Polymorphism of Methionine Synthase Gene in Nuclear Families of Congenital Heart Disease

Objective To investigate the relation of methionine synthase (MS) gene variation with congenital heart disease (CHD) phenotype. Methods One hundred and ninety three CHD patients (94 males and 99 females) and their biological parents (nuclear families) in Liaoning Province were selected as the case group, and another 104 normal persons (60 males and 44 females) and their parents without family history of birth defects as the control group. For all subjects the polymorphism of MS gene A2756G locus was examined by PCR-RFLP method. Results In offspring of the control group the frequencies of MS genotype (+/ -) and allele (+) were 10.7% and 5.3%, without existence of homozygote. The MS genotype distribution and allele frequencies of CHD patients and their mothers were not significantly different from the control (P > 0.05). The frequency of allele (+) in case fathers (5.0 %) was apparently lower than that in the control (9.1%, P=0.060), and the odds ratio (OR) was 0.53 (95% CI: 0.25-1.09). There was no difference in parents' genotype combination between the two groups, and in genotype distribution among different types of CHD. Analysis of genetic transmission indicated that mutation allele (+) existed transmission disequilibrium in CHD nuclear families. The percentage of allele (+) transmitted from parents was lower than that allele (-) with OR 0.26 (95% CI: 0.11-0.60). Conclusion MS gene variation in parents is associated with occurrence of CHD in offspring, and mutation allele (+) in parents may be related with the decrease of CHD risk in offspring.

Relationship Between Polymorphism of Methylenetetrahydrofolate Dehydrogenase and Congenital Heart Defect

To investigate the relationship between G1958A gene polymorphism of methylenetetrahydrofolate dehydrogenase (MTHFD) and occurrence of congenital heart disease (CHD) in North China. Methods One hundred and ninety-two CHD patients and their parents were included in this study as case group in Liaoning Province by birth defect registration cards, and 124 healthy subjects (age and gender matched) and their parents were simultaneously selected from the same geographic area as control. Their gene polymorphism of MTHFD G1958A locus was examined with PCR-RFLP, and serum folic acid and homocysteine (Hcy) levels were tested with radio-immunoassay and fluorescence polarization immunoassay (FPIA). Results There existed gene polymorphism at MTHFD G1958A locus in healthy subjects living in North China. The percentages of GG, GA, and AA genotype were 57.98%, 35.57%, and 6.45% respectively, and the A allele frequency was 24.23%, which was significantly different from Western population. No difference was observed when comparing genotype distribution and allele frequency between the case and control groups, so was the result from the comparison between genders. The A allele frequency of arterial septal defect patients’ mothers (10.87%) was significantly lower than that of controls (28.15%) (P=0.014), with OR=0.31 (95% CI: 0.09-0.84), and no difference in the other subgroups. The percentage of at least one parent carrying A allele in arterial septal defect subgroup (43.48%) was significantly lower than that in controls (69.64%) (P=0.017), with OR=0.34 (95% CI: 0.12-0.92). The analysis of genetic transmission indicated that there was no transmission disequillibrium in CHD nuclear families. Their serum folic acid level was significantly higher than that of controls (P=0.000), and Hcy level of the former was higher than that of the latter with no statistical significance (P>0.05). Serum Hcy and folic acid levels of mothers with gene mutation were lower than those of mothers with no mutation. Conclusion No significant difference of genotype distribution and allele frequency existed between CHD patients and healthy population. MTHFD G1958A mutation in parents (particularly in mother) can decrease the risk of arterial septal defect in offspring. The possible mechanism of protection might be mutation, which can increase MTHFD enzyme activity, folic acid metabolism and homocysteine remethylation, and decrease Hcy level.

