Tanasit Techanukul

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

HB-EGF induces COL7A1 expression in keratinocytes and fibroblasts: Possible mechanism underlying allogeneic fibroblast therapy in recessive dystrophic epidermolysis bullosa

TO THE EDITOR Recessive dystrophic epidermolysis bullosa (RDEB) is a mechanobullous disease caused by mutations in the COL7A1 gene that encodes type VII collagen (C7) at the dermal–epidermal junction (DEJ) (Fine et al., 2008). The C7 protein is synthesized by both keratinocytes and fibroblasts (Stanley et al., 1985). We demonstrated previously that intradermal injections of allogeneic fibroblasts in RDEB can increase C7 expression at the DEJ, although that study did not disclose how long the benefits were sustained for or indicate a therapeutic mode of action (Wong et al., 2008). Allogeneic fibroblasts could not be detected 2 weeks after injection but, in some individuals, there was increased C7 protein at the DEJ for at least 3 months. To investigate this further, we injected 5 10 per cm normal control allogeneic fibroblasts (ICX-RHY, Vavelta, Intercytex; Manchester, UK) into one RDEB subject and took biopsies at days 7, 15, 30, 90, 180, 270, and 360. Each injection volume was 0.25 ml cm ; we also injected a similar volume of normal saline to adjacent skin (noting observations by Venugopal et al. (2010) that saline can also increase C7 in RDEB skin) and took biopsies at days 15 and 90. Clinicopathological details of the subject studied have been published previously (case 5; Wong et al., 2008). This patient is a compound heterozygote for the COL7A1 mutations c.2044C4T (p.Arg682X) and IVS87þ 4A4G. This particular donor splice-site mutation creates a leaky splice site, which leads to either in-frame skipping of exon 87 (69-bp) or wild-type sequence; this also allows for tracking of the mutant allele in skin biopsy complementary DNA. We assessed C7 immunolabeling at the DEJ (as described previously; Wong et al., 2008), COL7A1 gene expression by quantitative real-time RT-PCR (using a primer pair upstream of the splice-site mutation and another pair spanning the potential exon skip), and gene expression profiling using Sentrix Human-6 Whole Genome Expression Beadchips (Illumina, San Diego, CA); see Supplementary Materials and Methods online for full details. Quantification of immunofluorescence microscopy intensities for C7 labeling (see Wong et al., 2008 for Materials and Methods) is detailed in Supplementary Table 1 online. C7 labeling increased 15 days after fibroblast injection and was maintained for at least 270 days but returned to baseline levels by 360 days. For the saline control, at day 15, we noted a slight increase in labeling at the DEJ but a return to baseline by 90 days. COL7A1 gene expression, using both sets of primers (Figure 1a and b) showed that following fibroblast injection, there was a 420-fold increase at days 15 and 90, but this returned to baseline at day 180 and thereafter. Saline injection also resulted in a approximately 5-fold increase in patient skin COL7A1 gene expression at day 15, but levels at day 90 were similar to baseline (Figure 1a and b). For the COL7A1 primers spanning the splicesite mutation, at baseline, the ratio of wild-type to in-frame exon skip of exon 87 transcripts was approximately 2:3 (Figure 1b): this expression ratio persisted in all biopsy material. With regard to mechanism, no differences were noted in gene expression for cytokines already known to increase COL7A1 (full details are shown in Supplementary Tables 2–10 online). Of note, however, we observed a 43-fold increase in expression of the gene for heparin-binding epidermal growth factor-like growth factor (HB-EGF) (Iwamoto and Mekada, 2000), and quantitative real-time RT-PCR showed a similar temporal pattern to the COL7A1 quantitative real-time RTPCR data (linear correlation, r1⁄40.978; Po0.0001; compare HB-EGF data in Figure 1c with COL7A1 expression in Figure 1a and b). We also noted that gene expression profiles of FOS (linear correlation, r1⁄40.864; Po0.0006) and JUN (linear correlation, r1⁄40.945; Po0.0001) were also highly similar to the pattern of increased COL7A1 expression at the different time points (Supplementary Tables 2–10 online). JUN and FOS form the AP-1 transcription complex, which can bind to the COL7A1 promoter and enhance gene expression (Nakano et al., 2001). To investigate whether the upregulation of HB-EGF might be related to the increased expression of COL7A1, subconfluent-cultured keratinocytes and fibroblasts, both normal control and from two subjects with RDEB (this study individual and an unrelated subject with the COL7A1 mutations c.1732C4T (p.Arg578X) and c.7786delG (p.Gly2593fsX4), were treated with 100 ng ml 1 recombinant HB-EGF protein (R&D Systems,

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.

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.

