The term EBS includes all forms of EB in which cutaneous fragility is confined to the epidermis. In the vast majority of EBS forms, cleavage is detected histologically at the basal level and the disease follows a pattern of autosomal dominant transmission. Autosomal recessive transmission in EBS accounts for only 5% of cases. Although onset of clinical manifestations occurs, in general, at birth or during the neonatal period (Fig. 2), the first manifestations may appear in adults. In general, blistering in EBS is induced by trauma and, may rarely occur spontaneously. Scarring, milia, and dystrophic nails are usually less frequent in EBS than in JEB and DEB.

Figure 2.

Clinical manifestations of basal EBS. The keratin-encoding genes (KRT5, KRT14) are causative of the disease in all patients.19,20 Photographs obtained with signed informed consent (Source: u714-CIBERER-CIEMAT-UC3M-IISFJD).

A, Palmoplantar involvement of a patient with localized EBS during infancy. B, Generalized involvement of 2 siblings with EBS, of consanguineous parents, with autosomal recessive transmission19. C, Mottled pigmentation in a patient with EBS-MP.21 D-G, Extensive involvement in a neonate with generalized severe EBS. E-F-H-I, Progression during infancy. E, Widely distributed herpetiform lesions on the patient's back.

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Localized EBS is the most common form and it is characterized by blisters confined to the palms and soles and, in some cases, by oral erosions or blisters during infancy. Lesions can be present in any part of the body as a result of skin trauma. Severe generalized EBS, a less frequent subtype, is usually associated with marked morbidity and mortality during the neonatal period or early infancy. Its most distinctive feature is the formation of intact blisters grouped in an arch-shaped distribution (herpetiform). Patients with this form usually develop skin thickening on the palms and soles, giving rise to keratosis that may resolve on reaching adulthood. Some patients have nail distrophy atrophic scarring, milia, mucosal involvement (laryngeal stenosis), anemia, and retarded growth. Unlike the severe form, generalized intermediate EBS presents with nonherpetiform blisters, and anemia and growth retardation are uncommon.2

Other less frequent basal EBS subtypes are EBS with mottled pigmentation, migratory circinate EBS, autosomal recessive EBS (EBS-AR), EBS with muscular dystrophy, EBS with pyloric atresia, EBS with BP230 deficiency, and EBS with exophilin 5 deficiency.2 Recently, a new protein (kelch-like protein 24) has been identified, associated with a basal EBS subtype with distinctive clinical features13 compared with the classic forms of EBS (alopecia, follicular and cutaneous atrophy).

Currently, the term EBS also encompasses those forms in which mechanical fragility, unlike those described previously, occurs at the suprabasal level. This suprabasal EBS subtype includes the acral form, EBS superficialis, the acantholytic form, and skin fragility syndromes arising from desmoplakin, plakoglobin, and plakophilin deficiencies4 respectively (see Tables 1 and 3).

The most prominent clinical feature in all subtypes of JEB is enamel hypoplasia, a useful distinguishing characteristic for diagnosis of this type of EB (Fig. 3F). JEB can also be subdivided into several subtypes, all of which are of autosomal recessive transmission.

Figure 3.

Clinical manifestations of JEB. Photographs obtained with signed informed consent (Source: U714-CIBERER-CIEMAT-UC3M-IISFJD). A-B, Severe generalized JEB caused by mutations in the LAMC2 gene with absence of Lam 332, specifically the γ2 chain, which led to neonatal death in one of the patients. C, Intermediate generalized JEB. D, Localized JEB. In both patients, mutations were found in the COL17A1 gene, leading to reduced expression and complete absence of Col17, respectively. Note the postinflammatory hypopigmentation. E-F, JEB with pyloric atresia caused by mutations in the ITGB4 gene; protein expression was comparable to that of control in patient F and absent in the lethal form (not shown).22 Both patients underwent corrective surgery for pyloric atresia.

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The most severe subtype is the severe generalized which is present from birth and can affect the entire skin (Fig. 3A-B). A very significant finding in this subtype is the appearance of abundant symmetrically distributed granulation tissue in the mouth, central region of the face and nose, the upper part of the back, axillas, and inguinal folds. The clinical picture includes oral blisters, microstomia, and ankyloglossia, although these latter manifestations are not as marked as in the recessive forms of DEB. Extracutaneous manifestations can also affect other organs such as the esophagus, eyes, upper respiratory tract, and genitals. Patients may also present with growth retardation and multifactorial anemia. There is a high risk of death during infancy (up to 2 years of age) as a result of growth retardation, infections, pneumonia, and airway obstruction.