Transactivating effect of complete S protein of hepatitis B virus and cloning of genes transactivated by complete S protein using suppression subtractive hybridization technique

AIM: To investigate the transactivating effect of complete S protein of hepatitis B virus (HBV) and to construct a subtractive cDNA library of genes transactivated by complete S protein of HBV by suppression subtractive hybridization (SSH) technique and to clone genes associated with its transactivation activity, and to pave the way for elucidating the pathogenesis of hepatitis B virus infection.METHODS: pcDNA3.1(-)-complete S containing full-length HBV S gene was constructed by insertion of HBV complete S gene into BarmH-I/Kpn I sites. HepG2 cells were cotransfected with pcDNA3.1(-)-complete S and pSV-lacZ.After 48 h, cells were collected and detected for the expression of β-galactosidase (β-gal). Suppression subtractive hybridization and bioinformatics techniques were used.The mRNA of HepG2 cells transfected with pcDNA3.1(-)-complete S and pcDNA3.1(-) empty vector was isolated,and detected for the expression of complete S protein by reverse transcription polymerase chain reaction (RT-PCR)method, and cDNA was synthesized. After digestion with restriction enzyme RcaI, cDNA fragments were obtained.Tester cDNA was then divided into two groups and ligated to the specific adaptors 1 and 2, respectively. After tester cDNA had been hybridized with driver cDNA twice and underwent nested PCR twice, amplified cDNA fragments were subcloned into pGEM-Teasy vectors to set up the subtractive library. Amplification of the library was carried out within E. coli strain DH5α. The cDNA was sequenced and analyzed in GenBank with BLAST search after polymerase chain reaction (PCR) amplification.RESULTS: The complete S mRNA could be detected by RT-PCR in HepG2 cells transfected with the pcDNA3.1(-)-complete S. The activity of β-gal in HepG2 cells transfected with the pcDNA3.1(-)-complete S was 6.9 times higher than that of control plasmid. The subtractive library of genes transactivated by HBV complete S protein was constructed successfully. The amplified library contains 86 positive clones. Colony PCR showed that 86 clones contained DNA inserts of 200-1 000 bp, respectively.Sequence analysis was performed in 35 clones randomly,and the full length sequences were obtained with bioinformatics method and searched for homologous DNA sequence from GenBank, altogether 33 coding sequences were obtained. These cDNA sequences might be target genes transactivated by complete S protein of HBV. Moreover, two unknown genes were discovered, full length coding sequences were obtained by bioinformatics techniques,one of them was named complete S transactivated protein 1 (CSTP1) and registered in GenBank (AY553877).CONCLUSION: The complete S gene of HBV has a transactivating effect on SV40 early promoter. A subtractive cDNA library of genes transactivated by HBV complete S protein using SSH technique has been constructed successfully. The obtained sequences may be target genes transactivated by HBV complete S protein among which some genes coding proteins are involved in cell cycle regulation, metabolism, immunity, signal transduction, cell apoptosis and formation mechanism of hepatic carcinoma.

Genes transactivated by hepatitis C virus core protein, a microarray assay

AIM: To explore the new target genes transactivated by hepatitis C virus (HCV) core protein and to elucidate the pathogenesis of HCV infection.METHODS: Reverse transcribed cDNA was subjected tomicroarray assay. The coding gene transactivated by HCV core protein was cloned and analyzed with bioinformatics methods.RESULTS: The expressive vector of pcDNA3.1(-)-core was constructed and confirmed by restriction enzyme digestion and DNA sequencing and approved correct. mRNA was purified from HepG2 and HepG2 cells transfected with pcDNA3.1(-)-core, respectively. The cDNA derived was subjected to microarray assay. A new gene namedHCTP4 was cloned with molecular biological method in combination with bioinformatics method.CONCLUSION: HCV core is a potential transactivator.Microarray is an efficient and convenient method for analysis of differentially expressed genes.

