PMC9770794_01
Identified mutation locus of ZNF462 in the patient. (A) Locus of the adenosine insertion in ZNF462 and the exome sequencing reads at the locus. The reads marked with purple "I"s are those with the adenosine insertions. (B) Insertion of adenosine occurs in the seventh exon of ZNF462 (NM_021224.6). (C-E) Sanger sequencing results of the patient and his parents. Red ovals indicate the insertion site of adenosine. (F) Amino acid sequences of ZNF462 without (up) and with the adenosine insertion (down). In the mutated ZNF462 amino acid sequence, the adenosine insertion causes a variation from Val to Gly and different subsequent amino acids, and introduces premature translation terminations. (G) When compared with the dbSNP (r153) in the UCSC Genome Browser, the identified insertion site of adenosine was not a known SNP, as indicated by the red rectangle.
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Figure 1A shows the high-throughput sequencing reads for the seventh exon of the patient's ZNF462 gene, where 54% of these sequences carried the A insertion (or duplication), suggesting a heterozygous mutation at the A insertion site in the patient. Using GATK to analyze the exon sequencing data revealed an insertion of adenosine at the seventh exon of the ZNF462 gene [NM_021224.6: c.6311dup; p. (Val2105GlyfsTer32)] (Figure 1B). Then, we sequenced the same genomic regions in the patient and his parents with Sanger sequencing (Figures 1C-E). In the Sanger sequencing results for the patient, there were continuous secondary peaks from the insertion site of adenosine (red oval in Figure 1C), suggesting two different sequences for the heterozygous insertion site of adenosine. However, as shown in Figures 1D,E, the Sanger sequencing of the mother and father clearly indicated no mutations. As shown in Figure 1F, due to a code shift introduced by the additional adenosine, valine (Val) at the 2105th amino acid was transformed into glycine (Gly); the subsequent 32 amino acids were encoded incorrectly and terminated prematurely. The mutation (NM_021224.6:c.6311dup) was not a reported SNP after being compared to dbSNP (v153) Figure 1G.
Proteins interacting with ZNF462 and mutations in ZNF462. (A) Proteins interacting with ZNF462. STRING (v 11.5) was used to prepare the figure. (B) Enriched GO terms for genes in (A). Only top GO terms were shown, and details are provided in Materials and Methods. (C) Reported mutations in ZMF462 that are related to WSKA. The blue mutation is the one newly identified in this study. See Supplementary Table S1 for details. (D) Diseases and symptoms caused by genetic mutations in ZNF462, VPS13B, and ASXL2. Common symptoms of at least two diseases are shown in blue.
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We next examined the interacting proteins of ZNF462 with STRING (v11.5), as shown in Figure 2A. The top GO terms for these genes are "polyubiquitin modification-dependent protein binding," "RNA polymerase II complex binding," "proteasome binding," "metal ion-binding," "peroxisome proliferator-activated receptor binding," "3'-5' exonuclease activity," "DNA binding," and "RNA polymerase II intronic transcription regulatory region sequence-specific DNA binding," as shown in Figure 2B. We also manually retrieved the previously reported ZNF462 mutations related to WSKA, then visualized them with the mutation newly identified in this study (Figure 2C). ASXL2 gene could lead to Shashi-Pena syndrome, which is characterized by facial abnormalities and developmental delays (Figure 2D). ASXL2-deficient mice showed weight loss, enlarged hearts, and skeletal abnormalities. VPS13B is another protein interacting with ZNF462 (Figure 2A). Mutations in the VPS13B gene have been associated with Cohen's syndrome, a rare autosomal recessive syndrome associated with a variety of clinical manifestations including growth retardation, hypotonia, joint hyperactivity, microcephaly, intellectual impairment, and craniofacial and limb abnormalities (Figure 2D). VAX1 interacts with both ZNF462 and ASXL2 (Figure 2A). WSKA (Figure 2D). We hypothesize that ZNF462 and its interacting proteins may form a protein complex that is important for normal development and which awaits more studies to reveal its detailed functions.