Pathomechanisms of epidermolysis bullosa: Beyond structural proteins (2023)


Skin fragility disorders, a phenotypically diverse group of diseases, are characterized by blistering and erosions as a result of trauma of varying degree to the skin and the mucous membranes. The prototype of such conditions is epidermolysis bullosa (EB), characteristically presenting with blisters and erosions at birth or shortly thereafter. There is a tremendous variability in the spectrum of severity of EB, varying from early postnatal demise to life-long blistering tendency but without impact on the longevity of the affected individuals.

Initially, EB was divided into three broad categories based on the level of tissue separation leading to blister formation within the skin, as determined by diagnostic transmission electron microscopy of a skin biopsy [1]: (a) In the simplex forms of EB (EBS), tissue separation occurs within the epidermis, primarily at the level of basal keratinocytes (KC(s)) which disintegrate as a result of trauma to the skin leading to relatively superficial blisters and erosions; (b) in the junctional forms (JEB), tissue separation occurs within the epidermal basement membrane, primarily within the lamina lucida, due to abnormalities in hemidesmosomes and anchoring filaments, attachment complexes that traverse the basal lamina; and (c) in the dystrophic forms (DEB), blister formation occurs below the lamina densa within the upper papillary dermis, due to deficient anchoring fibrils which stabilize the association of the lamina densa to the underlying dermis (Table 1). In addition to these three major subtypes, Kindler EB (KEB, previously known as the Kindler syndrome), a rare syndromic form of EB, was recognized as a separate diagnostic entity (Table 1) [1]. In KEB, the blistering, which can be demonstrated at different levels of skin, is present during the early years of life but subsides later on, and the characteristic skin manifestations consist of poikiloderma, atrophy of the skin, pigmentary changes, and diffuse scarring [2]. These four subtypes of EB can be distinguished by immune epitope mapping which allows, by the use of immunofluorescent analyses utilizing different antibodies, to determine the level of blistering and subsequent subcategorization of the patients with prognostic implications. It should be noted, however, that there is a tremendous variability in the severity of the blistering within each of these categories of EB, and there can be an overlap leading to diagnostic difficulties when the patients are examined clinically, particularly during the early postnatal period when sub-classification is difficult. In addition, in many of these conditions, there are a number of extracutaneous manifestations which allow subclassification of different forms of the EB either into syndromic or non-syndromic types [3].

In addition to the four major subtypes of EB described above, there are several, relatively rare conditions within the spectrum of skin fragility disorders, which were initially included in the broad diagnostic classification of EB. Collectively, all these conditions were associated with mutations in as many as 21 distinct genes, and the phenotypic heterogeneity was explained by the topographic level of expression of the candidate genes within the cutaneous basement membrane zone (BMZ), the types of mutations, and the consequences of the mutations at the RNA and protein levels (Fig. 1) (Table 1). The latest classification of skin fragility disorders separates EB to a group designated as the “classic” forms, currently known to be associated with mutations in 17 different genes (Fig. 1) (Table 1). In addition to EB, there are skin fragility disorders characterized by peeling skin, erosions, hyperkeratosis, and associated connective tissue abnormalities (Table 1) [4].

The candidate genes for mutations in different forms of EB were initially identified on the basis of clinical, histopathologic, immunofluorescent and electron microscopic observations. For example, immunofluorescent analyses demonstrated the absence of a type VII collagen in the dermo-epidermal junction of the normal-appearing skin in patients with severe forms of DEB, and electron microscopy documented the absence of anchoring fibrils, critical attachment complexes at the dermo-epidermal junction composed of type VII collagen [5,6]. Subsequent cloning of the COL7A1 gene provided the basis for mutation detection, which identified pathogenic sequence variants in this gene both in the autosomal recessive and autosomal dominant forms of EB (RDEB and DDEB, respectively) [6]. Similarly, electron microscopic demonstration of clumped intermediate filaments in the basal KCs of the epidermis of EBS suggested that the genes encoding the basal keratins were the candidate genes for mutations. Indeed, subsequent genetic analyses identified mutations in KRT5 and KRT14, encoding keratins 5 (K5) and 14 (K14), respectively [7], [8], [9], [10], [11], [69], [70]. Demonstrations of abnormalities in the hemidesmosomes, critical attachment complexes that extend from the intracellular milieu of basal KCs to the basement membrane in the extracellular matrix (ECM), and consequently stabilize the association of epidermis to the underlying lamina lucida, suggested a number of new candidate genes in the junctional forms of EB. First, mutations in the COL17A1 gene, which encodes type XVII collagen, were identified in a subset of patients earlier diagnosed as generalized atrophic benign EB but now belonging to the intermediate category of JEB [4,12]. Subsequently, pathogenic sequence variants were documented in the LAMA3, LAMB3 and LAMC2 genes, which encode the subunit polypeptides α3, β3 and γ2 of laminin-332, as well as in ITGA6 and ITGB4 coding for the subunit polypeptides of the α6β4 integrin, protein components of the hemidesmosomes [[1], [4]]. In addition, mutations in the PLEC gene encoding plectin, a multifunctional structural protein, part of the hemidesmosomal complex, were identified [13], [14], [15]. The consequences of the PLEC mutations included, in addition to skin blistering, late-onset muscular dystrophy, pyloric or duodenal atresia, and neurological manifestations, thus defining plectin deficiency as a distinct syndromic form of EB [3]. Collectively, these demonstrations attested to the critical importance of the proteins encoded by the structural genes in providing physiological stability to the dermal-epidermal junction and their perturbations leading to the simplex, junctional or dystrophic forms of EB. In particular, the consequences of the mutations in specific collagens, types VII and XVII, in DEB and JEB, respectively, have been emphasized [16,17].

