Generation of a Patient-Derived Inducible Pluripotent Stem Cell Model for Studying the YARS2-Related Genetic Disease

Abstract

The YARS2 variant, harboring the compound heterozygous pathogenic mutations F185L/E264del, was identified in the gene for mitochondrial tyrosyl-tRNA synthetase in a proband that suffered a neonatal phenotype. To facilitate studies to better understand the severity of the mutations, we created a patient-derived inducible pluripotent stem cell (iPSC) model. We first derived iPSCs from fibroblasts of the patient Q1818, which contain two mutations, c.553T>C (p.F185L) and c.792_794delAGA (p.E264del) in the YARS2 gene. We then generated three isogenic control iPSC lines with one or both mutations corrected by using CRISPR-Cas9 technology. The correction of mutations in YARS2 was confirmed by Sanger sequencing. The stemness of iPSC lines was demonstrated by the expression of stem cell markers in the iPSCs, as determined using qPCR, immunostaining, and trilineage differentiation. Moreover, three positive clones of each iPSC line were extensively characterized, confirming that they originated from Q1818 fibroblasts, had normal karyotypes, and did not contain off-targets in the YARS2 coding sequence; genome wide off target effects were not a major concern. Subsequently, Q1818 iPSCs and the three isogenic control iPSCs were differentiated into clinically relevant motor neurons. In addition, we demonstrated that the patient fibroblasts and the derived iPSCs are heterozygous for either c. 553T>C or c.792_794delAGA, and that the two mutations are located on different alleles of the YARS2 gene, providing critical information for studying the mutation-associated disease. In conclusion, we have generated a set of four iPSC lines, which can be used as a model to study a clinically severe case of YARS2 disease.