MOLECULAR DIAGNOSTICS OF DUCHENNE/BECKER MUSCULAR DYSTROPHY PATIENTS BY MULTIPLEX LIGATION-DEPENDENT PROBE AMPLIFICATION ANALYSIS AND DIRECT SEQUENCING
Todorova A1,*, Guergueltcheva V2, Genova J3, Mihaylova V2, Todorov T1, Tchamova T2, Georgieva B1, Kremensky I4, Tournev I,2,5 Mitev V1
*Corresponding Author: Albena Todorova, Department of Chemistry and Biochemistry, Medical University Sofia, 2 “Zdrave” str., Sofia 1431, Bulgaria; Tel./Fax: +359-2-9530715; E-mail: todorova_albena@abv.bg
page: 3

MATERIALS AND METHODS

Patients. Fifty-one patients and four families with no living index patient available were analyzed by MLPA/ direct sequencing. All patients were diagnosed clinically only on the basis of clinical symptoms, family history, electromyography (EMG) data and creatine kinase levels. In some, the result of muscular biopsy was also available. Thirty-six patients were severely affected, diagnosed as DMD, 14 had BMD and one patient was considered to represent an intermediate form (IMD). The affected members of the four families with no living index patient were suspected to be severely affected (there was one affected patient in each family).

Method. DNA samples were obtained from peripheral blood, using a DNA extraction kit (QIAamp DNA Mini Kit, QIAGEN, Hilden, Germany). Multiplex PCR of 18 exons for deletions detection along the deletion hot-spot was performed by the classical protocols of Chamberlain et al. [2] and Beggs et al. [3].

The SALSA MLPA P034/P035 (MRC-Holland, Amsterdam, The Netherlands) kit was used in accordance with the manufacturer’s instructions [6]. Fifty to 200 ng of DNA were diluted with TE (Tris/EDTA) (1 M Tris, 0.5M EDTA, pH 8.0) buffer to a volume of 5 mL. The diluted samples were subjected to hybridization with the DMD gene-specific probes for all 79 exons and to 13 control probes (along Y, Xq and Xp chromosome regions) situated in both sets P034 and P035 [6], at 60°C overnight. The hybridized probes were ligated with a specific ligase mix, provided by the manufacturer. The final step represents PCR amplification of the ligated fragment products. The PCR buffer, PCR primers 6-FAM (6-carboxyfluorescein) labeled, the enzyme dilution buffer and the polymerase were provided in the kit.

The PCR products obtained were analyzed on an ABI PRISM™ 310 genetic analyzer (Applied Biosystems, Foster City, CA, USA) in the presence of ROX500 size standard (Applied Biosystems). Each patient sample was analyzed simultaneously with at least two normal male samples. In order to assess copy number changes (duplications) in comparison to the normal controls, MLPA data interpretation was performed by MLPA software - Coffalyser [6].

Sequencing. The DNA from patients who tested negative by MLPA were sequenced for the entire coding sequence of the DMD gene, including exon/intron borders. Each exon was amplified by primers, designed for sequencing in our laboratory (the primer sequence is available upon request). The PCR product was purified by PCR Product Pre-Sequencing Kit [United States Biochemicals (USB); Affymetryx Inc., Santa Clara, CA, USA] and sequenced by the BigDye Terminator Cycle Sequencing Kit v.3.1 (Applied Biosystems). The sequenced sample were subjected to a standard ethanol precipitation. The pellet was air-dried and kept at room temperature in the dark. Before run, the pellet was resuspended in 13 mL Hi-Di Formamide (Applied Biosystems) and loaded into the ABI PRISM™ 310 genetic analyzer (Applied Biosystems). Sequencing results were interpreted by Sequence Scanner v.1 (Applied Biosystems).




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