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Log-PCR: A New Tool for Immediate and Cost-Effective Diagnosis of up to 85% of Dystrophin Gene Mutations

¹ Dipartimento di Patologia Generale, Seconda Università degli Studi di Napoli, Naples; ² Telethon Institute of Genetics and Medicine (TIGEM), Naples; ³ Dipartimento di Medicina Sperimentale, Servizio di Cardiomiologia e Genetica Medica, Seconda Università degli Studi di Napoli, Naples; ⁴ Centro di Eccellenza per le Malattie Cardiovascolari, Seconda Università degli Studi di Napoli, Naples, Italy.

^aAddress correspondence to this author at: Dipartimento di Patologia Generale, Seconda Università degli Studi di Napoli, S. Andrea delle Dame, via L. De Crecchio 7-80138 Napoli, Italy. Fax 39 0815665704; e-mail vincenzo.nigro{at}unina2.it, nigro{at}tigem.it.

Background: Duchenne (DMD) and Becker (BMD) muscular dystrophies are caused by mutations in the dystrophin gene. Despite the progress in the technologies of mutation detection, the disease of one third of patients escapes molecular definition because the labor and expense involved has precluded analyzing the entire gene. Novel techniques with higher detection rates, such as multiplex ligation-dependent probe amplification and multiplex amplifiable probe hybridization, have been introduced.

Methods: We approached the challenge of multiplexing by modifying the PCR chemistry. We set up a rapid protocol that analyzes all dystrophin exons and flanking introns (57.5 kb). We grouped exons according to their effect on the reading frame and ran 2 PCR reactions for DMD mutations and 2 reactions for BMD mutations under the same conditions. The PCR products are evenly spaced logarithmically on the gel (Log-PCR) in an order that reproduces their chromosomal locations. This strategy enables both simultaneous mapping of all the mutation borders and distinguishing between DMD and BMD. As a proof of principle, we reexamined samples from 506 patients who had received a DMD or BMD diagnosis.

Results: We observed gross rearrangements in 428 of the patients (84.6%; 74.5% deletions and 10.1% duplications). We also recognized a much broader spectrum of mutations and identified 14.6% additional cases.

Conclusions: This study is the first exhaustive investigation of this subject and has made possible the development of a cost-effective test for diagnosing a larger proportion of cases. The benefit of this approach may allow more focused efforts for discovering small or deep-intronic mutations among the few remaining undiagnosed cases. The same protocol can be extended to set up Log-PCRs for other high-throughput applications.