J Korean Med Sci.  2007 Jun;22(3):572-576. 10.3346/jkms.2007.22.3.572.

Preimplantation Genetic Diagnosis for Ornithine Transcarbamylase Deficiency by Simultaneous Analysis of Duplex-nested PCR and Fluorescence In Situ Hybridization: A Case Report

• Lee HS
• Jun JH
• Choi HW
• Lim CK
• Yoo HW
• Koong MK
• Kang IS

• KMID: 1778375
• DOI: http://doi.org/10.3346/jkms.2007.22.3.572

Abstract

Ornithine transcarbamylase (OTC) deficiency is an X-linked co-dominant disorder. A couple, with a previous history of a neonatal death and a therapeutical termination due to OTC deficiency, was referred to our center for preimplantation genetic diagnosis (PGD). The female partner has a nonsense mutation in the exon 9 of the OTC gene (R320X). We carried out nested polymerase chain reaction (PCR) for R320X mutation and fluorescence in situ hybridization (FISH) for aneuploidy screening. Among a total of 11 embryos, two blastomeres per embryo from 9 embryos were biopsied and analyzed by duplex-nested PCR and FISH, and one blastomere per embryo from 2 embryos by only duplex-nested PCR. As a result of PCR and restriction fragment length polymorphism analysis, four embryos were diagnosed as unaffected embryos having the normal OTC gene. Among these embryos, only one embryo was confirmed as euploidy for chromosome X, Y and 18 by FISH analysis. A single normal embryo was transferred to the mother, yielding an unaffected pregnancy and birth of a healthy boy. Based on our results, PCR for mutation loci and FISH for aneuploidy screening with two blastomeres from an embryo could provide higher accuracy for the selection of genetically and chromosomally normal embryos in the PGD for single gene defects.

Keyword

Preimplantation Genetic Diagnosis (PGD); Ornithine Transcarbamylase (OTC) Deficiency; Nested PCR; FISH; Single Gene Defects

MeSH Terms

Adult
Aneuploidy
Codon, Nonsense
DNA Primers
Exons
Female
Humans
In Situ Hybridization, Fluorescence/*methods
Infant, Newborn
Male
Ornithine Carbamoyltransferase/*deficiency
Polymerase Chain Reaction/*methods
Pregnancy
Pregnancy Outcome
Preimplantation Diagnosis/*methods

Figure

• 

  Fig. 1 Identification of the maternal mutation (heterozygote type) in exon 9 of the OTC gene. After digesting the PCR product with BclI, the 319 or 218 bp PCR products of the affected mother were digested into 223 and 96 bp (lane 1) and 178 and 40 bp products (lane 4), respectively. The 40 bp product was hardly seen on an agarose gel. Lane 1 and 4; the first and nested PCR products of the affected mother, respectively, lane 2 and 3; BclI-digested products from the first PCR products of the father. M: molecular weight marker.

• 

  Fig. 2 Results of agarose gel electrophoresis of the restriction fragment length polymorphism (RFLP) analysis with BclI restriction enzyme in the preimplantation genetic diagnosis for ornithine transcarbamylase deficiency. In normal embryos (No. 1, 2, 3 and 8), the 218-bp PCR products were not digested with BclI.

• 

  Fig. 3 Results of the fluorescence in situ hybridization (FISH) analysis on the embryos diagnosed to be normal by PCR in the PGD for OTC deficiency. Panels A-D show the photographs for the results of FISH on the embryos No. 1, 2, 3, and 8 in Table II, respectively. Aqua, green and orange signals indicate the presence of the chromosome X, Y and 18 in the nucleus of the blastomeres, respectively. PGD, preimplantation genetic diagnosis; OTC, ornithine transcarbamylase; PCR, polymerase chain reaction

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