Neonatal junctional epidermolysis bullosa: treatment conundrums and ethical decision making.

Junctional epidermolysis bullosa (JEB), generalized severe (previously called JEB, Herlitz-type) has an extremely poor prognosis, with a mean age of death at 5 months old and most dead before age 3 years. We describe a typical case of a neonate with JEB who developed failure to thrive before his death from fungal septicemia at 4 months of age. This case highlights the ethical considerations of invasive treatments such as gastrostomy tube placements, intubations, and central line placements in neonates with JEB. We review the literature as well as discuss the ethical conundrums in the care of patients with JEB and other severe forms of epidermolysis bullosa.

A Japanese-specific recurrent mutation and a novel splice site mutation in the LAMC2 gene identified in two Japanese families with Herlitz junctional epidermolysis bullosa.

BACKGROUND: Herlitz junctional epidermolysis bullosa (H-JEB) is an extremely rare genodermatosis characterized by lethality owing to severe blister formation. We report two unrelated Japanese patients with H-JEB. Genetic analyses detected a single nonsense mutation on the LAMC2 gene in these two patients. AIM: To identify the mutation involved and describe the first reported Japanese recurrent mutation in the LAMC2 gene. METHODS: Direct sequencing was performed of DNA from either peripheral blood or fetal cells in amniotic fluid. Reverse transcriptase PCR was used to confirm that an aberrant transcript resulted from the splice site mutation. A haplotype analysis was performed to define the origin of the recurrent mutation. RESULTS: Both patients had blisters and erosions on the trunk and limbs at birth, with nail dystrophy. Patient 1 died as a result of sepsis at 30 weeks of age, and patient 2 died as a result of disseminated intravascular coagulation at 20 weeks of age. Mutation analysis of the LAMC2 gene revealed that patient 1 was compound heterozygous for a nonsense mutation (p.Cys553X) and a novel splice site mutation (c.2868+1delG), and patient 2 was a homozygous for p.Cys553X. Prenatal diagnosis performed during a subsequent pregnancy in family 2 revealed that this second child was heterozygous for p.Cys553X, and was thus not affected. Haplotype analysis suggested that a p.Cys553X allele derived from the same origin had been independently inherited by these two unrelated families. CONCLUSIONS: p.Cys553X in the LAMC2 gene may be a Japanese-specific recurrent mutation as a result of a founder effect, and it may therefore be useful for initial screening in the mutation analysis of H-JEB.

A mouse model of generalized non-Herlitz junctional epidermolysis bullosa.

Epidermolysis bullosa (EB) is a class of intractable, rare, genetic disorders characterized by fragile skin and blister formation as a result of dermal-epidermal mechanical instability. EB presents with considerable clinical and molecular heterogeneity. Viable animal models of junctional EB (JEB), that both mimic the human disease and survive beyond the neonatal period, are needed. We identified a spontaneous, autosomal recessive mutation (Lamc2(jeb)) due to a murine leukemia virus long terminal repeat insertion in Lamc2 (laminin gamma2 gene) that results in a hypomorphic allele with reduced levels of LAMC2 protein. These mutant mice develop a progressive blistering disease validated at the gross and microscopic levels to closely resemble generalized non-Herlitz JEB. The Lamc2(jeb) mice display additional extracutaneous features such as loss of bone mineralization and abnormal teeth, as well as a respiratory phenotype that is recognized but not as well characterized in humans. This model faithfully recapitulates human JEB and provides an important preclinical tool to test therapeutic approaches.

Bone mineralization in children with epidermolysis bullosa.

