Original Articles
Cloning and sequence analysis of SLC25A13 transcripts in human amniocytes
Abstract
Objective: To amplify the entire ORF of SLC25A13 cDNA which encodes citrin, a liver-type mitochondrial aspartateglutamate carrier, and to investigate sequence feature of the transcripts for this gene in cultured human amniocytes. This study will provide laboratory evidences for prenatal diagnosis of NICCD at mRNA level.
Methods: Total RNA was extracted from cultured amniocytes, and cDNA was synthesized, and then nest PCR was performed to amplify the entire ORF sequences of SLC25A13. The PCR products were purified, cloned, sequenced, and aligned with the genomic DNA of SLC25A13 to analyze the alternative splicing pattern.
Results: The entire ORF of SLC25A13 gene was successfully amplified. Three splice variants of SLC25A13, i.e., SLCA (normal mRNA), SLCB (CAG insertion between exon 9-10) and SLCC (exon 5-11 skipping), were identified in the subjects. However, no abnormal mRNA from the allele with mutation 851del4 was detected in the amniocytes cultured from a carrier fetus of this mutation.
Conclusions: This study demonstrated that the entire ORF of SLC25A13 cDNA can be successfully amplified from cultured human amniocytes, and revealed exon 5-11 skipping as a novel SLC25A13 transcript. Normal mRNA occupied majority of the transcripts in normal control and heterozygous amniocytes which contained normal allele and 851del4
mutation, indicating that the two fetuses wouldn't suffer from NICCD. These SLC25A13 transcription features provided laboratory evidences for prenatal diagnosis of NICCD.
Methods: Total RNA was extracted from cultured amniocytes, and cDNA was synthesized, and then nest PCR was performed to amplify the entire ORF sequences of SLC25A13. The PCR products were purified, cloned, sequenced, and aligned with the genomic DNA of SLC25A13 to analyze the alternative splicing pattern.
Results: The entire ORF of SLC25A13 gene was successfully amplified. Three splice variants of SLC25A13, i.e., SLCA (normal mRNA), SLCB (CAG insertion between exon 9-10) and SLCC (exon 5-11 skipping), were identified in the subjects. However, no abnormal mRNA from the allele with mutation 851del4 was detected in the amniocytes cultured from a carrier fetus of this mutation.
Conclusions: This study demonstrated that the entire ORF of SLC25A13 cDNA can be successfully amplified from cultured human amniocytes, and revealed exon 5-11 skipping as a novel SLC25A13 transcript. Normal mRNA occupied majority of the transcripts in normal control and heterozygous amniocytes which contained normal allele and 851del4
mutation, indicating that the two fetuses wouldn't suffer from NICCD. These SLC25A13 transcription features provided laboratory evidences for prenatal diagnosis of NICCD.
