DNA sensors to assess the effect of VKORC1 and CYP2C9 gene polymorphisms on warfarin dose requirement in Chinese patients with atrial fibrillation.

The optimal dose of warfarin depends on polymorphisms in the VKORC1 (the vitamin K epoxide reductase complex subunit (1) and CYP2C9 (cytochrome P450 2C9) genes. To minimize the risk of adverse reactions, warfarin dosages should be adjusted according to results from rapid and simple monitoring methods. However, there are few pharmacogenetic-guided warfarin dosing algorithms that are based on large cohorts from the Chinese population, especially patients with atrial fibrillation. This study aimed to validate a pharmacogenetic-guided warfarin dosing algorithm based on results from a new rapid electrochemical detection method used in a multicenter study. Three SNPs (CYP2C9 *2, *3 and VKORC1 c.-1639G > A) were genotyped by electrochemical detection using a sandwich-type format that included a 3' short thiol capture probe and a 5' ferrocene-labeled signal probe. A total of 1285 samples from four clinical hospitals were evaluated. Concordance rates between the results from the electrochemical DNA biosensor and the sequencing test were 99.8%. The results for gene distribution showed that most Chinese patients had higher warfarin susceptibility because mutant-type and heterozygotes were present in the majority of subjects (99.4%) at locus c.-1639G > A. When the International Warfarin Pharmacogenetics Consortium algorithm was used to estimate therapeutic dosages for 362 patients with AF and the values were compared with their actual dosages, the results revealed that 56.9% were similar to actual dosages (within the 20% range). A novel electrochemical detection method of CYP2C9 *2, *3and VKORC1 c.-1639G > A alleles was evaluated. The warfarin dosing algorithm based on data gathered from a large patient cohort can facilitate the reasonable and effective use of warfarin in Chinese patients with AF.

Non-invasive prenatal diagnosis of trisomy 21 by reverse transcriptase multiplex ligation-dependent probe amplification.

BACKGROUND: Obtaining fetal DNA or RNA by either chorionic villus sampling (CVS) or amniocentesis is currently, the gold standard prenatal diagnosis. However, these invasive procedures carry risk of miscarriage. A reliable method for non-invasive prenatal diagnosis (NIPD) has long been sought to reduce the risk of miscarriage. METHODS: Cell-free fetal RNA was extracted from the plasma of peripheral blood from 121 women 9-20 weeks of pregnancy. Five single nucleotide polymorphism (SNP) loci in PLAC4 gene were analyzed by reverse transcriptase multiplex ligation-dependent probe amplification (RT-MLPA), followed by capillary electrophoresis. Karyotype analysis was used for confirmation of prenatal diagnosis of trisomy 21. RESULTS: Of 121 samples, 23 were diagnosed with trisomy 21, 87 with normal ploidy, nine had all five SNP loci homozygous and two had one heterozygous SNP locus. Comparing with karyotype analysis, the diagnostic sensitivity and specificity of RT-MLPA were 92% and 100%, respectively. CONCLUSIONS: RT-MLPA is a convenient and reliable method for the diagnosis of trisomy 21. We have shown that this method has good specificity, high sensitivity, and high throughput, making this technique applicable for NIPD in clinical practice.

Development of a multiplex MethyLight assay for the detection of multigene methylation in human colorectal cancer.

In peripheral blood, cell-free methylated DNA has been reported to be a useful biomarker of noninvasive blood screening for the detection of colorectal cancer (CRC), including the genes ALX homeobox 4 (ALX4), septin 9 (SEPT9), or transmembrane protein with EGF-like, and two follistatin-like domains 2 (TMEFF2). Here we report a multiplex MethyLight polymerase chain reaction (PCR) assay that simultaneously detected the methylation status of ALX4, SEPT9, and TMEFF2, as well as quantifying methylation level of these genes in a total of 127 fresh tissue samples and 182 peripheral blood samples from CRC patients. Using the multiplex MethyLight assay, methylated ALX4, SEPT9, and TMEFF2 occurred in 56, 78, and 75% of CRC tissue samples and in 48, 75, and 71% of peripheral blood samples from CRC patients. The sensitivities of the combined study using the three genes as biomarkers for the detection of CRC in primary tissues and peripheral blood samples were 84 and 81%, with specificities of 87 and 90%, respectively. Combining the specificity of real-time PCR, the high throughput of multiplex PCR, and the high sensitivity of multigene detection, this multiplex MethyLight PCR assay may allow for future screening programs with large-scale noninvasive blood testing for early-stage CRC.

The manual Polybrene test has limited sensitivities for detecting the Kidd blood group system.