The strategies for detection of sequence variants in candidate genes were initially based on PCR amplification of genomic segments, as for example distinct exons and flanking intronic sequences, of the candidate genes, followed by nucleotide sequencing either by the Maxam-Gilbert or Sanger methods. It soon became evident, however, that many of the candidate genes, as for example, the type VII collagen gene, COL7A1, are large and complex requiring multiple PCR and sequencing reactions, thus being time consuming and costly. For example, COL7A1 was shown to be extremely complex consisting of 118 distinct exons, the largest number of exons in any gene having been identified at the time [18]. Also, many of the aberrant proteins on which the assessment of the candidate genes was based, such as laminin-332 or α6β4 integrin had multiple polypeptide subunits each of them being encoded by a distinct gene. Finally, some of the EB subtypes, particularly JEB, can be associated with pathogenic sequence variants in a number of different genes [4]. Consequently, mutation detection by the candidate gene approach was found to be cumbersome and inefficient. These limitations were overcome by adoption of next generation sequencing approaches that target concomitantly multiple genes or the entire exome or genome of an affected individual. For EB, a number of gene targeted sequencing arrays were developed which examined many or all of the 21 EB-associated genes in one reaction (Fig. 1) [19]. The limitation of this approach is that only a defined number of established candidate genes will be examined, and this methodology does not allow identification of novel, previously undescribed genes. The mutation detection strategies have recently moved to utilization of whole exome sequencing (WES) which allows interrogation of over 20,000 genes in the human genome, with emphasis on exons and flanking intronic sequences. Limitations of this approach include inability to identify pathogenic sequence variants which reside outside of the exons, such as deep intronic sequences distant from the exon-intron borders and those in the regulatory regions, such as in the promoter. Such mutations can be identified by application of whole genome sequencing (WGS) which identifies sequence variants in the entire genome consisting of approximately 3 billion base pairs. In comparison to WES, which examines one to two percent of the entire genome, WGS requires more sophisticated bioinformatics pipelines for sequence evaluations with increased databases and computer performance capacity. Also, DNA based WES and WGS do not identify the consequences of the mutations, particularly those affecting the splicing of the pre-mRNA and they can miss synonymous sequence variants at the exon-intron borders.

The consequences of mutations on splicing can be examined by whole transcriptome sequencing by RNA-Seq which, besides identifying the primary mutations in the DNA, also documents alterations in the splicing profiles by Sashimi plots and quantitates the gene expression at the RNA level by heatmap analysis [20]. A limitation of the whole transcriptome sequencing is that RNA has to be isolated from tissues or cells expressing the corresponding genes. However, in the case of skin disorders, a 4 mm biopsy, which contains KCs, fibroblasts, melanocytes, and skin intrinsic lymphocytes as the principal cell types, is sufficient for isolation of RNA for mutation analysis and gene expression profiling in heritable skin diseases. Based on these considerations, it has been suggested that whole transcriptome sequencing by RNA-Seq could serve as the first-tier approach to examine the genetic basis of heritable skin diseases [20].

In addition to RNA-Seq, the pathophysiological consequences of the sequence variants in skin fragility diseases have been investigated by mass spectrometry (MS) based proteomic techniques. Initially, robust peptide-sequence protein identification analysis of skin-samples via polyacrylamide gel electrophoresis, liquid chromatography, and electrospray ionization-MS/MS analysis allowed for characterization of proteins in keratinocytes, fibroblasts, and the extracellular matrix, however, revolutionization of proteomic approaches led to the development of techniques focused on accurate quantitative data rather than protein identification [21]. Therefore, the generation of chemical and metabolic labeling via Stable Isotope Labeling by Amino Acids in Cell Culture techniques and isobaric tags for relative and absolute quantification and tandem mass tags, respectively, were used to quantify protein abundance, identify post-translational modifications, and examine the dynamic proteome [21]. However, in the last few years label free approaches, such as sequential window acquisition of all theoretical mass spectra (SWATH-MS), have been used to study the proteomic physiological response of skin cells. These techniques have allowed identification of the structural proteins in the dermal-epidermal junction, followed by pathomechanisms of the pathogenic variants. In fact, proteomic studies elucidated the disease mechanism in RDEB and identified dysregulation of TGF-β signaling resulting in scarring due to loss of collagen VII [21, 22].

With the advent of next generation sequencing approaches, a number of new mutated genes have been identified in patients with different forms of skin fragility syndromes, particularly with EB. Many of these genes encode proteins that are not structural, but serve in enzymatic processing, cellular transport and other functions critical for physiological integrity of skin as a protective organ against external trauma (Table 1) (Fig. 1).

Post-translational modifications of the basement membrane structural proteins, particularly collagens, are critical for functional integrity of the skin. Consequently, genetic alterations affecting genes that modify these proteins often lead to heritable disorders, as illustrated by similar phenotypes in case of COL7A1 and PLOD3 mutations. Collagen, a family of structurally related macromolecules, is the most abundant protein in the body and is vital for the integrity of bones, muscles, and tendons, as well as skin [6]. In the dermis, the major interstitial collagen fibers, consisting of types I, III, and V, provide resilience to the skin as a protective organ against external trauma. Type VII collagen is a minor collagen, yet it is the key element of the anchoring fibrils at the dermal-epidermal junction (Fig. 1). COL7A1 encodes a 290-kDa type VII collagen polypeptide, synthesized and secreted by epidermal KCs and dermal fibroblasts [6]. All collagens are trimeric molecules and have a triple-helical collagenous domain made up of Gly-X-Y amino acid repeats, where the X and Y positions are often occupied by a proline or lysine [6,71]. These amino acids, when in the Y-position, can be hydroxylated by prolyl and lysyl hydroxylases to yield hydroxyproline and hydroxylysine, respectively. Type VII collagen consists of two adjacent collagenous domains separated by a 39 amino acid “hinge” region and flanked by N- and C-terminal non-collagenous domains, the latter one being critical for the formation of a homotrimeric type VII collagen which then folds into a characteristic triple-helix [6]. The stability of the triple helix at physiological temperatures is dependent on the presence of hydroxyproline residues. Following secretion, the collagen VII molecules are cleaved at the C termini and the molecules are oriented as antiparallel dimers, which continue through lateral aggregation to assemble into the anchoring fibrils [6].

