YMDD variants of HBV DNA polymerase gene: Rapid detection and clinicopathoiogical analysis with long-term Iamivudine therapy after liver transplantation

AIM: To look for a rapid low-cost technique for the detection of HBV variants.METHODS: Two patients who underwent orthotopic liver transplantation (OLT) for HBV infection were treated with lamivudine (100 mg daily) and HBV infection recurred in the grafted livers. The patients were monitored intensively for liver enzymes, hepatitis B surface antigen (HBsAg) and HBV DNA in serum. Liver biopsy was performed regularly. HBV DNA in a conserved polymerase domain (the YMDD locus) was amplified from serum of each patient by PCR and sequenced. HBV genotypes were analyzed by restriction fragment length polymorphism (RFLP) of the PCR products generated from a fragment of the polymerase gene.RESULTS: YMDD wild-type HBV was detected in one patient by PCR-RFLP and DNA sequencing 19 mo after OLT, and YIDD mutant-type HBV in the other patient, 16 mo after OLT.CONCLUSION: PCR-RFLP assay is an accurate and simple method for genotyping lamivudine-resistant HBV variants.

Development and Clinical Application of a Single-tube Nested PCR Method to Amplify the DNA Polymerase Ⅰ Gene of Treponema Pallidum

Objective: To develop a sensitive, specific and simple method for detection of extremely low numbers of T. pallidum in clinical specimens, as a significant addition to the serologic tests for syphilis diagnosis. Methods: Double-tube nested PCR(DN-PCR) and single-tube nested PCR(SN-PCR) assays were performed to amplify specific fragments of the DNA poly-merase I gene(polA) of T. pallidum. Sensitivity and specificity of the two PCR assays were tested. Eighty-six whole blood specimens from persons with suspected syphilis were detected by the two nested PCR methods. The TPPA test was used as a comparison for detecting syphilis in sera from corresponding patients. Results: Only specific amplicons could be obtained during amplification of the T. pallidum polA gene and the detection limit was approximately 1 organism when analyzed on gel by the two PCR methods. Of 86 clinical specimens, 62 were positive by TPPA. Of these, 54 and 51 were positive by the DN-PCR and SN-PCR, respectively, which does not represent a statistically significant difference between the two PCR tests. Of 24 TPPA-negative specimens, 5 were positive by both DN-PCR assay and SN-PCR assay. Conclusion: The SN- polA PCR method is extremely sensitive, specific and easy to perform for detecting low numbers of T. pallidum in clinical blood specimens as a complementary to serology for syphilis diagnosis.

The Study of Apolipoprotein B Gene Polymorphisms by Polymerase Chain Reaction.

Two polymorphic sites of apolipoprotein B（apoB） gene-Xbal and EcoRI were examined in a sample of 103 patients with documented coronary heart disease （CHD） and 100 healthy individuals. It was demonstrated: （1） The frequencies of rare Xallele （presence of Xbal cutting site） and E-allele （absence of cutting site） were significantly higher in CHD patients than those in controls. It was suggested that these genetic variations were associated with CHD. （2） The patients with genotype of XXhad significantly lower

The role of sexual related Y gene detection in the diagnosis of patients with gonadal dysgenesis

Objective To clarify the role of sexual related Y (SRY) gene detection in the diagnosis of gonadal dysgenesis.Methods Sixteen cases of gonadal dysgenesis were included in this study: 5 with androgen insensitivity syndrome, 1 with 17-α-hydroxylase deficiency, 4 with true hermaphrodite, 2 with 45,X/46,XY gonadal dysgenesis, 1 with 45,X gonadal dysgenesis, 1 with XY pure gonadal dysgenesis, 1 with testicular regression, and 1 XY female who gave birth to a normal baby. SRY gene was detected by using polymerase chain reaction (PCR) in blood and gonad samples and by direct sequencing of the SRY motif.Results Among the 16 cases, 15 were blood SRY positive, among which 13 (86.7%) showed the presence of testicular tissue, and 2 showed ovaries without testicular tissue. One SRY negative case showed the presence of testicular tissue. In 3 cases, SRY detection in gonadal tissue correlated with pathological findings but not with blood karyotype. The correlation between peripheral blood SRY and the pathology of the gonads was 81.25% and the correlation between the presence of peripheral blood Y chromosome and pathology of the gonads was 68.75%. Sequencing of the SRY motif in an XY female who gave birth to a normal baby showed no mutation.Conclusions SRY detection is more sensitive and specific than blood karyotype in the prediction of the presence of testicular tissue. Peripheral blood karyotype does not necessarily reflect gonadal type. There may be testicular related factors other than the SRY gene.

