Suzanne E. Clements

Revertant Mosaicism in Recessive Dystrophic Epidermolysis Bullosa

TO THE EDITOR Revertant mosaicism refers to the presence of two genetically heterogeneous populations of cells as a result of spontaneous genetic correction during mitosis (Hall, 1988; Jonkman et al., 1997). This phenomenon has been reported in several inherited diseases, including severe combined immunodeficiency, Bloom’s syndrome, Fanconi’s anemia, X-linked Wiscott–Aldrich syndrome, Duchenne muscular dystrophy, and tyrosinemia type I (Hirschhorn, 2003). With regard to genodermatoses, cutaneous revertant mosaicism has been described in epidermolysis bullosa (EB) (Fine et al., 2008). Notably, in vivo reversion of mutations in LAMB3, COL17A1, and KRT14 has underscored cutaneous mosaicism in non-Herlitz junctional EB and EB simplex, respectively (Darling et al., 1999; Schuilenga-Hut et al., 2002; Smith et al., 2004; Pasmooij et al., 2005, 2007; Jonkman and Pasmooij, 2009). Of potential clinical interest, such genetic events may not be that rare— perhaps occurring in up to one-third of cases of non-Herlitz junctional EB (Jonkman and Pasmooij, 2009). Multiple corrective mechanisms have been proposed or observed, including back mutations, intragenic crossovers, mitotic gene conversions, and second-site mutations (Jonkman et al., 1997; Pasmooij et al., 2005; Frank and Happle, 2007). Indeed, several different corrective processes can occur in the same patient (Jonkman and Pasmooij, 2009). The implications for phenotype, however, depend on several factors, including the timing and extent of the revertant mosaicism. Here we report a further example of revertant mosaicism in a different sub-type of EB, with probable intragenic crossover in the COL7A1 gene leading to restoration of basement membrane collagen VII and anchoring fibrils in a patch of skin in an individual with recessive dystrophic EB. The proband is a 41-year-old Caucasian British man with severe generalized recessive dystrophic EB (Fine et al., 2008). He has mutilating scars with bilateral mitten deformities and a history of recurrent squamous cell carcinomas. His skin is prone to traumainduced blistering, although for as long as he can remember, two small patches of skin on his left wrist and right shin never seem to blister despite repeated trauma. Examination of these sites revealed areas approximately 8 5 cm that resembled the normal skin in appearance and texture (Figure 1a and b). To explain the phenotypic heterogeneity, and following ethics committee approval (St Thomas’ Hospital Ethics Committee: 07/H0802/104) and informed consent and in accordance with the Declaration of Helsinki principles, skin from the left wrist was investigated with biopsy specimens taken from both the blister-prone area (unreverted) and the normal-appearing skin (reverted). Immunolabeling for collagen VII (clone LH 7.2; Sigma-Aldrich, Poole, UK) and transmission electron microscopy were performed as described elsewhere (McGrath et al., 1993) and the results are illustrated in Figure 1c–e. Sequencing of peripheral leukocyte genomic DNA revealed that the patient is a compound heterozygote for two loss-of-function mutations in COL7A1, c.1732C4T (p.Arg578X) in exon 13 (maternal) and c.7786delG (p.Gly2593fsX4) in exon 104 (paternal) (Figure 2a). Both of these are recurrent mutations within the white British population (Mellerio et al., 1997). To explain the heterogeneous skin phenotype, genomic DNA and RNA were extracted from whole skin from both unreverted and reverted areas using standard kits (DNA Extraction Minikit and RNAEasy Minikit, Qiagen, Crawley, UK), as well as from cultured fibroblasts from both sites (TRIzol, Invitrogen, Paisley, UK) using the manufacturer’s protocols. The fibroblast cultures were performed using standard methods (Wong et al., 2008). cDNA was generated using commercial kits and protocols (IScript cDNA generation kit, Biorad, Hemel Hempstead, UK). Reverse transcriptase-PCR was performed across the sites of both mutations and the results are illustrated in Figure 2b. Real-time reverse transcriptase-PCR was also carried out to assess COL7A1 gene expression in the different skin and cell samples with primers (details available on request) specifically designed to amplify a 200-bp region of the COL7A1 30UTR (SyberGreen Master Mix, Applied Biosystems, Warrington, UK). Reverse transcriptasePCR for each cDNA sample was carried out in triplicate and the results are illustrated in Figure 2c. Collectively, these investigations indicated that the reverted skin expressed collagen VII and that anchoring fibrils were present. In addition, the reverse transcriptasePCR data demonstrated that there was expression of wild-type cDNA spanning the frameshift mutation in exon 104 and that COL7A1 gene expression levels were similar to those seen in the patient’s brother who was heterozygous for one mutant COL7A1 allele (Figure 2c). Moreover, the gene correction in the patient’s reverted skin appeared to have occurred in keratinocytes rather than fibroblasts. To explore the mechanism of this correction, we performed long-range sequencing of the patient’s reverted skin cDNA using LongAmp Taq DNA polymerase (New England Biolabs, Hitchin, UK). We first searched for polymorphisms to distinguish between maternal and paternal alleles and identified differences for a common PvuII polymorphism in exon

