Determining the Pathogenicity of a Genomic Variant

Circulation ORIGINAL RESEARCH ARTICLE

Determining the Pathogenicity of a Genomic Variant of Uncertain Significance Using CRISPR/Cas9 and Human-Induced Pluripotent Stem Cells Downloaded from http://circ.ahajournals.org/ by guest on June 18, 2018

BACKGROUND: The progression toward low-cost and rapid next-generation sequencing has uncovered a multitude of variants of uncertain significance (VUS) in both patients and asymptomatic “healthy” individuals. A VUS is a rare or novel variant for which disease pathogenicity has not been conclusively demonstrated or excluded, and thus cannot be definitively annotated. VUS, therefore, pose critical clinical interpretation and risk-assessment challenges, and new methods are urgently needed to better characterize their pathogenicity. METHODS: To address this challenge and showcase the uncertainty surrounding genomic variant interpretation, we recruited a “healthy” asymptomatic individual, lacking cardiac-disease clinical history, carrying a hypertrophic cardiomyopathy (HCM)-associated genetic variant (NM_000258.2:c.170C>A, NP_000249.1:p.Ala57Asp) in the sarcomeric gene MYL3, reported by the ClinVar database to be “likely pathogenic.” Humaninduced pluripotent stem cells (iPSCs) were derived from the heterozygous VUSMYL3(170C>A) carrier, and their genome was edited using CRISPR/Cas9 to generate 4 isogenic iPSC lines: (1) corrected “healthy” control; (2) homozygous VUSMYL3(170C>A); (3) heterozygous frameshift mutation MYL3(170C>A/fs); and (4) known heterozygous MYL3 pathogenic mutation (NM_000258.2:c.170C>G), at the same nucleotide position as VUSMYL3(170C>A), lines. Extensive assays including measurements of gene expression, sarcomere structure, cell size, contractility, action potentials, and calcium handling were performed on the isogenic iPSC-derived cardiomyocytes (iPSC-CMs). RESULTS: The heterozygous VUSMYL3(170C>A)-iPSC-CMs did not show an HCM phenotype at the gene expression, morphology, or functional levels. Furthermore, genome-edited homozygous VUSMYL3(170C>A)- and frameshift mutation MYL3(170C>A/fs)-iPSC-CMs lines were also asymptomatic, supporting a benign assessment for this particular MYL3 variant. Further assessment of the pathogenic nature of a genome-edited isogenic line carrying a known pathogenic MYL3 mutation, MYL3(170C>G), and a carrier-specific iPSC-CMs line, carrying a MYBPC3(961G>A) HCM variant, demonstrated the ability of this combined platform to provide both pathogenic and benign assessments. CONCLUSIONS: Our study illustrates the ability of clustered regularly interspaced short palindromic repeats/Cas9 genome-editing of carrier-specific iPSCs to elucidate both benign and pathogenic HCM functional phenotypes in a carrierspecific manner in a dish. As such, this platform represents a promising VUS riskassessment tool that can be used for assessing HCM-associated VUS specifically, and VUS in general, and thus significantly contribute to the arsenal of precision medicine tools available in this emerging field. Circulation. 2018;138:00–00. DOI: 10.1161/CIRCULATIONAHA.117.032273

Ning Ma, PhD* Joe Z. Zhang, MD, PhD* Ilanit Itzhaki, PhD* Sophia L. Zhang, BS Haodong Chen, PhD Francois Haddad, MD Tomoya Kitani, MD, PhD Kitchener D. Wilson, MD, PhD Lei Tian, PhD Rajani Shrestha, MS Haodi Wu, PhD Chi Keung Lam, PhD Nazish Sayed, MD, PhD Joseph C. Wu, MD, PhD

*Drs Ma, Zhang, and Itzhaki contributed equally. Key Words: cardiomyopathy, hypertrophic ◼ clustered regularly interspaced short palindromic repeats ◼ CRISPR-Cas systems ◼ mutations ◼ gene editing ◼ induced pluripotent stem cells Sources of Funding, see page XXX © 2018 American Heart Association, Inc. http://circ.ahajournals.org

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Determining VUS Using CRISPR/Cas9 and iPSC

ORIGINAL RESEARCH ARTICLE

Clinical Perspective What Is New? • Our study demonstrates for the first time the unique potential of combining iPSC-based disease modeling and CRISPR/Cas9-mediated genome editing technology as a personalized risk-assessment platform for determining the pathogenicity of a variant of uncertain significance for hypertrophic cardiomyopathy in a patient-specific manner.

What Are the Clinical Implications?

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• This personalized risk-assessment platform will help improve the study and diagnostics of a variant of uncertain significance specifically and promote implementation of precision medicine in general, better guiding clinicians in their choice of therapy and providing a clearer result for variant of uncertain significance carriers.

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ontinued advancement in the field of medical genetics toward low-cost, and rapid next-generation sequencing, has enabled increased genetic screening for disease-causing genetic mutations, not only in patients but also in “healthy” individuals. Because of this, genetic testing has uncovered a multitude of novel unclassified genetic variants, also known as variant(s) of uncertain significance (VUS).1,2 A VUS is a genetic change for which pathogenicity has not been confirmed.3 Consequently, clinical genetic testing often reports VUS with ambiguous and uncertain pathogenicity. This can cause anxiety and stress in otherwise asymptomatic individuals and families who are found to carry a VUS, especially when a poorly characterized variant is erroneously reported as “likely pathogenic” or even “pathogenic.”4 VUS therefore pose serious clinical interpretation and risk-assessment challenges that limit the ability of healthcare providers to effectively counsel and treat individuals with unclear genetic predisposition to disease. Hypertrophic cardiomyopathy (HCM), a common cause of sudden cardiac death (SCD), is a heterogeneous disease with vast genetic variability in the human genome, including a high degree of novel (“private”) mutations.5 The findings of numerous VUS associated with HCM in “healthy” populations have created ambiguity in genetic test reports, posing a growing and unresolved challenge for cardiovascular precision medicine.6 In silico tools, such as PolyPhen2,7 AlignGVGD,8 and SIFT,9 have been developed to enable the prediction of the pathogenic likelihood of gene mutations by attempting to calculate the possible impact of an amino acid substitution on protein damage. However, these are only in silico predictions, and a variant classified as 2

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“likely pathogenic” or even “pathogenic” may actually be benign.10 Hence, it is imperative to develop new experimental systems, ideally of human origin, that can help assess the functional pathogenic potential of a VUS in a patient-specific manner. In the case of heart disease, recent advances in induced pluripotent stem cell (iPSC) biology are generating groundbreaking opportunities for the study of genetic cardiomyopathy and familial arrhythmogenic syndromes, such as long QT,11 catecholaminergic polymorphic ventricular tachycardia,12 Brugada syndrome,13 HCM,14 and left ventricular noncompaction.15 Cardiomyocytes derived from iPSCs (iPSC-CMs) enable recapitulation of abnormal pathogenic cardiac phenotypes in a diseaseand patient-specific manner.16 Further strengthening this “disease in a dish” platform, the field of genome editing now allows interrogation of genetic mutations via genotype-phenotype correlation in a patient-specific manner using iPSCs.17–20 With the aid of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 and single-stranded oligodeoxynucleotide–mediated genome editing technologies, a genetic mutation can be readily introduced or corrected in iPSCs to create isogenic patient-specific iPSC lines in a dish.21,22 The generated isogenic iPSC lines share the same genetic background and differ exclusively at the mutation site. This advantage eliminates confounding factors due to different genetic backgrounds, allowing the genotype-phenotype relationship to be deciphered in a more precise and measurable manner. Taken together, the powerful combination of iPSC-based disease modeling and tools such as CRISPR/Cas9-mediated genome editing present an unprecedented opportunity to study VUS functional phenotypes in a patient-specific manner. To highlight the potential application of CRISPR/ Cas9 and iPSC disease models to clinical genetic testing, we have chosen to study a VUS with contradictory genotype-phenotype reports. The VUS is a missense variant (NM_000258.2:c.170C>A, NP_000249.1:p. Ala57Asp) in MYL3, a protein-coding gene that is a prominent component of the sarcomere complex.23 The VUS was discovered in an asymptomatic carrier with neither clinical nor family history (3 generations) associated with suspicion of cardiac disease and sudden cardiac death (SCD). Despite the asymptomatic nature of the individual, this VUS is surprisingly predicted with confidence, using in silico tools, to be pathogenically damaging to protein function. Moreover, conflicting interpretations of pathogenicity are reported in the ClinVar database.24 Some genetic testing resources classify this variant as a VUS, while others classify it as a likely pathogenic mutation having the potential to cause HCM. In this study, we use a combined CRISPR/ Cas9–iPSC approach to evaluate the pathogenicity of this HCM-associated VUS. These approaches will be critical in the future to mitigate patient and family anxiCirculation. 2018;138:00–00. DOI: 10.1161/CIRCULATIONAHA.117.032273

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METHODS Detailed methods are available in the Data Supplement. The data, analytic methods, and study materials for the purposes of reproducing the results or replicating procedures can be made on request to the corresponding author who manages the information.

Genomic Variant Detection in Heart Disease–Related Genes


This study was carried out under the Stanford Institutional Review Board (Palo Alto, California). Fifty-four healthy subjects without cardiac disease clinical history, and presenting with normal ECG and echocardiography findings, were recruited by the Stanford Cardiovascular Institute Biobank. Genetic testing of these subjects for this study was approved by Stanford University Institutional Review Board. Genomic DNA was isolated using DNeasy Blood and Tissue kits (Qiagen). Ion Torrent libraries from the 54 individual DNA samples were prepared with the Ion AmpliSeqKit for Chef DL8 (Thermo Fisher Scientific) using a custom AmpliSeq panel (Thermo Fisher Scientific), that covers 135 cardiomyopathy and congenital heart disease DNA panel genes associated with sudden cardiac death.25 Next, the libraries were sequenced on an Ion PIChip (Thermo Fisher Scientific) with the Ion Proton system using the Ion PI™ Hi-QSequencing 200 Kit (Thermo Fisher Scientific). Sequence reads were aligned to human genome hg19 using Ion Torrent Suite Software (v5.0) and genomic variants were detected by Torrent Variant Caller plugin. Variant call format files of all samples were then annotated by Ion Reporter (ionreporter.thermofisher.com). Annotations of genetic variants were from ClinVar.24

iPSC Generation The study was carried out under Stanford Institutional Review Board and Stem Cell Research Oversight Committee guidelines. Patients’ peripheral blood mononuclear cells were collected and reprogrammed from a MYL3(170C>A) VUS carrier, a MYBPB3(961G>A) variant carrier, and 2 healthy individuals26 using the CytoTuneiPS 2.0 Sendai Reprogramming Kit (Thermo Fisher Scientific).

