Background

CL/P is a serious congenital birth defect happened in about 1.7 per 1000 liveborn babies [1]. It has become one of the most common developmental malformations in the world. In addition to facial deformities, functions including mastication, swallowing, pronunciation and respiration of patients would be affected to varying degrees, and even psychological development could be abnormal. Approximately 70% of CL/P occur as an isolated abnormality which was called NSCL/P [2].

CL/P is a disorder caused by the combination of genes and environmental interactions. At present, a variety of genetic pathogenic factors of CL/P are identified, including chromosome 8q24.21, 17q22 and 10q25.3, TGFα, TP63, IRF6, Runx2, etc. through different methods such as CMA, WES and so on [3,4,5,6,7,8]. However, the specific pathogenesis of NSCL/P is still unclear. Among these reported genes, TP63 is a key factor in the regulation of epithelial cell development, encoding a large number of p63 subtypes. It has been reported that the variation of TP63 could affect the binding ability of protein to DNA. Thomason et al. showed that the loss of TP63 function led to the phenotype of CL/P in mice [9].

In this study, we report the clinical findings detected in a Chinese pedigree (Fig. 1). Cytogenetic analysis and CMA of the pedigree members were normal. The proband was diagnosed by WES to be heterozygous for a C>T point mutation at nucleotide position 1324 (1324C>T) of TP63. The mutation was confirmed by sanger sequencing and it was not reported before.

Fig. 1
figure 1

Pedigree of the family. I1: grandmother of the proband with phenotype of CL/P. II2: uncle of the proband with phenotype of CL/P. III4: the proband with the phenotype of CL/P. III2 and III3: aborted fetuses with the phenotype of CL/P. III5: fetus of the proband’s mother with no CL/P phenotype. I2, II1, II3, II4 and III1: members of the pedigree with no CL/P phenotype

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Case presentation

The proband is a 3-year-old boy with healthy parents. He was full-term vaginally delivered with a birth weight of 3450 g and a length of 50 cm. Ultrasound examination revealed the signs of cleft lip and cleft palate during pregnancy of the proband's mother. Repair of cleft lip was performed 4 months after birth and palatoplasty was performed at 18 months old. At present, the proband's development and intelligence are normal without other deformities. The mother of the proband had a history of two terminations of pregnancy because of cleft lip and cleft palate of the fetus. The proband's uncle and grandmother both have the phenotype of cleft lip and cleft palate.

Cytogenetic analysis and CMA of the proband (Fig. 2) and other family members of the pedigree (data not shown) were normal. Results of WES analysis showed that the proband, the proband’s mother, uncle and grandmother carried the same c.1324C>T (p.Q442*) mutation in TP63 gene, possibly producing a truncated protein (Table 1). However, this mutation was absent in other family members as shown in the pedigree (Fig. 1) as well as healthy population controls. This c.1324C>T (p.Q442*) mutation of TP63 was not reported in literature before and also not found in NCBI dbSNP, genome AD, ExAC, and 1000 human genome dataset. Confirmation of Sanger sequencing was consistent with that of the WES (Fig. 3). In summary, this mutation can be interpreted as Likely Pathogenic according to ACMG guidelines [11].

Fig. 2
figure 2

The results of cytogenetic analysis (a) and CMA (b) of the proband were normal

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Table 1 Genotype of the six family members
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Fig. 3
figure 3

The mutation c.1324C>T in TP63 gene of the proband (a), the grandmother (b), the uncle (c), and the mother (d) was detected by WES and confirmed by sanger sequencing

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Discussion and conclusion

All patients with the phenotype of CL/P had the same mutation site of TP63 in this pedigree. It has been reported that mutations in TP63 underlie several monogenic malformation syndromes manifesting CL/P including Rapp-Hodgkin syndrome, ankyloblepharon-ectodermal dysplasia-cleft lip/palate syndrome, ectrodactyly-ectodermal dysplasia-cleft lip/palate syndrome, limb-mammary syndrome, and acro-dermato-ungual-lacrimal-tooth syndrome [12]. Leoyklang et al. investigated the possibility of TP63 mutation causing NSCL/P [2]. Variation of TP63 was revealed to affect the binding ability of protein to DNA. Animal experiments in mice have found that loss of TP63 function led to the phenotype of CL/P [9]. Study of Khandelwal et al. revealed that 6 of TP63 heterozygous variants detected in CL/P patients were inherited from an unaffected parent, suggesting reduced penetrance of such loss-of-function alleles. Incomplete penetrance has also been observed for other TP63 variants, including common variants such as those affecting arginine 319 (280accordingtoformernomenclature) [13]. Therefore, it explained why the mother of the proband did not show the phenotype of CL/P, although she carried the same mutation in TP63. Clinical variability and reduced penetrance are typical for TP63 variants.

TP63 plays an important role in the regulation of limb, epithelial, and craniofacial development [14]. This gene has at least six different isoforms, three of which containing the transactivating (TA) domain (TA isotypes) and the other three, none (△N isotypes). Alternative splicing at the 3′ end of the gene results in mainly three different C-terminal ends (α, β and γ). All isoforms contain a DNA-binding domain (DBD) and an OD, differing in the TA domain, sterilealpha-motif domain (SAM), auto-regulatory TA inhibiting domain (TI domain), and a second TA domain (TA2 domain) [15]. Kantaputra et al. implied that mutation in SAM domain of TP63 is associated with NSCL/P [16]. Khandelwal’s study indicated that missense variants affecting the OD might impair the capacity of p63 oligomerization [13]. The 1324C>T mutation of TP63 in the pedigree is a nonsense mutation. The amino acid at 442 of TP63 was changed from glutamine to a termination codon (p.Q442 *), leading to the premature termination of peptide chain synthesis. TP63 mutation discussed in this report was localized in the OD (Fig. 4). This variant may abrogate the oligomerization of mutant p63 protein into oligomeric complexes, resulting in the phenotype of CL/P. Therefore, c.1324C>T variant of TP63 likely represents a loss-of-function allele rather than a dominant-negative pattern [13]. The frequency of the mutation was not included in the NCBI dbSNP, genome AD, ExAC and 1000 Genomes databases. We speculate that the mutations of the TP63 gene (c.1324C>T) found in the Chinese family may be responsible for the phenotype of NSCL/P.

Fig. 4
figure 4

TA- and DN-p63 isoforms derived from alternative promoters

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Although a variety of mutations in TP63 have been reported in patients with SCL/P or NSCL/P, in-depth studies are needed to explore the specific pathogenesis. Leoyklang et al. recognized that a mutation in TP63 causing NSCL/P may occur at a site different from those underlying mendelian malformation syndromes. The phenotypic differences depend on variability of TP63 expression (2). This report demonstrates that this novel 1324C>T mutation is associated with NSCL/P, which highlights the wide phenotypic spectrum of TP63 mutations.

Our study not only helps to reveal the pathogenic gene of the pedigree but also provides the research basis for the molecular mechanism of TP63 gene causing the phenotype of NSCL/P.