Spectrum of mutations in the ANTXR2 (CMG2) gene in infantile systemic hyalinosis and juvenile hyaline fibromatosis.

oped a greater skin colour change at the final stage of irradiation. Although the vitiligo-involved site also shows an increase of the area under the curve, the small magnitude of change was not significant to detect pigment formation clinically. Figure 2(b) displays the correlation of the constitutive melanin content of normal skin to the degree of formation of IPD. The amount of constitutive melanin is quantified by an area of differential apparent absorbance between normal and vitiligoinvolved skin at baseline in the spectral range of 390–450 nm in which the soluble fraction of epidermal melanin predominantly contributes to the apparent absorbance. The result shows that the degree of IPD response appears to be related to the constitutive pigment expressed at short wavelengths. In this study, we found that VIS-NIR radiation produces IPD only in normally pigmented skin and that the presence of constitutive pigment is required to induce IPD response. We conclude that the degree of formation of IPD from VIS-NIR radiation is related to the content of constitutive pigment expressed at short wavelengths (390–450 nm). This relation has been confirmed in an ongoing study on healthy subjects with various skin types.

Rapp-Hodgkin and Hay-Wells ectodermal dysplasia syndromes represent a variable spectrum of the same genetic disorder.

Background  Rapp–Hodgkin syndrome (RHS) and Hay–Wells [also known as ankyloblepharon–ectodermal defects–cleft lip/palate (AEC)] syndrome have been designated as distinct ectodermal dysplasia syndromes despite both disorders having overlapping clinical features and the same mutated gene, TP63.

PORCN gene mutations and the protean nature of focal dermal hypoplasia

Focal dermal hypoplasia (FDH) is an X‐linked dominant disorder featuring developmental abnormalities of ectodermal and mesodermal tissues. Pathogenic mutations in the PORCN gene (locus Xp11.23) were identified in 2007 and thus far 27 different mutations have been reported. PORCN encodes a putative O‐acyltransferase which facilitates secretion of Wnt proteins required for ectomesodermal tissue development. We investigated PORCN gene pathology and pattern of X‐chromosome inactivation analysis in two unrelated Caucasian female patients who presented with multiple developmental abnormalities consistent with FDH. We also reviewed the clinical and molecular data for all reported PORCN mutations and assessed genotype–phenotype correlation for sporadic and familial cases of FDH. DNA sequencing revealed two new PORCN gene mutations: p.W282X and c.74delG (p.G25fsX51). X‐chromosome inactivation analysis revealed a random pattern in one case but was uninformative in the other. Collectively, point/small mutations account for 24 out of the 29 PORCN mutations and are typically seen in sporadic cases; larger deletions are more common in familial cases. Identification of two new PORCN gene mutations confirms the importance of PORCN‐associated Wnt signalling in embryogenesis. Both new cases showed Blaschko‐linear dermal hypoplasia and extensive ectomesodermal abnormalities, including severe limb developmental anomalies and a giant cell tumour of bone in one patient. Clinical variability can be attributed to the degree of lyonization and postzygotic genomic mosaicism, which are important mechanisms in determining the clinical presentation.

Loss-of-Function FERMT1 Mutations in Kindler Syndrome Implicate a Role for Fermitin Family Homolog-1 in Integrin Activation

Kindler syndrome is an autosomal recessive disorder characterized by skin atrophy and blistering. It results from loss-of-function mutations in the FERMT1 gene encoding the focal adhesion protein, fermitin family homolog-1. How and why deficiency of fermitin family homolog-1 results in skin atrophy and blistering are unclear. In this study, we investigated the epidermal basement membrane and keratinocyte biology abnormalities in Kindler syndrome. We identified altered distribution of several basement membrane proteins, including types IV, VII, and XVII collagens and laminin-332 in Kindler syndrome skin. In addition, reduced immunolabeling intensity of epidermal cell markers such as beta1 and alpha6 integrins and cytokeratin 15 was noted. At the cellular level, there was loss of beta4 integrin immunolocalization and random distribution of laminin-332 in Kindler syndrome keratinocytes. Of note, active beta1 integrin was reduced but overexpression of fermitin family homolog-1 restored integrin activation and partially rescued the Kindler syndrome cellular phenotype. This study provides evidence that fermitin family homolog-1 is implicated in integrin activation and demonstrates that lack of this protein leads to pathological changes beyond focal adhesions, with disruption of several hemidesmosomal components and reduced expression of keratinocyte stem cell markers. These findings collectively provide novel data on the role of fermitin family homolog-1 in skin and further insight into the pathophysiology of Kindler syndrome.

