Deletions and loss-of-function variants in TP63 associated with orofacial clefting

Abstract

We aimed to identify novel deletions and variants of TP63 associated with orofacial clefting (OFC). Copy number variants were assessed in three OFC families using microarray analysis. Subsequently, we analyzed TP63 in a cohort of 1072 individuals affected with OFC and 706 population-based controls using molecular inversion probes (MIPs). We identified partial deletions of TP63 in individuals from three families affected with OFC. In the OFC cohort, we identified several TP63 variants predicting to cause loss-of-function alleles, including a frameshift variant c.569_576del (p.(Ala190Aspfs*5)) and a nonsense variant c.997C>T (p.(Gln333*)) that introduces a premature stop codon in the DNA-binding domain. In addition, we identified the first missense variants in the oligomerization domain c.1213G>A (p.(Val405Met)), which occurred in individuals with OFC. This variant was shown to abrogate oligomerization of mutant p63 protein into oligomeric complexes, and therefore likely represents a loss-of-function allele rather than a dominant-negative. All of these variants were inherited from an unaffected parent, suggesting reduced penetrance of such loss-of-function alleles. Our data indicate that loss-of-function alleles in TP63 can also give rise to OFC as the main phenotype. We have uncovered the dosage-dependent functions of p63, which were previously rejected.

Fig. 1

Schematic representation of the deletions and point variants in TP63 identified in affected individuals and controls. The upper part of the figure depicts the complete deletions and the genes covered by deletions in the three families, W11–4934, W12–0831, and W16–065, respectively. The middle part of the figure presents a zoom-in image of the TP63 gene depicting the deletions in the families at the exon level and the splicing routes for various isoforms. The lower part of the figure shows the protein structure highlighting the various domains between different isoforms, with the variants in affected individuals and controls indicated

Fig. 2

Pedigree structures of the three orofacial clefting (OFC) families carrying a deletion affecting the TP63 gene. Black symbols indicate those individuals who are clinically affected with OFC and/or other features of ectodermal dysplasia. A complete overview of clinical features for each individual is provided in Table 1. Individuals used for SNP microarray analysis are indicated by the arrow. Individuals with underlined identifiers were tested by quantitative PCR (qPCR) (Table S2) for the presence of the deletion, which was confirmed in all cases

Fig. 3

Melting points of different tetramerization domain mutants and wild type. The melting point of the wild-type p63 tetramerization domain (blue line) as well as the p.Val405Met (orange line) and the p.Arg408His (gray line) variants was determined by a thermal shift assay. Due to the extraordinary stability of the tetramerization domains, the pH had to be shifted to 4.5 to ensure melting in a temperature range, which is accessible to this assay. Under these conditions, the measured melting temperatures were 85.6 ± 0.1 °C for the wild-type tetramerization domain, 51.6 ± 2.5 °C for the p.Val405Met variant, and 82.1 ± 0.1 °C for the p.Arg407His variant. The reduction in stability of the p.Val405Met variant is also reflected in a shift of ΔNp63α from a clear tetramer in wild-type protein towards a dimer-tetramer mix in the p.Val405Met variant on analytical SEC

Fig. 4

Transient transfection assay (luciferase induction) for the oligomerization domain (OD) variants. The fold inductions for the different mutant constructs are calculated relative to the WT isoform. p.Arg343Trp, an EEC causing mutant was used as control. p.Val405Met and p.Arg408His are the two OD variants tested. p.Arg352Gly, the only variant previously found responsible for nsOFC was also tested. The data are represented as the mean ± SEM

Fig. 5

Model explaining the functional effects of different kind of variants in p63 on the transactivation (TA) activity. The normal complex with all wild-type p63 molecules leads to regular level of TA. A heterozygous dominant-negative variant in TP63 will lead to half mutant copies of p63 molecules. Theoretically, 6.25% of the total tetrameric complexes will be formed by all wild-type monomer, which will have normal TA activity. The rest of the complexes will have at least one (or more) mutant monomer, which will block TA. The loss-of-function variants will lead to production of only half the total wild-type copies of p63 molecules. With only half the protein molecules, complexes formed will be normal but 50% less abundant. The heterozygous oligomerization domain (OD) variants will produce only half normal copy of the protein, leaving the other half incapable to oligomerize. As a result, there will be normal protein complexes but they will be almost half in number. These OD variants will therefore lead to partial loss-of-function