Generalized intermediate JEB, evident from birth, is the most common of the junctional variants. This generalized disorder presents as blisters, atrophic scars, and dystrophic or absent nails (Fig. 3D). The patients usually also present with postinflammatory hypopigmentation or depigmentation and, in some cases, scarring alopecia of the scalp. The risk of death due to airway occlusion or other complications is much lower than in the generalized form, as is the risk of anemia and growth retardation. Some patients, however, may develop squamous cell carcinoma.

Another clinically important subtype is JEB inversa, which presents as severe blisters confined to intertriginous sites of the skin, esophagus, and vagina. JEB-LOC syndrome is characterized by the formation of localized blisters on the face and neck with granulation tissue, upper airway abnormalities, nail abnormalities, and tooth enamel hypoplasia.2 JEB with pyloric atresia presents with generalized blisters from birth and congenital atresia of pylorus sphincter. Congenital abnormalities of the genitourinary tract may also be present with variable prognosis from a milder phenotype to neonatal death (Fig. 3E-F). It is worth mentioning that there is a form of EB associated with pyloric atresia in which skin cleavage occurs in the basal epidermal layer and not in the dermal-epidermal junction. This form is therefore considered a subtype of EBS (EBS with pyloric atresia; Tables 1 and 3).

DEB is divided into 2 large groups, dominant and recessive, according to the pattern of genetic of transmission (Table 3, Fig. 4).1,2

Figure 4.

Clinical Manifestations of DEB. Clinical photographs of Spanish patients obtained with signed informed consent (Source: U714-CIBERER-CIEMAT-UC3M-IISFJD). Mutations in the gene that encodes collagen type VII (COL7A1) is the cause of the disease in all patients.23,24 A, Dominant DEB, nails only. B, Dominant DEB, generalized. C, Recessive DEB, localized. D, Recessive DEB, pretibial. E, Recessive DEB, generalized (non Hallopeau-Siemens syndrome). F, Recessive DEB, generalized severe (Hallopeau-Siemens). F, Recessive DEB, generalized severe (Hallopeau-Siemens). G-I, Other complications in patients with severe generalized RDEB. G, Microstomia with dental abnormalities and caries. H, Scarring and ocular blisters (lower eyelid). I, Squamous cell carcinoma on the stump of the left hand.

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The most severe subtype of dominant DEB (DDEB) is the generalized, which is characterized by the presence of blisters distributed throughout the body from birth. Milia atrophy, scarring, and dystrophic nails are also associated at a later age (Fig. 4B). Recurrent blisters and erosions in the esophagus is common. Other less severe types of DDEB are the nails only form (Fig. 4A), the acral form, localized DDEB with blisters confined to the hands and feet (Fig. 4C), the pretibial form, with minor plaque-like lesions, often of violaceous appearance (Fig. 4D), pruriginous form, which gives rise to untreatable pruritus, and the newborn form with generalized blisters accompanied by focal scarring which usually disappears after between 6 and 24 months of life.

Recessive DEB (RDEB), also divided into several subgroups, can show a wide range of severity, from simple nail dystrophy to generalized blistering and hand and feet deformities (pseudosyndactyly). The disease can also affect the mucosa and, in the most severe forms, is associated with a high risk of developing squamous cell carcinoma. The most frequent subtype is the generalized severe which is evident from birth, with blisters that progressively spread throughout the body and result in atrophic scarring. This subtype presents with granulation tissue, above all in chronic wounds, as well as joint contractures and development of pseudosyndactyly (Fig. 4F). Patients usually develop extensive caries, severe ankyloglossia, and microstomia (Fig. 4G) associated with deficient food intake. Other extracutaneous manifestations are corneal blisters, esophageal stenosis, chronic renal failure, multifactorial anemia, and retarded growth. This subtype, which is the most severe form of RDEB, is often complicated by the development of metastatic squamous cell carcinomas (Fig. 4I) and premature death may occur. The generalized intermediate subtype is less severe (Fig. 4E), and generally does not present with retarded growth or anemia, and the frequency of esophageal stenosis as well as skin involvement is lower. However, there is still a significant risk of developing squamous cell carcinoma. The less common subtypes are RDEB centripetalis, which starts in the acral areas and extends to the trunk, and RDEB inversa, in which the appearance of blisters is limited to intertriginous areas, the base of the neck, the top of the back, and the lumbosacral area. Patients with RDEB inversa are prone to develop blisters in the oral cavity, esophagus, and genitourinary tract, and may end up developing debilitating stenosis of the esophagus and vagina.