Genome Editing iPSCs were transfected with CRISPR/Cas9 and single-stranded oligodeoxynucleotide using the Lipofectamine 3000 Reagent (Thermo Fisher Scientific). For each well of a 6-well plate, 1 μg CRISPR/Cas9 vectors and 4 μg single-stranded oligodeoxynucleotide were used for transfection. For each genome edited line (isogenic corrected, isogenic homozygous VUSMYL3(170C>A), frameshift mutation MYL3(170C>A/fs), and isogenic heterozygous MYL3(170C>G)), several clones were generated, 3 of which were continuously propagated and used for cardiomyocytes differentiation and characterization. Circulation. 2018;138:00–00. DOI: 10.1161/CIRCULATIONAHA.117.032273

Statistical Analysis For statistical analysis, Student‘s t test was used to compare the difference between 2 data sets. For comparison among multiple groups, 1-way or 2-way ANOVA was used, where appropriate, and Holm-Sidak or Tukey after tests were used for all pairwise comparisons, depending on the properties of the data sets. A P value A, NP_000249.1:p. Ala57Asp) in the sarcomeric gene MYL3 (Figure 1A, 1B). It is important to note that this variant has not been previously reported in the literature and presents conflicting ClinVar reports, with some classifying the variant as a VUS and others as likely pathogenic (Table 1).24 MYL3 gene encodes MYL3 protein, which is an important modulator of sarcomeric myosin cross-bridge kinetics essential for the modulation of cardiac muscle contraction.23 The A57D variant is a nonconservative amino acid substitution (Figure 1C), which is likely to affect secondary protein structure as these residues differ in polarity, charge, size, and other properties.27 Missense variants in nearby residue (E56G) and at the xxx xxx, 2018

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ety when confronted with unclear genetic predisposition to a disease, allowing more targeted therapies to reduce unnecessary harmful medical interventions and testing while cutting cost.




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Downloaded from http://circ.ahajournals.org/ by guest on June 18, 2018 Figure 1. Confirmation of the likely pathogenic VUSMYL3(170C>A) uncovered in an asymptomatic carrier. Schematic illustration of the nucleotide and amino acid position of the VUS (NM_000258.2:c.170C>A, NP_000249.1:p.Ala57Asp) and two known pathogenic mutations (A57G, E56G) in the MYL3 gene. The red rectangles represent exons 1 to 7. Asterisks displays VUSMYL3(170C>A) position. Numbers 54 to 66 annotate amino acids positions (A). Sanger sequencing confirmation of the VUSMYL3(170C>A). Upper panel: Sequencing result of a healthy control individual, lower panel: sequencing result of the heterozygous VUSMYL3(170C>A) carrier. Nucleotide position of the VUSMYL3(170C>A) is displayed by a framing square (B). Alignment of regions flanking the VUSMYL3(170C>A) (red font) in MYL3 protein showing evolutionary conservation of the mutated residue across species and isoforms (C). A prediction score of the VUSMYL3(170C>A) predicted by the in silico tool PolyPhen-2 (D). The VUSMYL3(170C>A) carrier’s ECG recording (E). VUS indicates variant of uncertain significance.

same residue (A57G) have been reported in the Human Gene Mutation Database in association with HCM (Figure 1A), further supporting the functional importance of this region of the protein and a strong association of this particular locus with the HCM disease.28 The VUSMYL3(170C>A) was then evaluated using an in silico 4

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analysis tool, PolyPhen2, on a damaging score scale of 0 to 1, 0 being benign and 1 being pathogenic (damaging to the protein function).7 It is surprising that despite the asymptomatic cardiac history of the 46 years old VUSMYL3(170C>A) carrier, the in silico analysis tool displayed a high score of 0.998 (Figure 1D), confidently Circulation. 2018;138:00–00. DOI: 10.1161/CIRCULATIONAHA.117.032273

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Table 1. ClinVar Report for VUSMYL3(170C>A) Clinical Significance (Last Evaluated)

Collection Method

Condition(s) (Mode of Inheritance)

Submitter–Study Name

Description

Uncertain significance (August 29, 2013)

Clinical testing

Not specified [MedGen]

Laboratory for Molecular Medicine, Partners HealthCare Personalized Medicine

None

Uncertain significance (May 18, 2017)

Clinical testing

Not specified [MedGen]

GeneDx

Based on the currently available information, it is unclear whether this variant is a pathogenic variant or a rare benign variant

Uncertain significance (November 30, 2015)

Clinical testing

Cardiomyopathy [MedGen Orphane]

Division of Genomic Diagnostics, The Children’s Hospital of Philadelphia

None

Likely pathogenic (October 31, 2016)

Clinical testing

Not provided [MedGen]

Praxis Fuer Humangenetik Tuebingen

Ala57Asp variant may be pathogenic, additional studies are needed to fully assess its clinical significance

Uncertain significance (July 7, 2014)

Clinical testing

Primary familial hypertrophic cardiomyopathy [MedGen Orphanet OMIM]

Blueprint Genetics

Found together with likely pathogenic MYBPC3 (NM_000256.3:c.3800G>A)


predicting the VUSMYL3(170C>A) to be pathogenic and very damaging to the function of the MYL3 protein. This was further validated using additional in silico tools (SIFT and AlignGVGD). Moreover, interrogation of this VUSMYL3(170C>A) using the Exome Aggregation Consortium database showed that this mutation is observed in only 14 out of 121 338 individuals (Table II in the onlineonly Data Supplement), indicating again this variant is infrequent and likely pathogenic. At the carrier level, an electrocardiogram (ECG) assessment was conducted and did not detect an abnormal phenotype (Figure 1E). Similarly, echocardiogram did not show any evidence of hypertrophic cardiomyopathy (Table 2). To further assess the asymptomatic nature of this HCM-associated VUSMYL3(170C>A), 7 family members representing 3 generations underwent Sanger

sequencing. Four of the 7 family members (70, 48, 46, 11 years of age), including the studied proband, were found to carry the “likely pathogenic” VUSMYL3(170C>A) in their genomes (Figure 1B, 1C in the online-only Data Supplement). Similar to the proband, none of them displayed any phenotypic evidence of HCM, as is depicted by their echocardiograph results in Table 2, nor proarrhythmic clinical history (eg, syncope or resuscitation).

Generation of an Isogenic-Corrected “Healthy” Control iPSC Line Using CRISPR/Cas9 One crucial limitation of patient-specific iPSCs as a unique tool for the study of human disease has been the inability to perform experiments under genetically

Table 2. Echocardiography Results for Individuals With MYL3 c.170C>A or MYBPC3 c.961G>A

Left ventricular diastolic diameter, cm

MYL3 c.170C>A Individual I.2

MYL3 c.170C>A Individual II.2

MYL3 c.170C>A Individual II.1

MYL3 c.170C>A Individual III.2

MYBPC3 c.961G>A Patient

4.5

4.7

5.4

4.4

4.5

0.85

0.82

0.92

0.67

1.8

Wall thickness, cm Septal Posterior

0.87

0.88

0.85

0.74

0.92

Relative

0.38

0.36

0.33

0.32

0.41

Left ventricular mass index, g/m2

75

66

84

65

151

Left ventricular ejection fraction, %

70

68

61

67

48.7

E, cm/s

69

62

51

90

58.7

E/A

1.3

0.9

1

2

1.4

e’ average, cm/s

6.5

7.0

10

13

7.1

E/e’

11

8.9

5.1

6.9

8.5

Left atrial volume index, mL/m2

28

23

21

24

41.2

Estimated RAP, mm Hg Septal curvature

3

3

3

3

10

Normal

Normal

Normal

Normal

Reverse curvature

E indicates peak early diastolic transmitral flow velocities; E/A, peak early and late diastolic transmitral flow velocities ratio; E/e’, peak early diastolic transmitral flow velocities and peak early diastolic mitral annular velocities ratio; and RAP, right atrial pressure. MYL3 c.170C>A individuals: I.2 70-year-old mother; II.1 48-year-old brother; II.2 46-year-old proband; III.2 11-year-old daughter.

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defined conditions. This is particularly relevant for the study of this specific VUSMYL3(170C>A) case, as heterogeneous genetic background among individuals may disguise the subtle phenotype in vitro because of the asymptomatic nature of the VUSMYL3(170C>A) carrier. Accordingly, in addition to the control iPSC lines derived from 2 nonconsanguineous healthy individuals,26 an isogenic genetically corrected control line was generated from iPSCs of the heterozygous VUSMYL3(170C>A) carrier to help evaluate disease-relevant changes. The VUSMYL3(170C>A) carrier’s peripheral blood mononuclear cells were collected and reprogrammed to iPSCs (Figure 2 in the online-only Data Supplement). CRISPR/ Cas9 were then used to generate the isogenic corrected control cell line (Figure 2A and 2B). Three independent isogenic corrected clones were generated and confirmed by Sanger sequencing (Figure 2C; Figure 3A in the online-only Data Supplement). To exclude potential off-target cuttings by CRISPR/Cas9, 10 genomic loci with the highest sequence homology to the guide RNA’s binding site were checked by Sanger sequencing, separately for each 1 of the 3 clones. Sequencing results showed that the generated isogenic control cell line did not harbor any insertions or deletions at these loci (Table III in the online-only Data Supplement).

Cardiomyocytes derived from HCM patients upregulate natriuretic peptide precursor A expression and have high β-myosin/α-myosin ratios.14,29 In addition, HCM patient-specific iPSC-CMs were previously reported to display upregulated expression of a group of genes that include NPPA, TNNT2, MYL2, MYL4, and MYH7.14,15 Accordingly, gene expression analysis was performed on the VUSMYL3(170C>A)-iPSCCMs at 45 to 50 days postdifferentiation. The heterozygous VUSMYL3(170C>A)-iPSC-CMs displayed normal gene expression similar to their control counterparts, exhibiting no increased expression of the aforementioned genes, nor an elevation of the β-myosin/αmyosin ratio (Figure 3E; Figure 4 in the online-only Data Supplement). Next, we further investigated the potential effect of the VUSMYL3(170C>A) on the expression level of additional myosin light chain (MYL) genes that are highly expressed in cardiomyocytes (MYL3, MYL6, MYL7 and MYL9).30 It is interesting to note that the heterozygous VUSMYL3(170C>A)-iPSCCMs expressed normal gene expression for all the tested myosin light chain genes (Figure 3E; Figure 4 in the online-only Data Supplement). Altogether, these data show that the VUSMYL3(170C>A)-iPSC-CMs do not display the HCM-associated phenotype at the gene expression level.