BACKGROUND AND OBJECTIVES: The Kidd system antibodies, if undetected, can cause immediate or delayed hemolytic transfusion reactions as well as hemolytic disease of the newborn. There have been anecdotal reports about the inefficiency of the manual Polybrene test in detecting these antibodies. Here, we sought to determine the sensitivity of the manual Polybrene test in detecting anti-Jk(a) and anti-Jk(b) antibodies and Jk(a) and Jk(b) antigens. MATERIALS AND METHODS: Ten archived anti-Jk(a)/Jk(b) antibody positive human sera were examined by both the manual Polybrene test and the indirect antiglobulin test using polyspecific antibodies, monospecific anti-IgG antibodies and anti-C3 antibodies. Furthermore, 40 randomly selected donor blood samples were collected and phenotyped for the frequencies of Jk(a) and Jk(b) antigens using the manual Polybrene test and the indirect antiglobulin test. The results from these tests were further confirmed by saline tube tests. RESULTS: The manual Polybrene test displayed an overall sensitivity of 60% in detecting anti-Jk(a) and anti-Jk(b) antibody. Specifically, it had a sensitivity of 57.14% for anti-Jk(a) antibody and a sensitivity of 66.7% for anti-Jk(b) antibody. Furthermore, the manual Polybrene test exhibited a sensitivity of 46.15% for Jk(a) antigen and a sensitivity of 77.42% for Jk(b) antigen. CONCLUSION: The manual Polybrene test has a very low sensitivity in detecting anti-Jk(a) and anti-Jk(b) antibody, especially anti-Jk(a) antibody. It is also a very insensitive test for detecting Jk(a) antigen.

Simultaneous detection of different respiratory virus by a multiplex reverse transcription polymerase chain reaction combined with flow-through reverse dot blotting assay.

Cell culture and immunofluorescence (IF) assays have been traditionally used for the laboratory diagnosis of respiratory viral infections, but these assays have a low sensitivity and are time consuming. We developed a multiplex reverse transcription polymerase chain reaction combined with flow-through reverse dot blotting (mRT-PCR-FT-RDB) assay for the simultaneous detection of influenza virus type A including H5 subtype and H9 subtype, influenza virus type B, parainfluenza virus types 1 and 3, respiratory syncytial virus, human rhinovirus, and human coxsackievirus. In comparison with viral culture and IF assay as the gold standard method, the mRT-PCR-FT-RDB assay gave a sensitivity and a specificity of 100% and 98%. The high sensitivity and specificity, the rapid result turnaround time, and the reduced expense of the mRT-PCR-FT-RDB assay compared with viral culture and IF assay suggest that this assay would be a significant improvement over traditional ones for the detection of respiratory viruses in a clinical laboratory.

Development of a multiplex quantitative fluorescent PCR assay for identification of rearrangements in the AZFb and AZFc regions.

The azoospermia factor b (AZFb) and azoospermia factor c (AZFc) regions in the human Y chromosome consist of five palindromes constructed from six distinct families of amplicons and are prone to rearrangement. Partial deletion and duplication in the region can cause azoospermia or oligozoospermia and male infertility. The aim of the study was to establish a quantitative fluorescent PCR (QF-PCR) assay to classify AZFb and AZFc rearrangements. A single pair of fluorescent primers was designed to amplify simultaneously the amplicon in AZFc and the length-variant homologous sequences outside of the region as control. Since the copy number of the control sequences is fixed in the human genome, dosage of the target could be easily obtained through comparing the height of the fluorescent peaks between the target and the control after amplification with limited PCR cycles. Most types of rearrangements in AZFb and AZFc regions could be classified with QF-PCR containing four such primer pairs. Eleven types of rearrangement in AZFb and AZFc regions were well discriminated with QF-PCR. In conclusion, QF-PCR is a simple and reliable method to detect rearrangements in AZFb and AZFc.

Development of a multiplex real-time polymerase chain reaction for the detection of influenza virus type A including H5 and H9 subtypes.

Avian influenza viruses (AIVs) are endemic in wild birds and, if transmitted to poultry, can cause serious economic losses. The aim of this study was to develop a multiplex real-time reverse transcriptase polymerase chain reaction (RT-PCR) for rapid detection of influenza virus type A, including H5 and H9 subtypes. The selected primers and various labeled TaqMan reporter probes corresponding to matrix, H5, and H9 genes were used in a multiplex real-time RT-PCR to simultaneously detect triple fluorescent signals. The results showed that the multiplex real-time RT-PCR assay can be applied to detect RNA of influenza virus type A including H5 and H9 subtypes with a high specificity and a sensitivity of 10 copies per reaction. As a result of its short turnaround times and a high specificity and sensitivity, the assay is very suitable for large-scale screening during AIV outbreaks.

Determination of hepatitis B virus genotype by flow-through reverse dot blot.

