Epidermolysis bullosa (EB) is an inherited mechanobullous disorder characterized by skin fragility and blister formation occurring spontaneously or after minor trauma. It is classified into four main types based on the level of separation of the skin: EB simplex (EBS), junctional EB (JEB), dystrophic EB (DEB), and Kindler EB (KEB) (Has et al., 2020a). EB is known to exhibit high genetic heterogeneity.

EBS is the most common form of EB and is characterized by blister formation within the epidermis arising from cleavage within the basal layer. Most cases of EBS are caused by dominant mutations of KRT5 (keratin 5), KRT14 (keratin 14), and KLHL24, with a few cases linked to recessive variants of PLEC, KRT5, KRT14, and other genes (Fine et al., 2014; Has et al., 2020b; Mariath et al., 2020). JEB involves separation between the dermis and the epidermis (lamina lucida) and is mostly inherited in an autosomal recessive mode. DEB is characterized by separation within the uppermost dermis (sublamina densa) and has two subtypes, autosomal dominant DEB (DDEB) and autosomal recessive DEB (RDEB), that both arise from mutations in COL7A1 (collagen type VII). KEB affects mixed layers of the skin and is associated with photosensitivity (Has et al., 2020a).

EB with congenital absence of skin (CAS), previously named Bart syndrome, is considered a clinical finding under the umbrella of EB (Fine et al., 2014; Has et al., 2020b). EB with CAS can be diagnosed based on clinical findings. However, identification of the layer of skin cleavage and precise classification of the EB type/subtype require immunofluorescence mapping and/or transmission electron microscopy, preferably on newly-induced blisters (Has et al., 2020a).

The most common EB type associated with CAS is DEB (58–64%) arising from COL7A1 mutations, followed by JEB (linked to ITGB4) and EBS in that order (Mariath et al., 2020; Martinez-Moreno et al., 2020). Recessive variants of LAMC2 (laminin-332), ITGA6 and PLEC are less frequently found in these cases (Mariath et al., 2020; Martinez-Moreno et al., 2020). Autosomal dominant EB with CAS is mainly caused by monoallelic variants of KRT5 and KLH24. EBS-severe with pyloric atresia (EBS-PA) is linked to mutations in PLEC, while JEB-PA is caused by mutations in ITGB4 and rarely ITGA6 (Pfendner and Uitto, 2005; Natsuga et al., 2010a; Natsuga, 2015; Mariath et al., 2020).

Herein, we describe EB with CAS in five unrelated patients, revealing rare phenotypes of congenital cloudy cornea, esophagogastric obstruction, and anuria and the underlying genetic defects.

We identified pathogenic/likely pathogenic variants in five different genes as the causes of EB with CAS in five unrelated Thai families, including four novel recessive alleles in PLEC, LAMC2, ITGB4, and COL7A1, and a known dominant mutation in KRT5.

A handful of EB cases have been reported in Southeast Asian populations including Thai, Malaysian, Singaporean, and Indonesian (Singalavanija et al., 1994; Wessagowit et al., 2007; Tang et al., 2013; Bishnoi et al., 2021) with only two studies providing molecular data limited to COL7A1.(Wessagowit et al., 2007; Bishnoi et al., 2021). One likely reason for the small number of reports on EB with its type/subtype diagnosis in Thailand, at least, is the lack of availability of sophisticated immunohistopathological analyses specific for the disorder.

According to the recent 2020 clinical practice guidelines for laboratory diagnosis of EB in affected neonates, a skin pathologic study is recommended as the first diagnostic step with a genetic analysis performed in parallel (Has et al., 2020b). With the advance of next-generation sequencing (NGS) technology and its rapidly decreasing price, extended genetic testing has become affordable and easier to access for confirmation of disorders with high genetic heterogeneity, such as EB. When applied to our local context, WES was chosen as the first test in the present study.

Several studies using NGS multigene panels for EB diagnosis have reported diagnostic yields of 83–97%, with the variation arising from the characteristics of the examined populations such as ethnicity and presence of consanguinity, as well as the cohort size (40–91 families) and the number (11–49 genes) and details of the genes on the NGS panels (Vahidnezhad et al., 2017; Has et al., 2018; Lucky et al., 2018; Mariath et al., 2019). A study using WES had a 100% detection rate in a cohort of 57 families affected by EB of various types (Yu et al., 2021). The present study yielded 100% detection frequency and revealed genetic heterogeneity in the results, despite the very small cohort. The data support the use of WES as a reasonable alternative method for diagnosis of EB and confirm that it provides very high coverage of target exons/genes. However, the major limitation of genetic testing alone is the lack of histological confirmation of EB, and thus further insights into the dermatopathogenic mechanisms are crucial for deeper research on this disease.

