p63 Gene Mutations in EEC Syndrome, Limb-Mammary Syndrome, and Isolated Split Hand–Split Foot Malformation Suggest a Genotype-Phenotype Correlation

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.     

A novel c.1037C > G (p.Ala346Gly) mutation in TP63 as cause of the ectrodactyly-ectodermal dysplasia and cleft lip/palate (EEC) syndrome

Ectrodactyly – ectodermal dysplasia and cleft lip/palate (EEC) syndrome (OMIM 604292) is a rare disorder determined by mutations in the TP63 gene. Most cases of EEC syndrome are associated to mutations in the DNA binding domain (DBD) region of the p63 protein. Here we report on a three-generation Brazilian family with three individuals (mother, son and grandfather) affected by EEC syndrome, determined by a novel mutation c.1037C > G (p.Ala346Gly). The disorder in this family exhibits a broad spectrum of phenotypes: two individuals were personally examined, one presenting the complete constellation of EEC syndrome manifestations and the other presenting an intermediate phenotype; the third affected, a deceased individual not examined personally and referred to by his daughter, exhibited only the split-hand/foot malformation (SHFM). Our findings contribute to elucidate the complex phenotype-genotype correlations in EEC syndrome and other related TP63-mutation syndromes. The possibility of the mutation c.1037C > G being related both to acro-dermato-ungual-lacrimal-tooth (ADULT) syndrome and SHFM is also raised by the findings here reported.     

Split-hand/split-foot malformation is caused by mutations in the p63 gene on 3q27

Split-hand/split-foot malformation (SHFM), a limb malformation involving the central rays of the autopod and presenting with syndactyly, median clefts of the hands and feet, and aplasia and/or hypoplasia of the phalanges, metacarpals, and metatarsals, is phenotypically analogous to the naturally occurring murine Dactylaplasia mutant (Dac). Results of recent studies have shown that, in heterozygous Dac embryos, the central segment of the apical ectodermal ridge (AER) degenerates, leaving the anterior and posterior segments intact; this finding suggests that localized failure of ridge maintenance activity is the fundamental developmental defect in Dac and, by inference, in SHFM. Results of gene-targeting studies have demonstrated that p63, a homologue of the cell-cycle regulator TP53, plays a critically important role in regulation of the formation and differentiation of the AER. Two missense mutations, 724A-->G, which predicts amino acid substitution K194E, and 982T-->C, which predicts amino acid substitution R280C, were identified in exons 5 and 7, respectively, of the p63 gene in two families with SHFM. Two additional mutations (279R-->H and 304R-->Q) were identified in families with EEC (ectrodactyly, ectodermal dysplasia, and facial cleft) syndrome. All four mutations are found in exons that fall within the DNA-binding domain of p63. The two amino acids mutated in the families with SHFM appear to be primarily involved in maintenance of the overall structure of the domain, in contrast to the p63 mutations responsible for EEC syndrome, which reside in amino acid residues that directly interact with the DNA.     

Heterozygous germline mutations in the p53 homolog p63 are the cause of EEC syndrome.

EEC syndrome is an autosomal dominant disorder characterized by ectrodactyly, ectodermal dysplasia, and facial clefts. We have mapped the genetic defect in several EEC syndrome families to a region of chromosome 3q27 previously implicated in the EEC-like disorder, limb mammary syndrome (LMS). Analysis of the p63 gene, a homolog of p53 located in the critical LMS/EEC interval, revealed heterozygous mutations in nine unrelated EEC families. Eight mutations result in amino acid substitutions that are predicted to abolish the DNA binding capacity of p63. The ninth is a frameshift mutation that affects the p63alpha, but not p63beta and p63gamma isotypes. Transactivation studies with these mutant p63 isotypes provide a molecular explanation for the dominant character of p63 mutations in EEC syndrome.     

