OBJECTIVETo study the mechanisms of dystrophin gene deletion, the junction fragment with exons 45-54 deletion were cloned and sequenced.

METHODSA Duchenne muscular dystrophy (DMD) patient with exons 45-54 deletion has been substantiated by PCR amplification of the exons. Then we used a PCR-based genome-walking method for localizing the breakpoints in introns 44 and 54. At last, the deletion-junction fragment was directly amplified by PCR approach with forward and reverse primers annealing to a DNA sequence as close as possible to the breakpoints in introns 45 and 54. The sequencing result of the deletion-junction fragment was compared with the normal intronic sequences.

RESULTSA total of 2716 bp sequence containing the junction fragment was obtained. The 5' breakpoint was located in LINE/L1 element of intron 44 and close to a matrix attachment region (MAR). The 3' breakpoint was located in the minor potential MAR with topoisomerase II cleavage sites around. Beside the 3' breakpoint there was a 6 bp palindromic sequence. A 4 bp microhomologous sequence (AGAG) was in the joint of the deletion-junction fragment.

CONCLUSIONThe nonhomologous recombination caused by L1 repeated element, topoisomerase II cleavage sites, MARs and the nonhomologous end joining of microhomologous sequence may be the important factors in this huge gene fragment deletion.