p63 Gene mutations in eec syndrome, limb-mammary syndrome, and isolated split hand-split foot malformation suggest a genotype-phenotype correlation

Abstract

p63 mutations have been associated with EEC syndrome (ectrodactyly, ectodermal dysplasia, and cleft lip/palate), as well as with nonsyndromic split hand-split foot malformation (SHFM). We performed p63 mutation analysis in a sample of 43 individuals and families affected with EEC syndrome, in 35 individuals affected with SHFM, and in three families with the EEC-like condition limb-mammary syndrome (LMS), which is characterized by ectrodactyly, cleft palate, and mammary-gland abnormalities. The results differed for these three conditions. p63 gene mutations were detected in almost all (40/43) individuals affected with EEC syndrome. Apart from a frameshift mutation in exon 13, all other EEC mutations were missense, predominantly involving codons 204, 227, 279, 280, and 304. In contrast, p63 mutations were detected in only a small proportion (4/35) of patients with isolated SHFM. p63 mutations in SHFM included three novel mutations: a missense mutation (K193E), a nonsense mutation (Q634X), and a mutation in the 3' splice site for exon 5. The fourth SHFM mutation (R280H) in this series was also found in a patient with classical EEC syndrome, suggesting partial overlap between the EEC and SHFM mutational spectra. The original family with LMS (van Bokhoven et al. 1999) had no detectable p63 mutation, although it clearly localizes to the p63 locus in 3q27. In two other small kindreds affected with LMS, frameshift mutations were detected in exons 13 and 14, respectively. The combined data show that p63 is the major gene for EEC syndrome, and that it makes a modest contribution to SHFM. There appears to be a genotype-phenotype correlation, in that there is a specific pattern of missense mutations in EEC syndrome that are not generally found in SHFM or LMS.

Figure 1

Distribution of p63 gene mutations in EEC syndrome, SHFM, and LMS. Except for one frameshift mutation in exon 13, all mutations in EEC syndrome are amino acid substitutions in the DNA-binding domain that are predicted to abrogate interaction with the DNA and, hence, reduce transactivation activity. LMS mutations are frameshifts in exons 13 and 14. The EEC syndrome patient with the exon 13 frameshift mutation also has clinical characteristics of LMS. The mutations identified in patients with SHFM are distributed along the gene. Substitution of arginine 280 may give rise to either classical EEC syndrome or SHFM.

Figure 2

Predicted effect of the splice-site mutation in the p63 gene. a, Predicted consequence of the 3′ splice-site mutation. The strictly conserved adenosine of the acceptor splice site has been substituted by a cytosine. An excellent alternative 3′ splice site is located just three nucleotides in front of the original one. Splicing at this alternative site results in an insertion of one proline residue for the mutant allele. The adherence to the 3′ splice-site consensus sequence was calculated according to the method of Shapiro and Senapathy (1987). b, The IVS4-2A→C mutation gives rise to alternative splicing. Sequence analysis of the RT-PCR product obtained in an exon-trapping assay in which COS-1 cells were transfected with either a wild-type construct (top panel) or a construct containing the splice-site mutation (bottom panel). The reverse complement sequence is shown. The mutation results in incorporation of an additional proline codon in the transcript.

Figure 3

Partial karyotype of a patient with a terminal deletion of the long arm of chromosome 3 [46,XX,del(3)(q27→qter)]. The patient has multiple congenital abnormalities, but none of the symptoms is typical of EEC syndrome (Chitayat et al. 1996). Shown is the staining of a typical cell after FISH. Only the nondeleted chromosome 3 shows staining of the YAC containing the p63 gene (closed arrow), whereas both chromosomes 3 show centromeric staining (open arrow).