Patricia J.C. Dopping-Hepenstal

Lack of plakophilin 1 increases keratinocyte migration and reduces desmosome stability

Ablation of the desmosomal plaque component plakophilin 1 underlies the autosomal recessive genodermatosis, skin fragility-ectodermal dysplasia syndrome (OMIM 604536). Skin from affected patients is thickened with increased scale, and there is loss of adhesion between adjacent keratinocytes, which exhibit few small, poorly formed desmosomes. To investigate further the influence of plakophilin 1 on keratinocyte adhesion and desmosome morphology, we compared plakophilin 1-deficient keratinocytes (vector controls) with those expressing recombinant plakophilin 1 introduced by retroviral transduction. We found that plakophilin 1 increases desmosomal protein content within the cell rather than enhancing transcriptional levels of desmosomal genes. Re-expression of plakophilin 1 in null cells retards cell migration but does not alter keratinocyte cell growth. Confluent sheets of plakophilin 1-deficient keratinocytes display fewer calcium-independent desmosomes than do plakophilin 1-deficient keratinocytes expressing recombinant plakophilin 1 or keratinocytes expressing endogenous plakophilin 1. In addition electron microscopy studies show that re-expression of plakophilin 1 affects desmosome size and number. Collectively, these results demonstrate that restoration of plakophilin 1 function in our culture system influences the transition of desmosomes from a calcium-dependent to a calcium-independent state and this correlates with altered keratinocyte migration in response to wounding. Thus, plakophilin 1 has a key role in increasing desmosomal protein content, in desmosome assembly, and in regulating cell migration.

Striate palmoplantar keratoderma arising from desmoplakin and desmoglein 1 mutations is associated with contrasting perturbations of desmosomes and the keratin filament network

Background  Several hereditary human diseases are now known to be caused by distinct mutations in genes encoding various desmosome components. Although the effects of some of these mutant genes have been analysed by targeted disruption experiments in mouse models, little is known about the cell and tissue changes in affected human patients.

A keratin 14 'knockout' mutation in recessive epidermolysis bullosa simplex resulting in less severe disease.

Epidermolysis bullosa simplex (EBS) is a blistering skin disease caused in most cases by mis‐sense mutations in genes encoding the basal epidermal keratin (K) 5 and K14. The inheritance is usually autosomal dominant and the mutant keratin proteins appear to exert a dominant negative effect on the keratin intermediate filament cytoskeleton in basal keratinocytes. We report a child with a homozygous K14 mutation resulting in the complete absence of K14 protein in the epidermis; remarkably, he only had mild to moderate disease. Electron microscopy of a skin biopsy showed a marked reduction in numbers of keratin intermediate filaments in the basal keratinocytes. Immunofluorescence microscopy using monoclonal antibody LL001 against K14 showed no staining, suggesting a functional knockout of K14. Sequence analysis of genomic DNA revealed a homozygous mutation in codon 31 of K14 that resulted in a premature stop codon further downstream in exon 1. The childs mother, who is unaffected by the disease, is heterozygous for the mutation. The consanguineous father was unaffected and unavailable for testing. The resulting mRNA is predicted to encode a protein of 116 amino acids, of which the first 30 are identical to the normal K14 sequence, and the remaining 86 residues are mis‐sense sequence. Four previously reported cases of autosomal recessive EBS with functional knockout of K14 were severely affected by blistering, in contrast to our patient in whom the predicted protein has only the first 30 amino acids of K14 and is therefore the closest to a true knockout of K14 protein yet identified.

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

Germline Mutation in EXPH5 Implicates the Rab27B Effector Protein Slac2-b in Inherited Skin Fragility

The Rab GTPase Rab27B and one of its effector proteins, Slac2-b (also known as EXPH5, exophilin-5), have putative roles in intracellular vesicle trafficking but their relevance to human disease is not known. By using whole-exome sequencing, we identified a homozygous frameshift mutation in EXPH5 in three siblings with inherited skin fragility born to consanguineous Iraqi parents. All three individuals harbor the mutation c.5786delC (p.Pro1929Leufs(∗)8) in EXPH5, which truncates the 1,989 amino acid Slac2-b protein by 52 residues. The clinical features comprised generalized scale-crusts and occasional blisters, mostly induced by trauma, as well as mild diffuse pigmentary mottling on the trunk and proximal limbs. There was no increased bleeding tendency, no neurologic abnormalities, and no increased incidence of infection. Analysis of an affected persons skin showed loss of Slac2-b immunostaining (C-terminal antibody), disruption of keratinocyte adhesion within the lower epidermis, and an increased number of perinuclear vesicles. A role for Slac2-b in keratinocyte biology was supported by findings of cytoskeletal disruption (mainly keratin intermediate filaments) and decreased keratinocyte adhesion in both keratinocytes from an affected subject and after shRNA knockdown of Slac2-b in normal keratinocytes. Slac2-b was also shown to colocalize with Rab27B and β4 integrin to early adhesion initiation sites in spreading normal keratinocytes. Collectively, our findings identify an unexpected role for Slac2-b in inherited skin fragility and expand the clinical spectrum of human disorders of GTPase effector proteins.

