title: Identification of a novel single nucleotide polymorphism of HADHA gene at a referred primer-binding site during pre-diagnostic tests for preimplantation genetic diagnosis.
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.
authors.affiliation: Laboratory of Reproductive Biology and Infertility, Cheil General Hospital and Women's Healthcare Center, Sungkyunkwan University School of Medicine, Seoul, Korea.
authors.forename: Hyoung-Song
authors.initials: HS
authors.lastname: Lee
authors.forename: Hye Won
authors.initials: HW
authors.lastname: Choi
authors.forename: Chun Kyu
authors.initials: CK
authors.lastname: Lim
authors.forename: Mi Kyoung
authors.initials: MK
authors.lastname: Koong
authors.forename: Inn Soo
authors.initials: IS
authors.lastname: Kang
authors.forename: Han-Wook
authors.initials: HW
authors.lastname: Yoo
authors.forename: Jin-Ho
authors.initials: JH
authors.lastname: Choi
authors.forename: Jin Hyun
authors.initials: JH
authors.lastname: Jun
mesh_terms: D015094:3-Hydroxyacyl CoA Dehydrogenases / Q000172:deficiency; D000328:Adult; D001665:Binding Sites; D005260:Female; D006801:Humans; D063989:Long-Chain-3-Hydroxyacyl-CoA Dehydrogenase; D008297:Male; D063991:Mitochondrial Trifunctional Protein; D063992:Mitochondrial Trifunctional Protein, alpha Subunit; D009097:Multienzyme Complexes / Q000235:genetics*; D009154:Mutation; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D019836:Preimplantation Diagnosis
chemical_list: D009097:Multienzyme Complexes; D015094:3-Hydroxyacyl CoA Dehydrogenases; C576205:HADHA protein, human; D063989:Long-Chain-3-Hydroxyacyl-CoA Dehydrogenase; D063992:Mitochondrial Trifunctional Protein, alpha Subunit; D063991:Mitochondrial Trifunctional Protein