Korean J Reprod Med.  2008 Jun;35(2):99-110.

Establishment and Application of Molecular Genetic Techniques for Preimplantation Genetic Diagnosis of Osteogenesis Imperfecta

• Kim MJ
• Lee HS
• Choi HW
• Lim CK
• Cho JW
• Kim JY
• Song IO
• Kang IS

Abstract

OBJECTIVES
Preimplantation genetic diagnosis (PGD) has become an assisted reproductive technique for couples carrying genetic conditions that may affect their offspring. Osteogenesis imperfecta (OI) is an autosomal dominant disorder of connective tissue characterized by bone fragility and low bone mass. At least 95% of cases are caused by dominant mutations in the COL1A1 or COL1A2. In this study, we report on our experience clinical outcomes with 5 PGD cycles for OI in two couples.
METHODS
Before clinical PGD, we assessed the amplification rate and allele drop-out (ADO) rate of alkaline lysis and nested PCR protocol using heterozygous patient's single lymphocytes in the pre-clinical diagnostic tests for OI. We performed 5 cycles of PGD for OI by nested PCR for the causative mutation loci, COL1A1 c.2452G>A and c.3226G>A, in case 1 and case 2, respectively. The PCR products were analyzed by agarose gel electrophoresis, restriction fragment length polymorphism (RFLP) analysis with HaeIII restriction enzyme in the case 1 and direct DNA sequencing.
RESULTS
We confirmed the causative mutation loci, COL1A1 c.2452G>A in case 1 and c.3226G>A in case 2. In the pre-clinical tests, the amplification rate was 94.2% and ADO rate was 22.5% in case 1, while 98.1% and 1.9% in case 2, respectively. In case 1, a total of 34 embryos were analyzed and 31 embryos (91.2%) were successfully diagnosed in 3 PGD cycles. Eight out of 19 embryos diagnosed as unaffected embryos were transferred in all 3 cycles, and in the third cycle, pregnancy was achieved and a healthy baby was delivered without any complications in July, 2005. In case 2, all 19 embryos (100.0%) were successfully diagnosed and 4 out of 11 unaffected embryos were transferred in 2 cycles. Pregnancy was achieved in the second cycle and the healthy baby was delivered in March, 2008. The causative locus was confirmed as a normal by amniocentesis and postnatal diagnosis.
CONCLUSIONS
To our knowledge, these two cases are the first successful PGD for OI in Korea. Our experience provides a further demonstration that PGD is a reliable and effective clinical techniques and a useful option for many couples with a high risk of transmitting a genetic disease.

Keyword

Preimplantation genetic diagnosis (PGD); Osteogenesis imperfecta (OI); COL1A1; COL1A2; Allele drop-out (ADO)

MeSH Terms

Alleles
Amniocentesis
Carotenoids
Collagen Type I
Connective Tissue
Diagnostic Tests, Routine
DNA
Electrophoresis, Agar Gel
Embryonic Structures
Family Characteristics
Korea
Lifting
Lymphocytes
Molecular Biology
Osteogenesis
Osteogenesis Imperfecta
Oxygenases
Polymerase Chain Reaction
Polymorphism, Restriction Fragment Length
Pregnancy
Preimplantation Diagnosis
Prostaglandins D
Reproductive Techniques, Assisted
Carotenoids
Collagen Type I
DNA
Oxygenases
Prostaglandins D