In the present study, four patients, except for the patient with the mildest manifestations, were born prematurely. Furthermore, greater severity of CAS and skin erosion appeared to be associated with greater prematurity. We have noted substantial numbers of premature births among previously reported cases with available data, especially for patients with extensive cutis aplasia and erosion of the skin (Charlesworth et al., 2003; Natsuga et al., 2010b; Chen et al., 2018; Han et al., 2019).

We have described, for the second time, the presence of congenital corneal cloudiness as a primary abnormality of the cornea in two EB patients: Patient 1 with a PLEC mutation and Patient 4 with an ITGB4 mutation. The first case of congenital corneal haziness in EB was recently described in a neonate with RDEB due to COL7A1 mutations (Sawka et al., 2021). Ocular manifestations in EB usually represent late complications following exposure keratitis and scarring of the eyelids. Our Patient 1 did not show any eye lid abnormality or corneal scarring, while our Patient 4 showed ectropion on one eye but bilateral cloudy cornea, suggesting an underlying mechanism other than physical/rubbing trauma of the cornea.

The skin, oral mucosa, and outer surface of the cornea are stratified squamous epithelium containing type I hemidesmosomes composed of plectin, integrin α6β4, BP230, and BP180 (collagen type XVII) that link laminin-332 in the basement membrane and lamina densa. The skin-type keratin pair is K5/K14, while the cornea-type keratin pair is K3/K12 (Figure 4) (Walko et al., 2015; McKay et al., 2020). Collagen types VII and IV are also expressed in the epithelial basement membrane (EpBM) of the cornea and skin dermis. Integrin α6β4 was shown to mediate adhesion of basal cells to the corneal EpBM as well as to collagen, laminin, and other extracellular matrix proteins including fibronectins and vitronectin (McKay et al., 2020). Decreased integrin expression led to reduced proliferation of corneal epithelial cells and impaired adhesion to the underlying basement membrane, resulting in ocular surface defects (McKay et al., 2020). A report on the histopathological findings for the anterior cornea in an adult patient with EBS described normal superficial and intermediate corneal epithelia but abnormal structures in the basement membrane, namely fibrocellular pannus deep to the thickened EpBM and multilaminar basement membrane with faulty adhesion complexes (Adamis et al., 1993). Taken together, these data suggest that the congenital corneal cloudiness in EB could arise through primary malformation of the corneal epithelium.

The partial obstruction of the esophagogastic junction in our Patient 4 represents another rare gastrointestinal anomaly in EB, as it has been previously reported in a few cases.(Doi et al., 1986; Cetinkursun et al., 1995; Maman et al., 1998). These gastrointestinal anomalies might have been underdiagnosed because of the early demise of the patients. The PA and esophagogastic atresia could be a result of intraluminal mechanical and/or chemical irritation, leading to mucosal destruction and followed by cicatrization. We further demonstrated the very rare occurrence of a vesicoureteric obstruction with anuria in our Patient 4 with an ITGB4 mutation (Dang et al., 2008).

PLEC encodes plectin protein, which directly interacts with keratin K5/K14 intermediate filaments and also binds to integrin α6β4 and other related proteins to form a multiprotein complex that stabilize the adhesion of basal keratinocytes (epithelial cells) to the underlying basement membrane (Walko et al., 2015). The p. Glu956Ter variant could lead to a severely truncated protein that lack the entire rod domain and the carboxy-terminal globular domain of normal plectin, the latter of which functions as a β4-integrin binding segment and a binding site for keratin intermediate filaments. Therefore, the mutant protein is likely to be nonfunctional or even not produced through activation of nonsense-mediated mRNA decay, resulting in a fatal phenotype (Natsuga et al., 2010a).

Premature truncation (PTC) mutations in PLEC were shown to be associated with early lethality in patients with EBS-PA with CAS (Charlesworth et al., 2003; Nakamura et al., 2005; Pfendner and Uitto, 2005; Natsuga, 2015). Three affected siblings described by Charlesworth et al. (Charlesworth et al., 2003) showed similar patterns of extensive skin involvement in scalp/facial regions, PA, hypotonia, and early demise due to a PTC mutation in PLEC (c.2727del14 in exon 22 of NM 00445.5. The extreme phenotypes in these siblings were similar to the findings in our Patient 1, who harbored a pathogenic variant in the adjacent exon of PLEC (c.2536G > T, last nucleotide in exon 21 (NM_00,445.5). Molecular and histopathologic studies in the siblings demonstrated that the c.2727del14 allele led to PTC and total absence of plectin protein in the skin annexes (Charlesworth et al., 2003). The unusual severity of our Patient 1 and the patients reported by Charlesworth et al. (Charlesworth et al., 2003) could be due to the critical position of the mutations that cause PTC within the plakin globular domain of plectin.