Molecular basis of EEC (ectrodactyly, ectodermal dysplasia, clefting) syndrome: Five new mutations in the DNA-binding domain of the TP63 gene and genotype–phenotype correlation

EEC (ectrodactyly, ectodermal dysplasia, clefting; OMIM 604292) syndrome is an autosomal dominant developmental disorder. Characteristic clinical features comprise abnormalities in several ectodermal structures including skin, hair, teeth, nails and sweat glands as well as orofacial clefting and limb defects. Pathogenic mutations in the TP63 transcription factor have been identified as the molecular basis of EEC syndrome and to date 34 mutations have been reported. The majority of mutations involve heterozygous missense mutations in the DNA‐binding domain of TP63, a region critical for direct interactions with DNA target sequences. In this report, we present an overview of EEC syndrome, discuss the role of TP63 in embryonic development and skin homeostasis, and report five new TP63 gene mutations. We highlight the significant intra‐ and interfamilial phenotypic variability in affected individuals and outline the emerging paradigm for genotype–phenotype correlation in this inherited ectodermal dysplasia syndrome.

The molecular skin pathology of familial primary localized cutaneous amyloidosis

Please cite this paper as: The molecular skin pathology of familial primary localized cutaneous amyloidosis. Experimental Dermatology 2010; 19: 416–423.

Mutations in AEC syndrome skin reveal a role for p63 in basement membrane adhesion, skin barrier integrity and hair follicle biology

Background  AEC (ankyloblepharon–ectodermal defects–clefting) syndrome is an autosomal dominant ectodermal dysplasia disorder caused by mutations in the transcription factor p63. Clinically, the skin is dry and often fragile; other features can include partial eyelid fusion (ankyloblepharon), hypodontia, orofacial clefting, sparse hair or alopecia, and nail dystrophy.

International Research Symposium on Ankyloblepharon-Ectodermal Defects-Cleft Lip/Palate (AEC) syndrome.

Ankyloblepharon‐ectodermal defects‐cleft lip/palate (AEC) syndrome (Hay–Wells syndrome, MIM #106220) is a rare autosomal dominant ectodermal dysplasia syndrome. It is due to mutations in the TP63 gene, known to be a regulatory gene with many downstream gene targets. TP63 is important in the differentiation and proliferation of the epidermis, as well as many other processes including limb and facial development. It is also known that mutations in TP63 lead to skin erosions. These erosions, especially on the scalp, are defining features of AEC syndrome and cause significant morbidity and mortality in these patients. It was this fact that led to the 2003 AEC Skin Erosion Workshop. That conference laid the groundwork for the International Research Symposium for AEC Syndrome held at Texas Childrens Hospital in 2006. The conference brought together the largest cohort of individuals with AEC syndrome, along with a multitude of physicians and scientists. The overarching goals were to define the clinical and pathologic findings for improved diagnostic criteria, to obtain tissue samples for further study and to define future research directions. The symposium was successful in accomplishing these aims as detailed in this conference report. Following our report, we also present 11 manuscripts within this special section that outline the collective clinical, pathologic, and mutational data from 18 individuals enrolled in the concurrent Baylor College of Medicine IRB‐approved protocol: Characterization of AEC syndrome. These collaborative findings will hopefully provide a stepping‐stone to future translational projects of TP63 and TP63‐related syndromes.

Classifying ectodermal dysplasias: Incorporating the molecular basis and pathways (Workshop II)†

Hereditary conditions are traditionally classified based either on physical/physiological attributes or using the names of the individuals credited with identifying the condition. For the 170 plus conditions classified as ectodermal dysplasias (EDs), both of these nosological systems are used, at times interchangeably. Over the past decade our knowledge of the human genome and the molecular basis of the EDs have greatly expanded providing the impetus to consider alternative classification systems. The incorporation of the molecular basis of hereditary conditions adds important information allowing effective transfer of objective genetic information that can be lacking from traditional classification systems. Molecular information can be added to the nosological system for the EDs through a hierarchical‐ and domain‐based approach that encompasses the conditions name, mode of inheritance, molecular pathway affected, and specific molecular change. As new molecular information becomes available it can be effectively incorporated using this classification approach. Integrating molecular information into the ED classification system, while retaining well‐recognized traditional syndrome names, facilitates communication at and between different groups of people including patients, families, health care providers, and researchers.