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English: Figure 2. Characterization of the ZC4H2 de novo mutation (c.352C>T). (A) Identification of the de novo mutation with Sanger sequencing. The c.352C>T mutation in exon 3 of ZC4H2 was a de novo mutation identified only in the proband and not in her parents. (B) Analysis of X-chromosome inactivation. Two peaks of 262 and 294 bp were defined in the proband, indicating those inherited from her mother and father. The area under the peak inherited from the proband’s parents before enzyme digestion were 28,157 and 44,595, while those after enzyme digestion were 25,589 and 36,565, respectively. (C) Construction of the expression vectors. Wild-type and mutant proteins of ZC4H2 could be expressed in pCMV6-Flag-ZC4H2-wt and pCMV6-Flag-ZC4H2-mt c. 352C>T. The tagged protein (Flag) was inserted into the vectors, and the fusion protein was identified with antibodies targeting either Flag or ZC4H2. GFP was expressed by the normal control vector pLVHM-GFP, and therefore the transfection efficiency was determined by examining the fluorescence intensity. (D) Identification of relative expression levels of ZC4H2 by RT-qPCR. The relative expression levels of ZC4H2 were found to be 0.88 ± 0.058 and 0.91 ± 0.034, respectively. No significant differences were therefore identified between the two groups (p = 0.72). (E) Identification of the ZC4H2 protein in the 293T cells. Western blotting results showed that ZC4H2 and Flag could be detected in the cells transfected with pCMV6-Flag-ZC4H2-wt, whereas these were not seen in the cells transfected with pLVHM-GFP or pCMV6-Flag-ZC4H2-mt c. 352C>T. By contrast, β-actin was detected in all transfected cells.