During the processing of collagen polypeptides, a number of post-translational modifications are required to create a functional protein. In one of them, the lysyl residues in the Y position of the Gly-X-Y repeats are targets for post-translational modifications by lysyl hydroxylases (LH), procollagen-lysine, 2-oxoglutarate 5-dioxygenases (PLOD)(Fig. 1). Lysyl hydroxylases consist of four distinct isoforms: LH1, LH2a, LH2b, and LH3 [23]. LH3/PLOD3 encodes a multidomain polypeptide, PLOD3, which contains two functional domains: (i) a C-terminal lysyl hydroxylase domain, which hydoxylates collagen lysyl residues (LH activity), and (ii) a catalytically active N-terminal glycosyltransferase domain, which glycosylates hydroxylysine residues by adding a galactose (GT activity) (Fig. 2a) [23]. The AC domain, which does not appear to be biologically active, is positioned between these two catalytically active domains (Fig. 2a) [23]. Lysyl hydroxylation and O-glycosylation of the hydroxylysyl residues are critical post-translational modifications providing physiological properties to collagens and their fiber structures. Unlike other lysyl hydroxylases which are catalytically active in rough endoplasmic reticulum during collagen synthesis by cells, LH3 is secreted into the extracellular millieu and has been detected in the sub-lamina densa, where it hydroxylates and glycosylates the lysines in the collagenous domain of type VII collagen (Fig. 1) (Table 1) [23]. In this context, it is important to note that hydroxylysyl residues play a critical role in the formation of stable intermolecular collagen cross-links which stabilize the supramolecular assembly of collagen fibers. Therefore, alterations in the PLOD3 gene can result in destabilized collagen networks and affect cell-matrix signaling for adhesion. In keeping with the role of LH3 in the biosynthesis of type VII collagen, mutations in PLOD3 can lead to the severe pathological manifestations seen in PLOD3 associated EB.

The clinical manifestations of PLOD3 mutations vary. Until now, only 16 cases of autosomal recessive PLOD3 variants have been described, however, only two of the reported cases have described cutaneous involvement with syndromic manifestations [24, 25]. One patient harbored a homozygous missense mutation in the LH catalytic domain (c.1880T>C; p.Leu627Pro) (Fig. 2b). The other patient was compound heterozygous for a missense mutation in the GT domain and a premature termination codon due to a deletion within the LH catalytic site (c.668A>G; p.Asn223Ser/C.2071delT;p.Cys691AlafsX9) (Fig. 2b) [24, 25].

The patients with PLOD3 mutations have a clinically variable presentation including craniofacial dysmorphism, short stature, musculoskeletal abnormalities, hearing loss, vascular aneurysms, and trauma-induced blistering and erosions noted at birth [24, 25]. Histopathologically, there is sub-lamina densa separation at the dermal-epidermal junction and fibrosis within the papillary dermis, consistent with DEB [25]. Immunohistochemistry for LH3 at the basement membrane zone was greatly reduced in the skin of these patients [25, 26]. Furthermore, there was diminished type VII collagen expression, as well as reduced quantity and altered morphology of anchoring fibrils, all contributing to the clinical manifestations and pathogenesis consistent with a syndromic form of RDEB [25].

U6 snRNA biogenesis gene (USB1) encodes a 3′−5′exoribonuclease, U6 snRNA phosphodiesterase (USB1), which modifies the 3′ polyuridine tract of the U6 snRNA, one of the five subunits (U1, U2, U4, U5 and U6) of the spliceosome complex that catalyzes the removal of introns from pre-mRNA (Fig. 1) (Table 1) [27]. It cleaves the phosphodiester bond at the 3′ end of the uridine and adenosine tract of U6 snRNA, and it catalyzes a terminal 2′,3′-cyclic phosphate group (>p) at the end of a non-templated uridine (Fig. 1) [27]. These 3′ modifications protect the spliceosomal U6 snRNA from adenylation by the polyA polymerase Trf4. This adenylation makes the U6 snRNA a target for degradation by nuclear exosomes. However, when USB1 is mutated, U6 snRNA is polyadenylated, making it a target for degradation [28]. This modification induces changes in spliceosome activity, which affects the expression of a number of genes, including those responsible for the structural integrity of the basement membrane zone [29].

Aberrations in the USB1 protein were first associated with the rare autosomal recessive genodermatosis, poikiloderma with neutropenia (PN), in the Navajo population, and more recently with Kindler EB [29], [30], [31]. Currently, there are 29 reported mutations in USB1, 15 of which are nonsense mutations or insertion/deletions causing premature termination codons (Fig. 2c, Fig. 2d). Five of them reside within canonical splice sites (+2/−2) at exon-intron junction, and one of them is a non-canonical intronic splice site variant, all resulting in predicted loss-of-function (Fig. 2c). The remaining three are missense variants, noted to be variants of unknown significance (VUS) (Fig. 2c) [29].

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PN is initially characterized by an inflammatory eczematous rash that develops within the first six to 12 months of life and resolves with post-inflammatory poikiloderma [31]. Additionally, these patients present with telangiectasia, palmoplantar hyperkeratosis, nail dystrophy, skin atrophy, and photosensitivity associated with three reported cases with skin blistering [29, [31], [32], [33]]. Neutropenia typically develops in the first two years of life and puts the patients at risk for recurrent sinopulmonary bacterial infections, such as pneumonia, otitis media, and sinusitis. These patients also exhibit systemic symptoms of skeletal defects including growth hormone deficiency resulting in short stature, craniofacial dysmorphism, such as saddle nose and midface hypoplasia, as well as dental defects, myelodysplasia and hematologic malignancies [31]. Skin histopathology of PN is characterized by atrophic and flattened epidermis.

Clinically, there is a significant overlap between PN and KEB. KEB is characterized by trauma-induced blistering and progressive poikiloderma, photosensitivity and mucosal blistering. Additionally, these patients suffer from dental defects such as gingivitis and periodontitis, recurrent infections including conjunctivitis and severe colitis, as well as nonmelanoma skin cancers [34]. KEB is associated with mutations in FERMT1, encoding fermitin family homolog 1 (Kindlin-1), an integral protein of focal adhesions (Fig. 1). In addition to the clinical manifestations, ultrastructural abnormalities and reduced immunolabeling for Kindlin-1 at the dermal-epidermal junction are characteristic diagnostic findings in KEB. Additionally, transmission electron microscopy typically shows widening of intercellular spaces between KCs and reduplication of the lamina densa [29, 34].

