Microarrays for high-throughput genotyping of MICA alleles using allele-specific primer extension.

The role of major histocompatibility complex (MHC) class I chain-related gene A (MICA), a ligand of NKG2D, has been defined in human diseases by its allele associations with various autoimmune diseases, hematopoietic stem cell transplantation (HSCT) and cancer. This study describes a practical system to develop MICA genotyping by allele-specific primer extension (ASPE) on microarrays. From the results of 20 control primers, strict and reliable cut-off values of more than 30,000 mean fluorescence intensity (MFI) as positive and less than 3000 MFI as negative, were applied to select high-quality specific extension primers. Among 55 allele-specific primers, 44 primers could be initially selected as optimal primer. Through adjusting the length, six primers were improved. The other failed five primers were corrected by refractory modification. MICA genotypes by ASPE on microarrays showed the same results as those by nucleotide sequencing. On the basis of these results, ASPE on microarrays may provide high-throughput genotyping for MICA alleles for population studies, disease-gene associations and HSCT.

Allele Specific Polymerase Chain Reaction (AS-PCR) Assay to Detect Knockdown and Acetylcholinesterase Mutations in Anopheles superpictus

Objective: The study aims to determine the presence of L1014F, L1014S, L1014C alleles, which are responsible for knockdown resistance and Ace-1 G119S alleles, which are responsible for acetylcholinesterase insensitivity in Anopheles superpictus, the secondary vector of malaria in Turkey. Methods: In this study, 60 Anopheles superpictus adult females were collected from Aydin, Denizli, and Mugla provinces. Then, allele-specific primers for kdr L1014F, L1014S, and L1014C alleles, and the Ace-1 G119S allele were designed. The presence of these alleles was screened in three Anopheles superpictus populations by allele-specific polymerase chain reaction. Results: Although L1014S allele frequency was too low in Aydin, Mugla, and Denizli populations, neither kdr L1014F and L1014C nor Ace-1 G119S mutations were found in any population. Conclusion: In this study, kdr L1014S mutation was detected for the first time in the Aegean Anopheles superpictus populations.

Rapid Multiplex Genotyping of 20 HLA-A*02:01 Restricted Minor Histocompatibility Antigens.

A subset of MHC-associated self-peptides presented by the recipient's cells and immunologically foreign to the donor can induce an allogeneic immune response after hematopoietic stem cell transplantation (HSCT). These immunogenic peptides originate from the genomic polymorphisms and are known as minor histocompatibility antigens (MiHA). MiHA mismatches trigger the post-transplant immune response, which could manifest in both the deleterious "graft-vs.-host" disease and the beneficial "graft-vs.-leukemia" effect. Importantly, some MiHAs are considered to be promising targets for posttransplant T-cell immunotherapy of hematopoietic malignancies. This creates a demand for a robust and fast approach to genotyping MiHA-encoding polymorphisms. We report a multiplex real-time PCR method for the genotyping of 20 polymorphisms that are encoding HLA-A*02:01-restricted MiHAs. This method uses allele-specific primers and gene-specific hydrolysis probes. In 1 h it allows for the detection of MiHA mismatches in a donor-recipient pair without the need for electrophoresis, sequencing, or other time-consuming techniques. We validated the method with Sanger and NGS sequencing and demonstrated good performance over a wide range of DNA concentrations. We propose our protocol as a fast and accurate method of identifying mismatched MiHAs. The information on the MiHA mismatches is useful for studying the allogeneic immune response following HSCT and for selecting the targets for post-transplant T-cell therapy.

Detection of resistance to fluoroquinolones and injectable drugs among antituberculosis drugs by allele-specific primer extension on a microsphere-based platform.

Molecular drug susceptibility testing (DST) for antituberculosis drugs is important for improving the efficacy of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) treatment. In this study, we developed a molecular high-throughput assay system based on allele-specific primer extension (ASPE) and MagPlex-TAG microspheres, referred to here as TAG-ASPE, which can detect mutations related to resistance to injectable second-line drugs and fluoroquinolones. Target genes were amplified by multiplex PCR using DNA from H37Rv and 190 clinical Mycobacterium tuberculosis strains and extended by ASPE using 22 ASPE primers. ASPE products were then sorted on the TAG-ASPE array and detected using a Luminex 200 system. The performance of the TAG-ASPE method was compared with that of sequencing and phenotypic DST. Comparison of the TAG-ASPE method with sequencing showed that the sensitivity and specificity of the TAG-ASPE method were 100% [95% confidence interval (CI), 96.38-100%] and 100% (95% CI, 95.70-100%) for the rrs gene and 100% (95% CI, 96.90-100%) and 100% (95% CI, 95.07-100%) for the gyrA gene, respectively. Compared with phenotypic DST, the sensitivity and specificity of the TAG-ASPE method for detecting drug-resistance mutations against injectable second-line drugs were 92.52% (95% CI, 85.8-96.72%) and 98.7% (95% CI, 92.98-99.97%), respectively. Additionally, the sensitivity and specificity for fluoroquinolone-resistance detection were 85.4% (95% CI, 78.36-90.85%) and 100% (95% CI, 92.38-100%), respectively. The results of this study demonstrate that the TAG-ASPE method can effectively detect mutations conferring resistance to second-line antituberculosis drugs in numerous clinical specimens.

