Document Detail

Preimplantation genetic diagnosis (PGD) for Duchenne muscular dystrophy (DMD) by triplex-nested PCR.
MedLine Citation:
PMID:  16353285     Owner:  NLM     Status:  MEDLINE    
OBJECTIVES: Duchenne muscular dystrophy (DMD) is a lethal X-linked recessive disorder with an incidence of approximately 1 in 3500 males, caused by mutation in the DMD gene. About 2/3 of DMD cases are caused by gross DMD gene deletion mutations. The purpose of this study was to develop a series of single-cell multiplex-nested PCR protocols for preimplantation genetic diagnosis (PGD) of the most prevalent DMD deletions. METHODS: The protocols were developed on single blood leukocytes from normal males and females and patients with known DMD gene deletion. In the first reaction, 2 of 11 different primer sets (exons 4, 8, 12, 13, 17, 46, 47, 49, 50, 52 and intron 52) were used to allow the simultaneous amplification of different DMD loci and the SRY gender marker, in a single triplex-nested polymerase chain reaction (PCR). Aliquots of this reaction were then subjected to nested PCR in which each locus was amplified individually. Following the successful establishment of single-cell triplex-nested PCR in single leukocytes, the technique was employed in five clinical PGD cases. RESULTS: For each DMD locus, more than 50 single leukocytes from healthy controls and more than 100 single leukocytes from affected individuals with known deletions were analyzed. Amplification efficiency for each tested locus was 98-100%. The false-negative rates for each analysis taken separately was <1%. Taken together, however, the results of the triplex-nested PCR analysis had a false-negative rate of 0%. No contamination was detected in all wash-drop blanks tested. We subsequently performed 18 PGD cycles in 5 DMD carriers. A total of 156 embryos were biopsied and successfully analyzed. Of these, 39 affected embryos were detected and 50 unaffected embryos were transferred (mean = 2.9 +/- 1.1 embryos per cycle). These resulted in three biochemical pregnancies and three clinical pregnancies, all of which have culminated in the birth of normal offspring. CONCLUSION: Triplex-nested PCR using 2 of 11 DMD loci and the SRY gender marker allow PGD for >90% of DMD families with known deletions. These protocols are associated with a high amplification efficiency and accuracy.
Mira Malcov; Dalit Ben-Yosef; Tamar Schwartz; Nava Mey-Raz; Foad Azem; Joseph B Lessing; Ami Amit; Yuval Yaron
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Publication Detail:
Type:  Case Reports; Journal Article    
Journal Detail:
Title:  Prenatal diagnosis     Volume:  25     ISSN:  0197-3851     ISO Abbreviation:  Prenat. Diagn.     Publication Date:  2005 Dec 
Date Detail:
Created Date:  2005-12-20     Completed Date:  2007-04-12     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  8106540     Medline TA:  Prenat Diagn     Country:  England    
Other Details:
Languages:  eng     Pagination:  1200-5     Citation Subset:  IM    
Copyright Information:
Copyright 2005 John Wiley & Sons, Ltd.
Sara Racine in vitro Fertilization Unit, Lis Maternity Hospital, Tel Aviv, Israel.
Data Bank Information
Bank Name/Acc. No.:
OMIM/300376;  310200
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MeSH Terms
Case-Control Studies
Cytogenetic Analysis / methods*
Embryo Transfer
False Negative Reactions
Gene Deletion*
Muscular Dystrophy, Duchenne / blood,  diagnosis*,  genetics
Polymerase Chain Reaction / methods*,  standards
Preimplantation Diagnosis* / ethics

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

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