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

Abstract

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." Human-induced pluripotent stem cells (iPSCs) were derived from the heterozygous VUS MYL3_(170C>A) carrier, and their genome was edited using CRISPR/Cas9 to generate 4 isogenic iPSC lines: (1) corrected "healthy" control; (2) homozygous VUS MYL3_(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 VUS MYL3_(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 VUS MYL3_(170C>A)-iPSC-CMs did not show an HCM phenotype at the gene expression, morphology, or functional levels. Furthermore, genome-edited homozygous VUS MYL3_(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 carrier-specific manner in a dish. As such, this platform represents a promising VUS risk-assessment 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.

Keywords: CRISPR-Cas systems; cardiomyopathy, hypertrophic; clustered regularly interspaced short palindromic repeats; gene editing; induced pluripotent stem cells; mutations.

Figure 1. Confirmation of the likely pathogenic VUSMYL3_(170C>A) uncovered in an asymptomatic carrier

(A) 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 1-7. Asterisks displays VUSMYL3_(170C>A) position. Numbers 54-66 annotate amino acids positions. (B) 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. (C) Alignment of regions flanking the VUSMYL3_(170C>A) (red font) in MYL3 protein showing evolutionary conservation of the mutated residue across species and isoforms. (D) A prediction score of the VUSMYL3_(170C>A) predicted by the in silico tool PolyPhen-2. (E) The VUSMYL3_(170C>A) carrier’s ECG recording.

Figure 2. Genome-editing of an isogenic corrected iPSC line using CRISPR

(A) Schematic view of CRISPR/Cas9 and ssODN mediated genome editing. (B) Sequence of gRNA and ssODN 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 lower-case letters display nucleotides positions of the VUSMYL3_(170C>A), while the red capitalized ‘A’ represents a silent mutation. (C) Sanger sequencing confirmation of the isogenic corrected iPSC line (clone 1). Additional clones are presented in Supplemental Figure S3.

Figure 3. Cell morphology and gene expression analysis of the heterozygous VUSMYL3_(170C>A) iPSC-CMs

(A) Sarcomere immunostaining analysis of iPSC-CMs differentiated from the two 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. (B) A schematic representation of cell size measurements using Phalloidin. Scale bars represents 150 μm. (C) Distribution of iPSC-CMs size. (D) iPSC-CMs size statistical analysis (n≥200 cells per line). The heterozygous VUSMYL3_(170C>A)-iPSC-CMs 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), (E) Gene expression analysis of differentiated iPSC-CMs at day 45-50 dpd. The heterozygous VUSMYL3_(170C>A)-iPSC-CMs revealed no significant difference in gene expression compared with the aforementioned tested iPSC-CM lines.

Figure 4. Comprehensive functional analysis of the heterozygous VUSMYL3_(170C>A) iPSC-CMs

(A) Representative contractility traces of iPSC-CMs from the two healthy control, isogenic corrected, and the heterozygous VUSMYL3_(170C>A) lines. (B-D) 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. (E) Representative action potential traces. (F) Number of iPSC-CMs displaying proarrhythmic activity. (G) Representative Ca²⁺ transients. (H) Number of iPSC-CMs showing proarrhythmic calcium transients.

Figure 5. Comprehensive assays for assessing the homozygous VUSMYL3_(170C>A) and the heterozygous frameshift mutation MYL3^(170C>A/fs) iPSC-CMs

(A) Sanger sequencing confirmation of the isogenic homozygous VUSMYL3_(170C>A) iPSC (clone1). Additional clones are presented in Supplemental Figure S6. (B) Sanger sequencing confirmation of the isogenic heterozygous frameshift mutation MYL3^(170C>A/fs)-iPSC (clone1). Additional clones are presented in Supplemental Figure S6. The red capitalized ‘A’ represents the VUSMYL3_(170C>A). The blue lower-case ‘t’ represents non-homologous end joining (NHEJ)-mediated 1bp insertion. The red lower-case letters (tga) display the premature stop codon. (C) 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. (D) 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). (E) Sarcomere immunostaining analysis displaying sacromeric-α-Actinin (red) and troponin T (green) stainings. Scale bars represents 10 μm. (F-H) Beating rate, contraction velocity, and relaxation velocity analysis, respectively. The homozygous 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). (I) Representative action potential traces. (J) Number of iPSC-CMs displaying proarrhythmic activity. (K) Representative calcium transients. (L) Number of iPSC-CMs showing proarrhythmic calcium transients.

Figure 6. Comprehensive assays for assessing the MYL3_(170C>G) and MYBPC3_(961G>A) iPSC-CMs

(A) Sanger sequencing confirmation of the isogenic MYL3_(170C>G) clone1,(Additional clones are presented in Supplemental Figure S8) and MYBPC3_(961G>A) iPSC line. (B) 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). (C) HCM-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. (D-F) 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 corrected and heterozygous VUSMYL3_(170C>A). (G-H) Representative action potentials traces displaying DADs (highlighted by red vertical arrows) and varying inter-beats intervals (indicated by red horizontal arrows) recorded from MYL3_(170C>G) and MYBPC3_(961G>A) iPSC-CMs, respectively. (I) Number of MYL3_(170C>G) and MYBPC3_(961G>A) iPSC-CMs displaying proarrythmic activity (J) Representative Ca²⁺ transients displaying DADs. (K) Number of MYL3_(170C>G) and MYBPC3_(961G>A) iPSC-CMs depicting proarrhythmic calcium transients. *p < 0.1, ** p < 0.01, *** p < 0.001, **** p < 0.0001, compared with isogenic corrected and heterozygous VUSMYL3_(170C>A) lines.