A novel de novo nonsense mutation in ZC4H2 causes Wieacker‐Wolff Syndrome

Figure 1. Schematic diagram of ZC4H2 with mutations. The structure of the human ZC4H2 gene is schematically represented. All known mutations are shown at the top of the picture, while the position where the chromosome is deleted or inverted is shown at the bottom of the figure. Nucleotide numbering designates the A of the translation start codon ATG as +1. The numbered boxes represent exon. The “/”sign stands for deletion, while “×”signs shows the break point area.“*” (c.199C>T) indicates the patient in the present study

Figure 2. Clinical features of the female patient. (a) Images of the patient at 3 days, 7 months, and 22 months after birth showed overlapping and camptodactyly of fingers, right club foot, short neck, narrow shoulder, and short toes. (b) At 10 days, brain MRI is normal, pelvic MRI at 2 months showed right hip dislocation, and X‐ray of limb at 3 months shows fixed contractures of fingers and vertical talus

Figure 3. Identification of a de novo nonsense mutation R67X in ZC4H2 gene. (a) Pedigree of the patient with ZC4H2 p.R67X mutation. (b) Sanger sequencing of ZC4H2 gene in genomic DNA from the proband and her father and mother. The c.199C>T (p. R67X) was found in the patient but not in her parents, suggesting that the mutation was de novo. (c) Schematic diagram of ZC4H2 gene structure with mRNA and protein products. Wild type and mutant ZC4H2 coding sequences surrounding the mutation were shown. (d) HEK293T cells were transfected with plasmids expressing either N‐terminal EGFP‐tagged wild‐type ZC4H2 or R67X mutant ZC4H2m. Anti‐GFP antibody was used to detect the N‐terminal EGFP‐tagged proteins by Western blot using 15%Tris‐Glycine SDS‐PAGE gel. β‐actin or β‐tubulin was used as control. (e) HEK293T cells were transiently transfected with plasmids expressing either empty vector, or C‐terminal Flag‐tagged wild‐type ZC4H2 and R67X mutant ZC4H2m. EGFP expression plasmid was co‐transfected for transfection efficiency control. Anti‐ZC4H2 antibody was used to detect the wildtype ZC4H2 protein. EGFP and β‐tubulin was used as control

Figure 4. Confocal microscopic analysis. HEK293T cells were transfected with plasmids expressing either the wild‐type ZC4H2 (e‐h) or mutant ZC4H2 c.199 C>T (p. R67X) protein (i‐l) with N‐terminally tagged EGFP. The empty EGFP vector was used as the negative control (a‐d). Phalloidin 555 and DAPI were used to stain the cytoskeleton and nucleus, respectively. Merged images of EGFP, DAPI, and phalloidin 555 are observed in d, h, and l. (m) An increased presence of wild‐type N‐EGFP‐ZC4H2 protein punctuate aggregated in the cytosol while the truncated protein is in the cytosol and nuclei at the same time, similar to EGFP protein only

Figure 5. X‐chromosome inactivation analysis of the patient with the R67X ZC4H2 mutation (proband). Amplification peaks of the smaller and larger CAG repeat alleles of the androgen receptor gene before (a) and after (b) digestion with the enzyme HhaI. The Y‐axis shows the amplification peak values corresponding to the number of PCR products of each allele. The amplification values of the smaller size allele (279 bp), undigested and digested, were 1,286 and 782, respectively. For the larger size allele (287 bp), undigested and digested amplification values were 605 and 2,884, respectively. The calculated X‐chromosome inactivation ratio was 22:78 according to the method described before

Figure 6. Protein structure by I‐TASSER modeling. (a) The secondary structural of ZC4H2 protein, helix are shown in red, and the coil is in black. “＊” represents the previous reported pointed, nonsense and frameshift mutations, and the present mutation is shown in blue “＊”. (b) The 3‐dimensional atomic model based on the protein sequence generated by the iterative threading assembly refinement server