USE OF EPITOPE LIBRARIES TO IDENTIFY EXON-SPECIFIC MONOCLONAL-ANTIBODIES FOR CHARACTERIZATION OF ALTERED DYSTROPHINS IN MUSCULAR-DYSTROPHY

The majority of mutations in Xp21-linked muscular dystrophy (MD) can b e identified by PCR or Southern blotting, as deletions or duplications of groups of exons in the dystrophin gene, but it is not always possi ble to predict how much altered dystrophin, if any, will be produced. Use of exon-specific monoclonal antibodies (mAbs) on muscle biopsies f rom MD patients can, in principle, provide information on both the amo unt of altered dystrophin produced and, when dystrophin is present, th e nature of the genetic deletion or point mutation. For this purpose, mAbs which recognize regions of dystrophin encoded by known exons and whose binding is unaffected by the absence of adjacent exons are requi red. To map mAbs to specific exons, random ''libraries'' of expressed dystrophin fragments were created by cloning DNAseI digestion fragment s of a 4.3-kb dystrophin cDNA into a pTEX expression vector. The libra ries were then used to locate the epitopes recognized by 48 mAbs to fr agments of 25-60 amino acids within the 1,434-amino-acid dystrophin fr agment used to produce the antibodies. This is sufficiently detailed t o allow further refinement by using synthetic peptides and, in many ca ses, to identify the exon in the DMD (Duchenne MD) gene which encodes the epitope. To illustrate their use in dystrophin analysis, a Duchenn e patient with a frameshift deletion of exons 42 and 43 makes a trunca ted dystrophin encoded by exons 1-41, and we now show that this can be detected in the sarcolemma by mAbs up to and including those specific for exon 41 epitopes but not by mAbs specific for exon 43 or later ep itopes.