PRENATAL DIAGNOSIS IN MACEDONIAN DUCHENNE MUSCULAR DYSTROPHY FAMILIES
Kocheva SA1,2, Trivodalieva S1, Plaseska-Karanfilska D1, Vlaski-Jekic S3, Kuturec M2, Efremov GD1,*
*Corresponding Author: Professor Dr. Georgi D. Efremov, Macedonian Academy of Sciences and Arts, Research Center for Genetic Engineering and Biotechnology, Aven Krste Misirkov 2, POB 428, 1000 Skopje, Republic of Macedonia; Tel.: +3892-120253; Fax: +3892-115434; E-mail: gde@manu.edu.mk
page: 59

MATERIALS AND METHOD

Fifteen families at risk of having a child with DMD were referred to the Research Center for Genetic Engineering and Biotechnology at Skopje in the Republic of Macedonia for genetic counseling and prenatal diagnosis. Nine of the families were of Macedonian ethnic origin, while six were of Albanian origin living in Macedonia. Probands in 11 families were previously found to have deletions of different exons of the dystrophin gene, one had a gene duplication and in three the mutation was unknown.

DNA was extracted from chorionic villus samples (CVS) in seven pregnancies (in the first trimester of pregnancy), and amnionic fluid in eight pregnancies (in the second trimester of pregnancy). DNA was extracted from CVS or amniocytes following a standard phenol/chloroform extraction procedure [10]. Prenatal diagnosis was based on a combination of the following: 1) DNA analysis of the dystrophin gene by direct detection of deletions by multiplex polymerase chain reaction (mPCR) [11-13]; 2) Multiplex ligation-dependent probe analysis (MPLA) for detection of deletions and duplications [14] and 3) DNA linkage analysis, using eight highly polymorphic intragenic short tandem repeats [STR-(CA)n] for the detection of abnormal X chromosomes in the families with unknown mutation [15,16]. To exclude maternal cell contamination, DNA analysis was performed on CVS and on amniocytes by quantitative fluorescent (QF)-PCR analysis of 15 STR markers on chromosomes 13, 18, 21 and X, as well as the amelogenine gene for sex determination.

1) Multiplex Polymerase Chain Reaction. More than 98% of the deletions of the dystrophin gene are readily detectable using an mPCR approach, based on an exon by exon analysis strategy within two hot-spot regions (exons 2-22 and 44-53). Multiplex PCR was performed in three assays (Set A: exons 4, 8, 12, 17, 19, 44, 45, 48 and 51; Set B: exons Pm, 3, 6, 13, 43, 47, 50, 52 and 60; Set C: exons 16, 32, 41, 42 and 49) allowing the amplification of 23 exons [11-13]. Fetal DNA was amplified using specific oligonucleotide primers for 23 exons of the dystrophin gene. Multiplex PCR was carried out in a final volume of 50 μl containing 100 ng of genomic DNA, 30 pmol of each primer, 200 μM each dNTP, standard PCR buffer, 1.5 mM MgCl2 and 2 U Taq DNA polymerase (AmpliTaq Gold, Applied Biosystems, Branchburg, NJ, USA). Samples were subjected to 30 cycles of amplification, each consisting of 1 min. at 94°C, 1 min. at 53-55°C and 4 min. at 72°C, and a final extension of 72°C for 10 min. The PCR products were electrophoresed on 3% agarose gel in a TBE buffer and visualized under UV light after ethidium bromide staining. Deletions were easy to visualize by PCR-based procedures in males, because of their hemizygosity for this gene. The analysis is based on discrimination between the presence and absence of a PCR product. The duplications of exons, however cannot be visualized by a PCR-based assay.

2) Multiplex Ligation-Dependent Probe Amplification Analysis (MLPA). This assay for the DMD gene was performed on nine samples and required two different reactions due to the large number of exons [14]. One reaction contained probe mix PO34 that covered exons 1-20, 21-30, 41-50 and 61-70. The second reaction contained probe mix PO35 and covered exons 11-22, 31-40, 51-60, and 71-79. The assay conditions were essentially according to the manufacturer’s recommendations (MRS Holland, Amsterdam, The Netherlands). The PCR products were analyzed by capillary electrophoresis on an ABI PRISM™ 310 Genetic Analyzer (Applied Biyosystems).

3. DNA Linkage Analysis. For DNA polymorphism within or flanking the dystrophin gene [15,16], the PCR was carried out in a final volume of 50 μL containing 100 ng of genomic DNA, 30 pmols of each primer, 200 μM each dNTP, standard PCR buffer, 1.5 mM MgCl2 and 2 U Taq DNA polymerase (Ampli Taq Gold, Applied Biosystems). Samples were subjected to 35 cycles of amplification consisting each one of 1 min. at 94°C, 1 min. at 55-60°C and 1 min. 30 sec. at 72°C and a final extension at 72°C for 10 min. Aliquots of PCR products were mixed in a half volume of formamide dye solution (98% formamide, 10 mMEDTA, 0.1 bromphenol blue and 0.1% xylen cyanol), heated to 94°C for 3 min., and electrophoresed on a 6% denaturing polyacrylamide sequencing gel. Following electrophoresis the samples were transferred onto the membrane and analyzed after hybridization with a (CA)n repeat probe.





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