T cell receptor Vβ gene bias in rheumatoid arthritis

Objectives To explore the pathogenesis of rheumatoid arthritis (RA) by studying the expression of T cell receptors (TCRs).Methods T cell receptor Vβ (TCR Vβ) gene usage and expression were analyzed from synovial membrane and peripheral blood of 8 RA patients, 2 osteoarthritis patients and 2 accident amputees. The complementary determining region 3 (CDR3) of 25 TCR Vβ subfamily genes in unselected T cell populations were amplified semi-quantitatively by reverse transcription-polymerase chain reaction (RT-PCR). The products were further studied by genescan for frequency of Vβ usage.Results The numbers of Vβ subfamilies expressed by T cells from RA peripheral blood and synovial membrane were not significantly restricted. More importantly, biased Vβ gene expression in RA synovium was observed and Vβ6, Vβ17, and Vβ22 genes were the predominant subfamilies. It was noteworthy that the expression of Vβ17 in RA synovium was significantly increased. Conclusion Our data were consistent with the hypothesis that several antigen or superantigen-driven processes may be involved in the pathogenesis of RA.

Relationship of large multifunctional proteasome 7 gene polymorphism with susceptibility to type 1 diabetes mellitus and DR3 gene

Objective To study the relationship of the large multifunctional proteasome 7 (LMP7) gene polymorphism with susceptibility to type 1 diabetes mellitus (DM-1) and the DR3 gene in south Chinese Han population.Methods LMP7 genotypes and the DR3 gene were identified in 71 DM-1 patients and 86 healthy persons (as controls) by polymerase chain reaction-restriction fragment length polymorphism. DM-1 patients and controls were divided into DR3-positive and DR3-negative subjects. The frequencies of LMP7 genotypes and alleles were compared between DM-1 patients and controls respectively in the random subjects and in the DR3-matched subjects. Furthermore, DM-1 patients were divided into 3 groups according to the age of diabetic onset: group A≤14 years, group B 15-30 years, group C≥31 years.Results In the random subjects, the frequency of LMP7-B/B was lower (39% vs 58%, P<0.05) and that of LMP7-B/A was higher (54% vs 31%, P<0.01) in DM-1 patients than that in controls. In DR3-positive subjects, the frequencies of LMP7 genotypes and alleles showed no differences between DM-1 patients and controls. In DR3-negative subjects, the frequency of LMP7-B/B was decreased (40% vs 61%) and that of LMP7-B/A was increased (55% vs 28%, P<0.01) in DM-1 patients. The frequencies of LMP7 genotypes and alleles showed no significant differences among different ages of diabetic onset.Conclusions LMP7-B/B may be the protective genotype, and LMP7-B/A may be the susceptible genotype of DM-1, and this may not be affected by the DR3 gene. Persons with LMP7-B/B may have a decreased risk, and those with LMP7-B/A have an increased risk suffering from DM-1. The LMP7 gene may not be associated with the age of diabetic onset.

PCR assay for the inversion causing severe Hemophilia A and its application

Objective To develop a new technique based on long distance polymerase chain reaction (LD PCR) to replace Southern blotting method to detect Factor Ⅷ (FⅧ) gene inversion leading to severe Hemophilia A (HA) and carrier.Methods Four primers P, Q, A&B were designed and synthesized. P&Q is sp ecific for 5' and 3' flanking regions of F8A 1 respectively. A&B is specific fo r 5' and 3' flanking regions of F8A 2/F8A 3 respectively. LD PCR with 3 p rime rs and 3 temprature was set up, optimized and used to detect the inversion.Results The LD PCR with primers P,Q,A&B, P,Q&B and P,Q&A can be used t o detect the gene inversion and discriminate carrier from wild type. A blind ana lysis of 53 DNA samples from HA families was carried out by the LD PCR and Sout hern blotting respectively. Two sets of the results were completely identical. T hey were 23 cases of inversion, 27 cases of wild type and 3 cases of carriers. T he sensitivity and specificity of LD PCR are both 100%. Three inversion hem izygotes and 4 female carriers were identified from 5 HA families by the LD PCR technology.Conclusions The LD PCR with primer P,Q&B or P,Q,A&B can be used to det ect the gene inversion and the carrier of inversion. Compared with Southern blot ting, this technique is simple, rapid, inexpensive, more sensitive, accurate and non isotopic.

Expression of autotaxin mRNA in human hepatocellular carcinoma

Objective To investigate the expression of autotaxin (ATX) mRNA existed in the human hepatocellular carcinoma (HCC), and whether there is relation between the level of ATX expression and clinicopathological features of HCC.Methods Five normal liver tissues and 32 histologically verified HCC specimens were obtained. Semi quantitative reverse transcription polymerase chain reaction was used to detect mRNA expression of ATX.Results ATX was expressed in all 32 HCC and 5 normal liver tissues. The mean expression level of ATX gene in HCC samples was higher than that in normal liver tissues (68.23%±15.31% vs. 31.97%± 8.05%, P<0.001). Patients were divided into two groups: low ATX HCC (15 cases) and high ATX HCC (17 cases) by the cutoff point of median value. Intrahepatic metastasis, vascular invasion and poor differentiation were more frequently noted in HCC patients with high ATX expression than in patients with low ATX expression.Conclusion ATX gene was found to be overexpressed in some HCC and correlated with HCC development and metastases.