Analysis of Large Phenotypic Variability of EEC and SHFM4 Syndromes Caused by K193E Mutation of the TP63 Gene

EEC (ectrodactyly, ectodermal dysplasia, clefting; OMIM 604292) is an autosomal dominant developmental disorder resulting mainly from pathogenic mutations of the DNA-binding domain (DBD) of the TP63 gene. In this study, we showed that K193E mutation in nine affected individuals of a four-generation kindred with a large degree of phenotypic variability causes four different syndromes or TP63-related disorders: EEC, Ectrodactyly-ectodermal dysplasia (EE), isolated ectodermal dysplasia, and isolated Split Hand/Foot Malformation type 4 (SHFM4). Genotype-phenotype and DBD structural modeling analysis showed that the K193-located loop L2-A is associated with R280 through hydrogen bonding interactions, while R280 mutations also often cause large phenotypic variability of EEC and SHFM4. Thus, we speculate that K193 and several other DBD mutation-associated syndromes may share similar pathogenic mechanisms, particularly in the case of the same mutation with different phenotypes. Our study and others also suggest that the phenotypic variability of EEC is attributed, at least partially, to genetic and/or epigenetic modifiers

Small Deletions of SATB2 Cause Some of the Clinical Features of the 2q33.1 Microdeletion Syndrome

Recurrent deletions of 2q32q33 have recently been reported as a new microdeletion syndrome. Clinical features of this syndrome include severe mental retardation, growth retardation, dysmorphic features, thin and sparse hair, feeding difficulties and cleft or high palate. The commonly deleted region contains at least seven genes. Haploinsufficiency of one of these genes, SATB2, a DNA-binding protein that regulates gene expression, has been implicated as causative in the cleft or high palate of individuals with 2q32q33 microdeletion syndrome. In this study we describe three individuals with smaller microdeletions of this region, within 2q33.1. The deletions ranged in size from 173.1 kb to 185.2 kb and spanned part of SATB2. Review of clinical records showed similar clinical features among these individuals, including severe developmental delay and tooth abnormalities. Two of the individuals had behavioral problems. Only one of the subjects presented here had a cleft palate, suggesting reduced penetrance for this feature. Our results suggest that deletion of SATB2 is responsible for several of the clinical features associated with 2q32q33 microdeletion syndrome

Prediction of Human Disease Genes by Human-Mouse Conserved Coexpression Analysis

One of the most limiting aspects of biological research in the post-genomic era is the capability to integrate massive datasets on gene structure and function for producing useful biological knowledge. In this report we have applied an integrative approach to address the problem of identifying likely candidate genes within loci associated with human genetic diseases. Despite the recent progress in sequencing technologies, approaching this problem from an experimental perspective still represents a very demanding task, because the critical region may typically contain hundreds of positional candidates. We found that by concentrating only on genes sharing similar expression profiles in both human and mouse, massive microarray datasets can be used to reliably identify disease-relevant relationships among genes. Moreover, we found that integrating the coexpression criterion with systematic phenome analysis allows efficient identification of disease genes in large genomic regions. Using this approach on 850 OMIM loci characterized by unknown molecular basis, we propose high-probability candidates for 81 genetic diseases

Deletion of Porcn in Mice Leads to Multiple Developmental Defects and Models Human Focal Dermal Hypoplasia (Goltz Syndrome)

Focal Dermal Hypoplasia (FDH) is a genetic disorder characterized by developmental defects in skin, skeleton and ectodermal appendages. FDH is caused by dominant loss-of-function mutations in X-linked PORCN. PORCN orthologues in Drosophila and mice encode endoplasmic reticulum proteins required for secretion and function of Wnt proteins. Wnt proteins play important roles in embryo development, tissue homeostasis and stem cell maintenance. Since features of FDH overlap with those seen in mouse Wnt pathway mutants, FDH likely results from defective Wnt signaling but molecular mechanisms by which inactivation of PORCN affects Wnt signaling and manifestations of FDH remain to be elucidated.We introduced intronic loxP sites and a neomycin gene in the mouse Porcn locus for conditional inactivation. Porcn-ex3-7flox mice have no apparent developmental defects, but chimeric mice retaining the neomycin gene (Porcn-ex3-7Neo-flox) have limb, skin, and urogenital abnormalities. Conditional Porcn inactivation by EIIa-driven or Hprt-driven Cre recombinase results in increased early embryonic lethality. Mesenchyme-specific Prx-Cre-driven inactivation of Porcn produces FDH-like limb defects, while ectodermal Krt14-Cre-driven inactivation produces thin skin, alopecia, and abnormal dentition. Furthermore, cell-based assays confirm that human PORCN mutations reduce WNT3A secretion.These data indicate that Porcn inactivation in the mouse produces a model for human FDH and that phenotypic features result from defective WNT signaling in ectodermal- and mesenchymal-derived structures