Molecular epidemiological study on pre-X region of hepatitis B virus and identification of hepatocyte proteins interacting with whole-X protein by yeast two-hybrid

AIM: To identify the pre-X region in hepatitis B virus (HBV)genome and to study the relationship between the genotype and the pre-X region. To investigate the biological function of whole-X (pre-X plus X) protein, we performed yeast two-hybrid to screen proteins in liver interacting with whole-X protein.METHODS: The pre-X region of HBV was amplified by polymerase chain reaction (PCR) method, and was cloned to pGEM Teasy vector. After the target region was sequenced, Vector 8.0 software was used to analyze the sequences. The whole-X bait plasmid was constructed by using yeast two-hybrid system 3. Yeast strain AH109 was transformed. After expression of the whole-X protein in AH109 yeast strains was proved, yeast two-hybrid screening was performed by mating AH109 with Y187 containing liver cDNA library plasmid. The mated yeast was plated on quadruple dropout medium and assayed for α-gal activity. The interaction between whole-X protein and the protein obtained from positive colonies was further confirmed by repeating yeast two-hybrid. After extracting and sequencing of plasmid from blue colonies, we carried out analysis by bioinformatics. RESULTS: After sequencing, 27 of 45 clones (60%) were found encoding the pre-X peptide. Eighteen of twenty-seven clones (66.7%) of pre-X coding sequences were found from genotype C. Five positive colonies that interacted with whole-X protein were obtained and sequenced; namely, fetuin B, UDP glycosyltransferase 1 family-polypeptide A9, mannose-P-dolichol utilization defect 1, fibrinogen-B beta polypeptide, transmembrane 4 superfamily member 4CD81 (TM4SF4).CONCLUSION: The pre-X gene exists in HBV genome.Genes of proteins interacting with whole-X protein in hepatocytes were successfully cloned. These results brought some new clues for studying the biological functions of whole-X protein.

Screening of hepatocyte proteins binding to complete S protein of hepatitis B virus by yeast-two hybrid system

AIM: To investigate the biological function of complete S protein and to look for proteins interacting with complete S protein in hepatocytes.METHODS: We constructed bait plasmid expressing complete S protein of HBV by cloning the gene of complete S protein into pGBKT7, then the recombinant plasmid DNA was transformed into yeast AH109 (a type). The transformed yeast AH109 was mated with yeast Y187 (α type) containing liver cDNA library plasmid in 2xYPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium (SD/Trp-Leu-His-Ade) containing X-α-gal for selection and screening. After extracting and sequencing of plasmids from positive (blue) colonies, we underwent sequence analysis by bioinformatics.RESULTS: Nineteen colonies were selected and sequenced.Among them, five colonies were Homo sapiens solute carrier family 25, member 23 (SLC25A23), one was Homo sapiens calreticulin, one was human serum albumin (ALB)gene, one was Homo sapiens metallothionein 2A, two were Homo sapiens betaine-homocysteine methyltransferase,three were Homo sapiensNa+ and H+ coupled amino acid transport system N, one was Homo sapiens CD81 antigen (target of anti-proliferative antibody 1) (CD81), three were Homo sapiens diazepam binding inhibitor, two colonies were new genes with unknown function.CONCLUSION: The yeast-two hybrid system is an effective method for identifying hepatocyte proteins interacting with complete S protein of HBV. The complete S protein may bind to different proteins i.e., its multiple functions in vivo.

Cloning and identification of NS5A TP2gene and its spliced variant transactivated by hepatitis C virus non-structural protein 5A

AIM: To clone, identify and study new NSSATP2 gene and its spliced variant transactivated by hepatitis C virus nonstructural protein 5A. METHODS: On the basis of subtractive cDNA library of genes transactivated by NS5A protein of hepatitis C virus, the coding sequence of new gene and its spliced variant were obtained by bioinformatics method. Polymerase chain reaction (PCR) was conducted to amplify/VSSATP2 gene. RESULTS: The coding sequence of a new gene and its spliced variant were cloned and identified successfully. CONCLUSION: A new gene has been recognized as the new target transactivated by HCV NS5A protein. These results brought some new clues for studying the biological functions of new genes and pathogenesis of the viral proteins.