[Detecting Down syndrome with a novel dual-color competitive quantitative fluorescent polymerase chain reaction method].

OBJECTIVE: To develop a rapid method for the detection of Down syndrome (DS) using dual-color competitive quantitative fluorescent polymerase chain reaction (DCC-QF-PCR), and to assess its feasibility for the prenatal diagnosis of Down syndrome. METHODS: DNA was extracted from peripheral blood of 30 DS patients and 60 normal men, common primers for DSCR and USC2 genes and respective TaqMan probes were designed and synthesized. The results of DCC-QF-PCR were compared with those of QF-PCR which measured the ratio between DSCR and GAPDH. Forty-six amniotic fluid samples were assayed with DCC-QF-PCR. The results were compared with that of karyotyping. Monoclone fragments for DSCR and USC2 genes were obtained from direct cloning of PCR products. DCC-QF-PCR was carried out using different DNA ratios of DSCR and USC2 as the template. The dosage ratio between DSCR and USC2 was calculated. RESULTS: The gene dosage ratio of the DS patients was 1.41-1.74, which was significantly higher than that of normal men (0.93-1.15). The dosage ratio range of DSCR and GAPDH by QF-PCR was comparatively greater than that of DSCR and USC2. Three samples were diagnosed as DS, which was in good agreement with that of karyotyping analysis. There was no significant difference between the gene dosage ratio from DCC-QF-PCR and that of predetermined (P>0.05). CONCLUSION: DCC-QF-PCR is an accurate, rapid, and low cost method, which only requires tiny amount of sample and therefore has broad application in the genetic and prenatal diagnosis.

[Expression of glucocorticoid receptor alpha and beta mRNA in orbital tissues in thyroid associated ophthalmopathy].

OBJECTIVE: To evaluate the expression of glucocorticoid receptor alpha (GRalpha) and beta (GRbeta) messenger RNA (mRNA) in orbital tissues from thyroid associated ophthalmopathy (TAO). METHODS: Samples of extraocular muscle and orbital fat were obtained from 17 patients with TAO and 10 healthy individuals. Total RNA was extracted and reversely transcripted into cDNA. The expression of GRalpha and GRbeta mRNA was detected by means of fluorescent quantitative polymerase chain reaction (PCR). RESULTS: Expression of GRalpha mRNA was much higher than GRbeta mRNA in all extraocular muscle and orbital fat biopsies. The relative copy of GRalpha was 40.15 +/- 11.37 in TAO patients and 20.64 +/- 7.07 in the controls. GRalpha: GRbeta mRNA ratio of these two groups was 77.76 +/- 18.77 and 148.34 +/- 23.86, respectively. There was significant difference between these two groups (P < 0.05). No significant difference was noted between extraocular muscle and orbital fat biopsies, between glucocorticoid-treated and non-treated patients or among hyperthyroidism, hypothyroidism and euthyroidism (P > 0.05). CONCLUSIONS: The increased expression of GRalpha mRNA and decreased GRalpha: GRbeta ratio in orbital tissues may play an important role in the pathogenesis of TAO and the effects of glucocorticoid treatment.

[Effect of ligand pioglitazone activating PPAR-gamma on the invasiveness of cholangiocarcinoma cell in vitro and the expression regulation of related genes].

OBJECTIVE: To explore the effect and mechanism of ligand pioglitazone (PGZ) activating PPAR-gamma on the invasiveness of cholangiocarcinoma cell in vitro. METHODS: Human hilar cholangiocarcinoma cell line QBC939 was selected and cultured in vitro for this research. The rate of QBC939 cell growth inhibition was detected by MTT, and the influence of PGZ on the expression of MMP-7 mRNA and TIMP-1 mRNA was measured by using FQ-PCR. The in vitro invasiveness and mobility of QBC939 were quantified by Matrigel invasion assay and crossing-river test. RESULTS: Among the low concentration (5 micromol/L-40 micromol/L) groups at the point of 12-hours, PGZ did not show to inhibit significantly the growth of QBC939 cells (P>0. 05). However, the PGZ could down-regulate the expression of MMP-7 mRNA in QBC939 cells (P<0. 01), and up-regulate the TIMP-1 mRNA expression to be without obvious statistics significance (P>0. 05). At last, PGZ could reduce the number of QBC939 cell breaking through the matrigel and prolonging the time of crossing-river significantly (P< 0. 01) in a dose-dependent manner. CONCLUSION: For ligand PGZ to activate PPAR-gamma can inhibit the invasiveness of QBC939 cells in vitro via regulating the expression of MMP-7 and the mobility of QBC939 cells probably.

[Gene mutation analysis of coagulation factor VIII from a female patient with hemophilia A].

Hemophilia A affects male, whereas females are carriers and generally spared from this disease. However, we here reported a 65-year-old female with Hemophilia A while screening the gene mutation of coagulation factor VIII. The female went to hospital because of tripping to lead her right chest to be injured with subcutaneous hematoma. She had historically a hemorrhagic diathesis. The physical examination discovered her hip limited to bend and move, but no discrepancy length between her two legs. The initial laboratory tests showed that the activated partial thromboplastin time (APTT) was 61. 3 seconds (20-40 seconds), and the APTT corrected by mixing with normal plasma was 41.3 s, but the levels of PT, FIB and TT were normal. The plain radiographs revealed the hip joints to suffer from the acetabular dysplasia and osteoarthritis. The level of FVIII:C was 2%, F IX:C 200%, vWF:Ag 120%, vWF:Rcof 100%, vWF:CBA 128%, and the F VIII binding assay to vWF was normal. The primers for exon 14 of F VIII gene were designed according to the NM - 000132 gene sequence. DNA was abstracted from the patient blood. PCR were carried out and the DNA sequence was followed. A new mutation of 4111A-->C was discovered, which caused the amino acid sequence changed (T 1314 P). The mutation of T 1314 P may be the cause of this female patient to get the hemophilia A. This mutation was a novel one which has never been reported before.

[Applying competitive polymerase chain reaction to the detection of hepatitis B virus DNA].

OBJECTIVE: To reduce the rate of accidental false negative result in the HBV DNA PCR test on clinical serum samples. METHODS: A competitive polymerase chain reaction (C-PCR) was used to decrease the false negative ratio. In the C-PCR, a constructed inner control DNA was added for co-amplification with the HBV target DNA. RESULTS: In a 20 microl C-PCR system, about 60 to 200 copies of inner control DNA could give apparent co-amplification signal band after electrophoresis on a 2% agarose gel. Five of 120 samples of clinical serum (4.2%) could not be amplified. CONCLUSION: C-PCR has the advantage of yielding information on false negative in the HBV DNA PCR assay of clinical serum samples.