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Li F.Z.,Shantou University | Li F.Z.,Qingdao Agricultural University | Li L.B.,Key Laboratory of Birth Defects and Reproductive Health | Zhong Y.,Chengdu Jinjiang Hospital for Maternal and Child Health Care | And 7 more authors.
PLoS ONE | Year: 2013

Objective: Studying the methylation status of long terminal repeats (LTR) and its relationship to gag expression of HIV-1 in order to explore regulation mechanism of HIV-1 gene expression in vertical transmission from sperm to embryo. Methods/Principal Findings: Sperm samples were collected from a healthy donor and seven patients with HIV/AIDS. Zona-free hamster ova were fertilized by donor's spermatozoa transfected with pIRES2-EGFP-LTR-gag and patient's spermatozoa to obtain zygotes and 2-cell embryos, respectively. Interspecific in vitro fertilization, bisulfite sequencing PCR (BSP), RT-PCR, nested RT-PCR, nested real-time qRT-PCR and 2-△△Ct method, indirect immunofluoresence (IF) assay were performed. For donor's samples, the methylation rates of HIV-1 LTR were 0.56%, 1.67%, 0.56%, 0.56% in plasmid, spermatozoa, zygotes and 2-cell embryos, respectively while spermatozoa were transfected with unmethylated plasmid, and were 95.0%, 84.44%, 3.3%, 1.67% while transfected with methylated plasmid. The positive bands for HIV-1 gag cDNA were detected in spermatozoa and 2-cell embryos. The positive signals for HIV-1 p24 Gag protein were detected in 2-cell embryos but not in spermatozoa. For patient's samples, methylation rates of HIV-1 LTR were different in spermatozoa among patients. After fertilization, CpG sites in HIV-1 LTR were highly demethylated in zygotes and 2-cell embryos. The gag transcription levels increased with decreasing of methylation rates of HIV-1 LTR, which showed a strong negative correlations between gag transcription levels and methylation rates of HIV-LTR ether in the spermatozoa (r = -0.9877, P<0.0001) or in the sperm-derived 2-cell embryos (r = -0.9092, P = 0.0045). Conclusion: LTR methylation regulates expression of HIV-1 gag in vertical transmission from sperm to embryo. © 2013 Li et al.


Zhang D.,Key Laboratory of Birth Defects and Reproductive Health | Zhang D.,Chongqing Institute of Population and Family Planning | Li L.,Key Laboratory of Birth Defects and Reproductive Health | Li L.,Chongqing Institute of Population and Family Planning | And 15 more authors.
Gene | Year: 2013

Congenital heart disease (CHD) is the most frequently occurring congenital disorder in newborns and is the most frequent cause of infant death from birth defects. Human genetic studies have identified that numerous genes encoding transcription factors that regulate specific events in heart development are responsible for inherited and sporadic CHD. Nuclear factor-kappa B (NF-κB) is a major transcription regulator of immune response, apoptosis and cell-growth control genes. The aim of this study was to investigate whether the functional -94 insertion/deletion ATTG polymorphism (rs28362491) in the promoter of nuclear factor κB gene (NFKB1) is associated with susceptibility to CHD. Polymerase chain reaction (PCR)-polyacrylamide gel electrophoresis (PAGE) method was used to genotype rs28362491 in 122 atrial septal defect (ASD) patients, 114 ventricular septal defect (VSD) patients, and 412 controls. The frequencies of II (Insertion/Insertion) genotype in the ASD and VSD patients were significantly higher than that of controls (p= 0.004 for ASD Vs. controls, and p= 0.009 for VSD Vs. controls, respectively), and the frequencies for I allele in CHD patients were also significantly higher than that in controls (p= 0.01 for ASD Vs. controls, and p= 0.009 for VSD Vs. controls, respectively). This study suggests that the functional -94 insertion/deletion ATTG polymorphism in the promoter of NFKB1 is associated with CHD. © 2012 Elsevier B.V.

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