BACKGROUND: Various factors may have deleterious effects on bone health in patients with epidermolysis bullosa (EB). OBJECTIVES: In a retrospective notes review, to assess bone mineralization in children with EB and to identify the relative contributions of nutrition, activity and disease severity to low bone mass. METHODS: Thirty-nine children with EB [32 recessive dystrophic EB (RDEB), four Dowling-Meara EB simplex (DMEBS) and three junctional EB (JEB)] had lumbar spine bone mass measured using dual X-ray absorptiometry (GE Lunar Prodigy; GE Healthcare, Chalfont St Giles, U.K.). Seventy-six healthy children were also studied. Weight and height were recorded and mobility was rated. RESULTS: Children with RDEB and JEB, but not those with DMEBS, had lower bone mineral density SD scores than controls; differences remained after adjusting for the smaller body size of the patients. Bone mass was best predicted by mobility rating. CONCLUSIONS: Children with RDEB and JEB have low bone mass after adjusting for their smaller size, which may put them at risk for fragility fractures. Low bone mass was best predicted by the level of mobility, raising the hypothesis that improving activity or bone loading may be a potential preventive intervention in these children. However, as low bone mass may be multifactorial in these children, more detailed investigation of potential aetiological factors is required before interventions are planned.

The alpha-3 polypeptide chain of laminin 5: insight into wound healing responses from the study of genodermatoses.

Laminin 5 (kalinin/epiligrin/nicein) is an essential structural component of the dermal-epidermal junction, composed of three polypeptide subunits: laminin alpha3, beta3 and gamma2. Studies of the inherited skin fragility disorder junctional epidermolysis bullosa (JEB) have suggested that the major role of this heterotrimeric protein is to act as an adhesive ligand essential for binding the epidermis to the underlying dermis and thus maintaining the integrity of the skin. Protein interaction studies have shown that the C terminus of the alpha3 subunit binds to a range of integrin complexes depending on the motility status of keratinocytes. This allows laminin 5 to interact with either hemidesmosomes or the actin cytoskeleton. Recently we have reported that the absence of the N-terminal region of laminin alpha3a in laryngo-onchyo-cutaneous syndrome causes excessive granulation tissue production at wound sites. As granulation tissue production is also a problem in JEB, this implicates laminin 5 in control of this wound healing response.

Novel and recurrent mutations in the laminin-5 genes causing lethal junctional epidermolysis bullosa: molecular basis and clinical course of Herlitz disease.

Herlitz disease (H-JEB), the lethal form of junctional epidermolysis bullosa, is a rare genodermatosis presenting from birth with widespread erosions and blistering of skin and mucosae because of tissue cleavage within the epidermal basement membrane. Mutations in any of the three genes encoding the alpha3, beta3 and gamma2 chains of laminin-5 underlie this recessively inherited disorder. Here, we report the molecular basis and clinical course of H-JEB in 12 patients. Two novel nonsense mutations in the gene LAMA3 (E281X and K1299X) and a novel frame-shift mutation in the gene LAMB3 (1628insG) leading to a premature termination codon were identified by DNA sequencing and confirmed by restriction fragment length polymorphism analysis. In the four patients affected, neither the resulting truncated polypeptide chains nor assembled laminin-5 protein were detectable by immunofluorescence. Three patients were found to be heterozygous for the known hotspot mutation R635X and the recurrent mutations Q373X or 29insC in the gene LAMB3, whereas five others were homozygous for R635X. Significant variations in the disease progression and survival times between 1 and 30 months in this group of H-JEB patients emphasised the impact of modifying factors and the importance of immunostaining or mRNA assessment as parallel diagnostic methods. Interestingly, the only patients who survived for longer than 6 months were four females carrying the mutation R635X homozygously. In one of them, the clinical course may have been improved by treatment with artificial skin equivalents. These data may stimulate further investigation of genotype-phenotype correlations and facilitate mutation analysis and genetic counselling of affected families.

Epidermolysis bullosa: pathophysiology and nursing care.

Epidermolysis bullosa (EB) is defined as a group of skin disorders that presents as blistering of the skin in varying degrees of severity. Most cases are inherited, but rare cases may be acquired. There are three main types: simplex, junctional, and dystrophic. The dermal-epidermal junction of the newborn skin is a vital area of attachment. Any defects in the components that comprise this junction can lead to fragility of the skin. EB diagnosis and testing can be per formed both prenatally and postnatally. There is no cure for EB. Treatment revolves around supportive care and prevention of complications. The NICU nurse plays an important role on the multidisciplinary team as primary caregiver to the infant and educator to the family.

