A rapid screening with direct sequencing from blood samples for the diagnosis of Leigh syndrome

ABSTRAK Gen FTO berfungsi sebagai regulasi homeostasis, deposisi lemak dan pengaturan obesitas. Penelitian ini bertujuan untuk mengidentifikasi polimorfisme SNP g.125550A>T di ekson 3 gen FTO pada bangsa sapi potong Indonesia. Sampel darah diperoleh dari 209 ekor sapi, terdiri atas sapi bali (44), madura (20), Pesisir (20), katingan (20), Peranakan ongole (PO) (22), Pasundan (20), Sumba Ongole (SO) (11), brahman (20), simental (15), dan limousin (18). Polimorfisme gen FTO dianalisis menggunakan metode PCR-RFLP (HpyCH4III) dan direct sequencing. Hasil genotiping SNP g.125550A>T adalah polimorfik (genotipe AA, AT, dan TT) pada sapi madura, pesisir, katingan, PO, pasundan, SO, brahman, simental, dan limousin. Frekuensi alel A dan T masing-masing adalah 0,70, 0,68, 0,84, 0,89, 0,70, 0,86, 0,90, 0,73, 0,69 dan 0,30, 0,33, 0,16, 0,11, 0,30, 0,14, 0,10, 0,27, 0,31. Nilai Ho dan He masing-masing adalah 0,60-0,14 dan 0,44-0,18 serta dalam keseimbangan Hardy-Weinberg (P>0.05). Sementara pada sapi bali bersifat monomorfik hanya bergenotipe AA. Hasil sekuensing SNP g.125550A>T ditemukan mutasi tranvesi A menjadi T pada posisi nukleotida g.125550. Berdasarkan hasil penelitian ini, dapat disimpulkan bahwa SNP 125550A>T gen FTO beragam dan berpotensi dijadikan marka genetik untuk kualitas daging pada bangsa sapi potong Indonesia.Kata Kunci: gen FTO, PCR-RFLP, Sapi, SNP g.125550A>TABSTRACTThe FTO gene functions as regulation of homeostasis, fat deposition and regulation of obesity. This study aimed to identify the polymorphism of SNP g.125550A>T in exon 3 of FTO gene in Indonesian beef cattle. Blood samples were collected from 209 cattle, including bali (44), madura (20), pesisir (20), katingan (20), PO (22), pasundan (20), SO (11), brahman (20), simental (15), and limousin (18). Polymorphism of the FTO gene was analyzed using PCR-RFLP (HpyCH4III) and direct sequencing methods. The results of genotyping SNP g.125550A>T was polymorphic (AA, AT and TT genotypes) in madura, pesisir, katingan, PO, pasundan, SO, brahman, simental, and limousin cattle. The frequency of A and T alleles were 0,70, 0,68, 0,84, 0,89, 0,70, 0,86, 0,90, 0,73, 0,69 and 0,30, 0,33, 0,16, 0,11, 0,30, 0,14, 0,10, 0,27, 0,31 respectively. The values of Ho and He were 0,60-0,14 and 0,44-0,18 respectively and in Hardy-Weinberg equilibrium (P>0,05). While in Bali cattle was monomorphic (AA genotype). Results of sequencing SNP g.125550A>T of the FTO gene found a transverse mutation A to T at the nucleotide position g.125550. As a result of this study, it can be concluded that SNP 125550A>T of the FTO gene was diverse and potentially used as genetic markers for meat quality in Indonesian beef cattle.Keywords: cattle, FTO gene, PCR-RFLP, SNP g.125550A>T.