While FERMT1 is the candidate gene for KEB, a recent study found a link between KEB and USB1. Albeit not officially named a gene for EB, Vahidnezhad et al. discovered a relationship between a non-canonical splice site mutation, c.265+4A>G, in USB1 that had classic ultrastructural KEB abnormalities [29, 34]. In this case, Kindlin-1 expression was reduced, however no mutations in FERMT1 were found, implying the connection between USB1 and FERMT1. Additionally, transcriptional evaluation of the patient's mRNA expression showed that the transcript level of multiple BMZ genes associated with EB and poikiloderma were reduced by >2 fold, and immunofluorescence of several basement membrane structural proteins associated with EB were reduced [29]. The reduction of the expression of multiple genes, as documented by RNA-seq, emphasizes the power of next generation sequencing in identification of the candidate genes, when analysis of the prototypical causative gene, such as FERMT1 in this case, is unyielding.

Exophillin-5 (EXPH5) encodes synaptotagmin-like protein homolog lacking C2 domains b (Slac2-b), which is an effector protein for the GTPase ras-related protein Rab27b. Slac2-b is involved in the intracellular trafficking of multivesicular bodies (MVB), which helps to maintain the epidermal integrity (Fig. 1, Fig. 3b) (Table 1). Alterations in this protein have been linked to abnormal epidermal development and skin fragility [35, 36]. In a series of patients with skin fragility, ultrastructural analysis of the skin revealed breaks in the basal epidermal layer that were compatible with EBS. Clinically, EBS caused by EXPH5 (EBS-EXPH5) is characterized by trauma-induced blisters that heal with scales and hemorrhagic crusts [36], [37], [38], [39], [40], [41], [42], [43]. Additionally, some cases demonstrate progressive mottled pigmentary changes [36, 41]. To date, there have been 10 cases of EBS associated with mutations in EXPH5, with one case being digenic with mutations in both EXPH5 and COL17A1 [42]. There have been nine distinct mutations reported in EXPH5, all of which were either homozygous or compound heterozygous nonsense mutations or deletions in exon 6 causing premature termination codons resulting in predicted loss-of-function (Fig. 3a).

It is well established that Slac2-b is involved in cell-to-cell adhesion and extracellular vesicle trafficking and secretion but the interactions that regulate this system are not fully understood (Fig. 3b) [35, 36]. Therefore, to elucidate the etiology of the blistering in EBS-EXPH5, multiple studies have assessed the physiologic role that Slac2-b plays in the migration and adhesion of structural proteins of the basement membrane zone (Fig. 3b). Studies have found that aberrations in this protein demonstrated a reduction in KC adhesion and lead to an accumulation of perinuclear vesicles. In fact, Moteleon et al., determined that Slac2-b is necessary for the trafficking of lamellar bodies secretory vesicles, lysosome related organelles (LROs), which are responsible for secreting lipids into the ECM for epidermal differentiation (Fig. 3b) [44]. Indeed, EXPH5-knockdown KCs exhibited abnormal epidermal differentiation, but when co-cultured with normal KCs, LROs were able to rescue the epidermal differentiation defect [44]. Slac2-b is also implicated in its interaction with transmembrane 4 L six family member 5 (TM4SF5), a membrane protein that promotes cell migration [45]. This study discovered that Slac2-b and paxillin expression regulate the acetylation of microtubules, which facilitates the trafficking and translocation of TM4SF5 [45]. Further investigation into mutant Slac2-b KC adhesion also found that mutant and depleted Slac2-b KCs exhibited increased perinuclear vesicle accumulation and reduced extracellular vesicle release (Fig. 3b) [46]. This study also found that CD63 vesicle trafficking was reduced and found that CD63 vesicles were present for both focal adhesion assembly and disassembly in KCs, but its actions are unclear. Additionally, mutant Slac2-b KCs exhibited increased number of focal adhesions, however cell adhesion was reduced and focal adhesion complexes had slower assembly and disassembly rates. These mutant Slac2-b and depleted Slac2-b KCs also had reduced levels of Rab27a, a protein well known for processing vesicle transportation and critical in the transport of melanosomes, which could possibly explain the mottled pigmentary changes seen in these patients [46]. These findings suggest a potential role for Slab2-b in the regulation of cell migration and adhesion in KCs for epidermal differentiation.

The Kelch-like (KLHL) gene family encodes over 40 evolutionarily conserved proteins that are ubiquitously expressed at low levels. The multidomain KLHLs consists of a BACK domain, multiple Kelch-like repeats, and a BTB/POZ domain which is known to aid in protein binding and dimerization. KLHLs work in tandem with an E3 ubiquitin ligase, cullin 3 (CUL3), via binding of BTB domain, for targeted ubiquitination of cytoskeletal intermediate filaments and transcription factors (Fig. 2e) (Table 1) [47]. Specifically, the Kelch-like family member 24 (KLHL24 gene) encodes the protein KLHL24, which functions as a substrate-specific adaptor for CUL3, and together they create a ubiquitin-ligase complex that has been implicated in the ubiquitination and degradation of keratin intermediate filaments [47]. This model is tightly controlled, and therefore, any changes to this system could be expected to lead to pathological skin manifestations.

