All patients with the phenotype of SHFM had the same variant of TP63 in this pedigree. It has been reported that heterozygous pathogenic variants in TP63 are associated with a total of seven diseases: ectrodactyly, ectodermal dysplasia, and cleft lip/palate syndrome 3 (EEC3; OMIM #604292); split-hand/foot malformation 4 (SHFM4; OMIM #605289); ankyloblepharon-ectodermal defects-clefting syndrome (AEC; OMIM #106260); acro-dermato-ungual-lacrimal-tooth syndrome (ADULT syndrome; OMIM #103285); limb-mammary syndrome (LMS; OMIM #603543); Rapp-Hodgkin syndrome (RHS; OMIM #129400); and orofacial cleft 8 (OFC8; OMIM #618149) [11]. The phenotypes of TP63-related diseases vary greatly; indeed, phenotypic variability within each disease is also considerable. In cases of SHFM, the hands and feet often exhibit ectrodactyly, syndactyly, a central cleft, and aplasia of the phalanges, metacarpals, and metatarsals. Although SHFM is predominantly syndromic, it can be isolated; in these cases, it is called SHFM4, referring to a “pure” limb dysplasia without other malformations. The patients in our study showed features consistent with this condition and were therefore classified as having SHFM4.
Variants in multiple regions of the TP63 gene are responsible for these conditions and affect the function of p63 in different ways. There is a clear genotype–phenotype correlation for TP63 gene variants. In EEC3 and AEC syndromes, variants are clustered in the DNA binding domain (DBD), the sterile alpha motif, and/or the transactivation inhibitory domains. Variants causing SHFM4 are distributed throughout the TP63 gene, including the transactivation and transactivation inhibitory domains, the splice site, and the DBD [8, 26, 27]; however, it remains unclear how these widely dispersed variants cause limb defects. Some variants in SHFM4 have been reported to alter the activation and stability of p63 [28, 29]. It is possible that SHFM is caused by altered p63 degradation, although different protein degradation pathways are involved [10, 28, 30]. The altered residues in the disease-causing alleles of SHFM4 appear to maintain the overall domain structure, unlike those that cause EEC3, which directly interact with DNA.
The variant in our patient (c.921G > T; p.Met307Ile) lies within the DBD of p63 (Fig. 3), which is responsible for establishing DNA interactions [8, 31]. The variant identified here is present on all protein coding isoforms. This residue is highly conserved across species (Fig. 2a). According to Grantham scores, the physicochemical differences between methionine and isoleucine are small; this new variant may cannot affect the structure of the DBD of p63. Nevertheless, this novel variant was predicted to would be disease-causing in our study. Importantly, 3us0 structure of p63 DBD is complex with a 22 base pair A/T rich response element. This structure can check the effect of the variant. The variant affects DNA binding or protein interaction ability of TP63. This may, in turn, affect the formation and differentiation of the AER, possibly leading to limb dysplasia.
Of the 148 variants in TP63 identified to date (Fig. 3), more than 110 are associated with EEC3 and/or AEC syndrome [32, 33] and only 15 have been implicated in SHFM4 [12]. Despite the high phenotypic heterogeneity of SHFM, there is no clear literature or database reporting the proportion of TP63 variants. Additionally, there is no clear report of TP63 penetrance, although incomplete penetrance is reported for many diseases involving this gene, including SHFM4 [8, 34,35,36,37]. The incomplete penetrance in the grandmother (II-4) stays in line with similar reported cases.
Incomplete penetrance of phenotypic features and wide clinical variability are often seen within families sharing the same TP63 variant. Therefore, it is possible that some affected individuals have mild or nearly undetectable abnormalities [38]. Co-segregation analysis revealed, that the variant was present in 4 individuals of the examined family of whom only 3 were affected with the SHFM4. Despite carrying the gene variant, II-4 showed no symptoms of SHFM4, indicating that this variant may have incomplete penetrance. Incomplete penetrance for SHFM4 has been reported [8, 36]. Incomplete penetrance has also been observed for other TP63-related disorders, including ADULT syndrome [34, 35], EEC3 [8], and orofacial cleft 8 [37, 39]. p63 knockout mice(p63−/−) have striking developmental defects, and heterozygous p63 knockout mice (p63+/−) do not display ectodermal defects [11, 40]. However, heterozygous p63+/EEC mice generated by knocking in the R279H allele rarely display ectrodactyly [41], whereas p63EEC/EEC mice resemble p63−/− mice. The development of EEC in patients carrying this mutated allele [12] indicates that mouse models react somewhat differently from humans to disturbed balances of wild-type versus mutated p63 molecules. This difference in response could be due to differences in the stability of the mutated mouse and human p63 proteins [42]. There was not incomplete penetrance reported in mouse TP63 model.
Clinical spectrum of the SHFM differs from patient to patient in severity and even varies among individuals in the same family [3, 43]. The proband has bilateral SHFM while his mother and great uncle have unilateral split hand. The phenotypic variability in this family was consistent with those reported in previous studies [8, 36]. This variable phenotype within family suggests the involvement of a modifier allele that contribute the complete penetrance and full expression of the phenotype in the proband. Perhaps a polymorphism within the TP63 gene itself is responsible for this effect [44]. Just as reported, the absence of ectodermal phenotypes in patients segregating previously reported variant in TP63 highlights the importance of modifier genes causing variations in TP63-related SHFM phenotypes [45]. The phenotypic differences depend on variability of TP63 expression [46]. Further research is warranted to determine whether these findings are attributable to epigenetic processes or to genetic modifiers. In-depth studies are needed to explore the specific pathogenesis.
SHFM4 is inherited in an autosomal dominant manner. Each child of an individual with SHFM4 has a 50% chance of inheriting the pathogenic variant. In the absence of functional data, a variant with incomplete penetrance identified in this study should be interpreted cautiously for prenatal diagnosis. It is therefore reasonable to recommend that pregnancy monitoring by non-invasive ultrasound should be offered to all first degree relatives of an individual with the variant. Prenatal diagnosis for pregnancies diagnosed SHFM by ultrasound is recommended for molecular genetic testing of TP63 [47]. Differences in perspective may exist among medical professionals and within families regarding the use of prenatal testing, particularly if the testing is being considered for the purpose of pregnancy termination rather than early diagnosis. While most centers would consider decisions regarding prenatal testing to be the choice of the parents, discussion of these issues is appropriate. It is appropriate to offer genetic counseling (including discussion of potential risks to offspring and reproductive options) to young adults who are affected or at risk [47].