Download Full-text

High-Throughput Genotyping of Thiopurine S-Methyltransferase by Denaturing HPLC

Clinical Chemistry ◽

10.1093/clinchem/47.3.548 ◽

2001 ◽

Vol 47 (3) ◽

pp. 548-555 ◽

Cited By ~ 59

Author(s):

Elke Schaeffeler ◽

Thomas Lang ◽

Ulrich M Zanger ◽

Michel Eichelbaum ◽

Matthias Schwab

Keyword(s):

High Throughput ◽

Direct Sequencing ◽

False Negative ◽

Rapid Screening ◽

Tpmt Activity ◽

Polymorphism Analysis ◽

Specific Pcr ◽

Restriction Fragment Polymorphism ◽

Allele Specific ◽

Tpmt Gene

Abstract Background: The thiopurine S-methyltransferase (TPMT) genetic polymorphism has a significant clinical impact on the toxicity of thiopurine drugs, which are used in the treatment of leukemia and as immunosuppressants. To date, 10 mutant alleles are known that are associated with intermediate or low TPMT activity. To facilitate rapid screening of clinically relevant TPMT mutations, we developed a strategy of high-throughput genotyping by applying denaturing HPLC (DHPLC). Methods: To test the specificity and efficiency of the DHPLC method, 98 DNA samples from a selected population of patients receiving thiopurine therapy or with previous thiopurine withdrawal were analyzed for the most frequent mutant TPMT alleles, *2 and *3A, which contain key mutations in exons 5, 7, and 10 to identify clearly different elution profiles. All fragments were examined by direct sequencing. Additionally, to test the sensitivity of DHPLC analysis, genotyping for the *2 and *3A alleles of all 98 DNA samples was performed by PCR-based methods (PCR-restriction fragment polymorphism analysis and allele-specific PCR). Results: The presence of mutations discriminating for alleles *2, *3A, *3C, and *3D, as well as various silent and intron mutations, were correctly predicted by DHPLC in 100% of the samples as confirmed by direct sequencing. Comparison with PCR-based methods for alleles *2 and *3 produced an agreement of 100% with no false-negative signals. Conclusions: DHPLC offers a highly sensitive, rapid, and efficient method for genotyping of the relevant TPMT mutations, discriminating at least for alleles *2 and *3, in clinical and laboratory practice. Additionally, DHPLC allows a simultaneous screening for novel genetic variability in the TPMT gene.

Download Full-text

Incidence of the JAK2V617F Mutation In Mexican Patients with Myeloproliferative Neoplasms (MPNs)

Blood ◽

10.1182/blood.v118.21.5180.5180 ◽

2011 ◽

Vol 118 (21) ◽

pp. 5180-5180

Author(s):

Gregorio Ignacio ◽

Rosa María Arana-Trejo ◽

Verónica Gónzalez ◽

Maria Paula Hérnandez ◽

Yolanda Lugo ◽

...

Keyword(s):

