Korean J Obstet Gynecol.  2004 Apr;47(4):708-718.

Successful Preimplantation Genetic Diagnosis for Ornithine Transcarbamylase Deficiency, Junctional Epidermolysis Bullosa and Lactic Acidosis Using Duplex Nested PCR: Delivery of Healthy Baby by Specific Preimplantation Genetic Diagnosis for Ornithine Tran

• Lee HS
• Choi HW
• Lim CK
• Min DM
• Byun HK
• Kim JY
• Koong MK
• Yoo HW
• Kim SC
• Jun JH
• Kang IS

Abstract

OBJECTIVE
Preimplantation genetic diagnosis (PGD) is reserved for couples with a risk of transmitting a serious and incurable disease, and hence avoids the undesirable therapeutic abortion. Herein, we report the result of PGD to carriers at risk of transmitting ornithine transcarbamylase (OTC) deficiency, junctional epidermolysis bullosa (EB) and lactic acidosis (LA) due to defect of pyruvate dehydrogenase alpha1 gene, respectively.
METHODS
The ovarian stimulation, oocyte retrieval and ICSI procedure were undergone by conventional protocols. PGD for single gene disorders was carried out after biopsy of one or two blastomeres from the embryos on the third day. We performed the duplex nested PCR of the simultaneous amplification for the causative mutation loci as well as the SRY gene on Y chromosome in case of OTC deficiency and LA. Two different mutation loci of ITGB4 gene in EB case were amplified by the same protocol. The PCR products were analyzed by agarose gel electrophoresis, restriction fragment length polymorphism analysis or direct DNA sequencing.
RESULTS
A total of 26 embryos were analyzed by duplex nested PCR. One or two blastomeres were biopsied, and successful diagnosis rate of PGD with PCR was 92.3% (24/26). There was no contamination in all PCR samples of negative controls (n=67). Five embryos (19.2%) were diagnosed as normal embryos, which were transferred to the mothers' uterus in each cases. In OTC deficiency case, singleton pregnancy was established. At 17 weeks of gestation, genetic normality of OTC gene in fetus was confirmed by amniocentesis. A healthy baby was successfully delivered at 36 weeks of gestation in OTC deficiency case. Unfortunately, pregnancies were not achievement in cases of EB and LA.
CONCLUSION
This is the first report in Korea that healthy baby was born after specific PGD for OTC deficiency. Our results demonstrate that duplex nested PCR for single cell is an efficient method in identifying the gender and single gene mutation or two different mutation loci, simultaneously. This PGD procedure could provide normal healthy baby to the couple with a high risk of transmitting genetic diseases.

Keyword

Single gene disorder; Preimplantation genetic diagnosis (PGD); Ornithine transcarbamylase deficiency; Epidermolysis bullosa; Lactic acidosis

MeSH Terms

Abortion, Therapeutic
Acidosis, Lactic*
Amniocentesis
Biopsy
Blastomeres
Diagnosis
Electrophoresis, Agar Gel
Embryonic Structures
Epidermolysis Bullosa*
Epidermolysis Bullosa, Junctional
Family Characteristics
Female
Fetus
Genes, sry
Korea
Oocyte Retrieval
Ornithine Carbamoyltransferase Deficiency Disease*
Ornithine Carbamoyltransferase*
Ornithine*
Ovulation Induction
Oxidoreductases
Polymerase Chain Reaction*
Polymorphism, Restriction Fragment Length
Pregnancy
Preimplantation Diagnosis*
Prostaglandins D
Pyruvic Acid
Sequence Analysis, DNA
Sperm Injections, Intracytoplasmic
Uterus
Y Chromosome
Ornithine
Ornithine Carbamoyltransferase
Oxidoreductases
Prostaglandins D
Pyruvic Acid