EBS is caused by mutations in genes encoding structural proteins in hemidesmosomal and focal adhesion complexes, CD151, DST, and PLEC [[72], [73], [74]]. However, the most common causes of autosomal dominant EBS are mutations in genes encoding structural proteins in the basal KCs of the epidermis, KRT5 and KRT14 [69]. However, next generation sequencing contributed to the discovery of KLHL24 involvement in EBS. To date, there have been 73 reported cases of EBS caused by KLHL24 mutations. Among them, there have been 60 cases of heterozygous monoallelic gain-of-function variants (c.1A>G, c.1A>T, c.2T>C, c.3G>T, and c.3G>A) in the translation initiation codon of KLHL24 affecting methionine 1 (Fig. 2e, Fig. 2f) [48], [49], [50], [51], [52], [53], [54], [55], [56], [57], [58], [59], [60]. These variants have been indicated as the causal mutations in patients presenting with EBS (EBS-KLHL24), with associated neurological and developmental delay, and a unique cardio-cutaneous syndrome. Although the phenotypic characteristics of EBS-KLHL24 vary, it is clinically defined by severely denuded skin/congenital aplasia cutis on the extremities at birth, which heals with atrophic scars, and associated mild trauma-induced blistering that heals with dyspigmented scars. However, skin fragility is noted to improve with increasing age. Additionally, patients may present with nail dystrophy, alopecia, palmoplantar hyperkeratosis, and oral ulcerations.

The monoallelic variants in the translation initiation codon result in a truncated KLHL24 lacking the first 28 amino acids of the protein (ΔN28-KLHL24) (Fig. 2e). The loss of the first 28 amino acids abolishes an auto-ubiquitination site within the mutant KLHL24, leading to a stabilized form of the protein that has increased ubiquitination capacity compared to the wild-type (WT) [49]. Initially, it was suggested that this stabilized mutant form of KLHL24 caused increased ubiquitination and selective degradation of K14, therefore compromising the mechanical integrity of the basal KCs [48]. Indeed, Lin et al. showed that a knock-in mouse model harboring the mutation c.3G>T had significantly destabilized and reduced K14 levels, however, He et al. did not note a significant reduction in K14 immunoreactivity, but rather increased fragmentation of K14 and vimentin in mutant KCs. Moreover, follow-up studies also confirmed that immunolabeling of WT and mutant K14 in EBS-KLHL24 skin showed normal K14 staining and K14 protein levels were preserved in both in vivo and in vitro disease models [50, 51, 53, 59]. This presentation caused many to postulate that the EBS-KLHL24 clinical presentation is either not entirely dependent on the loss of K14 or there is an age-dependent change in K14 filament degradation due to the clinical improvement in patients. Indeed, Vermeer et al. examined fetal-like human induced pluripotent KCs (hiPSC-derived KC) and matured primary KCs K14 degradation [61]. They found that there was no reduction in K14 immunofluorescence and western blot protein levels in differentiated and undifferentiated matured primary KCs of patients, respectively. However, undifferentiated fetal hiPSC-derived KCs K5/K14 protein levels were low and knockdown of KLHL24 led to a 3-fold and 4-fold increase in K5 and K14, respectively. Moreover, K14 protein levels in differentiated cells were not affected by KLHL24 knockdown [61]. Subsequent examination by Logli et al., of fetal KCs of EBS-KLHL24 patients revealed the involvement of additional keratins in the EBS-KLHL24 phenotypes. They found that keratins 7, 8, 17, and 18 were all significantly reduced, owing to degradation by proteasome in KCs expressing ΔN28-KLHL24. Additionally, they found that 33% of the mutant ΔN28-KLHL24-transduced KCs that were heat treated, exhibited aberrant detachment and were not viable [59]. These observations could relate to the pathomechanisms of severely denuded skin seen in this patient subgroup at birth, possibly due to the higher physiologic temperature of the neonates. However, the exact intermediate filament(s) involved in the pathogenesis of EBS-KLHL24 are still unclear and further analysis of the age-dependent involvement of each keratin is needed.

Due to the abundant tissue distribution of KLHLs, several studies have investigated the connection between aberrations in EBS-KLHL24 and other systemic pathologies. Specifically, a number of cases of EBS-KLHL24 patients have been diagnosed with rapidly progressive dilated cardiomyopathy (DCM). These patients had elevated cardiac biomarkers and/or diagnostic echocardiogram confirmation of DCM by late adolescence. The co-incidence of DCM and EBS-KLHL24 have been reported in 25 patients so far, with two of the patients dying early as a result of DCM [[53], [54], [55], [56], [57], 62]. Interestingly, sequence variants in KLHL24 have also been implicated in patients exhibiting hypertrophic cardiomyopathy (HCM), however, these patients do not exhibit EBS phenotype [62]. These studies reported loss of function mutations in different genetic locations of KLHL24, rather than the common translation initiation codon variants seen in EBS-KLHL24 patients. Currently there are two mutations reported to lead to HCM-KLHL24, one heterozygous nonsense mutation, c.1048G>T; p.Glu350*, that is located within the Kelch-like repeat domain, and a homozygous mutation, c.917G>A; p.Arg306His, in a highly conserved region of the gene Fig. 2f) [62].

Several studies have investigated the pathomechanism of KLHL24 involvement in both DCM and HCM. Initially, it was found that KLHL24 was expressed in cardiomyocytes, in a pattern that resembled that of structural intercalated discs [53]. Further investigation discovered that the gain-of-function mutations in DCM EBS-KLHL24 lead to excessive degradation of desmin, the cardiac homolog of K14, through ubiquitination and proteasomal degradation [56]. Furthermore, in patients with HCM there was a 10-fold reduction of functional desmin levels, and while there was a compensatory increased expression of desmin at the mRNA level, the overall function remained reduced [62]. Additionally, HCM patients' cardiac and skeletal myocytes had increased accumulation of glycogen, however no pathogenic sequence variants in genes associated with glycogen storage diseases were found. These findings suggested an additional pathomechanistic consequence of KLHL24 inactivation and may provide a linkage to a new form of glycogen storage disease with primary manifestations in the heart [62].

