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Use of epitope libraries to identify exon-specific monoclonal antibodies for characterization of altered dystrophins in muscular dystrophy.
Nguyen TM
,
Morris GE
.
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The majority of mutations in Xp21-linked muscular dystrophy (MD) can be identified by PCR or Southern blotting, as deletions or duplications of groups of exons in the dystrophin gene, but it is not always possible to predict how much altered dystrophin, if any, will be produced. Use of exon-specific monoclonal antibodies (mAbs) on muscle biopsies from MD patients can, in principle, provide information on both the amount of altered dystrophin produced and, when dystrophin is present, the 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 required. To map mAbs to specific exons, random "libraries" of expressed dystrophin fragments were created by cloning DNAseI digestion fragments of a 4.3-kb dystrophin cDNA into a pTEX expression vector. The libraries were then used to locate the epitopes recognized by 48 mAbs to fragments of 25-60 amino acids within the 1,434-amino-acid dystrophin fragment used to produce the antibodies. This is sufficiently detailed to allow further refinement by using synthetic peptides and, in many cases, to identify the exon in the DMD (Duchenne MD) gene which encodes the epitope. To illustrate their use in dystrophin analysis, a Duchenne patient with a frameshift deletion of exons 42 and 43 makes a truncated 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 epitopes.
Figure I Multiple mAb screening of bacterial fusion proteins
by using a miniblotter. The bacterial clones indicated (see table 1)
were grown and induced as streaks across a nitrocellulose sheet, as
described in Material and Methods. mAbs of the MANDYS number
shown were applied to all clones in the vertical lanes of the miniblotter. The control lane (all clones negative) contained PBS instead of
mAb. mAbs which show the same pattern will map close to each
other (e.g., MANDYS102, 103, and 106 bind to clones 2, 13, 25, and
26 only).
Figure 2 Mapping of MANDYS1-24 epitopes by using
DNAseI fragment libraries. Results are consistent with earlier mapping by either chemical cleavage (Nguyen thi Man et al. 1990a) or
transposon mutagensis (Sedgwick et al. 1991). Exon boundaries are
taken from Koenig et al. (1988, 1989) and Roberts et al. (1991,
1992b).
Figure 3 Mapping of 25 mAbs by using DNAseI fragment
libraries. a, Overlapping fragments from relevant clones in table 1,
shown in relation to dystrophin amino acid sequence and exon structure. Exon boundaries are from Koenig et al. (1989). Vertical broken
lines correspond to the epitope limits shown below. b, Three most
finely mapped epitopes, shown in relation to predicted structures for
dystrophin in which helical regions (hatched boxes) are separated by
linkers (lines). Model 1 (from Koenig and Kunkel 1990) and model 2
(from Cross et al. 1990) differ significantly in their predicted helixlinker boundaries within repeats 14-16 (repeat numbers are those in
Koenig and Kunkel 1990).
Figure 4 Characterization of a truncated dystrophin encoded by exons 1-41 in a Duchenne MD patient with a deletion of exons 42 and
43 (Helliwell et al. 1992), by using exon-specific mAbs. The mAbs used in this experiment were MANDYS1, MANDYS101, MANDYS102, and
MANDRAL.
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