Interrogation of Cell Morphology and Gene Expression of the Heterozygous VUSMYL3(170C>A)-iPSC-CMs

Functional Assessments of the Heterozygous VUSMYL3(170C>A)-iPSC-CMs

Multiple studies have reported myocyte sarcomere disarray and increased cell size to be features of HCM.14,15 Therefore, to test whether the heterozygous VUSMYL3(170C>A)-iPSC-CMs display similar sarcomeric disarray and increased cell size, we conducted immunostaining of sarcomeric proteins and cell size measurements on the VUSMYL3(170C>A)-iPSC-CMs, and compared them to the 2 healthy controls and isogenic corrected control (consisting of 3 clones) iPSC-CMs (Figure 3B and 3C in the online-only Data Supplement). It is interesting to note that immunostaining using sarcomere protein antibody anti–cardiac troponin T and anti–sarcomeric-α-Actinin showed that the heterozygous VUSMYL3(170C>A)-iPSC-CMs display normal sarcomere distribution, similar to the 2 healthy controls and isogenic control iPSC-CM counterparts (Figure 3A; Figure 3D in the online-only Data Supplement). Furthermore, cell size analysis revealed no significant differences in cell size distribution and average cell area among the tested iPSC-CM lines (healthy controls, isogenic corrected control, and heterozygous VUSMYL3(170C>A)) (Figure 3B through 3D; Figure 3E in the online-only Data Supplement). Overall, iPSC-CMs carrying the HCM-associated VUSMYL3(170C>A) do not express HCM-relevant abnormal morphological features. 6

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HCM is caused by mutations in sarcomere genes. As such, HCM-related changes in the molecular sarcomeric motor apparatus impair cardiac function, in many cases leading to increased contractility.31,32 To test for potential MYL3 mutation-related functional alterations in the contractile apparatus of VUSMYL3(170C>A)iPSC-CMs, contractility assays were performed. Similar to the healthy control and isogenic corrected control lines, the heterozygous VUSMYL3(170C>A)-iPSC-CMs displayed comparable contractility phenotype (Figure 4A), without any abnormal changes in beating rate, contraction and relaxation velocities (Figure 4B through 4D; Figure 5A in the online-only Data Supplement), contraction-relaxation distance, or contractionrelaxation duration (Figure 5B in the online-only Data Supplement). Arrhythmia is a clinical hallmark of HCM.33,34 Recent studies demonstrated the ability of patient- and disease-specific iPSC-CMs to recapitulate this abnormal electrophysiological phenotype using the iPSC technology.14,15 Accordingly, VUSMYL3(170C>A)-iPSC-CMs were electrophysiologically assessed using a fluorescent voltage sensor with fast kinetics for imaging high-frequency electric activity, applied as a membrane voltage change indicator, known as accelerated sensor of action potentials.35 The VUSMYL3(170C>A)-iPSC-CMs did Circulation. 2018;138:00–00. DOI: 10.1161/CIRCULATIONAHA.117.032273

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Downloaded from http://circ.ahajournals.org/ by guest on June 18, 2018 Figure 2. Genome-editing of an isogenic corrected iPSC line using clustered regularly interspaced short palindromic repeats (CRISPR). Schematic view of CRISPR/Cas9 and single-stranded oligodeoxynucleotide mediated genome editing (A). Sequence of gRNA and single-stranded oligodeoxynucleotide for generating the isogenic corrected iPSC line. The blue capitalized “C” annotates the desired Cytidine to be introduced to the VUSMYL3(170C>A) allele. The red lowercase letters display nucleotides positions of the VUSMYL3(170C>A), while the red capitalized “A” represents a silent mutation (B). Sanger sequencing confirmation of the isogenic corrected iPSC line (clone 1) (C). Additional clones are presented in Figure 3 in the online-only Data Supplement.

not display a clear proarrhythmic potential (Figure 4E), with only 8% of the heterozygous VUSMYL3(170C>A)-iPSC-CMs displaying delayed after depolarizations, a percentage similar to that detected in 2 healthy controls and isogenic corrected control lines (4%, 10%, and 9%, respectively) (Figure 4F; Figure 5C in the onlineonly Data Supplement). In summary, in contrast with the in silico pathogenic prediction, the lack of proarrhythmic activity presented by the VUSMYL3(170C>A)-iPSC-CMs clearly reflects the asymptomatic nature of the VUSMYL(170C>A) carrier. Circulation. 2018;138:00–00. DOI: 10.1161/CIRCULATIONAHA.117.032273

Previous studies interrogating the various mechanisms underlying arrhythmia in patients with HCM have implicated abnormal Ca2+ homeostasis as one of the proarrhythmic mediators.36,37 A recent study conducted on patient-specific HCM iPSC-CMs also revealed high levels of diastolic Ca2+, contributing to the disease proarrhythmic nature.14 To test for a proarrhythmic Ca2+-handling phenotype, we next recorded Ca2+ transients. Compared with the 2 healthy controls and the isogenic corrected control iPSC-CMs lines, the heterozygous VUSMYL3(170C>A)-iPSC-CMs did not exhibit abxxx xxx, 2018

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Downloaded from http://circ.ahajournals.org/ by guest on June 18, 2018 Figure 3. Cell morphology and gene expression analysis of the heterozygous VUSMYL3(170C>A) induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Sarcomere immunostaining analysis of iPSC-CMs differentiated from the 2 healthy control, isogenic corrected, and heterozygous VUSMYL3(170C>A) lines, displaying sacromeric-α-Actinin (red) and troponin T (green) stainings. Scale bars represents 10 μm (A). A schematic representation of cell size measurements using Phalloidin. Scale bars represents 150 μm (B). Distribution of iPSC-CMs size (C). iPSC-CMs size statistical analysis (n≥200 cells per line). The heterozygous VUSMYL3(170C>A)-iPSCCMs revealed no significant difference (ns) in average cell area compared with the tested iPSC-CM lines (healthy controls-1, healthy control-2 and isogenic corrected control) (D), Gene expression analysis of differentiated iPSC-CMs at day 45 to 50 postdifferentiation. The heterozygous VUSMYL3(170C>A)-iPSC-CMs revealed no significant difference in gene expression compared with the aforementioned tested iPSC-CM lines (E).

normal proarrhythmic Ca2+ transients (Figure 4G and 4H, Figure 5D in the online-only Data Supplement), in accordance with the previous electrophysiological findings. In conclusion, the genetic, morphologi8

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cal, and functional assessments of the heterozygous VUSMYL3(170C>A)-iPSC-CMs all display an asymptomatic phenotype, similarly to that exhibited clinically by the VUSMYL3(170C>A) carrier. Circulation. 2018;138:00–00. DOI: 10.1161/CIRCULATIONAHA.117.032273

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Downloaded from http://circ.ahajournals.org/ by guest on June 18, 2018 Figure 4. Comprehensive functional analysis of the heterozygous VUSMYL3(170C>A) induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Representative contractility traces of iPSC-CMs from the two healthy control, isogenic corrected, and the heterozygous VUSMYL3(170C>A) lines (A). Beating rate, contraction velocity, and relaxation velocity, respectively. The heterozygous VUSMYL3(170C>A)-iPSC-CMs revealed no significant difference (ns) in beating rate, contraction velocity, and relaxation velocity, respectively, compared with the aforementioned tested iPSC-CM lines (B through D). Representative action potential traces (E). Number of iPSC-CMs displaying proarrhythmic activity (F). Representative Ca2+ transients (G). Number of iPSC-CMs showing proarrhythmic calcium transients (H).

Comprehensive Analysis of a Generated Isogenic Homozygous VUSMYL3(170C>A)iPSC Line The heterozygous VUSMYL3(170C>A)-iPSC-CMs did not show a pathogenic phenotype, despite a high in silico Circulation. 2018;138:00–00. DOI: 10.1161/CIRCULATIONAHA.117.032273

assessment score confidently predicting it to be pathogenically damaging. This predicament may be the result of the VUSMYL3(170C>A) being a disease-causing variant in the recessive state, and therefore, explains the lack of pathogenic phenotype observed at the iPSC-CMs level. Unfortunately, because this variant is a VUS by definixxx xxx, 2018

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tion, there is no support for recessive inheritance of this VUSMYL3(170C>A). To overcome this possible predicament, we next created a homozygous VUSMYL3(170C>A) line using a CRISPR/Cas9 (Figure 6A in the online-only Data Supplement), generating 3 independent clones for characterization. Sanger sequencing confirmed that the heterozygous VUSMYL3(170C>A) line was turned into a homozygous VUSMYL3(170C>A) line (Figure 5A; Figure 6B in the online-only Data Supplement), and no offtarget mutation was detected in the 3 homozygous VUSMYL3(170C>A) iPSC clones (Supplemental Table IV). It is interesting to note that genome-edited homozygous VUSMYL3(170C>A)-iPSC-CMs displayed similar genetic, morphological, and functional assessment results, compared with those displayed by the heterozygous VUSMYL3(170C>A) and isogenic corrected control lines. The genome-edited homozygous VUSMYL3(170C>A)-iPSC-CMs also did not exhibit the following: (1) elevation in HCM-related genes (Figure 5C; Figure 7A in the online-only Data Supplement); (2) alterations in the gene expression level of the aforementioned myosin light chain genes (Figure 5C; Figure 7A in the online-only Data Supplement); (3) increased cell size (Figure 5D; Figure 7B in the online-only Data Supplement); (4) disarray of sarcomere organization (Figure 5E, Figure 7C in the online-only Data Supplement); (5) alterations in contractility (Figure 5F through 5H; Figure 7D through 7F in the online-only Data Supplement); (6) electrophysiological proarrhythmic phenotype (Figure 5I and 5J; Figure 7G in the online-only Data Supplement); or (7) irregular Ca2+-handling (Figure 5K and 5L; Figure 7H in the online-only Data Supplement). In conclusion, the lack of a pathogenic phenotype in all 3 individually generated genome-edited homozygous VUSMYL3(170C>A)iPSC-CMs clones, further supports the interpretation that the VUSMYL3(170C>A) variant is benign. To further confirm the benign phenotype interpretation, we generated a premature stop codon (frameshift mutation) in the healthy MYL3 allele of the heterozygous VUSMYL3(170C>A) cell line, thus generating a cell line that lacks a healthy MYL3 allele (loss of heterozygosity) and thus, only expresses the variant allele. Three individual frameshift mutation clones were generated using the same gRNA as the generation of the homozygous VUSMYL3(170C>A) line. To test the integrity of the frameshift mutation line, the presence of a premature stop codon in the MYL3 gene was validated by Sanger sequencing of the genomic DNA (Figure 5B; Figure 6C in the online-only Data Supplement). Sanger sequencing of the reverse transcription polymerase chain reaction product validated the degradation of the frameshift allele mRNA. Thus only the mutated allele mRNA was identified (Figure 6D in the online-only Data Supplement). Next, 3 frameshift mutation clones were assessed at the genetic, morphological, and functional levels, and compared with the heterozygous VUSMYL3(170C>A) 10

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and isogenic corrected control phenotypes. Similar to the homozygous VUSMYL3(170C>A) line, we found no evidence of a HCM phenotype in the frameshift mutation cell line (Figure 5C through 5L, Figure 7A through 5H in the online-only Data Supplement), further strengthening our conclusion that VUSMYL3(170C>A) is a benign genomic variant.