Haploinsufficiency for AAGAB causes clinically heterogeneous forms of punctate palmoplantar keratoderma

Palmoplantar keratodermas (PPKs) are a group of disorders that are diagnostically and therapeutically problematic in dermatogenetics. Punctate PPKs are characterized by circumscribed hyperkeratotic lesions on the palms and soles with considerable heterogeneity. In 18 families with autosomal dominant punctate PPK, we report heterozygous loss-of-function mutations in AAGAB, encoding α- and γ-adaptin–binding protein p34, located at a previously linked locus at 15q22. α- and γ-adaptin–binding protein p34, a cytosolic protein with a Rab-like GTPase domain, was shown to bind both clathrin adaptor protein complexes, indicating a role in membrane trafficking. Ultrastructurally, lesional epidermis showed abnormalities in intracellular vesicle biology. Immunohistochemistry showed hyperproliferation within the punctate lesions. Knockdown of AAGAB in keratinocytes led to increased cell division, which was linked to greatly elevated epidermal growth factor receptor (EGFR) protein expression and tyrosine phosphorylation. We hypothesize that p34 deficiency may impair endocytic recycling of growth factor receptors such as EGFR, leading to increased signaling and cellular proliferation.

Autosomal recessive epidermolysis bullosa simplex due to loss of BPAG1-e expression.

TO THE EDITOR In 2010, we identified a homozygous nonsense mutation in the dystonin (DST) gene in a Kuwaiti individual with a new form of autosomal recessive epidermolysis bullosa (EB) simplex (Groves et al., 2010). The mutation occurred within the coiled-coil rod domain of the BPAG1-e isoform (the hemidesmosomal 230-kDa bullous pemphigoid antigen) and the main symptom was mild trauma-induced predominantly acral blistering, although there was also some generalized skin fragility. However, additional neurological features were noted, including episodes of collapse, headaches, numbness, and weakness. Molecular studies also revealed a heterozygous mutation in the NOTCH3 gene consistent with concomitant autosomal dominant cerebral small-vessel arteriopathy (CADASIL; MIM125310; Joutel et al., 1996). Given that some DST isoforms may be expressed in the central nervous system (Young and Kothary, 2007; see Groves et al., 2010, for details of alternative splicing of DST), it was unclear whether the overall clinical features were solely due to the DST mutation or whether NOTCH3 might be involved. To address this further, we have now identified a second unrelated family with a different homozygous nonsense mutation in the same region of the DST gene. Notably, affected individuals have mild skin blistering but no neurological abnormalities. The proband was a 34-year-old Iranian woman who had lifelong skin blistering that was worse during summer. Blisters occurred on the soles, at sites of friction from clothing, and following removal of adhesive dressings. The mouth, eyes, and genital areas were never involved. No extracutaneous symptoms were reported. Her father and two of her three children also had mild blistering. Blisters occurred on the feet of her 15-year-old son following sporting activities, and her 6-month-old son developed blisters on the great toes after starting to crawl; her husband reported no skin fragility. Some consanguinity was reported in the family, but precise details were not forthcoming. On examination, she had a solitary blister on the instep of one foot (Figure 1a). The rest of her skin appeared normal apart from subtle atrophic scars affecting the shins, ankles, elbows, dorsal aspects of the hands, and lower back. Teeth, nails, and hair were normal, and there was no palmoplantar hyperkeratosis. Following written and informed consent, blood and skin samples were obtained from the patient in adherence to the Declaration of Helsinki Principles. The skin sampled was noninflamed and nonlesional. Initially, we performed immunofluorescence microscopy, as described elsewhere (Groves et al., 2010). Labeling for plectin, a6 and b4 integrin subunits, keratin 14, laminin-332, and collagens IV, VII, and XVII, however, showed no abnormalities. Next, we undertook transmission electron microscopy, as described previously (Groves et al., 2010). The morphology of individual hemidesmosomes was abnormal with poorly formed inner plaques, leading to a lucent zone between keratin filaments and outer hemidesmosomal plaques (Figure 1b). The keratin filaments extended to where the inner plaques should be, but did not associate with any attachment structures; no other abnormalities were noted. Further skin immunostaining with a mAb against BPAG1-e (clone BPC319, directed against the carboxyl terminus, Okumura et al., 2002) showed a complete absence of immunoreactivity compared with bright, linear labeling at the dermal– epidermal junction in control skin (Figure 1c). Sequencing of the patient’s genomic DNA identified a homozygous A4T transversion (c.3853A4T; GenBank NM_001723.4) that converts arginine to a stop codon, designated p.Arg1249X (Figure 1d). This mutation occurs within the coiled-coil domain in a region similar to the previously reported DST mutation, p.Gln1124X (Groves et al., 2010). The new mutation was not identified in screening 200 ethnically matched control chromosomes. Sequencing of the genes encoding keratins 5 and 14 revealed no pathogenic mutations. Skin biopsies and DNA samples were not available from other family members, but the data are consistent with autosomal recessive EB simplex due to a homozygous lossof-function mutation in the DST gene with a pseudodominant pattern of inheritance in this consanguineous family. The similar nature and sites of the DST mutations in this and the previously published case allow for clinicopathological refinement of the subtype of EB. BPAG1-e, or BP230, is a structural component of hemidesmosomal inner plaques in basal keratinocytes (Guo et al., 1995; Yang et al., 1996). The rod domain comprises a coiled-coil region that is involved in homodimerization, whereas the amino terminus is important for the recruitment of BPAG1-e into hemidesmosomes (Koster et al., 2003), and the carboxy-terminal region binds to keratin intermediate filaments (Yang et al., 1996; Fontao et al., 2003). Lossof-function mutations in the coiled-coil region in both cases led to similar ultrastructural pathology with an absence of hemidesmosomal inner plaques and a ‘‘lucent zone’’ where keratin filaments normally attach. Both mutations also led to a complete absence of BPAG1-e on skin immunostaining. However, skin immunohistochemistry in the Kuwaiti case also reported abnormalities in labeling for plectin, the b4 integrin subunit, and collagen XVII—changes that were not noted in the current case (data not shown). These inconsistencies require Abbreviations: DST, dystonin; EB, epidermolysis bullosa