KS is a recessive autosomal genodermatosis that is included in the EB classification given its clinical and biological features. This means that patients with this syndrome (fewer than 200 individuals throughout the world) are attended in EB reference centers.1,2 KS appears at birth with the presence of blisters that can have a generalized distribution and often disappear as the patient gets older (Fig. 5). The course of this form is characterized by the progressive development of poikiloderma, photosensitivity, premature skin aging, and keratoderma.25 Patients with KS can present with atrophic scarring, nail dystrophy, contractures, and interdigital synechia. Complications such as severe colitis, esophagitis, urethral stenosis, gingival hyperplasia, and squamous cell carcinomas can be associated with KS.1,2 The pro-oxidative state, described recently in epithelial cells of patients with KS, may explain the pathognomonic features not associated with skin fragility.26,27

Figure 5.

Clinical Manifestations of KS. Clinical photographs of patients obtained with signed informed consent (Source: U714-CIBERER-CIEMAT-UC3M-IISFJD). Mutations in the gene that encodes kindlin 1 (KIND1) is the cause of the disease in all patients.26–28 A, Erosions and premature aging of the skin, incipient already in infancy, on the backs of the hands. B, Evident cutaneous aging and poikiloderma along with nail dystrophy in a young patient. C, Ocular involvement, poikiloderma, interdigital synechia, and contracture of the hands in a female patient in her sixties.

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The first step recommended in the diagnosis of EB is a clinical classification of patients with skin fragility and, thus suspicion of possible abnormalities in dermal-epidermal anchoring proteins.14,15 Diagnosis continues with initial classification in the 4 main types of EB through identification of the level at which cleavage occurs by immunofluorescence (Table 4).

Antigen mapping is performed by using histological sections from biopsies of cryopreserved skin that includes a blister just before sampling. These sections are incubated with a battery of antibodies against Col IV and different protein implicated in EB. The antibody against Col IV, protein present in the lamina densa of the dermal-epidermal junction, enables us to unequivocally identify DEB (labeling located in the roof of the blister) and KS (reduplication of the basement membrane). The use of this antibody does not distinguish between EBS and JEB, as the labeling is present in the floor of the blister in both cases (Fig. 6).

Figure 6.

Col IV Immunofluorescence. Col IV can be observed at the floor of the blister in both JEB and EBS. In DEB, Col IV is observed in the roof of the blister, whereas in KS, a reduplication of the basement membrane is observed, distinctive for this disease. Source: Dr. Marta García (U714-CIBERER-CIEMAT-UC3M-IISFJD).

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The use of specific antibodies for the proteins implicated in EB and their expression pattern in the roof or floor of the blister helps to differentiate between EBS, JEB, and DEB (Figs. 7 and 8) also to delimit candidate genes.

Figure 7.

Differential diagnosis of types of EB by immunophenotyping. A, Schematic representation of the commonly studied proteins. B, Differential expression pattern.

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Figure 8.

Classification of EB and selection of candidate gene by antigen mapping (first screening) in histological sections of biopsies of cryopreserved skin. Microphotographs: Dr. Marta García (U714-CIBERER-CIEMAT-UC3M-IISFJD). A, Immunophenotype of basal EBS. The K5 and K14 keratins mark the floor and roof of the blister as a result of intraepidermal cleavage. Labeling of the remaining proteins is localized in the floor of the blister. The result indicates that the candidate genes in the initial screening are KRT5 and KRT14. B, Immunophenotype of JEB. As a result of cleavage of the intralamina lucida, labeling of the keratins is restricted to the blister roof while that of collagen VII remains in the lister floor. In this case, the absence of labeling of laminin 332 indicates that the candidate genes are LAMA3, LAMB3, and LAMC3, and that the molecular subclassification is JEB (see Table 5). Note that the absence of Lam332 can affect expression of integrins. C, Immunophenotype of DEB. As a result of cleavage at the level of the papillary dermis, all markers map to the roof of the blister. This result indicates that the candidate gene is COL7A1. The almost complete absence of Col VII is consistent with generalized severe DEB (see Table 5).

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In some cases, the results of immunofluorescence enable to further refine the subclassification, as decreased intensity (Table 5) or complete lack of labeling of a given protein consistently points its encoding gene as the candidate gene. The complete lack of labeling generally corresponds to null mutations. In other cases, complete proteins are produced with an altered amino acid sequence and its expression may be similar to the one in a healthy individual. The process for selection of the candidate gene is described in Figure 7.

Although immunophenotyping is currently not essential thanks to massive sequencing, it is still recommended as first step prior to genetic study. Immunophenotyping helps identify the implicated protein and, therefore, the candidate gene.23,29,30Table 5 shows the different EB subtypes according to molecular criteria: affected protein, level of expression in the skin, and encoding genes.

