title: Intracellular degradation of beta4 integrin in lethal junctional epidermolysis bullosa with pyloric atresia.
abstract: BACKGROUND
  Junctional epidermolysis bullosa with pyloric atresia (PA-JEB) is a rare autosomal recessive genodermatosis that manifests with neonatal mucocutaneous blistering and gastric outlet obstruction. The disease, which is caused by mutations in the alpha6beta4 integrin genes (ITGA6, ITGB4), is usually lethal. However, nonlethal cases have also been reported. Mutation database analysis has suggested that premature termination codons predominantly result in lethal forms while missense mutations frequently associate with nonlethal variants. Nevertheless, it is becoming more and more evident that the disease phenotype is also influenced by the position of the mutation in the protein functional domains.
  
  
  OBJECTIVE
  To investigate the molecular basis of a novel PA-JEB lethal case.
  
  
  METHODS
  Reverse transcriptase-polymerase chain reaction and direct sequencing-based mutation screening were performed. Mutation consequences in the patient's keratinocytes were then analysed by Northern blot and immunoprecipitation. Immunofluorescence analysis of cultured keratinocytes treated with protein intracellular degradation pathway inhibitors was also carried out.
  
  
  RESULTS
  The phenotype was caused by the presence, in the homozygous state, of a novel 33 bp in-frame deletion (nucleotides 175-207) in the ITGB4 coding sequence. Despite the normal steady-state level of integrin beta4 mRNA, the mutation, designated DeltaR59-A69, results in the almost complete absence of alpha6beta4 integrin in the patient's skin and cultured keratinocytes. Exposure of the patient's keratinocytes to the proteasomal inhibitor clasto-lactacystin beta-lactone increased the expression of the mutated beta4 integrin chains indicating that the proteasome complex is involved in the degradation of the internally deleted beta4 polypeptides.
  
  
  CONCLUSIONS
  We report for the first time a homozygous in-frame deletion in the ITGB4 gene. Our results suggest that the deletion of amino acids R59-A69 interferes with the biosynthetic folding of the protein, leading to a rapid degradation of the mutated beta4 chains. These findings provide new insight into the pathogenic effects of mutations affecting different functional domains of the beta4 integrin molecule and their prognostic implications in PA-JEB patients.
authors.affiliation: Laboratory of Molecular and Cell Biology, Istituto Dermopatico dell'Immacolata, IRCCS, Via dei Monti di Creta 104, 00167 Roma, Italy.
authors.forename: A
authors.initials: A
authors.lastname: Micheloni
authors.forename: N
authors.initials: N
authors.lastname: De Luca
authors.forename: G
authors.initials: G
authors.lastname: Tadini
authors.forename: G
authors.initials: G
authors.lastname: Zambruno
authors.forename: M
authors.initials: M
authors.lastname: D'Alessio
mesh_terms: D001483:Base Sequence; D015152:Blotting, Northern; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000235:genetics; D016109:Epidermolysis Bullosa, Junctional / Q000235:genetics*; D017809:Fatal Outcome; D005260:Female; D017353:Gene Deletion; D006801:Humans; D047468:Immunoprecipitation; D007231:Infant, Newborn; D039663:Integrin beta4 / Q000235:genetics*; D015603:Keratinocytes / Q000187:drug effects / Q000473:pathology; D008969:Molecular Sequence Data; D010375:Pedigree; D011708:Pylorus / Q000002:abnormalities*
chemical_list: D018076:DNA, Complementary; D039663:Integrin beta4