Keratin 9 Is Required for the Structural Integrity and Terminal Differentiation of the Palmoplantar Epidermis

Keratin 9 (K9) is a type I intermediate filament protein whose expression is confined to the suprabasal layers of the palmoplantar epidermis. Although mutations in the K9 gene are known to cause epidermolytic palmoplantar keratoderma, a rare dominant-negative skin disorder, its functional significance is poorly understood. To gain insight into the physical requirement and importance of K9, we generated K9-deficient (Krt9−/−) mice. Here, we report that adult Krt9−/−mice develop calluses marked by hyperpigmentation that are exclusively localized to the stress-bearing footpads. Histological, immunohistochemical, and immunoblot analyses of these regions revealed hyperproliferation, impaired terminal differentiation, and abnormal expression of keratins K5, K14, and K2. Furthermore, the absence of K9 induces the stress-activated keratins K6 and K16. Importantly, mice heterozygous for the K9-null allele (Krt9+/−) show neither an overt nor histological phenotype, demonstrating that one Krt9 allele is sufficient for the developing normal palmoplantar epidermis. Together, our data demonstrate that complete ablation of K9 is not tolerable in vivo and that K9 is required for terminal differentiation and maintaining the mechanical integrity of palmoplantar epidermis.

Congenital muscular dystrophy, myasthenic symptoms and epidermolysis bullosa simplex (EBS) associated with mutations in the PLEC1 gene encoding plectin

Mutations in the PLEC1 gene encoding plectin have been reported in neonatal epidermolysis bullosa simplex with muscular dystrophy of later-onset (EBS-MD). A neuromuscular transmission defect has been reported in one previous patient. We report a boy presenting from birth with features of a congenital muscular dystrophy and late-onset myasthenic symptoms. Repetitive nerve stimulation showed significant decrement, and strength improved with pyridostigmine. Subtle blistering noticed only retrospectively prompted further genetic testing, revealing recessive PLEC1 mutations. We conclude that PLEC1 should be considered in the differential diagnosis of congenital muscular dystrophies and myasthenic syndromes, even in the absence of prominent skin involvement.

Autosomal dominant junctional epidermolysis bullosa

Background  Epidermolysis bullosa (EB) encompasses a heterogeneous group of inherited skin disorders associated with trauma‐induced blistering. The junctional forms of EB (JEB), Herlitz JEB, non‐Herlitz JEB and JEB associated with pyloric atresia have all been attributed to autosomal recessive inheritance. We describe a 7‐year‐old girl with defective dental enamel, trauma‐induced blistering and subsequent scarring. Her mother, a carrier of the mutation p.G627V in the collagen XVII gene (COL17A1) had evidence of hypoplastic dental enamel without skin blistering. Her grandmother had non‐Herlitz JEB as a result of a compound heterozygous mutation in COL17A1 (p.G627V and c.3514ins25).