After determining the affected protein in the patient, we can proceed to genetic analysis through sequencing of the candidate gene. The search begins with focus on the most common mutations and ends with complete sequencing of the gene, if necessary. Currently, the search for the mutation is performed by Sanger sequencing, although new techniques are already being incorporated, such as NGS, which can include the search for mutations on different genes at the same time.16 NGS is a very useful step forwards, as molecular study can be performed directly, regardless of the diagnosis of the immunophenotyping analysis of the skin biopsy. NGS techniques, through panels that include at least all the coding sequences of known EB-genes, enable diagnosis of a larger number of cases in what is a heterogeneous disease. Even so, and although this approach can increase the proportion of patients diagnosed with genetic diseases, NGS will not be able to identify the mutation in all cases, as occurs in some other genetic diseases. To find a solution in cases in which the pathogenic mutation is not identified through direct gene sequencing, complementary techniques are used for the search for large deletions and duplications, such as multiplex ligation probe amplification or microarray-based comparative genomic hybridization. The incorporation of the mutation into the final classification of EB is critical for providing a rigorous genetic counseling, as it determines the mode of genetic transmission and also enables a theoretical prognosis for the progression of the disease. Prognosis made according to genetic findings should be treated with caution as environmental factors and other genetic modifiers may play a key role in disease progression.2

As mentioned repeatedly, there are many genes implicated in EB and, furthermore, there are many possible mutations in each one of these15; therefore, EB shows heterogeneity in terms of genetic alleles (many possible mutations) and of loci (different genes). All types of mutation have been implicated in EB, from premature stop codons to changes in reading frames, among others. The location is also diverse and may affect both the coding sequence and promotor regions, and even intronic regions. For proper assessment and interpretation of the pathogenic potential of a mutation present in a patient, general genetic databases such as the HGMD (HGMD® Human Gene Mutation Database Professional; http://www.biobase-international.com/product/hgmd) should be searched. These databases include all mutations published as causative of genetic diseases in humans. There are also other more specific databases such as the database of COL7A1 mutations associated with the International Registry of DEB Patients and COL7A1 Mutations31 (http://www.deb-central.org/molgenis.do) or the Human Intermediate Filaments Database32 (http://www.interfil.org). In the event that a mutation has not been reported in the general or specific databases, nor has it been included as a polymorphism in the Short Genetic Variation database (DbSNP; http://www.ncbi.nlm.nih.gov/projects/SNP/Variations_Survey.html), the presence should be ruled out in a minimum of 100 control individuals from a population with the same genetic origin. Although not always possible, it is recommended to confirm the pathogenic effect of the mutation at the RNA messenger or protein level.

Starting from the premise that there are more than 1000 mutations described in at least 19 genes, it is very important to have all the information available to optimize the search and perform the risk calculations. The number of new mutations identified increases year after year: in the case of some genes, for example the COL7A1 gene, these mutations are mostly specific to each family. Other information to bear in mind is the frequency of mutations described according to a geographic location or according to a given ethnic origin. For instance in the COL7A1 gene, was observed that the pathogenic c.6527insC mutation is present in 46.3% of alleles of Spanish patients with RDEB being more prevalent in the south-west of Spain (Extremadura and Andalusia). This very frequent mutation, originates from a single common ancestor is currently being investigated in the initial screening for the study of Spanish patients with DEB.23,33–35 This same mutation has also been found with a high recurrence in Chile,36 possibly as a result of waves of Spanish migration to Latin America in different periods. There are other frequent mutations in Spain and, although their prevalence is much lower, screening for them is also included in the study of possible carriers of a COL7A1 mutation. Absence of these recurrent mutations does not rule out the possibility that the proband is a carrier of any other mutation in the COL7A1 gene, but it does drastically reduce the individual risk.