ErbB4 receptor polymorphism 2368A>C and risk of breast cancer.

PURPOSE: A number of single nucleotide polymorphisms (SNPs) in EebB4 gene have been studied, which has clarified their impact on breast cancer in different populations. Nevertheless, the importance of rs13423759 in breast cancer has not been studied and its effect remained almost unclear. In this paper, we evaluated the frequency of rs13423759 different alleles in Iranian population and statistically analyzed their association with breast cancer risk. MATERIALS AND METHODS: Allele-specific Primer PCR (ASP-PCR) was recruited in this study to genotype rs13423759 position in 172 breast cancer and 148 healthy control subjects. The genotypes of control and cases were analyzed statistically to find the association between rs13423759 alleles and breast cancer incidence and its clinicopathological characteristics. In silico studies were performed in order to find the mechanistic viewpoint of rs13423759 alleles in breast cancer. RESULTS: rs13423759 allele C was shown to be significantly associated with breast cancer risk, HER2 positivity and increased risk of metastasis. Reciprocally, allele A was correlated with the lowered risk of breast cancer. The in silico studies showed that rs13423759 allele C is capable to strengthen the interaction between miR-548as, an oncomiRNA, and ErbB4 mRNA, leading to its lowered concentration in the cells. CONCLUSION: rs13423759 allele C is significantly associated with the enhanced risk of breast cancer, elevated metastasis and HER2 positivity.

Establishment of a nested-ASP-PCR method to determine the clarithromycin resistance of Helicobacter pylori.

AIM: To investigate clarithromycin resistance positions 2142, 2143 and 2144 of the 23SrRNA gene in Helicobacter pylori (H. pylori) by nested-allele specific primer-polymerase chain reaction (nested-ASP-PCR). METHODS: The gastric tissue and saliva samples from 99 patients with positive results of the rapid urease test (RUT) were collected. The nested-ASP-PCR method was carried out with the external primers and inner allele-specific primers corresponding to the reference strain and clinical strains. Thirty gastric tissue and saliva samples were tested to determine the sensitivity of nested-ASP-PCR and ASP-PCR methods. Then, clarithromycin resistance was detected for 99 clinical samples by using different methods, including nested-ASP-PCR, bacterial culture and disk diffusion. RESULTS: The nested-ASP-PCR method was successfully established to test the resistance mutation points 2142, 2143 and 2144 of the 23SrRNA gene of H. pylori. Among 30 samples of gastric tissue and saliva, the H. pylori detection rate of nested-ASP-PCR was 90% and 83.33%, while the detection rate of ASP-PCR was just 63% and 56.67%. Especially in the saliva samples, nested-ASP-PCR showed much higher sensitivity in H. pylori detection and resistance mutation rates than ASP-PCR. In the 99 RUT-positive gastric tissue and saliva samples, the H. pylori-positive detection rate by nested-ASP-PCR was 87 (87.88%) and 67 (67.68%), in which there were 30 wild-type and 57 mutated strains in gastric tissue and 22 wild-type and 45 mutated strains in saliva. Genotype analysis showed that three-points mixed mutations were quite common, but different resistant strains were present in gastric mucosa and saliva. Compared to the high sensitivity shown by nested-ASP-PCR, the positive detection of bacterial culture with gastric tissue samples was 50 cases, in which only 26 drug-resistant strains were found through analyzing minimum inhibitory zone of clarithromycin. CONCLUSION: The nested-ASP-PCR assay showed higher detection sensitivity than ASP-PCR and drug sensitivity testing, which could be performed to evaluate clarithromycin resistance of H. pylori.

Detection of first-line anti-tuberculosis drug resistance mutations by allele-specific primer extension on a microsphere-based platform.

