MRAS Variants Cause Cardiomyocyte Hypertrophy in Patient-Specific Induced Pluripotent Stem Cell-Derived Cardiomyocytes: Additional Evidence for MRAS as a Definitive Noonan Syndrome-Susceptibility Gene

Erin M. Higgins, J. Martijn Bos, Steven M. Dotzler, C. S. John Kim, Michael J. Ackerman

Research output: Contribution to journalArticle

Abstract

BACKGROUND: MRAS was identified recently as a novel Noonan syndrome (NS)-susceptibility gene. Phenotypically, both patients with NS, harboring pathogenic MRAS variants, displayed severe cardiac hypertrophy. This study aimed to demonstrate both the necessity and sufficiency of a patient-specific variant (p.Gly23Val-MRAS) to cause NS through the generation and characterization of patient-specific, isogenic control, and disease modeled induced pluripotent stem cell (iPSC) lines. METHODS: iPSCs were derived from a patient with a p.Gly23Val-MRAS variant to assess the effect of MRAS variants on pathogenesis of NS-associated cardiac hypertrophy. CRISPR/Cas9 gene editing was used to correct the pathogenic p.Gly23Val-MRAS variant in patient cells (isogenic control) and to introduce the pathogenic variant into unrelated control cells (disease modeled) to determine the necessity and sufficiency of the p.Gly23Val-MRAS variant to elicit the disease phenotype in iPSC-derived cardiomyocytes (iPSC-CMs). iPSC-CMs were analyzed by microscopy and immunofluroesence, single-cell RNAseq, Western blot, room temperature-quantitative polymerase chain reaction, and live-cell calcium imaging to define an in vitro phenotype of MRAS-mediated cardiac hypertrophy. RESULTS: Compared with controls, both patient and disease modeled iPSC-CMs were significantly larger and demonstrated changes in gene expression and intracellular pathway signaling characteristic of cardiac hypertrophy. Additionally, patient and disease modeled iPSC-CMs displayed impaired Ca2+ handling, including increased frequency of irregular Ca2+ transients and changes in Ca2+ handling kinetics. CONCLUSIONS: p.Gly23Val-MRAS is both necessary and sufficient to elicit a cardiac hypertrophy phenotype in iPSC-CMs that includes increased cell size, changes in cardiac gene expression, and abnormal calcium handling-providing further evidence to establish the monogenetic pathogenicity of p.Gly23Val-MRAS in NS with cardiac hypertrophy.

Original languageEnglish (US)
Pages (from-to)e002648
JournalCirculation. Genomic and precision medicine
Volume12
Issue number11
DOIs
StatePublished - Nov 1 2019

Fingerprint

Noonan Syndrome
Induced Pluripotent Stem Cells
Cardiac Myocytes
Cardiomegaly
Hypertrophy
Genes
Phenotype
Clustered Regularly Interspaced Short Palindromic Repeats
Calcium
Gene Expression
Cell Size
Virulence
Microscopy
Western Blotting
Cell Line
Polymerase Chain Reaction
Temperature

Keywords

  • cell size
  • gene editing
  • induced pluripotent stem cell
  • Noonan syndrome
  • phenotype

ASJC Scopus subject areas

  • Genetics
  • Cardiology and Cardiovascular Medicine
  • Genetics(clinical)

Cite this

@article{9da6ad159ea34624841fb3ddbfadc032,
title = "MRAS Variants Cause Cardiomyocyte Hypertrophy in Patient-Specific Induced Pluripotent Stem Cell-Derived Cardiomyocytes: Additional Evidence for MRAS as a Definitive Noonan Syndrome-Susceptibility Gene",
abstract = "BACKGROUND: MRAS was identified recently as a novel Noonan syndrome (NS)-susceptibility gene. Phenotypically, both patients with NS, harboring pathogenic MRAS variants, displayed severe cardiac hypertrophy. This study aimed to demonstrate both the necessity and sufficiency of a patient-specific variant (p.Gly23Val-MRAS) to cause NS through the generation and characterization of patient-specific, isogenic control, and disease modeled induced pluripotent stem cell (iPSC) lines. METHODS: iPSCs were derived from a patient with a p.Gly23Val-MRAS variant to assess the effect of MRAS variants on pathogenesis of NS-associated cardiac hypertrophy. CRISPR/Cas9 gene editing was used to correct the pathogenic p.Gly23Val-MRAS variant in patient cells (isogenic control) and to introduce the pathogenic variant into unrelated control cells (disease modeled) to determine the necessity and sufficiency of the p.Gly23Val-MRAS variant to elicit the disease phenotype in iPSC-derived cardiomyocytes (iPSC-CMs). iPSC-CMs were analyzed by microscopy and immunofluroesence, single-cell RNAseq, Western blot, room temperature-quantitative polymerase chain reaction, and live-cell calcium imaging to define an in vitro phenotype of MRAS-mediated cardiac hypertrophy. RESULTS: Compared with controls, both patient and disease modeled iPSC-CMs were significantly larger and demonstrated changes in gene expression and intracellular pathway signaling characteristic of cardiac hypertrophy. Additionally, patient and disease modeled iPSC-CMs displayed impaired Ca2+ handling, including increased frequency of irregular Ca2+ transients and changes in Ca2+ handling kinetics. CONCLUSIONS: p.Gly23Val-MRAS is both necessary and sufficient to elicit a cardiac hypertrophy phenotype in iPSC-CMs that includes increased cell size, changes in cardiac gene expression, and abnormal calcium handling-providing further evidence to establish the monogenetic pathogenicity of p.Gly23Val-MRAS in NS with cardiac hypertrophy.",
keywords = "cell size, gene editing, induced pluripotent stem cell, Noonan syndrome, phenotype",
author = "Higgins, {Erin M.} and Bos, {J. Martijn} and Dotzler, {Steven M.} and {John Kim}, {C. S.} and Ackerman, {Michael J.}",
year = "2019",
month = "11",
day = "1",
doi = "10.1161/CIRCGEN.119.002648",
language = "English (US)",
volume = "12",
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TY - JOUR