In Spain, there were no reference centers for EB47 until this year, when the Hospital Universitario de la Paz in Madrid and the Hospital Clínic i Provincial de Barcelona and Hospital de Sant Joan de Déu in Catalonia were designated as such. The research group of Prof. Marcela del Río (termeg.uc3m.es) has, however, been performing genetic diagnosis of EB since 2006 (ORPHA361523, ORPHA361536, ORPHA362075, and ORPHA361538), is implicated in research projects aiming to develop treatments for RDEB (TRA-049, CELLCAM, ORPHA300805, GENEGRAFT; ORPHA437567; ORPHA437569) and the national EB registry (https://registroraras.isciii.es). This group, part of the CIBER on Rare Disease Network (www.ciberer.es), is a partner of the international EB CLINET-Clinical Network of EB Centres and Experts (http://www.eb-haus.org/en/eb-clinet.html;http://www.eb-clinet.org/maps/eb-clinical-contacts.html). The group conducts the genetic studies in collaboration with dermatology and genetics departments of several hospitals throughout Spain, and in particular with the Pediatric Dermatology Division of the Hospital La Paz in Madrid (Dr. Raúl de Lucas), recently designated as reference center and with the Genetics Department of the Fundación Jiménez Dáiz (Dr. María José Trujillo-Tiebas; Dr. Carmen Ayuso), in coordination with the Spanish EB patient association, DEBRA España (www.pieldemariposa.es; info@debra.es). This cooperative activity at national and international level has enabled the group to develop a profound knowledge of the disease.8,19,21–26,33,35,37–51 Thus, when EB is suspected, in addition to requesting information from DEBRA-España, it may be helpful to contact Dr. María José Escámez (mj.escamez@ciemat.es), the coordinator of molecular studies conducted by the group, to ensure the most appropriate approach to the genetic study.

To date dominant forms for EBS and DEB has been described. The dominant forms are the most common ones in EBS whereas they account for a limited number of cases of DEB, for example between 7% and 8% in the Spanish cohort (126 families with DEB have been diagnosed).23 As in any genetic disease with autosomal dominant transmission, a single mutation in one of the copies of the gene is sufficient to cause the disease. In these cases, the affected transmit the mutation (and the disease) to their offsprings with a probability of 50% in each pregnancy. Generally, in DDEB the affected individuals have inherited the disease from one of their progenitors, who is also affected (dominant mutation inherited). However, in dominant EBS, in many cases, the patient is the first case in the family due to a de novo mutation in the germline of unaffected parents (10 families with EBS with de novo KRT5 and KRT10 mutations out of a total of 23 families with mutations in these genes). This is because the genes that encode keratins K5 and K14 have sites well known for genetic instability (hotspots) that are more susceptible to mutations in the gametes of the parents. This phenomenon also occurs with the gene that encodes Col VII, although less frequently; 2 families with de novo DDEB out of 126 families with DEB in the Spanish cohort. The risk of recurrence in future siblings of the proband, this is not 50%, as is the case with inherited mutations, but estimated to be approximately 2% to 5%,52 given the possibility that one of the parents has a mosaic mutation in the germline. Of note is that the risk of recurrence in children of the proband is indeed 50%. In the case of patients with de novo dominant forms, the advice given by the expert clinical geneticist is essential.

We should also highlight that there is a tendency among physicians to make a genetic diagnosis of suspicion of de novo dominant transmission in patients with mild dystrophic forms and unaffected parents. Given the low frequency of appearance of de novo dominant mutations in the Col VII gene, this type of diagnosis should be avoided until the genetic result is obtained and the mode of transmission determined. This is because, in most cases, these patients actually have recessive mutations that result in a mild clinical presentation.

When a case of DEB appears for the first time in a family, we tend to think in terms of autosomal recessive transmission in which both gene copies (paternal and maternal) need to be mutated for the disease to become manifest. In these cases, each progenitor is a healthy carrier (heterozygotes) of one of the 2 mutations present in the DNA of their affected child. The family risk of new cases is 25% for successive siblings of the patient. In these cases, healthy siblings of affected individuals should undergo genetic study as well when they reach adulthood to determine whether they are carriers or not. It is important to point out that carriers can only have affected children if their partner is also a carrier. The probability that, by chance, the partner is a carrier is less than 1 in 50 (2%), unless there is a consanguineous relationship (or the partner is from a nearby region with a higher prevalence of an EB-associated mutation). Once the mutation has been identified in the carrier, it is possible to screen for this gene in the partner to rule out their status as a carrier with a reasonable degree of certainty.

This information, regarding the pattern of transmission, and its reproductive implications, including the options described below for prenatal and preimplantation diagnosis, among others, must be provided in a visit known as genetic counselling or advice. This type of visits are regulated and defined by the Spanish law of Biomedical Research ([LIB] 14/2007); and the services provided by the Spanish national health system are set in the Order SSI/1356/2015, dated July 2. Genetic counselling or advice is defined as the procedure intended to inform a person about the possible consequences (for themselves and their offspring) of the results of a genetic analysis or screening and the benefits and risks and, where appropriate, the advice given regarding possible alternatives derived from the analysis. Counselling may occur both before and after a genetic test or screening and even if such tests or screening did not take place. This advice should be provided in a nondirective fashion and complying with the principles of beneficence and nonmaleficence so that patients can make own decision.

The approach is common for any type of genetic or hereditary disease, and is applied in the same way in EB. Some recommendations on advising patients with EB and their families have been published,52,53 although the application of new techniques requires to review their applicability and scope.