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Direct Sequencing ◽

Jak2v617f Mutation ◽

Normal Blood ◽

Base Substitution ◽

Blood Samples ◽

Specific Pcr ◽

Dna And Rna ◽

Mexican Patients

Abstract Abstract 5180 Introduction: The V617F mutation in JAK2 gene has been described in approximately 50–90% of patients with ET, MF AND PV [essential trombocythaemia, idiopathic myelofibrosis and policithemia vera]; but has also been reported, albeit at a lower frequency, in patients with other myeloid malignancies such as atypical CML, CMML, AML, MDS, JMML and CNL. A single G>T base substitution in exon 12 results in the conversion of valine to a phenylalanine aminoacid at position 617 of the JAK2 gene. The identification in this study were using techniques such as allele-specific PCR, RFLP-PCR and direct sequencing; for to determine the incidence in Mexican patients with MPNs. Patients and Methods: The JAK2V617F mutation was determined in 88 patients and 5 normal blood samples for healthy individuals as controls. About the patients, 60 were cataloged like MPNs and 28 patients with features suggestive of MPNs vs CML. Samples for bone marrow or peripherical blood were taken either at time of diagnosis of MPNs or during treatment with cytoreductive or anti-thrombotic agents. DNA and RNA were extracted using the QIAamp DNA and RNeasy mini kit (Qiagen) and amplified by the three techniques mentioned for JAK2V617F and by nested RT-PCR for BCR/ABL. Results: The five normal blood samples for controls were negative for JAK2V617F mutation and to BCR/ABL. Patients had median age 65 years (47–85 years old), 46% male and 54% female. In de overall patients: 60 patients with MPNs all were BCR/ABL negative and 20 (33%) had JAK2V617F. In the 28 patients with likely MPNs vs CML, 23 were BCR/ABL positive/JAK2 negative, two had the coexistence of both genetics defects [BCR/ABL+ and JAK2V617F+] and 3 BCR/ABL and JAK2 negative. Finally the patiens with JAK2V617F+, were 12 ET, PV 1, MF 2, CML 2, and 5 continued like MPNs. Discussion: The incidence of the JAK2V617F in this study for MPNs patiens were 33% and the incidence varied between MPNs subtype. Less than ten cases of BCR/ABL+ CML with JAK2V617F have been published; we report two patients with the coexistence and we agree with previous reports that screening for JAK2V617F mutation should be considered in any BCR/ABL+ CML patients and the clinical outcome will be define in long period. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Analysis of pharmacogenetic biomarkers in rectal patients trated with chemoradiotherapy

Journal of Clinical Oncology ◽

10.1200/jco.2009.27.15_suppl.e15051 ◽

2009 ◽

Vol 27 (15_suppl) ◽

pp. e15051-e15051

Author(s):

M. J. Lamas ◽

E. Balboa ◽

G. Duran ◽

P. Rana ◽

A. Gomez ◽

...

Keyword(s):

Rectal Cancer ◽

Direct Sequencing ◽

Dihydropyrimidine Dehydrogenase ◽

Stage Ii ◽

High Expression ◽

Tumour Regression ◽

Study Cohort ◽

Tumour Regression Grade ◽

Blood Samples ◽

Intron 1

e15051 Background: 5FU-based chemoradiotherapy before total mesorectal excision (TME) is currently the gold standard treatment for stage II and III rectal cancer patients. Pathological complete response (pCR) is related with a longer survival. We have used known predictive pharmacogenetics biomarkers to identify in our series responders and non responders to preoperative RQ. Methods: 77 stage II/III rectal patients were genotyped using direct sequencing (TS VNTR) and SNAPshot (DPYD, EGFR) techniques. DNA was obtained from peripheral blood samples. We have studied Thymidylate synthetase (TS VNTR; high expression haplotypes: TSER 2R/3R, 3C/3G, 3G/3G and low expression: TSER 2R/2R, 2R/3C, 3C/3C; TS 1494del6: associated to a better efficay of 5Fu), dihydropyrimidine dehydrogenase (DPYD; DPYD*2 associated to worse toxicity), EGFR (CA repeats in intron 1: 16/16 associated to worse efficacy) polymorphisms. Median age of our study cohort was 65 years old (37–85). There were 24 female and 53 male patients. All of them were Caucasian. 21 patients (27.3%) had stage II and 56 (72.7%) stage III. They were staged by TC, colonoscopy and endorectal ultrasonography. The patients received 5fu 325 mg/m2/day continuous infusion along the hyperfractionated accelerated radiotherapy schedule (50,4 Gy). All were submitted to TME. Outcomes after surgery are measured by tumour regression grade (from TRG1= complete pathological response, to TRG5=no regression). Data were studied by univariate and multivariate analysis. Results: The sample was in Hardy-Weinberg equilibrium for all polymorphisms, irrespectively of the response status. 50 patients (64.9%) and 27 (35.1%) had low and high expression genotype for TS respectively. pCR (TRG1) was obtained in 24 patients (31,6%) and microscopic foci (TRG2) in 14 (18,2%), TRG 3–4 in 38 (49,3%), and 1 patient had no response (TRG5). We haven’t found a statistically significant relationship between TRG1 and TS status, or any other biomarker studied. There's no relationship also with initial clinical stage. Conclusions: Biomarkers EGFR (intron 1 CA repeats), TS (TS 1494del6, TS VNTR) and DPYD in blood samples, are not good enough to predict response to RQ in rectal cancer. No significant financial relationships to disclose.

