Molecular characterization of a rare TP63 variant associated with split-hand/split-foot malformation 4 and incomplete penetrance: disruption of the p63-Dlx signaling pathway.

Background: Split-hand/foot malformation (SHFM) is a congenital disability characterized by the absence or hypoplasia of the central ray of the hands and/or feet. This study reports a causative variant in the TP63 gene in a Chinese family exhibiting limb anomalies associated with SHFM4.

Methods: Enrolled in this study was a Chinese family with limb anomalies without any other clinical features. Karyotype analysis and chromosomal microarray analysis (CMA) were conducted to identify chromosomal abnormalities. Whole exome sequencing (WES) was utilized to investigate sequence variants, while RNA sequencing assessed differentially expressed genes, with findings confirmed through quantitative PCR (qPCR).

Results: Karyotype analysis and CMA revealed no chromosomal abnormalities in the family. Subsequently, WES identified a rare heterozygous variant of NM_003722.5: c.956G > A (p.Arg319His) in the TP63 gene in the proband, which was inherited from her father who also presented with limb deformities. However, both of the sister and grandfather of the proband had the same variant but exhibited normal limb morphology. RNA sequencing results demonstrated an increased expression level of TP63 and its downstream genes (PERP, CDH3, and DLX5) compared with the controls, indicating an enrichment of cell adhesion molecules the differentially expressed genes in the patient. However, significant differences were noted only for the CDH3 and DLX5 genes in qPCR analysis (p<0.05).

Conclusion: This study identifies, for the first time, the TP63 gene variant c.956G > A (p.Arg319His) as a causative factor for SHFM4 in Chinese individuals with incomplete penetrance. In addition, we hypothesize that the p.Arg319His variant functions as a gain-of-function variant, leading to the upregulation of cell adhesion target genes. Such upregulation then disrupts the p63-Dlx signaling pathway and causes AER stratification failure.