Evaluation of Patient‐Specific MTERF4 Variants in Gene‐Edited Human iPSC‐derived Cardiomyocytes
Authors: Marquez Maribel , McDermott‐Roe Chris , Grzybowski Mike , Helbling Daniel , Dimmock David P. , Verbsky James W. , Geurts Aron 
Published: June 2021

Mitochondrial Transcription Termination Factor 4 (MTERF4) is a transcription factor involved in mitochondrial ribosomal biogenesis and was identified as a gene of interest via whole exome sequencing in a pediatric patient with hypertrophic cardiomyopathy (HCM) at Children's Hospital of Wisconsin. The two key variants of interest (VOI) identified in MTERF4 were a premature stop in one allele and a nonsynonymous point mutation on the second allele. The patient's specific variants in MTERF4 have not been previously reported in the literature or associated to HCM. An existing MTERF4 mouse model by Cámara et al. (Cámara et al, 2011. PMID: 21531335) has shown that a global knock out (KO) of MTERF4 resulted in embryonic lethality at developmental stage E8.5 and that a heart‐specific conditional KO of MTERF4 resulted in mitochondrial cardiomyopathy. Here, we combined precision genome editing via CRISPR/Cas9 system with human iPSC‐derived cardiomyocytes (hiPSC‐derived CMs) to model the HCM patient‐specific MTERF4 variants to determine if they contribute to the patient's HCM phenotype. We hypothesize that MTERF4 variants are contributing to cardiomyocyte impairment, leading to the development of the hypertrophic phenotype. To overcome the low frequency homology‐directed repair (HDR) events in hiPSCs, we are using co‐targeting with selection (Mitzelfelt et al., 2017.PMID: 28238794), a method that improves our efficiency of CRISPR/Cas9 genome editing in these cells. Three MTERF4 mutant clones have been identified by Sanger sequencing, were assessed for pluripotency using immunostaining for SSEA‐4 and Oct4, and qRT‐PCR was done using Oct4, GATA4, and SOX2 primers. Three hiPSCs clones that have undergone our co‐targeting protocol, but were unmodified by CRISPR/Cas9, serve as controls for the MTERF4 mutant clones. MTERF4 clones and controls were subjected to our modified Palecek matrix‐overlay method for CM differentiation and assessed at 3–4 weeks. The hiPSC‐derived CMs cell size analysis by average pixel area of the MTERF4 mutant indicated that they are significantly (p=0.0012) larger compared to MTERF4 control. The assessment of mitochondrial function demonstrated that the maximal mitochondrial respiration may be reduced in MTERF4 mutant hiPSC‐derived CMs compared to control (p=0.056). In the cardiovascular disease qRT‐PCR array data, gene expression associated with HCM (NPPA, NPPB, GATA4, TNNT, MYL7, MYH7) were elevated. Preliminary data support our hypothesis through changes in CMs size, mitochondrial function, and transcriptional expression for one MTERF4 mutant clone and control. This approach has generated an in vitro tool to evaluate aspects of diseases, such as cardiomyopathy that can be used for diagnostic screening and therapy. Repeating our initial studies, and adding mechanistic studies, with all three MTERF4 mutant and control clones will further validate our hypothesis.Support or Funding InformationKECK foundation DP2‐OD08396This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.