Download Full-text

Rapid Diagnosis of Bacteremia in Adults Using Acridine Orange Stained Buffy Coat Smears

Canadian Journal of Infectious Diseases ◽

10.1155/1990/159215 ◽

1990 ◽

Vol 1 (1) ◽

pp. 7-10

Author(s):

Mark Miller ◽

Jack Mendelson

Keyword(s):

Blood Culture ◽

Acridine Orange ◽

Buffy Coat ◽

Blood Cultures ◽

Rapid Screening ◽

Blood Culture System ◽

Blood Samples ◽

Significant Difference ◽

Rapid Screening Test ◽

Low Sensitivity

The use of acridine orange stained buffy coat smears was assessed as a rapid screening test for bacteremia in adults. A total of 356 consecutive blood cultures were submitted with simultaneous anticoagulated blood samples, from which a buffy coat smear was prepared and stained with acridine orange (100 mg/L; pH 3.0). Forty-one of 356 blood samples (12%) yielded organisms in the blood culture system. Compared to blood culture, the overall sensitivity of acridine orange stained buffy coat smears was 16%, specificity 88%, and positive predictive value 13%. There was no statistically significant difference in performance of the test among patients who had fever greater than 39°C and/or shock. The low sensitivity and specificity of the test makes it unsuitable as a means of rapid screening for adults with suspected bacteremia.

Download Full-text

Quantitative Evaluation of the Mitochondrial DNA Depletion Syndrome

Clinical Chemistry ◽

10.1373/clinchem.2009.141549 ◽

2010 ◽

Vol 56 (7) ◽

pp. 1119-1127 ◽

Cited By ~ 89

Author(s):

David Dimmock ◽

Lin-Ya Tang ◽

Eric S Schmitt ◽

Lee-Jun C Wong

Keyword(s):

Mitochondrial Dna ◽

Real Time ◽

Muscle Tissue ◽

Skin Fibroblasts ◽

Rapid Screening ◽

Simple Method ◽

Blood Samples ◽

Mtdna Depletion ◽

Coa Ligase ◽

Mitochondrial Dna Depletion

Abstract Background: The mitochondrial DNA (mtDNA) depletion syndromes (MDDSs) are autosomal recessive disorders characterized by a reduction in cellular mtDNA content. Mutations in at least 9 genes [POLG, polymerase (DNA directed), gamma; DGUOK, deoxyguanosine kinase; TK2, thymidine kinase, mitochondrial; TYMP, thymidine phosphorylase; MPV17, MpV17 mitochondrial inner membrane protein; SUCLA2, succinate-CoA ligase, ADP-forming, beta subunit; SUCLG1, succinate-CoA ligase, alpha subunit; RRM2B, RRM2B, ribonucleotide reductase M2 B (TP53 inducible); and C10orf2, chromosome 10 open reading frame 2 (also known as TWINKLE)] have been reported to cause mtDNA depletion. In the clinical setting, a simple method to quantify mtDNA depletion would be useful before undertaking gene sequence analysis. Methods: Real-time quantitative PCR (qPCR) was used to measure the mtDNA content in blood, muscle, and liver samples and in skin fibroblast cultures from individuals suspected of mitochondrial disorders, with or without deleterious mutations in genes responsible for MDDS. Results: The mtDNA content was quantified in 776 tissue samples (blood, n = 341; muscle, n = 325; liver, n = 63; skin fibroblasts, n = 47) from control individuals. mtDNA content increased with age in muscle tissue, decreased with age in blood samples, and appeared to be unaffected by age in liver samples. In 165 samples (blood, n = 122; muscle, n = 21; liver, n = 15; skin fibroblasts, n = 7) from patients with molecularly proven MDDSs, severe mtDNA depletion was detected in liver and muscle tissue with high specificity and sensitivity. Blood samples were specific but not sensitive for detecting mtDNA depletion, and skin fibroblasts were not valuable for evaluating mtDNA depletion. Mutations in the POLG, RRM2B, and MPV17 genes were prospectively identified in 1 blood, 1 liver, and 3 muscle samples. Conclusions: Muscle and liver tissues, but not blood or skin fibroblasts, are potentially useful for rapid screening for mtDNA depletion with real-time qPCR.

