Preimplantation genetic diagnosis (PGD) for Duchenne muscular dystrophy (DMD) by triplex-nested PCR.
OBJECTIVE
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.


CONCLUSIONS
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.
Malcov
Mira
M
Sara Racine in vitro Fertilization Unit, Lis Maternity Hospital, Tel Aviv, Israel.
Ben-Yosef
Dalit
D
Schwartz
Tamar
T
Mey-Raz
Nava
N
Azem
Foad
F
Lessing
Joseph B
JB
Amit
Ami
A
Yaron
Yuval
Y
D016022:Case-Control Studies; D020732:Cytogenetic Analysis / Q000379:methods*; D004624:Embryo Transfer; D005188:False Negative Reactions; D005260:Female; D017353:Gene Deletion; D006801:Humans; D007962:Leukocytes; D008297:Male; D020388:Muscular Dystrophy, Duchenne / Q000097:blood / Q000175:diagnosis* / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D011247:Pregnancy; D019836:Preimplantation Diagnosis / Q000941:ethics