There are no curative therapies currently available for the different subtypes of EB. Traditional therapeutic approaches focus on symptomatic relief and skin barrier protection, via topical treatments, wound management, and trauma avoidance [63]. However, molecular genetics has revolutionized the identification of the genetic heterogeneity in EB and underscore the features of physiologically complex syndromic and non-syndromic consequences of mutations in individual genes. Therefore, interdisciplinary genetic and symptomatic approaches to develop future therapies have led to pre-clinical and clinical, targeted gene and cell-based trials with a curative aim. Such therapies have focused on gene therapy, premature-termination codon read-through, cell therapy, protein replacement therapy, and the use of antisense oligonucleotides to aid in-frame skipping of exons harboring the mutations [63, 64]. In addition to gene and cell-based therapies, proteomic pathophysiology analyses have helped to identify treatment modalities in various disease such as RDEB, where losartan has been employed to interfere with the secondary effects of TGF-β dysregulation, ameliorating the inflammation and fibrosis associated with RDEB, with the goal of improving the patients’ quality of life [22, [65], [66], [67], [68]]. With the advent of next generation sequencing, discovery of novel genes opens another avenue for future therapeutic manipulations. Understanding of the physiological kinetics of the actions of specific proteins, such as encoded by EXPH5, USB1, KLHL24, and PLOD3, will allow for the development of targeted therapeutic agents that will not only open the road to a cure for EB, but potentially many other inherited disorders.

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    Collagen type IV (COL IV) is a major component of basement membranes (BM) in all organs. It serves functions related to BM organization and modulates the passage of growth factors from one tissue compartment to another. COL IV binds transforming growth factor (TGF) beta-1 and TGF beta-2 and, therefore, is a major modulator of TGF beta pro-fibrotic functions. After fibrotic corneal injury, TGF beta enters into the stroma from the tears, epithelium, endothelium and/or aqueous humor and markedly upregulates COL IV production in corneal fibroblasts in the adjacent stroma far removed from BMs. It is hypothesized this non-BM stromal COL IV binds TGF beta-1 (and likely TGF beta-2) in competition with the binding of the growth factors to TGF beta cognate receptors and serves as a negative feedback regulatory pathway to mitigate the effects of TGF beta on stromal cells, including reducing the developmental transition of corneal fibroblasts and fibrocytes into myofibroblasts. Losartan, a known TGF beta signaling inhibitor, when applied topically to the cornea after fibrotic injury, alters this COL IV-TGF beta pathway by down-regulating COL IV production by corneal fibroblasts. Non-BM COL IV produced in response to injuries in other organs, including the lung, skin, liver, kidney, and gut, may participate in similar COL IV-TGF beta pathways and have an important role in controlling TGF beta pro-fibrotic effects in these organs.

  • Research article

    Recalcitrant Cutaneous Warts in a Family with Inherited ICOS Deficiency

    Journal of Investigative Dermatology, Volume 142, Issue 9, 2022, pp. 2435-2445

    Recalcitrant warts, caused by human papillomaviruses (HPVs), can be a cutaneous manifestation of inborn error of immunity. This study investigated the clinical manifestations, immunodeficiency, single-gene susceptibility, and HPV repertoire in a consanguineous family with severe sinopulmonary infections and recalcitrant warts. Clinical and immunologic evaluations, including FACS and lymphocyte transformation test, provided evidence for immunodeficiency. Combined whole-exome sequencing and genome-wide homozygosity mapping were utilized to disclose candidate sequence variants. Whole-transcriptome sequencing was used to concomitantly investigate the HPV genotypes and the consequences of detected sequence variants in the host. The proband, a male aged 41 years, was found to be homozygous for the c.6delG, p.Lys2Asnfs∗17 variant in ICOS, encoding the inducible T-cell costimulator. This variant was located inside the 5 megabase of runs of homozygosity on 2q33.2. RNA sequencing confirmed the deleteriousness of the ICOS variant in three skin biopsies revealing significant downregulation of ICOS and its ligand, ICOSLG. Reads unaligned to the human genome were applied to 926 different viruses, and α-HPV57, β-HPV107, β-HPV14, and β-HPV17 were detected. Collectively, we describe a previously unrecognized inborn error of T-cell immunity to HPVs, indicating that autosomal recessive ICOS deficiency can underlie recalcitrant warts, emphasizing the immunologic underpinnings of recalcitrant warts at the nexus of human and viral genomic variation.

  • Research article

    Pancreatic ductal adenocarcinoma cells employ integrin α6β4 to form hemidesmosomes and regulate cell proliferation

    Matrix Biology, Volume 110, 2022, pp. 16-39

    Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis due to its aggressive progression, late detection and lack of druggable driver mutations, which often combine to result in unsuitability for surgical intervention. Together with activating mutations of the small GTPase KRas, which are found in over 90% of PDAC tumours, a contributory factor for PDAC tumour progression is formation of a rigid extracellular matrix (ECM) and associated desmoplasia. This response leads to aberrant integrin signalling, and accelerated proliferation and invasion. To identify the integrin adhesion systems that operate in PDAC, we analysed a range of pancreatic ductal epithelial cell models using 2D, 3D and organoid culture systems. Proteomic analysis of isolated integrin receptor complexes from human pancreatic ductal epithelial (HPDE) cells predominantly identified integrin α6β4 and hemidesmosome components, rather than classical focal adhesion components. Electron microscopy, together with immunofluorescence, confirmed the formation of hemidesmosomes by HPDE cells, both in 2D and 3D culture systems. Similar results were obtained for the human PDAC cell line, SUIT-2. Analysis of HPDE cell secreted proteins and cell-derived matrices (CDM) demonstrated that HPDE cells secrete a range of laminin subunits and form a hemidesmosome-specific, laminin 332-enriched ECM. Expression of mutant KRas (G12V) did not affect hemidesmosome composition or formation by HPDE cells. Cell-ECM contacts formed by mouse and human PDAC organoids were also assessed by electron microscopy. Organoids generated from both the PDAC KPC mouse model and human patient-derived PDAC tissue displayed features of acinar-ductal cell polarity, and hemidesmosomes were visible proximal to prominent basement membranes. Furthermore, electron microscopy identified hemidesmosomes in normal human pancreas. Depletion of integrin β4 reduced cell proliferation in both SUIT-2 and HPDE cells, reduced the number of SUIT-2 cells in S-phase, and induced G1 cell cycle arrest, suggesting a requirement for α6β4-mediated adhesion for cell cycle progression and growth. Taken together, these data suggest that laminin-binding adhesion mechanisms in general, and hemidesmosome-mediated adhesion in particular, may be under-appreciated in the context of PDAC.