Animal models for skin blistering conditions: absence of laminin 5 causes hereditary junctional mechanobullous disease in the Belgian horse.

Recent achievements in the genetic correction of keratinocytes isolated from patients with junctional epidermolysis bullosa have paved the way to a gene therapy approach for the disease. Because gene therapy protocols require preclinical validation in animals, we have characterized spontaneous animal models of junctional epidermolysis bullosa. In this study we have elucidated the genetic basis of the hereditary junctional mechanobullous disease in the Belgian horse, a condition characterized by blistering of the skin and mouth epithelia, and exungulation (loss of the hoof). Immunofluorescence analysis associated the condition to the absent expression of the gamma2 chain of laminin 5 and designated Lamc2 as the candidate gene. Comparative analysis of the nucleotide sequence of the full-length gamma2 cDNA isolated by reverse transcription polymerase chain reaction amplification of total RNA purified from the epithelium of a junctional epidermolysis bullosa foal and a healthy control disclosed a homozygous basepair insertion (1368insC) in the affected animal. Mutation 1368insC results in a downstream premature termination codon and is predicted to cause absent expression of the laminin gamma2 polypeptide. Our results also show that: (i) the horse junctional epidermolysis bullosa genetically corresponds to the severe Herlitz form of junctional epidermolysis bullosa in man; (ii) the amino acid sequence and structure of the horse laminin gamma2 chain are virtually identical to the human counterpart; (iii) the moderate eruption of skin blisters in the affected animals with respect to the human Herlitz junctional epidermolysis bullosa patients correlates with the protection provided by hair. Our observations suggest that the affected foals are a convenient source of epithelial cells from tissues that cannot be obtained from human junctional epidermolysis bullosa patients, and imply that hairless strains of animals with recessive skin disorders would be the best models for in vivo gene therapy approaches to skin blistering diseases.

Junctional epidermolysis bullosa: diagnosis and management of a patient with the Herlitz variant.

A case of Herlitz-type epidermolysis bullosa (EB) is reported. The baby was born after a normal, full-term pregnancy with blisters on his extremities. Over the next several months, progressive skin sloughing involving 95% of his body surface area developed, including gastrointestinal, laryngeal, tracheobronchial, and corneal involvement. The diagnosis of junctional EB, Herlitz type, was made using clinical, electron microscopic, and immunohistochemical criteria. Despite meticulous wound care, aggressive nutritional support, and continuous antimicrobial therapy, the baby died at 9 months of age. This report discusses the presentation, complications, and management of this unusual and tragic disease.

Molecular basis of non-lethal junctional epidermolysis bullosa: identification of a 38 basepair insertion and a splice site mutation in exon 14 of the LAMB3 gene.

Epidermolysis bullosa (EB) is a group of genodermatoses characterized by fragility and easy blistering of the skin. In the junctional forms of EB (JEB), blisters occur at the level of the lamina lucida, and specific mutations have been detected in the genes encoding different components of the hemidesmosomal-anchoring filament complex. In the non-lethal form of JEB (NL-JEB), mutations in genes encoding two of the polypeptide chains of the anchoring filament protein laminin 5 have recently been described. In this study, we searched for mutations in a family using PCR amplification of exon 14 of LAMB3, the laminin 5 beta3 chain gene, followed by heteroduplex analysis and automated sequencing of the PCR products. We detected a novel combination of mutations in this family, consisting of an out-of frame insertion on one allele, and a splice site mutation on the other allele, representing the first report of a large insertion in LAMB3, together with a splice site mutation inherited in trans, which result in the NL-JEB phenotype.

Junctional epidermolysis bullosis: defects in expression of epiligrin/nicein/kalinin and integrin beta 4 that inhibit hemidesmosome formation.