Comprehensive Analysis of Symptomatic HCM iPSC-CMs Lines To rule out the possibility that the benign prediction for the VUSMYL3(170C>A) was simply because of a lack of sensitivity on part of the iPSC-CMs platform, we next assayed 2 different HCM disease-associated mutations, displaying severe clinical HCM-related symptoms at the carrier level, in iPSC-CMs as positive controls. To validate that the benign assessment is not the result of the inability of the iPSC-CM platform to recapitulate a pathogenic MYL3 mutation phenotype in vitro, a known autosomal dominant pathogenic mutation, MYL3 c.170C>G, at the same nucleotide position as the VUSMYL3(170C>A), was studied. This mutation was reported to develop a severe HCM phenotype, including SCD, with disease penetrance in 78% of the genotyped MYL3 c.170C>G mutation carriers, and an onset as early as 28 years of age.27 For this purpose, the MYL3 c.170C>G mutation was introduced into the heterozygous VUSMYL3(170C>A) iPSCs using CRISPR/Cas9 (Figure 8A in the online-only Data Supplement). Accordingly, 3 independent iPSC clones (named MYL3(170C>G)) were generated and were tested by Sanger sequencing to confirm that the MYL3 c.170C>A variant allele was replaced with the MYL3 c.170C>G mutation (Figure 6A; Figure 8B in the online-only Data Supplement) without off-target cutting (Table V in the online-only Data Supplement). Next, to further elucidate the capability of the iPSC-CMs platform to recapitulate a pathogenic phenotype of a symptomatic carrier, a heterozygous HCM missense genetic variant in the sarcomeric gene MYBPC3 (MYBPC3 c.961G>A, p.Val321Met) (Figure 6A) was studied using a carrier-specific iPSC-CMs line. Note that the carrier presented an early HCM onset at the young age of 23 years (Table 2), is implanted with a cardioverter defibrillator, and has a strong family history of SCD, as early as 34 years of age. The iPSC lines were then evaluated for pathogenic morphological changes and functional proarrhythmic potential. Similar to the heterozygous VUSMYL3(170C>A)iPSC-CMs, both MYL3(170C>G)- and MYBPC3(961G>A)-iPSCCMs exhibited no significant difference in cell size (Figure 6B; Figure 8C in the online-only Data Supplement) and had normal sarcomere distribution (Figure 8D in the online-only Data Supplement). However, while heterozygous VUSMYL3(170C>A)-iPSC-CMs displayed normal expression of HCM-related genes (NPPA, TNNT2, Circulation. 2018;138:00–00. DOI: 10.1161/CIRCULATIONAHA.117.032273

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Downloaded from http://circ.ahajournals.org/ by guest on June 18, 2018 Figure 5. Comprehensive assays for assessing the homozygous VUSMYL3(170C>A) and the heterozygous frameshift mutation MYL3(170C>A/fs) induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Sanger sequencing confirmation of the isogenic homozygous VUSMYL3(170C>A) iPSC (clone1). Additional clones are presented in Figure 6 in the online-only Data Supplement (A). Sanger sequencing confirmation of the isogenic heterozygous frameshift mutation MYL3(170C>A/fs)-iPSC (clone1). Additional clones are presented in Figure 6 in the online-only Data Supplement. The red capitalized ”A” represents the VUSMYL3(170C>A). The blue lowercase “t” represents nonhomologous end joining (NHEJ)-mediated 1bp insertion. The red lowercase letters (tga) display the premature stop codon (B). Gene expression analysis. The homozygous VUSMYL3(170C>A)-iPSC-CMs and the frameshift mutation MYL3(170C>A/fs)-iPSC-CMs revealed no significant difference in gene expression compared with the isogenic corrected and the heterozygous VUSMYL3(170C>A) iPSC-CMs (C). Cell size assessment (n≥200 cells per line). The homozygous VUSMYL3(170C>A)-iPSC-CMs and frameshift mutation MYL3(170C>A/fs)-iPSC-CMs revealed no significant difference (ns) in cell size distribution (left panel) and average cell area (right panel) compared with isogenic corrected and heterozygous VUSMYL3(170C>A) (D). Sarcomere immunostaining analysis displaying sacromeric-α-Actinin (red) and troponin T (green) stainings. Scale bars represents 10 μm (E). Beating rate, contraction velocity, and relaxation velocity analysis, respectively. The homozygous (Continued )


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MYL2, and MYH7), these HCM-related genes, including the β-myosin/α-myosin ratio, were elevated in the MYL3(170C>G)- and MYBPC3(961G>A)-iPSC-CMs (Figure 6C; Figure 8E in the online-only Data Supplement). While both MYL3(170C>G)- and MYBPC3(961G>A)-iPSC-CMs did not exhibit a change in expression level of the myosin light chain genes (MYL2, MYL3, MYL6, MYL7 and MYL9) in comparison to the isogenic corrected and heterozygous VUSMYL3(170C>A)-iPSC-CMs, a significant increase in the MYL4 expression was noted (Figure 6C), as has been previously reported by multiple HCM-related studies.38–40 Contractility measurements did not display a statistically significant difference in beating rate among the tested lines (Figure 6D; Figure 8F in the online-only Data Supplement). Both MYL3(170C>G)- and MYBPC3(961G>A)-iPSC-CMs displayed faster contraction velocity in comparison to the corrected isogenic control line and heterozygous VUSMYL3(170C>A)-iPSC-CMs (Figure 6E; Figure 8F in the online-only Data Supplement). MYL3(170C>G)-iPSC-CMs displayed faster relaxation velocity (Figure 6F; Figure 8F in the online-only Data Supplement), as previously documented for this specific MYL3(170C>G) gene mutation,41,42 and as validated in this study by 3 independent MYL3(170C>G)-iPSC-CMs clones. The MYBPC3(961G>A)-iPSC-CMs did not exhibit a significant difference in relaxation velocity compared with the isogenic corrected control and heterozygous VUSMYL3(170C>A)-iPSC-CMs (Figure 6F). It is notable that although only 8% of heterozygous VUSMYL3(170C>A)-iPSC-CMs displayed a proarrhythmic electrophysiological phenotype in the form of delayed after depolarizations (Figure 4F), 42% and 46% of the MYL3(170C>G)- and MYBPC3(961G>A)-iPSC-CMs, respectively, exhibited delayed after depolarizations, displaying higher amplitude and shorter coupling intervals (Figure 6G and 6I; Figure 8G in the online-only Data Supplement). In addition, both presented significant variability in beat-to-beat intervals of action potentials (Figure 6H), another notable proarrhythmic characteristic. In accordance with the electrophysiological findings, 37% and 47% of the MYL3(170C>G)- and MYBPC3(961G>A)-iPSC-CMs, respectively, exhibited an abnormal Ca2+ phenotype, presenting double-peaked Ca2+ transients (Figure 6J and 6K; Figure 8H in the online-only Data Supplement) that were previously shown to be a proarrhythmic manifestation in HCM iPSC-CM14, as well as in another familial arrhythmogenic syndrome iPSC-CM study.12 These events were virtually absent in the VUSMYL3(170C>A)-iPSC-CMs. It is important to note that Fura2 Ca2+ imaging further demonstrated a significant increase in diastolic Ca2+ in both MYL3(170C>G)- and MYBPC3(961G>A)-iPSC-CMs,

when compared with the corrected and heterozygous VUSMYL3(170C>A)-iPSC-CMs lines (Figure 8I in the onlineonly Data Supplement). Taken together, similarly to the symptomatic nature of both MYL3(170C>G) and MYBPC3(961G>A) carriers, an evident symptomatic functional phenotype was recapitulated by the MYL3(170C>G)- and MYBPC3(961G>A)-iPSC-CMs, respectively, highlighting the potential of the iPSC-CM platform to successfully assess both pathogenic and benign phenotypes.

DISCUSSION In this study, we combine the powerful strengths of iPSC-based disease modeling and CRISPR/Cas9-mediated genome editing technology to demonstrate their unique potential as a personalized VUS risk-assessment platform for determining the pathogenicity of VUS. To highlight the dilemma and uncertainty that VUS presents for patients and clinicians around the world, we screened dozens of “healthy” individuals in search of a VUS that can clearly showcase the uncertainty surrounding genomic variant interpretation: a variant in a healthy individual that is annotated as “likely pathogenic.” By combining both aforementioned technologies for the study of such a VUS (MYL3 c.170C>G associated with HCM), we were able to (1) recapitulate the asymptomatic phenotype of the VUSMYL3(170C>A) carrier by showing a lack of HCM-related gene expression profile, morphological phenotype, and proarrhythmic activity in the heterozygous VUSMYL3(C>A)-iPSC-CMs; (2) confirm the asymptomatic clinical phenotype as a benign assessment in a genome-edited isogenic homozygous VUSMYL3(170C>A) line, as well as in a heterozygous frameshift mutation MYL3(170C>A/fs) line; and (3) validate the ability of the combined iPSC-CMs and genome-editing platform to recapitulate a pathogenic phenotype of a symptomatic carrier. HCM is a prevalent hereditary cardiac disorder linked to arrhythmia and SCD by both animal models and patient-specific iPSC-CMs studies.14,15,43 In contrast to these models, the carrier-specific VUSMYL3(170C>A)iPSC-CMs did not display a proarrhythmic phenotype. To validate whether the observed asymptomatic nature of the VUSMYL3(170C>A)-iPSC-CMs is the result of a recessive missense mutation, we also generated a homozygous VUSMYL3(170C>A) line. It is interesting to note that similar to the heterozygous cells, the phenotype displayed by all 3 homozygous iPSC-CMs clones was an asymptomatic one as well. The asymptomatic assessment of the homozygous line was

Figure 5 Continued. VUSMYL3(170C>A) and frameshift mutation MYL3(170C>A/fs)-iPSC-CMs revealed no significant difference in beating rate, contraction velocity, and relaxation velocity, respectively, compared with isogenic corrected and heterozygous VUSMYL3(170C>A) (F through H). Representative action potential traces (I). The Number of iPSC-CMs displaying proarrhythmic activity (J). Representative calcium transients (K). Number of iPSC-CMs showing proarrhythmic calcium transients (L). VUS indicates variant of uncertain significance.

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Downloaded from http://circ.ahajournals.org/ by guest on June 18, 2018 Figure 6. Comprehensive assays for assessing the MYL3(170C>G) and MYBPC3(961G>A) induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Sanger sequencing confirmation of the isogenic MYL3(170C>G) clone1, (additional clones are presented in Figure 8 in the online-only Data Supplement) and MYBPC3(961G>A) iPSC line (A). Cell size assessment (n≥200 cells per line) displayed as mean±SD. The MYL3(170C>G) and MYBPC3(961G>A) iPSC-CMs revealed no significant difference (ns) in average cell area compared with isogenic corrected and heterozygous VUSMYL3(170C>A) (B). Hypertrophic cardiomyopathy -related gene expression analysis. The MYL3(170C>G) and MYBPC3(961G>A) iPSC-CMs revealed no significant difference in gene expression compared with the isogenic corrected and the heterozygous VUSMYL3(170C>A) iPSC-CM lines (C). Beating rate, contraction velocity, and relaxation velocity, respectively. The MYL3(170C>G) and MYBPC3(961G>A) iPSC-CMs revealed no significant difference in beating rate, contraction velocity, and relaxation velocity, respectively, compared with isogenic (Continued )