BACKGROUND: Resistance of Mycobacterium tuberculosis to anti-tuberculosis (TB) drugs is almost exclusively due to spontaneous chromosomal mutations in target genes. Rapid detection of drug resistance to both first- and second-line anti-TB drugs has become a key component of TB control programs. Technologies that allow rapid, cost-effective, and high-throughput detection of specific nucleic acid sequences are needed. This study was to develop a high-throughput assay based on allele-specific primer extension (ASPE) and MagPlex-TAG microspheres to detect anti-TB drug resistance mutations. METHODS: DNA samples from 357 M. tuberculosis clinical isolates and H37Rv were amplified by multiplex PCR using four primer sets, followed by multiplex ASPE using 23 TAG-ASPE primers. The products were sorted on the TAG-ASPE array and detected by using the Luminex xMAP system. Genotypes were also determined by sequencing. RESULTS: Genetic drug susceptibility typing by the TAG-ASPE method was 100% concordant with those obtained by sequencing. Compared with phenotypic drug susceptibility testing (DST) as a reference method, the sensitivity and specificity of the TAG-ASPE method were 83% (95% confidence interval [CI], 79-88%) and 97% (95% CI, 90-100%) for isoniazid. For rifampin testing, the sensitivity and specificity were 90% (95% CI, 86-93%) and 100% (95% CI, 99-100%). Also, the sensitivity and specificity were 58% (95% CI, 51-65%) and 86% (95% CI, 79-93%) for ethambutol. CONCLUSIONS: This study demonstrated the TAG-ASPE method is suitable for highly reproducible, cost-effective, and high-throughput clinical genotyping applications.

Rapid detection of CYP2C9, CYP2C19,CYP4F2,VKORC1 and ABCB1 gene polymorphisms by liquid phase chip technology / 中华检验医学杂志

Objective@#To establish a method for simultaneous and rapid detecting of the polymorphisms in Cytochrome P450 2C9 (CYP2C9), CYP2C19, CYP4F2, Vitamin K epoxide reductase (VKORC1) and ATP-binding cassette subfamily B member1 (ABCB1) gene, which were associated with warfarin and clopidogrel, based on liquid phase chip technology.@*Methods@#Method establishment. The eight gene sequences near targeted sites related to warfarin and clopidogrel were found in Genbank, and the specific primers and probes were designed. Through multiple PCR amplification, followed by allele specific primer extension (ASPE), and MagPlex-Tag microspheres hybridization, the suspension array Luminex 200 system step-by-step, the genotypes were determined by fluorescence signal. The reaction system was optimized and its methodological evaluation was performed. 260 patients with antithrombotic therapy from Dongguan houjie hospital were recruited in this study form June 2017 to December 2018. The eight genotypes of the 260 patients were detected by the established method, and the results were compared with the sequencing results.@*Results@#The results of 260 samples showed that allelic median fluorescence intensity (MFI) ratios of homozygotes (mutant/wild-type) were all greater than 0.9 or less than 0.1, and all the allelic MFI ratios of heterozygotes were between 0.3 and 0.6. The within run and between run coefficients of variance for allelic MFI ratios were lower than 6.4% and 10.9%, respectively. The minimum DNA template requirements was 0.75ng. The genotypes of 260 patients determined by the established method were completely concordant with the sequencing results.@*Conclusion@#A method was established successfully for rapid detecting the genotypes which associated with warfarin and clopidogrel based on liquid phase chip technology.

Rapid detection of CYP2C9, CYP2C19, CYP4F2, VKORC1 and ABCB1 gene polymorphisms by liquid phase chip technology / 中华检验医学杂志

Objective To establish a method for simultaneous and rapid detecting of the polymorphisms in Cytochrome P4502C9 (CYP2C9), CYP2C19, CYP4F2, Vitamin K epoxide reductase (VKORC1) and ATP-binding cassette subfamily B member1 (ABCB1) gene, which were associated with warfarin and clopidogrel, based on liquid phase chip technology. Methods Method establishment. The eight gene sequences near targeted sites related to warfarin and clopidogrel were found in Genbank, and the specific primers and probes were designed. Through multiple PCR amplification, followed by allele specific primer extension (ASPE), and MagPlex-Tag microspheres hybridization, the suspension array Luminex 200 system step-by-step, the genotypes were determined by fluorescence signal. The reaction system was optimized and its methodological evaluation was performed. 260 patients with antithrombotic therapy from Dongguan houjie hospital were recruited in this study form June 2017 to December 2018. The eight genotypes of the 260 patients were detected by the established method, and the results were compared with the sequencing results. Results The results of 260 samples showed that allelic median fluorescence intensity (MFI) ratios of homozygotes (mutant/wild-type) were all greater than 0.9 or less than 0.1, and all the allelic MFI ratios of heterozygotes were between 0.3 and 0.6. The within run and between run coefficients of variance for allelic MFI ratios were lower than 6.4％and 10.9％, respectively. The minimum DNA template requirements was 0.75ng. The genotypes of 260 patients determined by the established method were completely concordant with the sequencing results. Conclusion A method was established successfully for rapid detecting the genotypes which associated with warfarin and clopidogrel based on liquid phase chip technology.

