed ~ fold knockdown of KCNK1 transcript in the sirna group. Panel B shows between ~1.-. fold knockdown of REM transcript in patient derived ipsc-cm lines in the sirna treated group compared to scrambled control. * denotes p < 0.0 with unpaired Student s t-test used to assess significance in Panel A and ANOVA in Panel B

5 A B C D E 1

6 Supplemental Figure. Effect of REM sirna on APD and ICaL in patient derived ipsc-cm. Panels A-E illustrate the effects of REM sirna on both patient ipsc-cm trios (IV-1, III-, IV-1 and IV-1, IV- and IV-). Data is depicted as macroscopic action potential recording, APD summary data, and ICaL comparison between scrambled control and REM sirna. Between -11 cells were analyzed in the effects of REM sirna on APD and - cells for the effects of REM sirna on ICaL in Panels A-E. Please refer to Supplemental Table for numbers of replicate measures (n) and statistical analysis

7 Supplemental Figure. Immunohistochemistry of patient derived ipsc-cms. Immunostaining of ipsc-cm derived from III- reveal canonical markers of cardiomyocyte lineage (alpha actinin and the cardiac isoform of Troponin T) as well as normal sarcomeric orientation. Immunohistochemical assessment reproduces identical results from the mildly affected mutation-positive son and severely affected mutationpositive sister (data not shown). Scale bar is 0 m.

8 Supplemental Tables Supplemental Table 1. Cleveland LQT Family Summary Statistics. 9 10

9 Generation Family Member Number Sex Phenotype III 1 F Severely affected F Mildly affected M Mildly affected M Severely affected (deceased) M Mildly affected IV 1 F Mildly affected M Mildly affected M Mildly affected F Mildly affected M Mildly affected F Severely affected F Mildly affected F Mildly affected 9 M Mildly affected 10 F Mildly affected 1 F Mildly affected 1 M Mildly affected 1 M Mildly affected 1 M Mildly affected 1 F Mildly affected 1 V 1 F Mildly affected M Mildly affected F Mildly affected M Mildly affected M Mildly affected M Mildly affected M Mildly affected Supplemental Table. Phenotype Details from All living herg RW Mutation- Positive Individuals from Cleveland LQT Family. This table shows all mutation- positive family members distributed across three generations involved in the study (and 1 deceased, Gen III, #). Rows shaded in dark grey with bold font are the original family members characterized by ipsc-cm and exome sequencing in the main text. 9

10 The light shaded grey rows indicate additional mutation carriers in the family that were additionally genotyped for REM and KCNK1. Non-shaded rows are mutation carriers that remain uncharacterized. Note: these Generation + Family Member Number identifiers do not correspond to those in Figure 1 because for conciseness Figure 1 is a zoomed-in snapshot of the family pedigree and thus uses its own numbering system. Here in this table we are showing all the herg RW mutation carriers in the family starting from the first individual in each generation. 10

11 ipsc-cm Background n APD 90 (msec) APD 0 (msec) APA (mv) MDP (mv) IV-1 (Control) 1 ± 9 11 ± ± -0 ± 1 III- (Severely affected) IV-1 (Mildly affected) 0 1 ± 11 1 ± 1 10 ± 1 1 ± 9 1 ± 9 10 ± - ± - ± 1 IV-1 (Control) IV- (Severely affected) 1 IV- (Mildly affected) 9 1 ± 1 ± 9 ± ± 1 1 ± 1 10 ± 19 ± 10 ± 10 ± - ± 1 - ± 1 - ± 1 1 Supplemental Table. Comprehensive Action Potential Characteristics from Patient Specific ipsc-cms from Figure. MDP: mean diastolic potential, APD90: action potential duration at 90% of repolarization in milliseconds, APD0: action potential duration at 0% of depolarization in milliseconds, APA: action potential amplitude. 9 11

12 Supplemental Table. Summary data of action potential duration and ICaL characteristics from patient specific ipsc-cms with and without nisoldipine treatment from Figure. ICaL: L- type Calcium Current. * denotes significance with p-value assessed by paired Student s t-test (p < 0.0 considered significant). 1

13 9 10 Supplemental Table. Exome Sequencing Gene Candidates. This table shows the complete list of candidate genes identified by exome sequencing in either the severely affected herg RW mutation-positive or mildly affected herg RW mutationpositive individuals. Applying the prioritization strategy shown in Supplemental Figure we identified in the severely affected group SNPs in five genes and three genes with insertion/deletions. In the mildly affected group, SNPs were identified in seven genes along with one insertion/deletion. SNP: single nucleotide polymorphism, In/Del: insertion/deletion, Chr: chromosome, MAF: minor allele frequency (as reported by 1000 Genomes or ExAC Database), UTR: untranslated region

14 III- (Severely affected Homozygous WT:S1) MDP (mv) APA (mv) Scrambled Control (n = 1) -. ± ±.0 KCNK1 sirna treated (n =1) -. ± ±.9 IV- (Mildly affected Heterozygote S1/G1) MDP (mv) APA (mv) Scrambled Control (n = 1) -. ±.11. ±.1 KCNK1 sirna treated (n = ) -1. ± ±.* 1 Supplemental Table. Additional Action Potential Characteristics from Effects of KCNK1 sirna on patient ipsc-cm (III- and IV-) from Figure. Data shown as mean ± SEM, MDP: mean diastolic potential, APA: action potential amplitude, *pvalue=0.000 as determined by unpaired Student s t-test. 1

15 Supplemental Table. Summary data of action potential and ICaL characteristics from patient specific ipsc-cms - and + REM sirna from Supplemental Figure. ICaL: L-type Calcium Current. * denotes significance as assessed by unpaired Student s t-test (p < 0.0 considered significant). 1

16 Supplemental Table. Summary data of action potential and ICaL characteristics from IV- patient ipsc-cm - and + CRISPR-Cas9 genome editing from Figure. CRISPR-Cas9 corrected REM on IV- background, ICaL: L-type Calcium Current. p-value assessed by unpaired Student s t- test (p < 0.0 considered significant). 1