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Figure 6 Continued. corrected and heterozygous VUSMYL3(170C>A) (D through F). Representative action potentials traces displaying delayed after depolarizations (highlighted by red vertical arrows) and varying interbeats intervals (indicated by red horizontal arrows) recorded from MYL3(170C>G) and MYBPC3(961G>A) iPSC-CMs, respectively (G, H). Number of MYL3(170C>G) and MYBPC3(961G>A) iPSC-CMs displaying proarrhythmic activity (I). Representative Ca2+ transients displaying delayed after depolarizations (J). Number of MYL3(170C>G) and MYBPC3(961G>A) iPSC-CMs depicting proarrhythmic calcium transients (K). *PA)-iPSC-CMs was not the result of a recessive gene. HCM is a complex and heterogeneous disease encompassing a wide variety of mutations and variants.29,32 To validate whether the benign asymptomatic assessments of both the heterozygous VUSMYL3(170C>A) and the genome-edited homozygous VUSMYL3(170C>A)iPSC-CMs are not the result of a potential inability on part of the iPSC-CMs platform to recapitulate a pathogenic MYL3 mutation phenotype, we genomeedited iPSCs with a known disease-causing mutation in the MYL3 gene (MYL3 c.170C>G), at the same nucleotide position as the VUSMYL3(170C>A).27 Next, to further test the sensitivity of this cellular platform to recapitulate a HCM VUS, iPSCs from an HCM patient with a MYBPC3 c.961G>A variant were generated from a carrier presenting classical clinical HCM symptoms. We found that both the MYL3(170C>G)- and MYBPC3(961G>A)-iPSC-CMs did show a HCM-pathogenic phenotype; for example, upregulation of HCM-related genes expression, elevation in β-myosin/α-myosin ratio, irregular contractility, proarrhythmic electrophysiological phenotype (delayed afterdepolarization), and an abnormal Ca2+ phenotype, similar to the results from previous HCM iPSC-CMs and HCM animal studies.14,15,41,44,45 The HCM-related phenotypes observed in both the MYL3(170C>G)- and MYBPC3(961G>A)-iPSCCMs, effectively demonstrate that the lack of phenotype exhibited by both the heterozygous and homozygous VUSMYL3(170C>A)-iPSC-CMs was not due to a technical artifact or faulty detection sensitivity on part of the iPSC-CMs platform, but rather a reflection of a benign phenotype. Over the past decade, a large range of studies, using similar assays to those presented in the current study, have demonstrated the intriguing ability of the iPSCCMs platform to recapitulate disease phenotype presented by the patient at bedside at the patient-derived cellular level in the dish, as been previously reported for genetic cardiomyopathy and arrhythmogenic syndromes such as: long QT, catecholaminergic polymorphic ventricular tachycardia, Brugada syndrome, familial dilated cardiomyopathy, and HCM.11,12,14,15,46 Similarly, iPSC-CMs have been also shown to recapitulate a normal “healthy” phenotype in cells derived from “healthy” subjects, as documented by count14

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less studies. This striking recapitulation capability of the iPSC-CMs platform is also vivid in this study. The chosen carrier is a healthy 46- year-old, active, athletic individual, that although has been phenotyped with a “likely pathogenic” HCM MYL3 c.170C>A VUS, does not have a clinical cardiac history, presents normal ECG, and normal echocardiography results. Further, supporting the asymptomatic nature of the carrier is the carrier’s impressive family history, consisting of 3 generations of asymptomatic carriers as old as 70 years of age. It is impressive that the proband-derived cells reflect the proband’s asymptomatic nature. In a similar manner, in the case of the MYL3(170C>G) mutation, the cells also strikingly recapitulated the carriers’ clinical phenotype. MYL3(170C>G) mutation carriers have been shown to experience HCM-related symptoms including SCD, with disease penetrance of 78%, and an onset as early as 28 and as late as 38 years of age. The genome-edited MYL3(170C>G)-iPSC-CMs nicely recapitulate the symptomatic nature of the carriers. Reinforcing these findings, an additional carrier displaying a HCM variant that demonstrates a clinical HCM onset as early as 23 years of age and a family history of SCD at an age as young as 34 was studied. In this case, the carrier-derived iPSC-CMs were also able to recapitulate a pathogenic phenotype. Altogether, the presented evidence here demonstrates the capability of the carrier-specific iPSC-CMs platform to reveal both a benign and pathogenic phenotype of prospective variants in a manner that recapitulates the carrier’s clinical presentation. Collectively, our data demonstrate the unique potential of combining iPSC-based disease modeling and CRISPR/Cas9-mediated genome editing technology as a personalized VUS risk assessment platform that can facilitate the study of the growing VUS phenomenon. Linking together iPSC and genome editing technologies presents an unprecedented opportunity to elucidate the pathogenicity of VUS in a dish in a carrier-specific manner. It is important to note that these efforts will help improve the study and diagnostics of VUS specifically and the promotion of personalized and precision medicine in general, better guiding clinicians in their choice of therapy and providing a clearer result for the VUS carriers. ARTICLE INFORMATION Received October 16, 2017; accepted March 24, 2018. The online-only Data Supplement is available with this article at http://circ. ahajournals.org/lookup/suppl/doi:10.1161/CIRCULATIONAHA.117.032273/-/ DC1.


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Joseph C. Wu, MD, PhD, Stanford University School of Medicine, 265 Campus Drive, G1120B Stanford, CA 94305. E-mail [email protected]

Affiliations Stanford Cardiovascular Institute (N.M., J.Z.Z., I.I., S.L.Z., H.C., F.H., T.K., K.D.W., L.T., R.S., H.W., C.L., N.S., J.C.W.), Division of Cardiology, Department of Medicine (N.M., J.Z.Z., I.I., S.L.Z., H.C., F.H., T.K., K.D.W., L.T., R.S., H.W., C.L., N.S., J.C.W.), and Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, CA (N.M., J.Z.Z., I.I., S.L.Z., H.C., F.H., T.K., K.D.W., L.T., R.S., H.W., C.L., N.S., J.C.W.).

Acknowledgments The authors thank Blake Wu for editing the article. The authors thank Andrew Olson from Stanford Neuroscience Microscopy Service and Jon Mulholland and Cedric Espenel from the Stanford Cell Sciences Imaging Facility for their help with confocal imaging.

Sources of Funding Downloaded from http://circ.ahajournals.org/ by guest on June 18, 2018

This study was funded by the Stanford Frankenstein@200 grant (to Dr Chen), National Institutes of Health grant K01 HL135455 (to Dr Sayed) and K08 HL119251 (to Dr Wilson), American Heart Association 17MERIT33610009 (to Dr J. Wu), and National Institutes of Health grants R01 HL126527, R01 HL130020, R01 HL113006, and R01 HL141371 (to Dr J. Wu).

Disclosures None.

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Correspondence



Ma et al



28. Andersen PS, Hedley PL, Page SP, Syrris P, Moolman-Smook JC, McKenna WJ, Elliott PM, Christiansen M. A novel myosin essential light chain mutation causes hypertrophic cardiomyopathy with late onset and low expressivity. Biochem Res Int. 2012;2012:685108. doi: 10.1155/ 2012/685108. 29. Seidman JG, Seidman C. The genetic basis for cardiomyopathy: from mutation identification to mechanistic paradigms. Cell. 2001;104:557–567. 30. England J, Loughna S. Heavy and light roles: myosin in the morphogenesis of the heart. Cell Mol Life Sci. 2013;70:1221–1239. doi: 10.1007/s00018-012-1131-1. 31. Belus A, Piroddi N, Scellini B, Tesi C, D’Amati G, Girolami F, Yacoub M, Cecchi F, Olivotto I, Poggesi C. The familial hypertrophic cardiomyopathyassociated myosin mutation R403Q accelerates tension generation and relaxation of human cardiac myofibrils. J Physiol. 2008;586:3639–3644. doi: 10.1113/jphysiol.2008.155952. 32. Marsiglia JD, Pereira AC. Hypertrophic cardiomyopathy: how do mutations lead to disease? Arq Bras Cardiol. 2014;102:295–304. 33. Maron BJ. Hypertrophic cardiomyopathy: a systematic review. JAMA. 2002;287:1308–1320. 34. Maron BJ, Shirani J, Poliac LC, Mathenge R, Roberts WC, Mueller FO. Sudden death in young competitive athletes: clinical, demographic, and pathological profiles. JAMA. 1996;276:199–204. 35. Chamberland S, Yang HH, Pan MM, Evans SW, Guan S, Chavarha M, Yang Y, Salesse C, Wu H, Wu JC, Clandinin TR, Toth K, Lin MZ and St-Pierre F. Fast two-photon imaging of subcellular voltage dynamics in neuronal tissue with genetically encoded indicators. Elife. 2017;6:e25690. 36. Adabag AS, Maron BJ, Appelbaum E, Harrigan CJ, Buros JL, Gibson CM, Lesser JR, Hanna CA, Udelson JE, Manning WJ, Maron MS. Occurrence and frequency of arrhythmias in hypertrophic cardiomyopathy in relation to delayed enhancement on cardiovascular magnetic resonance. J Am Coll Cardiol. 2008;51:1369–1374. doi: 10.1016/j.jacc.2007.11.071. 37. Wolf CM, Moskowitz IP, Arno S, Branco DM, Semsarian C, Bernstein SA, Peterson M, Maida M, Morley GE, Fishman G, Berul CI, Seidman CE, Seidman JG. Somatic events modify hypertrophic cardiomyopathy pathology and link hypertrophy to arrhythmia. Proc Natl Acad Sci U S A. 2005;102:18123–18128. doi: 10.1073/pnas.0509145102.

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38. Schaub MC, Hefti MA, Zuellig RA, Morano I. Modulation of contractility in human cardiac hypertrophy by myosin essential light chain isoforms. Cardiovasc Res. 1998;37:381–404. 39. Morano M, Zacharzowski U, Maier M, Lange PE, Alexi-Meskishvili V, Haase H, Morano I. Regulation of human heart contractility by essential myosin light chain isoforms. J Clin Invest. 1996;98:467–473. doi: 10.1172/JCI118813. 40. Petzhold D, Lossie J, Keller S, Werner S, Haase H, Morano I. Human essential myosin light chain isoforms revealed distinct myosin binding, sarcomeric sorting, and inotropic activity. Cardiovasc Res. 2011;90:513–520. doi: 10.1093/cvr/cvr026. 41. Kazmierczak K, Paulino EC, Huang W, Muthu P, Liang J, Yuan CC, Rojas AI, Hare JM, Szczesna-Cordary D. Discrete effects of A57G-myosin essential light chain mutation associated with familial hypertrophic cardiomyopathy. Am J Physiol Heart Circ Physiol. 2013;305:H575–H589. doi: 10.1152/ajpheart.00107.2013. 42. Kazmierczak K, Yuan CC, Liang J, Huang W, Rojas AI, Szczesna-Cordary D. Remodeling of the heart in hypertrophy in animal models with myosin essential light chain mutations. Front Physiol. 2014;5:353. doi: 10.3389/fphys.2014.00353. 43. Geisterfer-Lowrance AA, Christe M, Conner DA, Ingwall JS, Schoen FJ, Seidman CE, Seidman JG. A mouse model of familial hypertrophic cardiomyopathy. Science. 1996;272:731–734. 44. Marsiglia JD, Pereira AC. Hypertrophic cardiomyopathy: how do mutations lead to disease? Arq Bras Cardiol. 2014;102:295–304. 45. Witjas-Paalberends ER, Ferrara C, Scellini B, Piroddi N, Montag J, Tesi C, Stienen GJ, Michels M, Ho CY, Kraft T, Poggesi C, van der Velden J. Faster cross-bridge detachment and increased tension cost in human hypertrophic cardiomyopathy with the R403Q MYH7 mutation. J Physiol. 2014;592:3257–3272. doi: 10.1113/jphysiol.2014.274571. 46. Sun N, Yazawa M, Liu J, Han L, Sanchez-Freire V, Abilez OJ, Navarrete EG, Hu S, Wang L, Lee A, Pavlovic A, Lin S, Chen R, Hajjar RJ, Snyder MP, Dolmetsch RE, Butte MJ, Ashley EA, Longaker MT, Robbins RC, Wu JC. Patient-specific induced pluripotent stem cells as a model for familial dilated cardiomyopathy. Sci Transl Med. 2012;4:130ra47. doi: 10.1126/scitranslmed.3003552.