Junctional epidermolysis bullosis (JEB) is a heterogeneous inherited blistering disorder of human epithelial basement membranes (BMs). Characteristically, the epidermis detaches from the BM between the basal cells and the lamina lucida due to reduced numbers of hemidesmosomes (HDs). Attempts to identify a candidate gene for JEB led to the characterization of nicein, a protein complex in normal BMs that is absent from BMs of patients with JEB gravis. In independent research, two related BM glycoproteins, epiligrin and kalinin, were identified as functional adhesion components of HDs. Epiligrin was characterized as a BM ligand for basal cell adhesion via integrins alpha 3 beta 1 in focal adhesions and alpha 6 beta 4 in HDs. Kalinin was characterized as an adhesive ligand and a component of anchoring filaments. Recent antibody and sequence studies on epiligrin/nicein/kalinin have identified limited homologies with laminin. Ongoing studies in multiple laboratories seek to identify mutations in one or more of the three subunits of epiligrin that are causal in JEB gravis. Consistent with the genetic heterogeneity of JEB, we have identified a patient with a variant form of JEB that is associated with pyloric atresia. This patient has negligible HDs, normal epiligrin, but reduced expression of integrin beta 4. A defect in the beta 4 expression may define a subset of JEB cases that also present with pyloric atresia. These results testify to the dual requirements for epiligrin in the BM and integrin beta 4 in the plasma membrane in regulating function of HDs in epithelium.

Keratinocytes from junctional epidermolysis bullosa do adhere and migrate on the basement membrane protein nicein through alpha 3 beta 1 integrin.

BACKGROUND: Junctional epidermolysis bullosa (JEB) encompasses several genodermatoses characterized by skin blistering, and possibly disturbed wound healing. Although the molecular defects underlying JEB are not known, we have demonstrated previously that nicein, an adhesive laminin-related basement membrane component, is immunologically altered in the very severe JEB of Herlitz type (H-JEB), and was expressed to a lesser extent in skin from patients with inversa JEB (I-JEB). In this study, we assessed adhesion and migration of H-JEB and I-JEB keratinocytes on exogenous nicein and laminin to get insights on the biologic function defective in JEB skin. EXPERIMENTAL DESIGN: Adhesion of cultured epidermal keratinocytes from H-JEB and I-JEB patients was assayed by quantitation of cell attachment 1 hour after seeding into microtiter wells coated with nicein or laminin. Cell migration and modulation by function-blocking antibodies to integrins was quantified by computer-assisted image analysis of the tracks left by the cells in a phagokinetic assay using gold particles coated with nicein or laminin. RESULTS: In spite of the fact that H-JEB keratinocytes do not produce normal immunoreactive nicein, they were able to adhere on exogenous nicein similarly to normal and I-JEB keratinocytes which produce nicein. Adhesion of both JEB and normal keratinocytes to laminin was weak compared with nicein. At low and high concentrations of nicein, a reduced migration response occurred with H-JEB keratinocytes whereas I-JEB cells behaved like their normal counterparts. Integrin alpha 3 beta 1 was dominantly involved in adhesion and migration of all these cells. Laminin did not support the migration of either JEB or normal keratinocytes. CONCLUSIONS: H-JEB and I-JEB keratinocytes which produce no or less nicein than normal keratinocytes are able to adhere and migrate on exogenous nicein. Integrin alpha 3 beta 1 which is specifically involved in migration and adhesion of keratinocytes on nicein does not appear altered in JEB. These data indicate that defective nicein rather than modifications of the nicein-recognizing receptor play a central role in the pathogenesis of H-JEB.

Pathogenesis of mechanobullous disorders.

Recent advances in the molecular biology of the dermo-epidermal basement membrane zone have contributed greatly to our understanding of the etiopathogenetic pathways underlying mechanobullous disorders. Genetic linkage was established between the keratin gene clusters and epidermolysis bullosa simplex, and keratin mutations were identified in several patients. Anchoring filaments and the alpha 6 beta 4 integrin are likely to be affected in junctional EB. Genetic linkage was established between the collagen VII gene and both dominant and recessive subtypes of dystrophic epidermolysis bullosa, and different molecular abnormalities of collagen VII leading to formation of non-functional, rudimentary anchoring fibrils were observed in several families. These discoveries that led to definition of mutations underlying EB also help us to understand the normal physiology and function of the affected structures. They may also point the way to new therapeutic strategies for common acquired blistering diseases and disturbances of epithelialization in general.