OBJECTIVETo assess the association of PEX10 gene and 1p36 copy number variations in 1p36 region with concurrent epilepsy through analyzing 3 cases.

METHODSThe karyotypes of 3 patients were determined by high resolution chromosome banding, multiplex ligation dependent probe amplification (MLPA), fluorescence in situ hybridization (FISH) combined with single nucleotide polymorphism array (SNP) technology. Real-time PCR was carried out to determine the mRNA levels of PEX10 gene in peripheral blood of the patients.

RESULTSNo abnormality was found upon high resolution karyotyping. MLPA analysis showed that all of the 3 patients had a copy number variation of subtelomeric region in the short arm of chromosome 1, which was confirmed by FISH and SNP chip analyses. Case 1 and case 2 both had an epilepsy phenotype, and their copy number variations have encompassed the PEX10 gene. On the other hand, case 3 has absent epilepsy, and its PEX10 gene copy number was normal. Family investigation confirmed that the chromosome abnormalities in all of the 3 cases were of de novo type. Compared with healthy controls, real-time PCR showed that mRNA of the PEX10 gene was increased in case 1 but decreased in case 2.

CONCLUSIONThe abnormal expression of PEX10 gene resulting from copy number variations of 1p36 region may be associated with the epilepsy phenotype.

Child ; Chromosomes, Human, Pair 1 ; DNA Copy Number Variations ; Epilepsy ; genetics ; Female ; Humans ; Peroxins ; Polymerase Chain Reaction ; Polymorphism, Single Nucleotide ; Receptors, Cytoplasmic and Nuclear ; genetics

OBJECTIVETo screen the hepatitis B virus PreS1 associated protein from normal human liver cDNA library by the yeast two-hybrid system and explore the role of PreS1 protein in the infection of hepatitis B virus (HBV).

METHODSPCR was preformed to amplify the PreS1 gene containing EcoRI and PstI from HBV positive serum, and the production was inserted into plasmid pAS2-1 after digesting with the former two restricted endonuclease, then the bait vector pAS2-1-PreS1 was verified by auto-sequencing assay. The PreS1-BD fusion protein expressed in the yeast cells was confirmed by western blot, after pAS2-1-PreS1 was transfected into the yeast cell AH109. Yeast cells co-transfected with pAS2-1-PreS1 and the normal human liver cDNA library grew in selective SC/-trp-leu-his-ade2 medium, and the second screening was performed with LacZ report gene. Furthermore, segregation analysis and mating experiment were done to eliminate the false positive, then the true positive clones were submitted for PCR and sequencing. The results were submitted to the BLAST notebook of World Wide Wed Site NCBI to seek homologous sequence.

RESULTSBait vector pAS2-1-PreS1 included the anticipated fragment of PreS1 gene. Western blot showed that pAS2-1-PreS1 could correctly express PreS1-BD fusion protein in the yeast cells. After yeast cells co-transfected with pAS2-1-PreS1 and the normal human liver cDNA library, 97 colonies grew in the selective SC/-trp-leu-his-ade2 medium, only one clone was positive and showed high homology with Homo sapiens nascent-polypeptide-associated complex alpha polypeptide.

CONCLUSIONBait vector pAS2-1-PreS1 is successfully constructed, and nascent-polypeptide-associated complex alpha polypeptide protein expressed in hepatocyte can interact with PreS1 by the yeast two-hybrid system.

Base Sequence ; Cloning, Molecular ; DNA, Complementary ; genetics ; Gene Library ; Hepatitis B Surface Antigens ; genetics ; metabolism ; Humans ; Molecular Sequence Data ; Peroxins ; Protein Precursors ; genetics ; metabolism ; Recombinant Fusion Proteins ; genetics ; Saccharomyces cerevisiae Proteins ; genetics ; metabolism ; Transfection ; Two-Hybrid System Techniques ; Viral Envelope Proteins ; genetics ; metabolism ; Yeasts ; genetics