Determining the Pathogenicity of a Genomic Variant of Uncertain Significance Using CRISPR/Cas9 and Human-Induced Pluripotent Stem Cells Ning Ma, Joe Zhang, Ilanit Itzhaki, Sophia L. Zhang, Haodong Chen, Francois Haddad, Tomoya Kitani, Kitchener D. Wilson, Lei Tian, Rajani Shrestha, Haodi Wu, Chi Keung Lam, Nazish Sayed and Joseph C. Wu Downloaded from http://circ.ahajournals.org/ by guest on June 18, 2018

Circulation. published online June 18, 2018; Circulation is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 2018 American Heart Association, Inc. All rights reserved. Print ISSN: 0009-7322. Online ISSN: 1524-4539

The online version of this article, along with updated information and services, is located on the World Wide Web at: http://circ.ahajournals.org/content/early/2018/06/12/CIRCULATIONAHA.117.032273

Data Supplement (unedited) at: http://circ.ahajournals.org/content/suppl/2018/06/12/CIRCULATIONAHA.117.032273.DC1

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SUPPLEMENTAL MATERIALS

Determining the Pathogenicity of a Genomic Variant of Uncertain Significance Using CRISPR/Cas9 and Human Induced Pluripotent Stem Cells

Ning Ma, PhD1,2,3*, Joe Zhang, MD, PhD1,2,3*, Ilanit Itzhaki, PhD1,2,3*, Sophia L. Zhang, BS1,2,3, Haodong Chen, PhD1,2,3, Francois Haddad, MD1,2, Tomoya Kitani, MD, PhD1,2,3, Kitchener D. Wilson, MD, PhD1,2,3, Lei Tian, PhD1,2,3, Rajani Shrestha, MS1,2,3,Haodi Wu, PhD1,2,3, Chi Keung Lam, PhD1,2,3, Nazish Sayed, MD, PhD1,2,3, Joseph C. Wu, MD, PhD1,2,3

1

Stanford Cardiovascular Institute; 2Division of Cardiology, Department of Medicine; 3Institute

for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA

SUPPLEMENTAL METHODS The data, analytic methods, and study materials for the purposes of reproducing the results or replicating procedures can be made upon request to the corresponding author who manages the information.

Cell Culture: iPSCs were maintained under feeder-free conditions in defined E8 media (Thermo Fisher Scientific) on tissue culture plates coated with hESC-qualified Matrigel (BD Biosciences). iPSC-CMs were maintained in a RPMI 1640 medium (Thermo Fisher Scientific) supplemented with B27 supplements (Thermo Fisher Scientific) as described in published paper.1 All cell types were maintained at 5% CO2 and 37°C.

PCR Detection: DNA was extracted from collected cells using QuickExtract solution (Epicenter). PrimeSTAR® GXL DNA Polymerase (Clontech) was used for PCR. The sequence of primers used for identifying targeted colonies is as follows: forward primer, 5’cccaagcaaagccagcctgact; and reverse primer, 5’- tgcccgaccttctcacttctcca.

Vector Construction: pSpCas9(BB)-2A-GFP (PX458) was a gift from Feng Zhang (Addgene plasmid # 48138). For the site-specific CRISPR/Cas9 construction, two reverse complementary guide oligos were annealed first and then ligated to the linearized PX458 vector.

Fluorescent Activated Cell Sorting (FACS): iPSC-CMs were treated with Accutase® solution (Sigma-Aldrich) at 37°C for 5 minutes. Afterwards, cells were collected for centrifugation, following which the supernatant was removed and the cell pellet was resuspended using PBS.

2

Fixation, permeabilization, and antibody staining were then performed following the instruction of the BD Cytofix/CytopermTM Fixation/Permeabilization Solution Kit (Thermo Fisher Scientific, BDB554714). After staining, cells were resuspended in 500 uL of FACS buffer. Cell solution was then filtered in Falcon® 5mL Round Bottom Polystyrene, 12 x 75 mm Test Tube, with Cell Strainer Snap Cap (Corning), and loaded on a BD Biosciences FACS Aria II instrument fitted with a 100 µm nozzle using FACSDiva software. For iPSC characterization, anti-OCT4 antibody (Abcam, ab181557) and anti-SSEA4 antibody (Abcam, ab16287) were used. For detection of iPSC-CMs cardiac-specific proteins, anti-cardiac troponin T antibody (Abcam, ab45932) and anti sarcomeric-α-actinin antibody (Sigma-Aldrich, A7811-100UL) were applied.

Quantitative PCR (qPCR): Total RNAs were isolated from all studied iPSC-CMs lines at day 45-50 post differentiation, using the miRNeasy Mini kit (QIAGEN). 1 µg of RNA was used for synthesizing cDNA with the iScriptTM cDNA Synthesis kit (Bio-Rad). 0.25 µL of the cDNA was used to quantify gene expression using TaqMan probes and TaqMan Universal PCR Master Mix (Thermo Fisher Scientific).

Cell Size Measurement: Cells were first fixed by 4% paraformaldehyde (PFA) for 10 minutes at room temperature. After washing twice with PBS, cells were incubated with 0.1% Triton® X-100 for 20 minutes. Cells were then stained using 0.1% Triton® X-100, 0.1% Hoechst (Thermo Fisher Scientific) and 0.2% Alexa Fluor® 488 Phalloidin (Thermo Fisher Scientific) for 1 hour at room temperature. After washing twice with PBS, cells were imaged using a Cytation 5 Imaging Reader.

3

Contractility Assay: iPSC-CMs were dissociated using Accutase for 5 minutes and then were collected for centrifugation. Afterwards, cells were resuspended and counted. Each well of a 24-well plate was seeded with 300k cells, forming a synchronously beating iPSC-CMs monolayer. After a week of recovery, contractility was measured using the Sony SI8000 cell motion imaging system, in the presence of 5% CO2 and at a temperature of 37°C. Prior to initiation of recording, the cells were given time to equilibrate for 15 minutes at the given setting. Video imaging of the beating iPSC-CMs monolayers were recorded from 2 separate locations in each well (center and lateral edge), spatially adjusted in an automated recognized fashion, for 10 sec, at a frame rate of 75 fps, a resolution of 1024 × 24 pixels, and a depth of 8 bits using a 10× objective.

Optical Imaging of ASAP2: Dissociated iPSC-CMs were seeded on Matrigel-coated 35 mm glass bottom dishes with 20 mm micro-well (Cellvis). iPSC-CMs were infected with the pLV-EF1A-ASAP2 lentiviral vector (gift from Dr. Michael Z. Lin at Stanford University) with the multiplicity of infection (MOI) of 3 in RPMI-B27 medium containing polybrene (1 µg/ml) (VectorBuilder). Optical imaging of ASAP2 was performed 7 days after infection. Cells were superfused with Tyrode’s solution at 37°C. The Tyrode solution consisted of NaCl (140 mmol/L), KCl (5.4 mmol/L), CaCl2 (1.8 mmol/L), MgCl2 (1 mmol/L), HEPES (10 mmol/L), and glucose (10 mmol/L); pH was adjusted to 7.4 with NaOH. ASAP2 was excited at 488 nm and emission was collected over 510 nm. Line scan images were acquired on a Zeiss LSM710 confocal microscope (Zeiss) equipped with a 40× oil lens (NA: 1.30). Data were analyzed using ImageJ.

Ca2+ Imaging: Dissociated iPSC-CMs were seeded on Matrigel-coated coverslips (CS-24/50,

4

thickness 1 mm, Warner Instruments). Cells were loaded with 5 µM Fura-2AM (Thermo Fisher Scientific) in the presence of 0.02% Pluronic F-127 (Thermo Fisher Scientific) in Tyrode’s solution for 10 minutes at room temperature, followed by washing with Tyrode’s solution. iPSC-CMs were field-stimulated at 0.5 Hz at 37°C. Single cell Ca2+ imaging was conducted on a Nikon Eclipse Ti-E inverted microscope mounted with 40× oil immersion objective (0.95 NA). Fura-2 was excited at 340 nm and 380 nm wavelength using a Lambda DG-4 ultra-high-speed wavelength switcher (Sutter Instrument), and the emission of Fura-2 was collected over 510 nm wavelength. Raw data were analyzed using customized Matlab scripts and Ca2+ signal was presented as 340/380 ratio.

5

SUPPLEMENTAL FIGURE LEGENDS Supplemental Figure S1. Pedigree of the VUSMYL3(170C>A) carrier’s family. (A) Pie chart depicting the overall annotation profile of genetic variants that were detected in 135 heart disease-related genes across 54 healthy individuals. Numbers in the pie chart present the number of variants per category. (B) Schematic pedigree of the proband’s family. The VUSMYL3(170C>A) carrying proband (II-1) is marked by a red asterisk. Female family member are marked by circles and male by squares.. Shaded symbols indicate members carrying the VUSMYL3(170C>A), whereas open symbols represent absence of the VUSMYL3(170C>A). (C) Sanger sequencing results of all family members of the VUSMYL3(170C>A) carrier. Nucleotides positions of the VUSMYL3(170C>A) are depicted by framing squares.

Supplemental Figure S2. Pluripotency identification of iPSCs derived from the heterozygous VUSMYL3(170C>A) carrier. (A) Bright field image of cultured iPSC colonies. Scale bar: 100 µm. (B) Pluripotency related gene expression in the heterozygous VUSMYL3(170C>A) iPSCs relative to human ESC line H7. (C) Immunostaining of the heterozygous VUSMYL3(170C>A) iPSCs using anti-OCT4 (green) and anti-SSEA4 antibody (red). Scale bar: 200 µm. (D) FACS analysis of OCT4 and SSEA4 expression in the heterozygous VUSMYL3(170C>A) iPSCs. Blue, isotype control; Red, antigen staining for SSEA4 (left panel) or OCT4 (right panel). (E) Gene expression analysis of pluripotency related genes and differentiation related genes after embryoid body formation. (F) Normal karyotype of the heterozygous VUSMYL3(170C>A) iPSCs. Error bar: three replicates, mean ± SD.