T1 - MRAS Variants Cause Cardiomyocyte Hypertrophy in Patient-Specific Induced Pluripotent Stem Cell-Derived Cardiomyocytes

T2 - Additional Evidence for MRAS as a Definitive Noonan Syndrome-Susceptibility Gene

AU - Higgins, Erin M.

AU - Bos, J. Martijn

AU - Dotzler, Steven M.

AU - John Kim, C. S.

AU - Ackerman, Michael J.

PY - 2019/11/1

Y1 - 2019/11/1

N2 - BACKGROUND: MRAS was identified recently as a novel Noonan syndrome (NS)-susceptibility gene. Phenotypically, both patients with NS, harboring pathogenic MRAS variants, displayed severe cardiac hypertrophy. This study aimed to demonstrate both the necessity and sufficiency of a patient-specific variant (p.Gly23Val-MRAS) to cause NS through the generation and characterization of patient-specific, isogenic control, and disease modeled induced pluripotent stem cell (iPSC) lines. METHODS: iPSCs were derived from a patient with a p.Gly23Val-MRAS variant to assess the effect of MRAS variants on pathogenesis of NS-associated cardiac hypertrophy. CRISPR/Cas9 gene editing was used to correct the pathogenic p.Gly23Val-MRAS variant in patient cells (isogenic control) and to introduce the pathogenic variant into unrelated control cells (disease modeled) to determine the necessity and sufficiency of the p.Gly23Val-MRAS variant to elicit the disease phenotype in iPSC-derived cardiomyocytes (iPSC-CMs). iPSC-CMs were analyzed by microscopy and immunofluroesence, single-cell RNAseq, Western blot, room temperature-quantitative polymerase chain reaction, and live-cell calcium imaging to define an in vitro phenotype of MRAS-mediated cardiac hypertrophy. RESULTS: Compared with controls, both patient and disease modeled iPSC-CMs were significantly larger and demonstrated changes in gene expression and intracellular pathway signaling characteristic of cardiac hypertrophy. Additionally, patient and disease modeled iPSC-CMs displayed impaired Ca2+ handling, including increased frequency of irregular Ca2+ transients and changes in Ca2+ handling kinetics. CONCLUSIONS: p.Gly23Val-MRAS is both necessary and sufficient to elicit a cardiac hypertrophy phenotype in iPSC-CMs that includes increased cell size, changes in cardiac gene expression, and abnormal calcium handling-providing further evidence to establish the monogenetic pathogenicity of p.Gly23Val-MRAS in NS with cardiac hypertrophy.

AB - BACKGROUND: MRAS was identified recently as a novel Noonan syndrome (NS)-susceptibility gene. Phenotypically, both patients with NS, harboring pathogenic MRAS variants, displayed severe cardiac hypertrophy. This study aimed to demonstrate both the necessity and sufficiency of a patient-specific variant (p.Gly23Val-MRAS) to cause NS through the generation and characterization of patient-specific, isogenic control, and disease modeled induced pluripotent stem cell (iPSC) lines. METHODS: iPSCs were derived from a patient with a p.Gly23Val-MRAS variant to assess the effect of MRAS variants on pathogenesis of NS-associated cardiac hypertrophy. CRISPR/Cas9 gene editing was used to correct the pathogenic p.Gly23Val-MRAS variant in patient cells (isogenic control) and to introduce the pathogenic variant into unrelated control cells (disease modeled) to determine the necessity and sufficiency of the p.Gly23Val-MRAS variant to elicit the disease phenotype in iPSC-derived cardiomyocytes (iPSC-CMs). iPSC-CMs were analyzed by microscopy and immunofluroesence, single-cell RNAseq, Western blot, room temperature-quantitative polymerase chain reaction, and live-cell calcium imaging to define an in vitro phenotype of MRAS-mediated cardiac hypertrophy. RESULTS: Compared with controls, both patient and disease modeled iPSC-CMs were significantly larger and demonstrated changes in gene expression and intracellular pathway signaling characteristic of cardiac hypertrophy. Additionally, patient and disease modeled iPSC-CMs displayed impaired Ca2+ handling, including increased frequency of irregular Ca2+ transients and changes in Ca2+ handling kinetics. CONCLUSIONS: p.Gly23Val-MRAS is both necessary and sufficient to elicit a cardiac hypertrophy phenotype in iPSC-CMs that includes increased cell size, changes in cardiac gene expression, and abnormal calcium handling-providing further evidence to establish the monogenetic pathogenicity of p.Gly23Val-MRAS in NS with cardiac hypertrophy.

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KW - Noonan syndrome

KW - phenotype

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