Laminins comprise a family to trimeric extracellular matrix proteins expressed in the basement membranes of many tissues, appearing first during the early stages of embryonic development, and later in a wide variety of tissue-specific isoforms. Laminin 5 is expressed in the basement membrane zone of specialized epithelia with secretory or protective functions, such as the skin. It is observed in the anchoring filaments of the lamina lucida, which connect the hemidosmosomes of the basal keratinocytes to the lamina densa of the dermal-epidermal junction. Laminin 5 is unique within the laminin family, in that it is the only human laminin which has a demonstrated role in a genetic disorder. The junctional forms of EB (JEB) are inherited in an autosomal recessive manner, and are characterized by blisters which form within the dermal- epidermal basement membrane zone, resulting in dysadhesion of the epidermis from the dermis. Junctional EB frequently results in the demise of affected individuals within the first few months of life. Several lines of evidence support the hypothesis that the three genes encoding the anchoring filament protein laminin 5 as candidate genes for harboring the mutations in junctional EB. Mutations in the beta3 chain gene (LAMB3) and gamma2 chain gene (LAMC2) of laminin 5 have recently been shown to underlie the junctional forms of epidermolysis bullosa in some families. In other families, however, both the LAMC2 and LAMB3 genes have been excluded by genetic linkage analysis or by the presence of normal levels of both LAMC2 and LAMB3 mRNAs in cultured keratinocytes. In these cases, the gene encoding the third polypeptide of laminin 5, the alpha3 chain gene (LAMA3) has become an attractive candidate gene for harboring additional mutations underlying JEB. Toward the overall goal of understanding the role of the LAMA3 gene in the junctional forms of epidermolysis bullosa, the principal objectives of this proposal are:
Specific Aim 1 : To elucidate the intron/exon organization of the LAMA3 gene. This will involve the isolation and characterization of cosmid clones encompassing the sequences encoded by the LAMA3 cDNA. It is anticipated that the gene structure of LAMA3 will be complex, since there is evidence for at least three 5' alternative transcripts and two 3' alternative transcripts. Segments of the LAMA3 cDNA will be used for the design of oligonucleotide primers for direct PCR based sequencing from the genomic DNA clones for the identification of exon borders, flanking intron sequences and the overall gene structure.
Specific Aim 2 : To design a rapid PCR amplification and mutation detection strategy for the LAMA3 gene. This will involve the design and optimization of PCR primers based on the sequences of the flanking introns. PCR amplimers will be subjected to heteroduplex analysis for the identification of bands with altered mobility in the DNA of unaffected unrelated control individuals. These amplimers will be subcloned and sequenced, and putative neutral polymorphisms will be verified in the normal population.
Specific Aim 3 : To identify pathogenetic mutations in the LAMA3 gene in junctional EB patients. This will be accomplished using the PCR strategy developed in specific aim 2, and applied to JEB patients in which the candidate gene for mutations is LAMA3. As sequence variants are detected, they will be analyzed to verify that they are indeed pathogenetic and not simple sequence polymorphisms, and that their segregation through JEB families is consistent with the inheritance of the disorder.