Disruption of SATB2 or its long-range cis-regulation by SOX9 causes a syndromic form of Pierre Robin Sequence

Heterozygous loss-of-function (LOF) mutations in the gene encoding the DNA-binding protein, SATB2, result in micrognathia and cleft palate in both humans and mice. In three unrelated individuals, we show that translocation breakpoints (BPs) up to 896 kb 3′ of SATB2 polyadenylation site cause a phenotype which is indistinguishable from that caused by SATB2 LOF mutations. This syndrome comprises long nose, small mouth, micrognathia, cleft palate, arachnodactyly and intellectual disability. These BPs map to a gene desert between PLCL1 and SATB2. We identified three putative cis-regulatory elements (CRE1–3) using a comparative genomic approach each of which would be placed in trans relative to SATB2 by all three BPs. CRE1–3 each bind p300 and mono-methylated H3K4 consistent with enhancer function. In silico analysis suggested that CRE1–3 contain one or more conserved SOX9-binding sites, and this binding was confirmed using chromatin immunoprecipitation on cells derived from mouse embryonic pharyngeal arch. Interphase bacterial artificial chromosome fluorescence in situ hybridization measurements in embryonic craniofacial tissues showed that the orthologous region in mice exhibits Satb2 expression-dependent chromatin decondensation consistent with Satb2 being a target gene of CRE1–3. To assess their in vivo function, we made multiple stable reporter transgenic lines for each enhancer in zebrafish. CRE2 was shown to drive SATB2-like expression in the embryonic craniofacial region. This expression could be eliminated by mutating the SOX9-binding site of CRE2. These observations suggest that SATB2 and SOX9 may be acting together via complex cis-regulation to coordinate the growth of the developing jaw

Clinical and molecular consequences of disease-associated de novo mutations in SATB2

Purpose: To characterize features associated with de novo mutations affecting SATB2 function in individuals ascertained on the basis of intellectual disability. Methods: Twenty previously unreported individuals with 19 different SATB2 mutations (11 loss-of-function and 8 missense variants) were studied. Fibroblasts were used to measure mutant protein production. Subcellular localization and mobility of wild-type and mutant SATB2 were assessed using fluorescently tagged protein. Results: Recurrent clinical features included neurodevelopmental impairment (19/19), absent/near absent speech (16/19), normal somatic growth (17/19), cleft palate (9/19), drooling (12/19), and dental anomalies (8/19). Six of eight missense variants clustered in the first CUT domain. Sibling recurrence due to gonadal mosaicism was seen in one family. A nonsense mutation in the last exon resulted in production of a truncated protein retaining all three DNA-binding domains. SATB2 nuclear mobility was mutation-dependent; p.Arg389Cys in CUT1 increased mobility and both p.Gly515Ser in CUT2 and p.Gln566Lys between CUT2 and HOX reduced mobility. The clinical features in individuals with missense variants were indistinguishable from those with loss of function. Conclusion: SATB2 haploinsufficiency is a common cause of syndromic intellectual disability. When mutant SATB2 protein is produced, the protein appears functionally inactive with a disrupted pattern of chromatin or matrix association

A mutation of the p63 gene in non‐syndromic cleft lip

Mutations in the p63 gene (TP63) underlie several monogenic malformation syndromes manifesting cleft lip with or without cleft palate (CL/P). We investigated whether p63 mutations also result in non‐syndromic CL/P. Specifically, we performed mutation analysis of the 16 exons of the p63 gene for 100 Thai patients with non‐syndromic CL/P. In total, 21 variant sites were identified. All were single nucleotide changes, with six in coding regions, including three novel non‐synonymous changes: S90L, R313G, and D564H. The R313G was concluded to be pathogenic on the basis of its amino acid change, evolutionary conservation, its occurrence in a functionally important domain, its predicted damaging function, its de novo occurrence, and its absence in 500 control individuals. Our data strongly suggest, for the first time, a causative role of a heterozygous mutation in the p63 gene in non‐syndromic CL/P, highlighting the wide phenotypic spectrum of p63 gene mutations