LAMC2 encodes the laminin γ2 subunit, which forms a heterotrimer with the laminin α3 and laminin β3 subunits to form laminin-332 (Walko et al., 2015). The variant LAMC2:c.3385C > T (p.Arg1129Ter) is predicted to lose the carboxy-terminal 64 amino acids, including the distal segment of the coiled-coil domain. LAMC2 is the only laminin gene found to have mutations in EBS with CAS, with just five pathogenic variants described in previous reports, (Schneider and Muehle, 2008; Hammersen et al., 2016; Mariath et al., 2020), making LAMC2 the rarest gene underlying the disorder.

Kertin 5 assembles with keratin 14 to form heterodimeric and tetrameric proteins as the principal components of the intermediate filaments in basal keratinocytes that are crucial for mechanical stability (Walko et al., 2015; Khani et al., 2018). KRT5:c.429G > A (p.Glu477Lys) was described as the most common mutation in patients affected by EBS-severe with CAS, and was a de novo event in the majority of cases (Lalor et al., 2019; Mariath et al., 2020). The mutation is located at the last amino acid in the highly conserved end of the α-helical termination peptides critical for intermediate filament formation. In silico modelling showed that the variant led to a conformational change and an altered charge on the surface of the keratin K5/K14 heterodimers, disrupting the stability of the dimerization and thus intermediate filament formation (Lalor et al., 2019). The variant was associated with a high mortality rate (Sathishkumar et al., 2016; Lalor et al., 2019).

The integrin β4 subunit (encoded by ITGB4) and integrin α6 subunit form a heterodimeric protein at the lamina lucida of the skin and at the corneal epithelium. ITGB4:c.794dupC is expected to result in substitution of alanine with serine at codon 266, followed by a short frameshift before premature termination five residues later, designated p. Ala266SerfsTer5. Thus, the mutant protein lacks more than 80% of the carboxy-terminus, and is likely nonfunctional or not synthesized due to mRNA decay. Mutations of ITGB4 have been detected throughout the gene. A patient described by Dang et al. (Dang et al., 2008) with c.3903dupC/p.Gly 273Asp (in exon 31/exon 8), exhibited widespread CAS and skin erosion over the scalp/facial/ear regions, severe vesicoureteric obstruction, and anuria, reminiscent of our Patient 4 who also had a mutation of exon 8 encoding the N-terminal extracellular domain of the integrin β4 subunit. These findings indicate possible correlations between mutations in exon 8 and extremely severe phenotypes.

COL7A1 encodes the α1 subunit of type VII collagen, a major collagenous component of the lamina densa and upper dermis. Type VII collagen forms anchoring fibrils between stratified squamous epithelia and stroma (Chung and Uitto, 2010). Many COL7A1 variants have been described to cause DDEB and RDEB, depending on the nature and position of the mutations. Specifically, PTC mutations in both COL7A1 alleles tend to cause the RDEB-severe subtype, while a missense mutation in at least one allele tends to lead to phenotypic variability: intermediate or severe subtypes. In the present study, the genetic compound between a missense allele and a PTC mutation, p. Arg1814Cys/p.Glu2035SerfsTer171, led to a nonlethal form of RDEB-intermediate (self-improving). Most COL7A1 variants related to EB with CAS are aggregated in the triple-helix domain (Mariath et al., 2020) encoded by exons 31–109 (Christiano et al., 1994). The p. Arg1814Cys was predicted to produce an additional disulfide bond and disturb the secretion of collagen type VII and the stability of the triple helix (Dang et al., 2007).

We did not validate the predicted deleterious effect of the relevant variants by gene expression at neither RNA nor protein level, representing limitation of the present study.

CAS is present in 23% of EB patients (Mariath et al., 2019). Although the exact pathogenesis of CAS in EB remains to be resolved (Chiaverini et al., 2014), an acceptable long-held hypothesis is that CAS results from in utero friction/trauma to the limbs (Kanzler et al., 1992). However, the occurrence of similar pattern and severity of CAS within the same family cannot be explained by this assumption. Our data support an alternative hypothesis, recently proposed by several authors, (Charlesworth et al., 2003; Dang et al., 2008; Chiaverini et al., 2014; Lalor et al., 2019; Mariath et al., 2020), that the mutation type and position are the major contributing factors leading to CAS in all EB type/subtype, as evidenced by specific mutations/regions of the genes found to be linked with recurrent patterns of manifestations in different families, and the same severity and patterns of phenotypes seen in affected individuals within the same families.

Consanguinity as a founder effect is not uncommon in upcountry areas of Thailand, as demonstrated by the presence of homozygous variant in three families (Patients 1, 2 and 4) in the present study. The identification of specific mutations allows not only subtype classification and prognostication, but also precise genetic counselling and reproductive choices.

In conclusion, the present study has revealed exceptionally rare phenotypes in EB with CAS, namely congenital corneal cloudiness, esophageal obstruction, and anuria, and extended the genotypic spectrum of EB-related genes. Our data from an underrepresented population in Southeast Asia can further broaden the knowledge and research on EB.