Supplemental Figure S3. Identification of iPSC-CMs derived from the two healthy control

6

lines, isogenic corrected line and heterozygous VUSMYL3(170C>A) line. (A) Sanger sequencing confirmation of the isogenic corrected iPSC clone 2 and clone 3. (B) Bright field morphology of iPSC-CMs from the two healthy control lines, three isogenic corrected clones, and heterozygous VUSMYL3(170C>A) line. Scale bars, 50 µm. (C) Flow cytometry analyses of specific iPSC-CMs markers (TNNT and sarcomeric-α-actinin). Blue, isotype control; Red, antigen staining for TNNT (upper panel) or α-Actinin (lower panel). (D) Immunostaining analysis of specific iPSC-CMs markers TNNT (green) and sarcomeric-α-actinin (red) of the isogenic corrected iPSC clone 2 and clone 3 derived CMs. (E) Cell size measurement of three isogenic corrected iPSC clones-derived CMs. Data from ≥ 200 cells (acquired from three differentiations performed at three separate occasions) of each clone, mean ± SD. ns: non-significant, compared with isogenic corrected clone 1.

Supplemental Figure S4. Gene expression analysis of three isogenic corrected iPSC clones-derived CMs. Gene expression profile (NPPA, MYH6, TNNT2, MYH7, MYL2, MYL3, MYL4, MYL6, MYL7 and MYL9) of three isogenic corrected iPSC clones-derived CMs. Data from three replicates (acquired from three differentiations performed on three separate occasions), mean ± SD. ns: non-significant, compared with isogenic corrected clone 1.

Supplemental Figure S5. Functional analysis of the isogenic corrected iPSC clones-derived CMs and heterozygous VUSMYL3(170C>A) iPSC-CMs. (A) Beating rate (left panel), contraction velocity (middle panel), and relaxation velocity (right panel) analysis of three isogenic corrected iPSC clones-derived CMs. Data from three replicates, mean ± SD. ns: non-significant, compared

7

with isogenic corrected clone1. (B) Contraction distance, relaxation distance, contraction duration, and relaxation duration analysis of iPSC-CMs from the two healthy control lines, isogenic corrected clone 1, and heterozygous VUSMYL3(170C>A) line. Data from three replicates, mean ± SD. ns: non-significant, compared with two healthy control lines, and the isogenic corrected clone 1. (C) Number of iPSC-CMs showing proarrhythmic activity in isogenic corrected clone 2 and clone 3-derived CMs lines. (D) Number of iPSC-CMs showing calcium transients with a proarrhythmic phenotype in isogenic corrected clone 2 and clone 3-derived CMs lines. Three replicates means three differentiations performed on three separate occasions.

Supplemental Figure S6. Sequence validation of homozygous VUSMYL3(170C>A) and frameshift mutation MYL3(170C>A/fs) iPSC clones. (A) Sequence of gRNA and ssODN used for generating the homozygous VUSMYL3(170C>A) iPSC line. (B) Sanger sequencing results of homozygous VUSMYL3(170C>A) iPSC clone 2 and clone 3. (C) Sanger sequencing results of genomic DNA isolated from frameshift mutation MYL3(170C>A/fs) clone 2 and clone 3 showing 1bp insertion in the genome of frameshift mutation MYL3(170C>A/fs) clone2 (upper panel) and 13bp deletion in the genome of frameshift mutation MYL3(170C>A/fs) clone3 (lower panel). Red lower-case letters (tga): premature stop codon, Red capitalized letter ‘A’: VUSMYL3(170C>A). (D) Sanger sequencing results of cDNA from frameshift mutation MYL3(170C>A/fs) clone1 (top panel), clone2 (middle panel) and clone3 (bottom panel). Nucleotides positions of the VUSMYL3(170C>A) are depicted by a framing square.

Supplemental Figure S7. Characterization of homozygous VUSMYL3(170C>A) and frameshift mutation MYL3(170C>A/fs) clones-derived CMs. (A) Gene expression profile of three

8

homozygous VUSMYL3(170C>A) (left panel) and three frameshift mutation MYL3(170C>A/fs) clones-derived CMs (right panel). (B) Cell size measurement of homozygous VUSMYL3(170C>A) (left panel) and frameshift mutation MYL3(170C>A/fs) (right panel) clones-derived CMs. Data from ≥ 200 cells, mean ± SD. (C) Immunostaining analysis.. Scale bars: 10 µm. (D-F) Beating rate, contraction velocity, and relaxation velocity analysis, respectively. (G) Number of homozygous VUSMYL3(170C>A) clone 2- and clone 3- (left panel) and frameshift mutation MYL3(170C>A/fs) clone 2- and clone 3- (right panel) derived CMs showing normal and proarrhythmic activity. (H) Number of homozygous VUSMYL3(170C>A) clone 2- and clone 3- (left panel) and frameshift mutation MYL3(170C>A/fs) clone 2- and 3- (right panel) -derived CMs showing calcium transients with a proarrhythmic phenotype. Measurements were conducted in three replicates (three differentiations performed at three separate occasions). ns: non-significant, compared with either homozygous VUSMYL3(170C>A) clone 1 or frameshift mutation MYL3(170C>A/fs) clone 1. Data presented as mean ± SD. Supplemental Figure S8. Characterization of MYL3(170C>G) and MYBPC3(961G>A) iPS-CMs. (A) Sequence of gRNA and ssODN used for generating the isogenic MYL3(170C>G) iPSC line. (B) Sanger sequencing results of MYL3(170C>G) iPSC clone 2 and clone 3. (C) Cell size measurement of three MYL3(170C>G) clones-derived CMs. Data from ≥ 200 cells, (D) Immunostaining analysis of three MYL3(170C>G) clones-derived CMs and MYBPC3(961G>A) iPSC-CMs. Scale bars: 10 µm. (E) HCM-related gene expression analysis of the three MYL3(170C>G) clones-derived CMs. (F) Beating rate (top panel), contraction velocity (middle panel), and relaxation velocity (bottom panel) analysis of three MYL3(170C>G) clones-derived CMs. (G) Number of MYL3(170C>G) clone 2and clone 3-derived CMs showing proarrhythmic activity. (H) Number of MYL3(170C>G) clone 2and clone 3-derived CMs showing calcium transients with a proarrhythmic phenotype. (I)

9

Diastolic calcium of the isogenic corrected, heterozygous VUSMYL3(170C>A), homozygous VUSMYL3(170C>A), frameshift MYL3(170C>A/fs), MYL3(170C>G) and MYBPC3(961G>A) iPSC-CMs. ns: non-significant, * pG) clone1. Data presented as mean ± SD.

10

Supplemental Table S1. 17 “likely pathogenic” variants detected in healthy individuals

Locus

gene

location

function

protein

coding

Polyphen-2

Associated disease

chr1:116275561

CASQ2

NM_001232.3

missense

p.Phe189Leu

c.567C>G

1

CPVT

chr2:220284876

DES

NM_001927.3

missense

p.Ala213Val

c.638C>T

0.896

ARVC/DCM

chr3:38645238

SCN5A

NM001160161.1

missense

p.Leu619Phe

c.1855C>T

0.975

LQT

chr3:46902303

MYL3

NM_000258.2

missense

p.Ala57Asp

c.170C>A

1

HCM

chr4:114288907

ANK2

NM_001148.4

missense

p.Leu3740Ile

c.11218C>A

0.905

VT /VF

chr4:114288920

ANK2

NM_001148.4

missense

p.Thr3744Asn

c.11231C>A

0.951

VT /VF

chr8:11614575

GATA4

NM_002052.4

missense

p.Ser377Gly

c.1129A>G

0.54

DCM

chr10:69881254

MYPN

NM_001256267.1

missense

p.Tyr20Cys

c.59A>G

1

HCM/DCM

chr11:123513271

SCN3B

NM_018400.3

missense

p.Val110Ile

c.328G>A

0.999

LQT

chr11:47359047

MYBPC3

NM_000256.3

missense

p.Ala833Thr

c.2497G>A

0.999

HCM

chr12:121175678

ACADS

NM_000017.3

missense

p.Arg171Trp

c.511C>T

0.994

SCAD

chr12:121176083

ACADS

NM_000017.3

missense

p.Gly209Ser

c.625G>A

0.664

SCAD

chr14:23892910

MYH7

NM_000257.3

missense

p.Met982Thr

c.2945T>C

0.985

HCM

chr19:49686146

TRPM4

NM_017636.3

missense

p.Trp525Ter

c.1575G>A

-

Brugada

chrX:31496398

DMD

NM_004006.2

missense

p.His2921Arg

c.8762A>G

0.83

DMD

chrX:31496426

DMD

NM_004006.2

missense

p.Asn2912Asp

c.8734A>G

0.08

DMD

chrX:31496431

DMD

NM_004006.2

missense

p.Glu2910Val

c.8729A>T

0.21

DMD

Catecholaminergic polymorphic ventricular tachycardia (CPVT); arrhythmogenic right ventricular cardiomyopathy (ARVC); hypertrophic cardiomyopathy (HCM); long QT (LQT); ventricular tachycardia/ventricular fibrillation (VT/VF); dilated cardiomyopathy (DCM); spontaneous coronary artery dissection (SCAD); duchenne muscular dystrophy (DMD). 11

Supplemental Table S2. Frequencies of the VUSMYL3(170C>A) in different populations

Population

Allele Count Allele Number Homozygous Number Allele Frequency

European (Non-Finnish) 5

66684

0

7.498e-05

South Asian

5

16512

0

0.0003028

East Asian

3

8646

0

0.000347

Latino

1

11574

0

8.64e-05

Other

0

906

0

0

African

0

10402

0

0

Finnish

0

6614

0

0

Total

14

121338

0

0.0001154

12

Supplemental Table S3. No indels were found at the potential off-target loci of the gRNA for generating three independent isogenic corrected clones (C1, C2 and C3).

Sequence

Mismatches

Locus

Indels C1 C2 C3

GGTCTTCCTTGCACTCTGCCGAG

2MMs [1:12]

chr14:-105241012

N

N

N

AGTTTTTATTGAACTCTGCCCAG

3MMs [4:7:8]

chr12:-45858141

N

N

N

ACTCTTCCAGGAACTCTGCCAAG

3MMs [2:9:10]

chr8:+139651269

N

N

N

AGGGCTCATTGAACTCTGCCAAG

4MMs [3:4:5:8]

chr2:-60510140

N

N

N

CTTCTTCATAGAACTCTGCCAAG

4MMs [1:2:8:10]

chr11:-77389083

N

N

N

TTTCTTCATAGAACTCTGCCTGG

4MMs [1:2:8:10]

chr14:-99019889

N

N

N

TGCTGTCCTTGAACTCTGCCGGG

4MMs [1:3:4:5]

chr20:+39783116

N

N

N

AGTGGTTGTTGAACTCTGCCTGG

4MMs [4:5:7:8]

chr18:+48814709

N

N

N

TTTCTCCTTTGAACTCTGCCCAG

4MMs [1:2:6:8]

chr9:+2359678

N

N

N

ACCCTTCTCTGAACTCTGCCCAG

4MMs [2:3:8:9]

chr13:-95203119

N

N

N

13

Supplemental Table S4. No indels were found at the potential off-target loci of the gRNA for generating three independent homozygous VUSMYL3(170C>A) clones (C1, C2 and C3).