C-reactive protein + 1059 G>C polymorphism in type 2 diabetes and coronary artery disease patients.

Human C-reactive protein (CRP) is an acute phase reactant involved in chronic and acute inflammation. CRP is associated with metabolic syndrome, obesity, atherosclerosis, unstable angina, insulin resistance and diabetes. The present study evaluates the association of + 1059 G>C silent polymorphism in exon 2 of CRP gene in 581 cases [CAD (206), T2D (266), T2D with CAD (109)] and 235 controls in the population of Punjab (North-West India). The frequency of + 1059 G allele is highest in CAD (98.3%) followed by T2D (98.1%), T2D + CAD cases (97.7%) and controls (94.7%). G-allele is associated with increased risk of T2D [P = 0.003, OR = 2.93 (1.39-6.17)] and CAD [P = 0.004, OR = 3.25 (1.39-7.60)] in comparison to controls. Recessive model shows that GG genotype increases the risk of CAD by 4 fold (P = 0.003, OR = 4.19, 1.62-10.80), T2D by 3 fold (P = 0.008, OR = 3.23, 1.36-7.60) and T2D + CAD by 3.5 fold (P = 0.029, OR = 3.64, 1.14-11.66). Factor analyses show that BMI, WC, and WHR are core predictors for CAD and T2D, whereas CHO, TG and VLDL for T2D + CAD. The present study concludes that GG genotype of CRP + 1059 G>C polymorphism and clustering of obesity and dyslipidemia underlie the risk towards CAD, T2D and T2D + CAD in the North-West Indian population of Punjab.

Detection of First-Line Anti-Tuberculosis Drug Resistance Mutations by Allele-Specific Primer Extension on a Microsphere-Based Platform

BACKGROUND: Resistance of Mycobacterium tuberculosis to anti-tuberculosis (TB) drugs is almost exclusively due to spontaneous chromosomal mutations in target genes. Rapid detection of drug resistance to both first- and second-line anti-TB drugs has become a key component of TB control programs. Technologies that allow rapid, cost-effective, and high-throughput detection of specific nucleic acid sequences are needed. This study was to develop a high-throughput assay based on allele-specific primer extension (ASPE) and MagPlex-TAG microspheres to detect anti-TB drug resistance mutations. METHODS: DNA samples from 357 M. tuberculosis clinical isolates and H37Rv were amplified by multiplex PCR using four primer sets, followed by multiplex ASPE using 23 TAG-ASPE primers. The products were sorted on the TAG-ASPE array and detected by using the Luminex xMAP system. Genotypes were also determined by sequencing. RESULTS: Genetic drug susceptibility typing by the TAG-ASPE method was 100% concordant with those obtained by sequencing. Compared with phenotypic drug susceptibility testing (DST) as a reference method, the sensitivity and specificity of the TAG-ASPE method were 83% (95% confidence interval [CI], 79-88%) and 97% (95% CI, 90-100%) for isoniazid. For rifampin testing, the sensitivity and specificity were 90% (95% CI, 86-93%) and 100% (95% CI, 99-100%). Also, the sensitivity and specificity were 58% (95% CI, 51-65%) and 86% (95% CI, 79-93%) for ethambutol. CONCLUSIONS: This study demonstrated the TAG-ASPE method is suitable for highly reproducible, cost-effective, and high-throughput clinical genotyping applications.

Comparing methods of detection and quantitation of RNA editing of rat glycine receptor alpha3.

BACKGROUND: Recently, it has become evident that RNA editing-related changes are important in the modulation of neuronal information processing. Alternatively edited transcripts, when meagerly present, are hard to detect. Significant functional consequences may result, however, from small differences in editing efficiency. Moreover, it is difficult to evaluate the ratio of edited transcripts. The glycine receptor alpha3 subunit (GlyR alpha3) is expressed in the spinal cord, and transcripts of GlyR alpha3 are susceptible to RNA editing. The physiological role of this editing is still unclear. To analyze changes in RNA editing in various animal models, we need reliable and practical ways to detect and quantitate GlyR alpha3 RNA editing. RESULTS: We identified and assessed different ways of detecting edited RNA transcripts, including direct sequencing, denaturing high performance chromatography (DHPLC), allele-specific real-time PCR with TaqMan probes, and PCR with allele-specific primers. Using PCR with allele-specific primers on standard PCR products for edited and nonedited GlyR alpha3, we were able to detect as little as a 0.5% incidence of edited transcripts. We were able to detect a 5% incidence of RNA editing using direct sequencing and 2% using DHPLC. We could accurately determine the ratio of edited to non-edited RNA using DHPLC, direct sequencing, and allele-specific real-time PCR with TaqMan probes. CONCLUSION: We demonstrated exact and sensitive methods of detecting RNA editing. In prepared samples, we showed means of quantitating the incidence of editing of a particular site. The demonstrated methodologies should be very useful when extended to the evaluation of other types of RNA editing and single base mutations.

