Impaired activity of the fusogenic micropeptide Myomixer causes myopathy resembling Carey-Fineman-Ziter syndrome
Andres Ramirez-Martinez, Yichi Zhang, Marie-Jose van den Boogaard, John R. McAnally, Cristina Rodriguez-Caycedo, Andreas C. Chai, Francesco Chemello, Maarten P.G. Massink, Inge Cuppen, Martin G. Elferink, Robert J.J. van Es, Nard G. Janssen, Linda P.A.M. Walraven-van Oijen, Ning Liu, Rhonda Bassel-Duby, Richard H. van Jaarsveld, Eric N. Olson
Andres Ramirez-Martinez, Yichi Zhang, Marie-Jose van den Boogaard, John R. McAnally, Cristina Rodriguez-Caycedo, Andreas C. Chai, Francesco Chemello, Maarten P.G. Massink, Inge Cuppen, Martin G. Elferink, Robert J.J. van Es, Nard G. Janssen, Linda P.A.M. Walraven-van Oijen, Ning Liu, Rhonda Bassel-Duby, Richard H. van Jaarsveld, Eric N. Olson
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Research Article Muscle biology

Impaired activity of the fusogenic micropeptide Myomixer causes myopathy resembling Carey-Fineman-Ziter syndrome

Abstract

Skeletal muscle fibers contain hundreds of nuclei, which increase the overall transcriptional activity of the tissue and perform specialized functions. Multinucleation occurs through myoblast fusion, mediated by the muscle fusogens Myomaker (MYMK) and Myomixer (MYMX). We describe a human pedigree harboring a recessive truncating variant of the MYMX gene that eliminates an evolutionarily conserved extracellular hydrophobic domain of MYMX, thereby impairing fusogenic activity. Homozygosity of this human variant resulted in a spectrum of abnormalities that mimicked the clinical presentation of Carey-Fineman-Ziter syndrome (CFZS), caused by hypomorphic MYMK variants. Myoblasts generated from patient-derived induced pluripotent stem cells displayed defective fusion, and mice bearing the human MYMX variant died perinatally due to muscle abnormalities. In vitro assays showed that the human MYMX variant conferred minimal cell-cell fusogenicity, which could be restored with CRISPR/Cas9–mediated base editing, thus providing therapeutic potential for this disorder. Our findings identify MYMX as a recessive, monogenic human disease gene involved in CFZS, and provide new insights into the contribution of myoblast fusion to neuromuscular diseases.

Authors

Andres Ramirez-Martinez, Yichi Zhang, Marie-Jose van den Boogaard, John R. McAnally, Cristina Rodriguez-Caycedo, Andreas C. Chai, Francesco Chemello, Maarten P.G. Massink, Inge Cuppen, Martin G. Elferink, Robert J.J. van Es, Nard G. Janssen, Linda P.A.M. Walraven-van Oijen, Ning Liu, Rhonda Bassel-Duby, Richard H. van Jaarsveld, Eric N. Olson

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Figure 3

Modeling the MYMX R46* variant in skeletal muscle cells differentiated from patient-derived iPSCs.

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Modeling the MYMX R46* variant in skeletal muscle cells differentiated f...
(A) Gingival fibroblasts from a male patient homozygous for the MYMX R46* variant were used to generate iPSCs, which were then induced to differentiate into skeletal muscle cells. To generate isogenic control cells, adenine base editing was used to edit T to C to obtain the wild-type genomic sequence in iPSCs. (B) Genomic sequence surrounding codon 46 of the human MYMX locus harboring the MYMX R46* variant (indicated by a black asterisk). The sequence of the sgRNA used for adenine base editing is shown, along with the PAM sequence. Adenine base editing results in conversion of the variant T to a C (T12, shown in red) and restoration of the open reading frame. The base editing window is shown in orange. A possible bystander nucleotide (T10) for adenine base editing is shown in blue. (C) Percentage of T-to-C base editing at T10 and T12 as determined by EditR analysis. (D) MYMX WT (edited) and MYMX R46*/R46* iPSC-derived skeletal muscle cells were stained for myosin heavy chain expression by My32 antibody and for nuclei with Hoechst 33342. Fusion was impaired in MYMX R46*/R46* muscle cells (bottom), whereas edited myoblasts formed multinucleated myotubes (top). Scale bar: 50 μm. (E) Expression of myogenic factors (MYOD1, MYOG), desmin (DES), and muscle fusogens (MYMX, MYMK) in iPSC-derived skeletal muscle cells as detected by qRT-PCR. n = 3 replicates per group. Statistical comparisons between groups were evaluated by unpaired and 2-sided Student’s t test. *P < 0.05. Error is expressed as SEM.