Identification of a novel single nucleotide polymorphism of HADHA gene at a referred primer-binding site during pre-diagnostic tests for preimplantation genetic diagnosis.
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.
Lee
Hyoung-Song
HS
Laboratory of Reproductive Biology and Infertility, Cheil General Hospital and Women's Healthcare Center, Sungkyunkwan University School of Medicine, Seoul, Korea.
Choi
Hye Won
HW
Lim
Chun Kyu
CK
Koong
Mi Kyoung
MK
Kang
Inn Soo
IS
Yoo
Han-Wook
HW
Choi
Jin-Ho
JH
Jun
Jin Hyun
JH
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
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