FMUC  2009|2010 Turma 7 e 8: Joana Calvão, Joana Laranjinha, Joana Maciel, João Nuno Pereira, Suheila Patel

E PIDERMOLYSIS BULLOSA (EB) EB simplex (epidermis; dominant) Junctional EB (dermal- epidermal junction; recessive) Distrophyc EB (dermis; dominant and recessive) group of heritable disorders skin and related epithelial tissues break and blister due to minor mechanical trauma Classification rare form - acquired BMZ – Basement Membrane Zone

S YMPTOMS They depend on the type of EB, but can include: Scalp blistering and alopecia (hair loss); Blistering around the eyes and nose; Blistering in or around the mouth and throat, causing dysphagia (swallowing difficulty); Skin thickening on palms and soles of the feet (hyperkeratosis) Formation of large, fluid-filled blisters that develop in response to minor trauma.

W HO IS AFFECTED BY EB? It mainly affects babies and children It affects boys and girls equally “butterfly children” EB in the world… 50 EB cases occur per 1 million live births United States – UK – % EB simplex 5% dystrophic EB 1% junctional EB 2% unclassified Ryan, 3 years

C OMPLICATIONS Caused by scarring scar tissue can build up between fingers or toes so that they join together - pseudosyndactyly In recessively inherited EB Dehydratation open wounds – loss of large amounts of fluid

more common in recessive dystrophic EB and junctional EB inflamed eyelids – blepharitis the eyelid turning outwards – ectropion blepharitis ectropion the most common cause of death Affecting the eyes Squamous cell carcinoma (SCC) Mainly affects recessive dystrophic EB Develops between 15 and 35 years It is invasive and has high metastatic potential C OMPLICATIONS

C AUSES Mutations of the genes coding for specific proteins, that maintain the mechanical integrity of tissues, such as: Mutation in these genes Epidermolysis bullosa simplex Plectin links the components of citoskeleton Gene: PLEC1 Keratin (14;5) forms the cytoskeleton of epithelial cells Gene: KRT14, KRT5 Microscopy of keratin filaments inside cells

Integrin Mediates attachment between a cell and the tissues surrounding it Genes: ITGB4, ITGA6, ITGB4 Laminin Important part of basal lamina Genes: LAMA3, LAMB3, LAMC2 Type VII Collagen anchor fibril between the external epithelia and the underlying stroma Gene: COL7A1 Junctional epidermolysis bullosa Dystrophic epidermolysis bullosa Mutation C AUSES

D IAGNOSIS 1 st step: Anamnesis 2 nd step: Skin biopsy 3 rd step: Microscopy analysis methods 4 th step: DNA mutation analysis

S KIN BIOPSY 2 ND STEP : S KIN BIOPSY Obtain a skin biopsy following a thorough history and physical examination.

M ICROSCOPY ANALYSIS METHODS 3 RD STEP : M ICROSCOPY ANALYSIS METHODS Two main analysis methods: - criterion standard for determining the level of blistering Electron Microscopy However : - slow and expensive procedure - not always available in smaller laboratories find the type of EB diagnosis - information on the BMZ morphology

It is used to make an immunomapping, performed in skin lesions. Immunofluorescent Microscopy panel of antibodies against each of the antigens known to be affected in epidermolysis bullosa identification of the underlying molecular defect exposure of sections of skin lesions, prepared in cryostat, to specific monoclonal antibodies against various molecules of the BMZ M ICROSCOPY ANALYSIS METHODS 3 RD STEP : M ICROSCOPY ANALYSIS METHODS

4 TH STEP : DNA MUTATION ANALYSIS Mutation screening Mutation screening: restriction fragment-length polymorphism analysis, hotspot analysis, and finally, direct DNA sequencing. There have been identified more than 1,000 mutations that can cause EB. blood - patient and family members DNA

T REATMENT No cure. ways of easing the symptoms Avoiding clothes that fit tightly or rub the skin; Blister puncturing and wound dressing; Topical antibiotics to treat infections; Good dental hygiene to prevent further blisters in the mouth; Diet high in calories and proteins; Surgical treatment: esophageal dilation, restoration of the hand. Currently...

T REATMENT Future therapies… Protein Therapy Gene Therapy the missing or defective protein is produced in vitro by recombinant methods EB subtypes involving a defect in type VII collagen (protein has a long half life in the body). genes targeted to restore normal protein production Two clinical trials in progress dystrophic epidermolysis bullosa 1. University of Minnesota bone marrow transplantation is used as the mechanism of delivery of corrective skin cells 2. Stanford University retroviral mediated type VII collagen gene transfer blistered skin most useful

Correction of junctional epidermolysis bullosa by transplantation of genetically modified epidermal stem cells Mavilio F, Pellegrini G, et al. | Nature Medicine, Vol. 12, No. 12 (19 November 2006), pp Epidermal stem cells LAM5- 3–deficient: 1. transduced with a retroviral vector encoding LAM5- 3; 2.used to prepare epidermal grafts. normal levels of functional LAM5; firmly adherent epidermis (1 year); absence of blisters, infections, inflammation or immune response. “ex vivo gene therapy of JEB is feasible and leads to full functional correction of the disease” blistered leg Results

P REVENTION Prenatal Diagnosis mutation analysis identification of the defective gene Fetal skin biopsies and fetoscopy increased risk of pregnancy loss Chorionic villus sample Chorionic villus sample  8-10 weeks Amniotic fluidAmniotic fluid  in the second trimester

R EFERENCES overview bullosa/Pages/Complications.aspx