    Proteomic data are available via ProteomeXchange with the identifiers PXD027803, PXD027823 and PXD027827.

  • Research article

    Kinase Inhibition by PKC412 Prevents Epithelial Sheet Damage in Autosomal Dominant Epidermolysis Bullosa Simplex through Keratin and Cell Contact Stabilization

    Journal of Investigative Dermatology, Volume 142, Issue 12, 2022, pp. 3282-3293

    Epidermolysis bullosa simplex (EBS) is a severe and potentially life-threatening disorder for which no adequate therapy exists. Most cases are caused by dominant sequence variations in keratin genes K5 or K14, leading to the formation of cytoplasmic keratin aggregates, profound keratinocyte fragility, and cytolysis. We hypothesized that pharmacological reduction of keratin aggregates, which compromise keratinocyte integrity, represents a viable strategy for the treatment of EBS. In this study, we show that the multikinase inhibitor PKC412, which is currently in clinical use for acute myeloid leukemia and advanced systemic mastocytosis, reduced keratin aggregation by 40% in patient-derived K14.R125C EBS-associated keratinocytes. Using a combination of epithelial shear stress assay and real-time impedance spectroscopy, we show that PKC412 restored intercellular adhesion. Molecularly, global phosphoproteomic analysis together with immunoblots using phosphoepitope-specific antibodies revealed that PKC412 treatment altered phosphorylated sites on keratins and desmoplakin. Thus, our data provide a proof of concept to repurpose existing drugs for the targeted treatment of EBS and showcase how one broad-range kinase inhibitor reduced keratin filament aggregation in patient-derived EBS keratinocytes and the fragility of EBS cell monolayers. Our study paves the way for a clinical trial using PKC412 for systemic or local application in patients with EBS.

© 2022 Elsevier B.V. All rights reserved.


What is the pathophysiology of epidermolysis bullosa? ›

Pathophysiology of Epidermolysis Bullosa

Genetically mediated defects in epithelial adhesion proteins result in skin and mucous membrane fragility, which predisposes the epithelium to easy bullae formation after minor trauma or sometimes spontaneously.

What protein does epidermolysis bullosa make? ›

The four major types of epidermolysis bullosa simplex can result from mutations in either the KRT5 or KRT14 gene. These genes provide instructions for making proteins called keratin 5 and keratin 14.

What gene is mutated in epidermolysis bullosa? ›

Mutations in the COL7A1 gene cause all forms of dystrophic epidermolysis bullosa. This gene provides instructions for making a protein that forms the pieces (subunits) of a larger protein called type VII collagen.

What are the facts about epidermolysis bullosa? ›

People with EB have a fault in their DNA which stops the production of proteins. These proteins are used to hold the layers of skin together and make it strong. EB affects the skin and internal organs, and is a genetic lifelong condition that cannot be cured.

What is the root cause of epidermolysis bullosa? ›

Causes of epidermolysis bullosa

EB is caused by a faulty gene (gene mutation) that makes skin more fragile. A child with EB might have inherited the faulty gene from a parent who also has EB. Or they might have inherited the faulty gene from both parents who are just "carriers" but don't have EB themselves.

What is the molecular basis of epidermolysis bullosa? ›

Mutations within the gene encoding the anchoring fibril protein type VII collagen (COL7A1) have recently been established as the pathogenetic basis for the inherited blistering skin disorder, dystrophic epidermolysis bullosa.

What is the keratin mutation in epidermolysis bullosa? ›

Epidermolysis bullosa simplex (EBS) is an inherited skin disorder caused by mutations in keratins K5 (keratin 5) and K14 (keratin 14), with fragility of basal keratinocytes leading to epidermal cytolysis and blistering.

What type of collagen is epidermolysis bullosa? ›

Abstract. Recessive dystrophic epidermolysis bullosa is a devastating blistering disease caused by mutations in the COL7A1 gene, which encodes type VII collagen, the major component of anchoring fibrils.

What is the relationship between epidermolysis bullosa and connective tissue? ›

Epidermolysis bullosa is a connective tissue disorder that causes your skin to blister and tear easily. Treatment helps prevent blisters from forming, care for blisters and skin, treat nutritional problems that arise from blisters in the mouth or esophagus and manage pain.

What chromosomes are affected in epidermolysis bullosa? ›

Recent reports have suggested that EBS mutations may relate to keratin abnormalities. In this study, we conducted RFLP analyses to test the hypothesis that EBS is linked to one of the keratin gene clusters on chromosome 12 or chromosome 17.

What is the differentiating factor in the types of inherited epidermolysis bullosa? ›

There are three classic types of inherited EB (simplex, junctional and dystrophic). They are differentiated by the level of blister cleavage and subdivided according to the pattern of genetic inheritance, morphology/topography of lesions and genetic mutation involved.

What famous person had epidermolysis bullosa? ›

Jonny Kennedy (4 November 1966 – 26 September 2003) was a British man who had a rare inherited condition known as dystrophic epidermolysis bullosa (EB or DEB). Kennedy ultimately died of skin cancer, a complication of EB.

What is the most common cause of death in epidermolysis bullosa? ›

Monitoring for cancer: Squamous cell carcinoma is the leading cause of death in EB usually occurring after the 2nd decade of life. Patients with RDEB and JEB are at increased risk of developing skin cancers during their lifetimes.

Is epidermolysis bullosa autoimmune? ›

Epidermolysis bullosa acquisita (EBA) is an acquired, autoimmune subepidermal blistering disease with an approximate prevalence of 0,2/million people.

Why do people with EB have deformed hands? ›

Blistering begins at birth or shortly afterwards. Much of the skin is covered in blisters and there is extensive internal blistering. Children can develop deformities caused by the recurrent scarring of the fingers and toes (pseudosyndactyly) and the hands and arms become fixed in stiff positions (contractures).

What is the life expectancy of a person with epidermolysis bullosa? ›

The disease appears at birth or during the first few years of life, and lasts a lifetime. Prognosis is variable, but tends to be serious. Life expectancy is 50 years, and the disease brings with it complications related to infections, nutrition and neoplastic complications.