BACKGROUND: The clinical outcome and response to therapy of hepatitis B virus infection differ depending upon viral genotype. Most methods of determining the viral genotype are relatively time-consuming and costly. Moreover, the results of some methods are influenced by single nucleotide mutations. OBJECTIVES: To develop a novel HBV genotyping process insensitive to single nucleotide mutations using an improved reverse dot blot method employing the principle of "flow-through hybridization". STUDY DESIGN: The flow through reverse dot blot (FT-RDB) method was developed using DNA from different HBV genotypes. HBV sequences from Genebank were used to design primers and probes. Specificity and sensitivity of the method were evaluated with clinical samples in which the HBV viral load was quantified by real-time PCR. Results were compared to those of multiplex PCR and sequencing. Another 59 clinical samples were used to test the clinical applicability of the method. RESULTS: We showed that FT-RDB could be made insensitive to single nucleotide mismatch by adjusting the hybridization temperature. All HBV-negative samples showed no signals in the assay. The detection sensitivity of the method was found to be between 10(3) and 10(4) DNA copies/ml. The results of FT-RDB were 84% concordant with those of multiplex PCR, and 96% concordant with sequencing results in 101 cases. The genotype all 59 clinical samples was accurately identified. CONCLUSIONS: We demonstrated that the FT-RDB method was rapid, reliable, accurate and inexpensive. It appears to be useful for routine clinical HBV genotyping even in non-specialized hospital laboratories.

Relationship between apolipoprotein E, D10S1225 polymorphisms and late-onset Alzheimer's disease.

BACKGROUND: There were some papers published in the Jonrnal of Science, December 2000 suggesting that one or more important susceptibility genes for late onset Alzheimer's disease (LOAD) were located on the long arm of chromosome 10. Linkage analysis showed maximum lod score close to D10S1225 loci, which indicated the loci might contribute to the etiology of Alzheimer's disease (AD). METHODS: Fifty-nine LOAD patients and 107 controls were recruited. Apolipoprotein E (ApoE) genotypes were determined by reverse dot blotting hybridization assay. The D10S1225 was genotyped by 12% nondenaturing polyacrylamide gels electrophoresis and analyzed by silver staining. Statistical analysis was used to compare genotype and allele distributions between LOAD group and control group for ApoE and D10S1225 polymorphisms. RESULTS: ApoE epsilon 4 was significantly higher in LOAD group in comparison with the control group (chi(2) = 6.530, P = 0.011). Seven different alleles of D10S1225 have been identified. The length of these gene fragments were 178 bp, 181 bp, 184 bp, 187 bp, 190 bp, 193 bp, and 196 bp, respectively. A total of 21 different genotypes were observed. There was no relationship between D10S1225 polymorphism and LOAD (chi(2) = 4.488, P > 0.05). Conclusion This study suggests that ApoE epsilon 4 is a risk factor for LOAD, however, the results indicated that there is not any possible linkage for disequilibria with a nearby AD risk gene near D10S1225.

Rapid and accurate genotyping of YMDD motif variants in the hepatitis B virus genome by an improved reverse dot blot method.

By using the "flow-through hybridization" principle, we developed a new, rapid and accurate reverse dot blot (RDB) method to detect lamivudine resistance-associated YMDD motif variants in hepatitis B virus (HBV) genome. The improved RDB method was very fast at simultaneously detecting HBV YMDD wild-type and mutant motifs. In a blind analysis, 100 samples previously genotyped by DNA clonal sequencing analysis were used to evaluate the sensitivity and specificity of this assay. Conventional restriction fragment length polymorphism (RFLP) data were also used to test our method. In blind experiments, our improved RDB method had an accuracy and specificity of 100%, which was much higher than RFLP, which had an accuracy and specificity of only 83.0%. In clinical detection practice, 49 patients highly suspected of lamivudine resistance were successfully diagnosed by this method. Our improved RDB assay is a simple, rapid, cheap, semiautomatic, accurate, sensitive, and contamination-proof method of detecting lamivudine resistance-associated mutants in the human hepatitis B virus genome.

[Six novel mutations in PAH gene detected by sequencing].

OBJECTIVE: To explore new mutation in phenylalanine hydroxylase (PAH) gene. METHODS: The PAH genes from 40 phenylketonuria (PKU) patients and 30 normal controls were screened by PCR-single strand conformation polymorphism (SSCP) and further sequencing. RESULTS: Eleven mutations and 3 polymorphisms in PAH gene were found. No abnormalities in the PAH gene from 30 controls were detected. CONCLUSION: M276K, M276R, 280insT, IVS10nt+32T-->A, IVS4nt+47C-->T were demonstrated as novel mutations in comparison with the PAH mutation database. One mission mutation (H290R) was first documented in Chinese PKU gene.

[Advances in the studies of molecular heredity of phenylketonuria].