Number of rare germline CNVs and <Emphasis Type="Italic">TP53</Emphasis> mutation types

Background

The Li-Fraumeni syndrome (LFS), an inherited rare cancer predisposition syndrome characterized by a variety of early-onset tumors, is caused by different highly penetrant germline mutations in the TP53 gene; each separate mutation has dissimilar functional and phenotypic effects, which partially clarifies the reported heterogeneity between LFS families. Increases in copy number variation (CNV) have been reported in TP53 mutated individuals, and are also postulated to contribute to LFS phenotypic variability. The Brazilian p.R337H TP53 mutation has particular functional and regulatory properties that differ from most other common LFS TP53 mutations, by conferring a strikingly milder phenotype.

Methods

We compared the CNV profiles of controls, and LFS individuals carrying either p.R337H or DNA binding domain (DBD) TP53 mutations by high resolution array-CGH.

Results

Although we did not find any significant difference in the frequency of CNVs between LFS patients and controls, our data indicated an increased proportion of rare CNVs per genome in patients carrying DBD mutations compared to both controls (p=0.0002***) and p.R337H (0.0156*) mutants.

Conclusions

The larger accumulation of rare CNVs in DBD mutants may contribute to the reported anticipation and severity of the syndrome; likewise the fact that p.R337H individuals do not present the same magnitude of rare CNV accumulation may also explain the maintenance of this mutation at relatively high frequency in some populations.

     

Splitting p63

Causative TP63 mutations have been identified in five distinct human developmental disorders that are characterized by various degrees of limb abnormalities, ectodermal dysplasia, and facial clefts. The distribution of mutations over the various p63 protein domains and the structural and functional implications of these mutations establish a clear genotype-phenotype correlation.     

Split-hand/foot malformation - molecular cause and implications in genetic counseling

Split-hand/foot malformation (SHFM) is a congenital limb defect affecting predominantly the central rays of the autopod and occurs either as an isolated trait or part of a multiple congenital anomaly syndrome. SHFM is usually sporadic, familial forms are uncommon. The condition is clinically and genetically heterogeneous and shows mostly autosomal dominant inheritance with variable expressivity and reduced penetrance. To date, seven chromosomal loci associated with isolated SHFM have been described, i.e., SHFM1 to 6 and SHFM/SHFLD. The autosomal dominant mode of inheritance is typical for SHFM1, SHFM3, SHFM4, SHFM5. Autosomal recessive and X-linked inheritance is very uncommon and have been noted only in a few families. Most of the known SHFM loci are associated with chromosomal rearrangements that involve small deletions or duplications of the human genome. In addition, three genes, i.e., TP63, WNT10B, and DLX5 are known to carry point mutations in patients affected by SHFM. In this review, we focus on the known molecular basis of isolated SHFM. We provide clinical and molecular information about each type of abnormality as well as discuss the underlying pathways and mechanism that contribute to their development. Recent progress in the understanding of SHFM pathogenesis currently allows for the identification of causative genetic changes in about 50 % of the patients affected by this condition. Therefore, we propose a diagnostic flow-chart helpful in the planning of molecular genetic tests aimed at identifying disease causing mutation. Finally, we address the issue of genetic counseling, which can be extremely difficult and challenging especially in sporadic SHFM cases.     

Molecular genetic characterization of a Korean split hand/split foot malformation (SHFM)

Split hand/split foot malformation (SHFM; ectrodactyly) is genetically heterogeneous, with mutations identified at five loci (SHFM1 at 7q21.3, SHFM2 at Xq26, SHFM3 at 10q24, SHFM4 at 3q27 and SHFM5 at 2q31). In this study, we attempted to identify and localize the causative allele of a Korean case of SHFM. Pedigree analysis showed that the Korean SHFM was autosomally dominant and its penetrance was high, indicating that it was not caused by SHFM2. Clinical features were variable, but limited to the four limbs unlike SHFM1, SHFM4 and SHFM5. G-banding and FISH failed to identify any chromosomal abnormalities. We also performed mutation screening by SSCP and DNA sequencing, as well as loss of heterozygosity (LOH) analysis, to exclude the possibility that SHFM4 or SHFM5 were involved; these revealed no mutations in gene p63 and no LOH on 2q31, respectively. It therefore appears that the Korean SHFM may be caused by mutation of SHFM3. In fact, linkage analysis using informative microsatellite markers indicated that SHFM3 was linked to D10S577 with a maximum LOD score of 1.15 at recombination fraction zero. Finally, we identified two novel alleles (191 and 211 bp) of D10S577 that have not been found in Western populations.     

