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

OBJECTIVEThe aim of this study was to explore the genetic features of a family with 2-methyl-3-hydroxybutyryl-CoA dehydrogenase deficiency (MHBDD) which may provide the basis for the diagnosis and genetic counseling.

METHODClinical data of the proband was collected, total RNA and genomic DNA were extracted from the peripheral blood. The whole coding region of the ACAT1 gene was amplified by RT-PCR. 5' noncoding region of the ACAT1 gene and all 6 exons and flanking intron regions of the HADH2 gene were amplified by PCR. All amplification products were directly sequenced and compared with the reference sequence.

RESULT(1) The patient was a one-year-old boy who presented with psychomotor retardation and astasia when he was admitted to the hospital. Biochemical test revealed slight hyperlactatemia (3.19 mmol/L) and magnetic resonance imaging showed delayed myelination. 2-Methylacetoacetyl-CoA thiolase deficiency was suggested by gas chromatography-mass spectrometry. (2) There was no mutation in the ACAT1 gene and a hemizygous missense mutation c.388C > T was found in the 4 exon of the HADH2 gene which resulted in p. R130C. Proband's mother was the heterozygote and the father was normal.

CONCLUSIONThis is the first report on MHBDD patient and HADH2 mutation in China. p.R130C is responsible for the pathogenesis of the disease in the infant.

3-Hydroxyacyl CoA Dehydrogenases ; genetics ; Acetyl-CoA C-Acetyltransferase ; deficiency ; genetics ; Acyl Coenzyme A ; genetics ; metabolism ; Base Sequence ; DNA Mutational Analysis ; Dyskinesias ; Heterozygote ; Humans ; Infant ; Intellectual Disability ; enzymology ; genetics ; pathology ; Lipid Metabolism, Inborn Errors ; genetics ; pathology ; Male ; Mental Retardation, X-Linked ; Mutation, Missense ; Pedigree ; Reverse Transcriptase Polymerase Chain Reaction