Does epidermolysis bullosa affect brain? ›

Abstract. Background: Children with Epidermolysis bullosa (EB) suffer from an intractable, burdensome skin disease that may result in cognitive as well as social and emotional problems.

Is epidermolysis bullosa a missense mutation? ›

The prevalence of epidermolysis bullosa simplex (EBS) is estimated to be approximately 6 to 30 per 1 million live births. The disease is usually caused by missense mutations in KRT5 and KRT14, encoding keratins mostly expressed in the epidermal basal layer.

What do epidermolysis bullosa simplex mutations in keratin genes interfere with the formation of? ›

RNA Interference (RNAi)

Which type of mutation results in the most severe form of junctional epidermolysis bullosa? ›

JEB generalized severe is the more serious form of the condition.

What are the three types of epidermolysis bullosa? ›

The main types of epidermolysis bullosa are:
  • Epidermolysis bullosa simplex. This is the most common type. ...
  • Junctional epidermolysis bullosa. This type may be severe, with blisters beginning in infancy. ...
  • Dystrophic epidermolysis bullosa. ...
  • Kindler syndrome.
Aug 20, 2022

What is the difference between Type and 1 and 3 and Type 2 collagen? ›

The main difference between collagen 1 2 and 3 is that collagen 1 is most abundant in bones, tendons, ligaments, and in the skin while collagen 2 occurs in hyaline and articular cartilages and collagen 3 is the main component of reticular fibers which make a supporting mesh in soft tissues and organs.

What are the subtypes of epidermolysis bullosa? ›

There are four main types of EB that are classified based on the layer of the skin affected. These are dystrophic epidermolysis bullosa (DEB), epidermolysis bullosa simplex (EBS), junctional epidermolysis bullosa (JEB), and Kindler syndrome (KS).

What is the difference between epidermolysis bullosa and pemphigus? ›

Bullous pemphigoid (BP) and epidermolysis bullosa acquisita are distinct autoimmune blistering disorders. BP is characterized by autoantibodies directed against the NC16A domain of collagen XVII, whereas patients with epidermolysis bullosa acquisita have autoantibodies against the NC1 domain of type VII collagen.

What is the recessive dystrophic form of epidermolysis bullosa? ›

Recessive dystrophic EB – severe (RDEB-S)

In this form of EB, the skin is extremely fragile, often with extensive blistering and wounds. There is usually no collagen VII present. Wounds tend to heal with a scar that can lead to contractures and impaired movement, particularly affecting the hands.

Is Marky with butterfly skin still alive? ›

Epidermolysis bullosa is a genetic condition that causes the skin to be fragile and blister easily. A well-known 21-year-old TikToker named Marky Jaquez has passed away from Epidermolysis bullosa, also known as butterfly skin disease. It's a genetic condition that causes the skin to be fragile and blister easily.

What Hollywood actor has lupus? ›

Diagnosed in 2012, Nick Cannon, a multitalented American rapper, actor, comedian, director, screenwriter, producer, and entrepreneur, first experienced severe symptoms of lupus, including kidney failure and blood clots in his lung.

What did Marky Jaquez have? ›

Mother of Son with Rare Skin Condition Speaks Out after Being Banned from TikTok. Melissa Jaquez's son Marky was born with the rare skin disease called “epidermolysis bullosa,” also known as butterfly syndrome. The genetic skin disorder causes Marky's skin to blister easily and become very fragile.

How close are we to a cure for epidermolysis bullosa? ›

There's currently no cure for epidermolysis bullosa (EB), but treatment can help ease and control symptoms. Treatment also aims to: avoid skin damage. improve quality of life.

How many people in the US have epidermolysis bullosa? ›

Epidermolysis Bullosa, or EB, is a rare genetic connective tissue disorder that affects 1 out of every 20,000 births in the United States (approximately 200 children a year are born with EB).

Why is epidermolysis bullosa so painful? ›

Because patients with epidermolysis bullosa are deficient in laminin-332, the transduction of the stimulus is unsuppressed. Their sensory neurons are excited much more strongly, and thus they react much more sensitively to mechanical stimuli,” Professor Lewin explained.

How many people in the world have epidermolysis bullosa? ›

Understanding EB & its classification

With a prevalence of only about 500,000 people worldwide, EB as a group of conditions is rare, and some subtypes are very rare.

Can you get epidermolysis bullosa later in life? ›

In its mildest form, the blisters usually occur only on the hands and feet of a newborn. Later in life, the skin may stop blistering, leaving a teen or adult with thickened, hard skin on the palms and soles. Epidermolysis bullosa simplex causes blistering in the outermost layer of skin, which is the epidermis.

How does epidermolysis bullosa work? ›

Epidermolysis bullosa (EB) is a group of genetic (inherited) disorders that causes your skin to be fragile and blister and tear easily. Blisters and sores form when clothing rubs against your skin, or you bump your skin. Mild cases of the disease usually cause painful blisters on the hands, elbows, knees and feet.

What is epidermolysis bullosa summary? ›

Epidermolysis bullosa (EB) is a group of rare diseases that cause the skin to blister easily. Epidermolysis bullosa causes blisters, which quickly burst and leave slow-healing wounds like the one on this baby's knee. The skin blisters because it's so fragile. The fragile skin is usually noticeable at birth.

What pathogen is epidermolysis bullosa? ›

Patients with the genetic blistering disease epidermolysis bullosa (EB) often have chronic wounds that can become colonized by different bacteria, especially the opportunistic pathogen Staphylococcus aureus.

What part of the cell are affected in epidermolysis bullosa? ›

In EB simplex, trauma-induced loss of tissue integrity consistently occurs within the basal layer of epidermal keratinocytes (Figures ​1 and ​

How does epidermolysis bullosa affect immune system? ›

A fifth type of the disease, epidermolysis bullosa acquisita, is a rare autoimmune disorder that causes the body's immune system to attack a certain type of collagen in the person's skin. Sometimes, it happens with another disease such as inflammatory bowel disease.


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