Prenatal diagnostic testing for infantile and late-infantile neuronal ceroid lipofusinoses (NCL) using allele specific primer extension (ASPE) / 北京大学学报(医学版)

SUMMARY Infantile (INCL, NCL1) and late-infantile (LINCL, NCL2) neuronal ceroid lipofuscinoses have been found to result from genetic deficiency of genes CLN1 and CLN2, respectively. The application of molecular analyses can facilitate prenatal diagnosis for families affected by NCL1 or NCL2, in which the familial mutation(s) have been identified. Molecular testing with allele-specific primer extension and DNA sequencing was performed in nine pregnancies, four from two NCL1 families and five from five NCL2 families. Lysosomal enzyme activity assays were carried out as well.Four fetuses from three pregnancies in NCL1 families were found to be carriers for a mutation 451C-T in the CLN1 gene and one was normal. Prenatal testing of three NCL2 families who carried mutation R208X in the CLN2 gene showed that all fetuses were carriers. In NCL2 families who carried either mutation IVS5-1C or/and IVS5-1A two normal pregnancies were detected. Our studies indicate that DNA testing, which may provide definitive prenatal diagnosis for NCL, may be used in combination with lysosomal enzyme activity analyses.

Genetype combination distribution and allele frequency distribution of single nucleotide polymorphisms of NOS2 C-1173T and G-954C in the Han Chinese population / 重庆医科大学学报

Objective:To study genetype combination distribution and allele frequency distribution of NOS2 C-1173T and G-954C single nucleotide polymorphisms (SNP) of inducible nitric oxide synthase(NOS2) in the Chinese Han nationality population.Methods:Genomic DNA of leukocytes in venous blood was obtained from 565 Han Chinese people(female 251 and male 314).The NOS2 G-954C and NOS2 C-1173T SNPs were genotyped by nested allele-specific primer (NASP)PCR.Results:The genotype frequencies of CC,CT and TT were 66.73%,26.37% and 6.90% respectively in the NOS2 C-1173T SNP.C and T allele haplotype frequencies of NOS2 C-1173T SNP was 79.88% and 20.12% respectively.The genotype frequencies of NOS2 GG,GC and CC were 61.77%,37.88% and 0.35% respectively in the NOS2 G-954C SNP.G and C allele haplotype frequencies was 80.71% and 19.29% respectively in the NOS2 G-954C SNP.The preceding 5 combinations in the natural combination distribution of two sites of NOS2 SNP were found as follows:(1)The genotype combination of 233 subjects was NOS2 C-1173C+G-954G,its probability being 41.24%.(2)The genotype combination of 143 subjects was NOS2 C-1173C+G-954C,its probability being 25.31%.(3)The genotype combination of 89 subjects was NOS2 C-1173T+G-954G,the probability being 15.75%.(4)The genotype combination of 59 subjects was NOS2 C-1173T+G-954C,the probability being 10.44%.(5)The genotype combination of 27 subjects was NOS2 T-1173T+G-954G,the probability being 4.78%.Conclusion:This study shows the features of combination distribution and frequency distribuion of NOS2 SNPs and provides the basic laboratory data for the further study relationships among NOS2 SNPs,its physiological function and diseases.

Authentication of TCM Carapax Trionycis by allele-specific diagnostic polymeras chain reaction / 中草药

Object\ To develop a convenient and practical method for the identification of Carapax Trionycis Methods\ Based on the sequence variations of 12S rRNA gene between Pelodiscus sinensis and other softshell turtles, a pair of allele specific primers was designed to distinguish P. sinensis from other species of Trionychidae. DNA were extracted and anplified and Carapax Trionycis could be identified accurately by polymerase chain reaction (PCR) using the primers Results\ Ten samples of turtle shell from different sources were indentified by the allele specific PCR with the primers The result indicated that three samples were substitutes of Carapax Trionycis, consilient with the result from DNA sequence analysis The mitochondrial 12S rRNA gene fragment of P. maculatus and a faked imitation had also been sequenced Conclusion\ The primers could be used as key components in Carapax Trionycis identification kit