Detection of deletions and duplications in the Duchenne muscular dystrophy gene by the molecular method MLPA in the first Argentine affected families

Abstract

AbstRACt. Deletions/duplications in the Duchenne muscular dystrophy (DMD) gene account for 60 to 70% of all alterations. A new technique, multiplex ligation-dependent probe amplification (MLPA), has been described that allows the detection of large genetic rearrangements by simultaneous amplification of up to 45 target sequences. The present article is based on the diagnosis of the first Argentine affected families by the application of MLPA. DNA samples from patients with and without a previous diagnosis were included. MLPA assays were performed according to manufacturer recommendations. Polymerase chain reaction and direct sequencing were performed when a single-exon deletion was detected. Results were analyzed using the Gene Marker v1.6 and Sequencing Analysis v5.2 software. In the samples with a previous diagnosis (as identified by short tandem repeat-polymerase chain reaction analysis), MLPA confirmed in some samples the same deletion and detected in others a larger deleted fragment. This enabled the prediction of the expected male phenotype. One deletion and one duplication were detected in patients without previous diagnosis. In this study, we investigated the applicability of MLPA in our country. Our results showed a 100% confirmation of the deleted fragments detected by short tandem repeat segregation analysis. Moreover, in some cases, the MLPA assay was able to refine the breakpoints involved. In addition, MLPA identified deletions/duplications in samples without previous diagnosis. In comparison to the available diagnosis strategies in Argentina, MLPA is less time-consuming, and spans the complete coding region of DMD. The application of MLPA will improve the genetic diagnosis of DMD/Becker muscular dystrophy in our country.

Identification of deletions and duplications in the Duchenne muscular dystrophy gene and female carrier status in western India using combined methods of multiplex polymerase chain reaction and multiplex ligation-dependent probe amplification

“…In the absence of MLPA evaluation, these patients would have remained unidentified without any genotypic confirmation, an essential step for identifying others in the family including female carriers. [12,13] With the introduction of the concept of exon skipping, knowing the exact mutation in a patient is necessary for prognostic evaluation. Studying all the 79 exons for any deletions or duplications is useful in predicting disruption of reading frame which in turn facilitates genotype-phenotype correlation.…”

MLPA mutation detection in Argentine HNPCC and FAP families

“…Our previous experiences of MLPA analysis had been with the X linked gene DMD [20], and as far as we had be able to ascertain, all the analyzed patients and carriermothers had more than one deleted exon. With this kind of electropherograms we could confirm the deletion of an exon by the absence of the consecutive one.…”

Implementación de la Prueba del Multiplex PCR para el Gen DMD en Pacientes con sospecha de Distrofia Muscular de Duchenne/Becker y la identificación de una deleción de los exones 48-51

“…La optimización de la prueba de multiplex PCR se realizó sa-tisfactoriamente en nuestro Centro. La estrategia de Multiplex PCR del gen DMD, se realiza por la amplificación vía PCR de dos regiones hot spots de deleciones recurrentes en el gen, y que corresponden a los exones 2 al 20 y 44 al 53 (24,32). El análisis por esta técnica tiene muchas ventajas sobre otras técnicas tradicionalmente usadas en el diagnóstico genético de DMD como las basadas en la técnica del Southern Blot en relación al tiempo y costo principalmente.…”