Sequence

Mismatches

Locus

Indels C1 C2 C3

AGTGAGGTTGAACAGCATGAGAG

3MMs [1:5:9]

chr12:-107792399

N

N

N

TGGGCAGTCAAACAGCATGAGAG

3MMs [3:6:10]

chr18:+52953785

N

N

N

TGTGCTTTCCAACAGCATGAGAG

3MMs [6:7:10]

chr10:+4729601

N

N

N

TGTCCTCTCCAACAGCATGACAG

4MMs [4:6:7:10]

chr1:+40222149

N

N

N

GATGCGATCGAACAGCATGTGAG

4MMs [1:2:7:20]

chr6:+41250150

N

N

N

TCTGTGGTAGCACAGCATGAGAG

4MMs [2:5:9:11]

chr5:-132089491

N

N

N

AGTGGGGTTGAAGAGCATGAGGG

4MMs [1:5:9:13]

chr11:+127348738

N

N

N

TGTGGGTTCCAAAAGCATGAAGG

4MMs [5:7:10:13]

chr5:-54405129

N

N

N

TGTCCAGTCCAACAGCTTGAGGG

4MMs [4:6:10:17]

chr3:+129239902

N

N

N

AGTGGGGTCGTACAGCATCAGAG

4MMs [1:5:11:19]

chr1:+156884123

N

N

N

14

Supplemental Table S5. No indels were found at the potential off-target loci of the gRNA for generating three independent MYL3(170C>G) clones (C1, C2 and C3).

Sequence

Mismatches

Locus

Indels C1 C2 C3

GGTCTTCCTTGCACTCTGCCGAG

2MMs [1:12]

chr14:-105241012

N

N

N

AGTTTTTATTGAACTCTGCCCAG

3MMs [4:7:8]

chr12:-45858141

N

N

N

ACTCTTCCAGGAACTCTGCCAAG

3MMs [2:9:10]

chr8:+139651269

N

N

N

AGGGCTCATTGAACTCTGCCAAG

4MMs [3:4:5:8]

chr2:-60510140

N

N

N

CTTCTTCATAGAACTCTGCCAAG

4MMs [1:2:8:10]

chr11:-77389083

N

N

N

TTTCTTCATAGAACTCTGCCTGG

4MMs [1:2:8:10]

chr14:-99019889

N

N

N

TGCTGTCCTTGAACTCTGCCGGG

4MMs [1:3:4:5]

chr20:+39783116

N

N

N

AGTGGTTGTTGAACTCTGCCTGG

4MMs [4:5:7:8]

chr18:+48814709

N

N

N

TTTCTCCTTTGAACTCTGCCCAG

4MMs [1:2:6:8]

chr9:+2359678

N

N

N

ACCCTTCTCTGAACTCTGCCCAG

4MMs [2:3:8:9]

chr13:-95203119

N

N

N

15

Reference: 1. Burridge PW, Li YF, Matsa E, Wu H, Ong SG, Sharma A, Holmstrom A, Chang AC, Coronado MJ, Ebert AD, Knowles JW, Telli ML, Witteles RM, Blau HM, Bernstein D, Altman RB and Wu JC. Human induced pluripotent stem cell-derived cardiomyocytes recapitulate the predilection of breast cancer patients to doxorubicin-induced cardiotoxicity. Nat Med. 2016;22:547-556.

16

A

B

17 112

118

* Drug response variants Unknown Benign or likely benign variants Variants of unknow significance (VUS) Likely pathogenic variants

343

Total=592 variants

C

I.1

I.2

II.1

II.3

III.1

III.2

A

B H7 heterozygous VUSMYL3(170C>A) iPSC

oct4

C

sox2

nanog

D Anti-SSEA4

E

F heterozygous VUSMYL3(170C>A) iPSC

1 SX M

6 PA X

7 X1 SO

O G

N AN

SO X2

O C

T4

heterozygous VUSMYL3(170C>A) iPSC-EB

Anti-OCT4

A

D isogenic corrected clone 2

isogenic corrected clone 2


B

control-1


control-2



isogenic corrected isogenic corrected clone 2 clone 3

heterozygous VUSMYL3(170C>A)

heterozygous VUSMYL3(170C>A)

Anti-α-Actinin

Anti-TNNT

C


control-2

control-1


E ns

3 co

rre iso ct ge ed ni cl c on e

2 rre iso ct ge ed ni cl c on e co

co


1

Cell size (μm²)

co


3

2 rre iso ct ge ed ni cl c on e

co

co


1

Cell size (μm²)

ns

MYL7

ns ns ns

MYL9

ns

MYL2 ns

MYL6 ns

is co og e cl rrec nic on te e d 3


is co og e r cl rec nic on te e d 1 is co og e cl rrec nic on te e d 2

Normalized to corrected clone1

ns


MYL4

co og e cl rrec nic on te e d 1 is co og e cl rrec nic on te e d 2

ns

is

MYH7/MYH6 Normalized to corrected clone1


is co og e r cl rec nic on te e d 1 is co og e cl rrec nic on te e d 2


ns



is co og e cl rrec nic on te e d 1 is co og e cl rrec nic on te e d 2





MYH6 ns


ns

is co og e r cl rec nic on te e d 3


ns ns


ns ns

is co og e cl rrec nic on te e d 1 is co og e r cl rec nic on te e d 2

MYL3 ns




MYH7 ns

ns





ns


is co og e cl rrec nic on te e d 2 is co og e r cl rec nic on te e d 3



NPPA TNNT2 ns

ns

ns

Number of cells

l-2

ro

nt

-1

ro l

Normal

4

3

46

45

isogenic isogenic corrected clone 2 corrected clone 3

Proarrhythmic

Normal

D

2

2

60

60

-2

Proarrhythmic

isogenic isogenic corrected clone 2 corrected clone 3 0C

>A )

is co og rre en ct ic ed he VU te SM ro z YL yg 3( ous 17

ro l

-1

Contraction duration (sec)

ns 0C

)

>A

co og rre en ct ic ed h VU ete SM ro z YL yg 3( ous 17

is

l-2

ro

nt

co

l-1

ro

nt

co

)

>A

0C

Relaxation distance (μm)

is co og e cl rrec nic on te e d 1 is co og e cl rrec nic on te e d 2 is co og e cl rrec nic on te e d 3



Contraction velocity (μm/sec)

Beating rate/min

Relaxation velocity (μm/sec)

ns

nt

ro l

)

>A

is co og e cl rrec nic on te e d 1 is o co g e cl rrec nic on te e d 2 is co og e cl rrec nic on te e d 3

ns

co

nt

co

0C

is co og rre en ct ic ed h VU ete SM ro z YL yg 3( ous 17

l-2

ro

nt

co

l-1

ro

nt

co

Contraction distance (μm)

ns

Number of cells

C is co og rre en ct ic ed h VU ete SM ro z YL yg 3( ous 17

co

nt

co

Contraction duration (sec)

A ns

ns ns

B ns

ns

ns

A

B homozygous clone 2 homozygous clone 3 myl3 wild type allele

C

VUSMYL3(170C>A) allele frameshift mutation allele

VUSMYL3(170C>A) allele frameshift mutation allele

D

Number of cells

Normal

Proarrhythmic

4

3

47

48

homozyogus homozyogus clone 2 clone 3 ns

ns

ns

ns

ns

H

Normal

Proarrhythmic

2

2

48

47

frameshift clone 2

frameshift clone 3

PP A M YH TN 6 N T2 M YH M 7 /M YH YH 7 6 M YL M 2 YL 3 M YL M 4 YL 6 M YL 7 M YL 9

frameshift clone 1

ns

Normal

ns ns

ns

2

2

57

58

ns ns

ns ns

homozyogus homozyogus clone 2 clone 3

cl me on sh e ift 1 fra cl me on sh e ift 2 fra cl me on sh e ift 3

ns

Normalized expression to frameshift clone1

ns ns

ns

fra

ns

nsns

N

ns ns

ns ns

Proarrhythmic

Number of cells

ns

ns ns

m cl ozy on o e gu 1 s ho m o cl zy on o e gu ho 2 s m cl ozy on o e gu 3 s

ns

nsns


ns ns

homozygous clone 3

ho

ns

homozygous clone 2

Number of cells

ns

fra cl me on sh e ift 1 fra cl me on sh e ift 2 fra cl me on sh e ift 3

ns

ns

Cell size (μm²)

ns ns

Beating rate/min

M

Normalized expression to homozygous clone1

ns ns

cl me on sh e ift 1 fra cl me on sh e ift 2 fra cl me on sh e ift 3

N

PP A YH TN 6 N T2 M YH M 7 /M YH YH 7 6 M YL M 2 YL 3 M YL M 4 YL 6 M YL 7 M YL 9

B ns

fra

m cl ozy on o e gu 1 s ho m cl ozy on o e gu ho 2 s m cl ozy on o e gu 3 s

ho

Cell size (μm²)

ns ns ns

fra cl me on sh e ift 1 fra cl me on sh e ift 2 fra cl me on sh e ift 3

m cl ozy on o e gu 1 s ho m cl ozy on o e gu ho 2 s m cl ozy on o e gu 3 s

Beating rate/min

homozygous ns clone 1

Number of cells

G

m cl ozy on o e gu 1 s ho m cl ozy on o g ho e 2 us m cl ozy on o e gu 3 s

ho

D

ho

E


A

frameshift clone 2 nsns

homozygous clone 2

ns ns

frameshift clone 2

frameshift clone 3

ns ns ns

ns

Normal

ns nsns ns ns

C homozygous clone 3

frameshift clone 3

F

ns ns

Proarrhythmic

1

2

59

58

frameshift clone 2

frameshift clone 3

MYL3(170C>G) clone 2


Normal

Proarrhythmic

20

18

30

32

61


38

21

39

32

Proarrhythmic


I

ns

ns

ns

e

on

cl

e

3

2

e

on

cl

e

on

cl

3

2

1

ns

on

1


cl

e

e

ns

on

cl

ns

on

MYBPC3(961G>A)


e

on

cl

e

on

cl

e

on

cl

3

2

1

Beating rate/min

F ns

ns

*

A)

H cl


ns

9

ns ns

YL

ns

G>

Normal ns ns

Diastolic Ca2+ (340/380)

Cell size (μm²)

ns

M


7


YL

ns ns

M

ns

6

ns

YL

M

YL cl 3(1 on 70 e C>G 1 ) M YL cl 3(1 on 70 e C>G 2 ) M YL cl 3(1 on 70 e C>G 3 )

cl

Cell size (μm²)

C

iso ge he corre nic VU tero cte SM zyg d o Y h L3 us VU omo (170C> SM zyg A) fra YL3 ous me (17 MY shif 0C>A) L t MY 3 (170C> A/f L3 s) MY (170C > G BP C3 ) (9

G ns ns

M

ns

4

ns

YL

ns ns

M

ns

3


YL


M

ns

Number of cells

E

YH 7 M /M YH YH 7 6 M YL 2


M

T2

N

ns

TN

ns

YH 6

M

PP A

N

M

YL 3 on (170 e C>G 1 ) M YL cl 3(1 on 70 e C>G 2 ) M YL cl 3(1 on 70 e C>G 3 )

D

Number of cells

Normalized expression to MUTMYL3(170C>G) clone 1

A B


MYL3(170C>G) clone 3 ns

ns

ns ns

*

Determining the Pathogenicity of a Genomic Variant

Determining the Pathogenicity of a Genomic Variant

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