Phenylketonuria (PKU) is one kinds of autosomal recessive disease caused by phenylalanine hydroxylase (PAH) gene mutation. This article reviews the recent molecular heredity progress on the phenylalanine hydroxylase gene's orientation, structure, and gene mutation and gene regulation. At same time, mutation gene in vitro expression and the character of 3D structure of PAH in PKU are involved. In this paper, also discussed the influence of vitro expression and 3D protein structure by gene mutations and the molecular mechanism of the relationship between genotype and phenotype in PKU patient.

[Molecular characterization of a new mutation E122G of human ornithine transcarbamylase gene].

OBJECTIVE: To determine the molecular basis of late onset ornithine transcarbamylase (OTC) deficiency in a Chinese family of Han nationality and the exon sequences of OTC gene of this patient. METHODS: Polymerase chain reaction-single strand conformation polymorphism and direct sequencing were used to identify the mutation type. RESULTS: A missense mutation E122G in the conserved residue of exon 4 was identified which is unreported before. CONCLUSION: The E122G mutation in human OTC gene may cause late onset OTC deficiency.

[Study of immunological indicators in peripheral blood for nasopharyngeal carcinoma].

OBJECTIVE: To find reliable diagnostic immunological indicators in peripheral blood for nasopharyngeal carcinoma, and to evaluate the possibility of cancer screening and early diagnosis of the malignancies in the future. METHODS: Plasma samples were obtained from 83 patients with nasopharyngeal carcinoma and from 105 patients with benign diseases. Nine cytokines were selected as the candidates for the indicators and their quantity in the plasma samples determined by enzyme-linked immunosorbent assay, the result of which was analyzed statistically. RESULTS: Interleukin (IL)-4 level was obviously increased, while IL-6, IL-12, transforming growth factor (TGF)-beta, interferon (IFN)-gamma levels significantly reduced (P<0.05) in the plasma from patients with nasopharyngeal carcinoma. CONCLUSION: These cytokins may serve as the indicators in the diagnosis of nasopharyngeal carcinoma.

[Detection of telomerase hTERT gene expression of exfoliated cell in broncho-alveolar lavage fluid].

BACKGROUND & OBJECTIVES: The patients with lung carcinoma usually have high telomerase activity, the pseudopositive result is happen frequently when the test is done with traditional method. Telomerase activity is more strongly correlated with hTERT mRNA. This study was designed to detect the expression of telomerase hTERT gene in broncho-alveolar lavage fluid of the patients with lung carcinomas by relative quantitative RT-PCR method, to afford a useful tool for early and differentiate diagnosis of lung cancer. METHOD: The exfoliated cells in broncho-alveolar lavage fluid from 20 human suspicion lung carcinomas and 10 health persons were analyzed for hTERT expression by relative quantitative RT-PCR method, in the same time a part of exfoliated cells were detected with pathologic method. After operation, pathologic detection was performed for all samples. RESULTS: Of 20 human suspicion lung carcinomas and, 19 patients were lung cancer, 1 patient was inflammatory pseudotumor in pathological diagnosis after operation. The relative expression of telomerase hTERT gene in 16 cases of 19 patients with lung carcinoma were detected with the relative quantitative RT-PCR, the positive rate is 84.2% (16/19), the expressions of telomerase hTERT gene were at various quantities, average levels is 0.42. The positive rate in exfoliated cells detected with pathologic method were 57.9% (11/19). There was a statistical difference between them(P < 0.01). Ten normal persons were hTERT gene expression negative. CONCLUSIONS: Relative quantitative RT-PCR were more sensitive than cell morphologic method in detecting telomerase hTERT gene expression in broncho-alveolar lavage fluid of patients with lung carcinomas. Detecting quantitatively the levels of telomerase hTERT gene expression in broncho-alveolar lavage fluid will may be useful for early and differentiate diagnosis of lung carcinomas, especially for peripheral lung carcinomas.

Development and clinical trial of fluorescence PCR diagnostic kit to detect Mycobacterium tuberculosis.

OBJECTIVE: To develop a new diagnostic kit for Mycobacterium tuberculosis (TB) detection on the basis of fluorescence PCR (F-PCR) and conduct clinical trial of this kit for assessing its performance in comparison with other diagnostic methods. METHODS: We collected 546 clinical sputum samples from patients with phthisis and normal subjects to examine TB by way of F-PCR in parallel with examination with modified R.culture, auramine smear with fluorescence staining and LCx DNA diagnostic kit from Abbott for comparison. RESULTS: A clinical diagnostic kit for TB detection was developed with F-PCR, and clinical trial showed that its positivity rate was 49.1%, sensitivity 89.2%, specificity 98.8% and efficiency 93.6%. CONCLUSION: F-PCR is obviously superior to culture method and smear method and comparable with LCx in terms of sensitivity, and can be used to monitor TB DNA in the secretions to facilitate clinical diagnose and therapeutic effects monitoring.