Ectodermal dysplasia-skin fragility syndrome due to a new homozygous internal deletion mutation in the PKP1 gene

Ectodermal dysplasia‐skin fragility syndrome (ED‐SFS) is a rare autosomal recessive genodermatosis resulting from mutations in the PKP1 gene, encoding the desmosomal plaque protein plakophilin‐1 (PKP1). Mutations in PKP1 may manifest with skin fragility and erosions, patches of scale crust on the trunk and limbs, peri‐oral cracking and inflammation, hypotrichosis, palmoplantar keratoderma with painful fissuring and other somewhat variable ectodermal anomalies. Ten cases of the syndrome have been reported. We report a further case of this desmosomal genodermatosis. A 14‐month old child, born to consanguineous parents, presented with a history of neonatal bullae and subsequent development of dystrophic nails, sparse eyelashes and eyebrows, woolly scalp hair, abnormal dental development and a desquamating erythematous rash at sites of trauma. A clinical diagnosis of ED‐SFS was supported by skin biopsy findings of suprabasal intraepidermal clefting and a loss of immunoreactivity for PKP1. Sequencing of genomic DNA revealed a homozygous 5 base pair deletion in exon 5 of the PKP1 gene, designated c.897del5 (CAACC). This new mutation creates a frameshift, leading to a downstream premature termination codon, p.Pro299fsX61. This case highlights the clinicopathological consequences of inherited mutations in the PKP1 gene and illustrates the key role of desmosomes in skin biology.

Novel and Recurrent FERMT1 Gene Mutations in Kindler Syndrome

Kindler syndrome (OMIM 173650) is an autosomal recessive condition characterized by skin blistering, skin atrophy, photosensitivity, colonic inflammation and mucosal stenosis. Fewer than 100 cases have been described in the literature. First reported in 1954, the molecular basis of Kindler syndrome was elucidated in 2003 with the discovery of FERMT1 (KIND1) loss-of-function mutations in affected individuals. The FERMT1 gene encodes kindlin-1 (also known as fermitin family homologue 1), a 77 kDa protein that localizes at focal adhesions, where it plays an important role in integrin signalling. In the current study, we describe five novel and three recurrent loss-of-function FERMT1 mutations in eight individuals with Kindler syndrome, and provide an overview of genotype-phenotype correlation in this disorder.

Common IL-31 gene haplotype associated with non-atopic eczema is not implicated in epidermolysis bullosa pruriginosa.

Epidermolysis bullosa pruriginosa (EBP; OMIM #604129) is an unusual variant of autosomal dominant (or occasionally recessive) dystrophic epidermolysis bullosa (DEB) in which intense itching and scratching impacts upon the phenotype (1, 2). Although trauma-induced blistering often occurs, and toenail dystrophy is almost universal, the skin lesions can often resemble nodular prurigo, lichen simplex chronicus, hypertrophic lichen planus, dermatitis artefacta or other acquired itchy dermatoses (1, 3, 4). EBP therefore can be difficult to diagnose clinically and its precise pathophysiology is not known. Of note, the nature of the underlying mutations in the type VII col-lagen gene, COL7A1, does not differ substantially from those delineated in other non-itchy cases of DEB (3–5). Moreover, parameters such as IgE levels, atopy, biochemical or endocrinological abnormalities, iron deficiency, filaggrin gene pathology, and matrix metalloproteinase-1 gene promoter polymorphisms, have all been excluded as potential disease-modifying factors (1, 3, 4, 6). Itch is a common, complex, and only partially understood clinical symptom (7). Nevertheless, recent data have demonstrated that interleukin-31 (IL-31), a cytokine belonging to the IL-6 family, may be relevant to some pruritic disorders (8–11). Notably, over-expression of IL-31 in transgenic mice (ubiquitous or lymphocyte-specific promoter) induces severe itching, normal IgE levels and a phenotype similar to non-atopic eczema (8). Expression of the IL-31 gene is also up-regulated in the skin in several itchy human skin disorders, including atopic dermatitis, allergic contact dermatitis, psoriasis and nodular prurigo (9–11). IL-31 signals via a receptor complex that is composed of IL-31 receptor A (IL-31RA) and oncostatin M receptor (OSMR) subunits (12), and naturally occurring mutations in both these receptor components may underlie familial primary localized cutaneous amyloidosis, a pruritic autosomal dominant disease (13). Polymorphisms in the IL-31 gene have also been linked to eczema susceptibility. Schulz et al. (14) have shown that a particular IL-31 gene haplotype may be associated with altered regulation of IL-31 gene expression, and that this can have functional consequences and be more common in subjects with non-atopic eczema. Schulz et al. (14) identified three principal IL-31 gene haplotypes, which they termed A, B and C, that are present in > 90% of the white Caucasian population. These could be distinguished by genotyping three single-nucleotide polymorphisms: IL-31 2057 G>A (rs6489188; chromosomal position 121226729), IL-31 1066 G>A (rs11608363; chromosomal position 121225738), and IL-31 IVS2+12 A>G (chromosomal position 121224332). The A, B and C haplotypes reflected the following combination of polymorphisms: GAA, AGA and GGG, …