CMT2W patients carrying the P134H mutation do not have a defect in tRNA aminoacylation. (A, Left) Western blot analysis of 4 PBMC-derived lymphoblast samples from 1 family: 2 isolated from CMT2W patients (CMT2W 1 and 2) and 2 from healthy members (control 1 and 2). (A, Right) Normalized relative levels of HisRS protein among all family members. (B, Upper) Northern blot analysis of total RNA extracted from PBMC-derived lymphoblast samples probed against nuclear-encoded tRNAHis, nuclear-encoded tRNAAla, and mitochondrial (mito)-encoded tRNAHis. Total RNA was loaded onto a denaturing sequencing gel to separate charged from uncharged tRNAs. deacyl, deacylated samples included as controls. (B, Lower) Charged fraction of investigated tRNAs was quantified as a ratio of charged-to-total tRNA. Quantification data are presented as mean ± SD (n = 2 biological replicates per group). (C) In vitro tRNA aminoacylation assay with lysates of PBMCs to detect HisRS activity. Quantification data are presented as mean ± SD (n = 3 technical replicates).