Differentially Expressed Genes in EEC and LMS Syndromes

Objectives

Ectrodactyly ectodermal dysplasia cleft lip/palate (EEC) syndrome and limb-mammary syndrome (LMS) share a similar phenotype and the same pathogenic gene, which complicates the ability to distinguish between these diagnoses. The current study aims to identify a potential and practical clinical biomarker to distinguish EEC from LMS.

Methods

Two EEC pedigrees and one LMS pedigree that have been previously reported were reanalyzed. After confirmation of the causative mutations for these new patients, whole-genome expression microarray analysis was performed to assess the molecular genetic changes in these families.

Results

Five new patients with classic symptoms were reported, and these individuals exhibited the same mutation as their relatives (c.812 G>C; c.611G>A; and c.680G>A). According to the whole genome expression results, the EEC patients exhibited different gene expression characteristics compared with the LMS patients. More than 5,000 genes were differentially expressed (changes >2 or <0.5-fold) among the EEC patients, LMS patients and healthy individuals. The top three altered pathways have been implicated in apoptosis, the hematopoietic cell lineage and the Toll-like receptor signaling pathway.

Conclusion

Our results provide additional clinical and molecular information regarding EEC and LMS and suggest that peripheral blood cytokines may represent a promising clinical biomarker for the diagnosis of these syndromes.     

Genetic characteristics of the "EEC" syndrome (ectrodactyly, ectodermal dysplasis, cheilognathopalatoschisis)]

The analysis of the literature and author's observations of the "EEC" syndrome (ectrodactyly, ectodermal dysplasia and cleft lip/palate) revealed that this is a disorder with an autosomal-dominant type of inheritance with an incomplete penetrance and varying expressivity. Both sexes are affected with the same frequency. The complete form of the syndrome was mentioned in 27 cases only; all other patients had incomplete forms. The combination of two out of 3 main features is enough for the diagnosis of this syndrome. The most common trait of the "EEC" syndrome is ectrodactyly (73/77), clefts of lip or palate were observed in 53 patients out of 77, the ectodermal dysplasia was mentioned in 44 cases. There is an increase of mutation frequency in older parents.     

TRPV4-associated skeletal dysplasias

Dominant mutations in the TRPV4 gene result in a bone dysplasia family and form a continuous phenotypic spectrum that includes, in decreasing severity, lethal, and nonlethal metatropic dysplasia (MD), spondylometaphyseal dysplasia Kozlowski type (SMDK), and autosomal dominant brachyolmia. Several rare variant phenotypes that have some overlap but deviate in some ways from the general pattern have also been described. The known variant phenotypes are spondyloepiphyseal dysplasia Maroteaux type (Pseudo-Morquio type 2), parastremmatic dysplasia, and familial digital arthropathy with brachydactyly. Interestingly, different TRPV4 mutations have been associated with dominantly inherited neurologic disorders such as congenital spinal muscular atrophy and hereditary motor and sensory neuropathy. Finally, a small number of patients have been identified in whom a TRPV4 mutation results in a phenotype combining skeletal dysplasia with peripheral neuropathy. The TRPV4 gene encodes a regulated calcium channel implicated in multiple and diverse cellular processes. Over 50 different TRPV4 mutations have been reported, with two codons appearing to be mutational hot spots: P799 in exon 15, mostly associated with MD, and R594 in exon 11, associated with SMDK. While most pathogenic mutations tested so far result in activation of the calcium channel in vitro, the mechanisms through which TRPV4 activation results in skeletal dysplasia and/or peripheral neuropathy remain unclear and the genotype-phenotype correlations in this group of disorders remains somewhat mysterious. Since the phenotypic expression of most mutations seems to be relatively constant, careful clinical and radiographic assessment is useful in directing molecular analysis.