J Korean Med Sci.  2006 Oct;21(5):794-799. 10.3346/jkms.2006.21.5.794.

Identification of a Novel Single Nucleotide Polymorphism of HADHA Gene at a Referred Primer-binding Site During Pre-diagnostic Tests for Preimplantation Genetic Diagnosis

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

• KMID: 1781894
• DOI: http://doi.org/10.3346/jkms.2006.21.5.794

Abstract

The pre-diagnostic test for preimplantation genetic diagnosis (PGD) of long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency was performed by polymerase chain reaction (PCR) and direct sequencing for hydroxyacyl-Coenzyme A dehydrogenase/3-ketoacyl-Coenzyme A thiolase/enoyl-Coenzyme A hydratase (HADHA) gene. We obtained unexpected genotyping results of HADHA gene by allele drop-out in the analysis of patients' genomic DNA samples with a referred PCR primer set. Upon further analysis with a re-designed primer set, we found a novel single nucleotide polymorphism (SNP) at the referred primer-binding site in the normal allele of HADHA gene (NT_022184, 5233296 a>t). We found that the frequency of this novel SNP was 0.064 in Korean population. Pre-diagnostic test using single lymphocytes and clinical PGD were successfully performed with the re-designed primer set. Nineteen embryos (95.0%) among 20 were successfully diagnosed to 5 homozygous mutated, 8 heterozygous carrier and 6 wild type. Among 6 normal embryos, well developed and selected 4 embryos were transferred into the mother's uterus, but a pregnancy was not achieved. We proposed that an unknown SNP at primer-binding sites would be a major cause of allele drop-out in the PGD for single gene dis-order.

Keyword

Preimplantation Diagnosis; long-chain 3-hydroxyacyl CoA dehydrogenase; LCHAD Deficiency; peroxysomal bifunctional enzyme; HADHA Gene; Allele Drop-out; Polymorphism, Single Nucleotide

MeSH Terms

*Preimplantation Diagnosis
*Polymorphism, Single Nucleotide
Polymerase Chain Reaction
Mutation
Multienzyme Complexes/*genetics
Male
Humans
Female
Binding Sites
Adult
3-Hydroxyacyl CoA Dehydrogenases/deficiency

Figure

• 

  Fig. 1 Pedigree of the family having LCHAD deficiency. Father, mother and their live daughter have both wild (W) and mutant (M) allele of the HADHA gene. Father and mother have the same HADHA gene mutation site (♦). Both mother and her daughter have a normal allele with an unidentified SNP (▴).

• 

  Fig. 2 Schematic representation for a part of the HADHA gene, together with the position of SNP and two pairs of primers used in this study. PCR primer sets were designed from the 5' and 3' flanking regions of the exon 16 in the HADHA gene. The right uppermost is the sequences of reverse primer (E16-R) and two lowers are the sequences of genomic DNA of wild and variant type alleles, respectively. The horizontal arrows indicate the direction and the location of each primer and the vertical arrow indicates the mutation loci (HADHA IVS16+2 t>g) in this family.

• 

  Fig. 3 Sequencing results showing the region of the HADHA-E16 reverse primer-binding site harboring the novel SNP. Arrows indicate the position of SNP. Male patient (A) was homozygous (A/A) whereas female patient (B) and her daughter (C) were identified as heterozygous (A/T), respectively.

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