Download Full-text

MTT FORMAZAN REPLACED WST-8 AS A BETTER SIMPLE SCREENING METHOD FOR DETECTION OF GLUCOSE-6-PHOSPHATE DEHYDROGENASE DEFICIENCY

Indonesian Journal of Tropical and Infectious Disease ◽

10.20473/ijtid.v7i6.13454 ◽

2019 ◽

Vol 7 (6) ◽

pp. 161

Author(s):

Indah Tantular

Keyword(s):

Screening Method ◽

Residual Activity ◽

Rapid Screening ◽

Blood Samples ◽

Naked Eye ◽

Rapid Screening Test ◽

Hydrogen Carrier ◽

Purple Color ◽

Glucose 6 Phosphate Dehydrogenase ◽

Simple Screening

We have previously developed the WST-8 method as a simple and rapid screening test for detection of glucose-6-phosphate dehydrogenase (G6PD) deficiency accomplished by the naked eye. However, it was little difficult to distinguish between faint orange colors developed by heterozygous females and pink colors of normal hemolyzed blood, since both have similar tones. To solve this problem, we established a new and simple screening method that utilizes another formazan substrate, MTT (3-(4,5-dimethyl-2- thiazolyl)-2,5-diphenyl-2H tetrazolium bromide) in combination with a hydrogen carrier, 1-methoxy phenazine methosulfate. MTT formazan exhibits a purple color, thus allowing for the ability to easily distinguish the pink colors of hemolyzed blood. However, MTT has been reported to react with hemoglobin non-specifically and to interfere with the interpretation of the color reaction. In our examinations by mixing MTT with hemolyzed blood, we found that the non-specific reaction was very slow, and that the addition of a small amount of blood (5~10 μl) into a reaction mixture (800 μl) did not interfere with the reaction of G6PD activity. In this new MTT method, a strong purple color was generated in normal blood samples at 20~30 min after incubation, which could be distinguished by the naked eye from G6PD-deficient blood samples with less than 50% residual activity. In addition, quantitative measurement using a spectrophotometer was also possible despite the fact that MTT formazan is water-insoluble.

Download Full-text

Rapid, ultrasensitive and highly specific biosensor for the diagnosis of SARS-CoV-2 in clinical blood samples

Materials Chemistry Frontiers ◽

10.1039/d0qm00294a ◽

2020 ◽

Vol 4 (7) ◽

pp. 2000-2005 ◽

Cited By ~ 3

Author(s):

Lu Zeng ◽

Yue Li ◽

Jie Liu ◽

Lingling Guo ◽

Zhongxing Wang ◽

...

Keyword(s):

Rapid Screening ◽

Immunochromatographic Strip ◽

Blood Samples ◽

Asymptomatic Carriers

IgG–IgM immunochromatographic strip for rapid screening of SARS-CoV-2 infection including confirmed patients, suspect and asymptomatic carriers in 15 min.

Download Full-text

Rapid screening of SNPs in metastatic colorectal cancer (mCRC) utilizing multiplex sequencing technology (Sequenom).

Journal of Clinical Oncology ◽

10.1200/jco.2012.30.4_suppl.418 ◽

2012 ◽

Vol 30 (4_suppl) ◽

pp. 418-418

Author(s):

Radhashree Maitra ◽

Jay B. Nayak ◽

Atrayee Basu-Mallick ◽

Arjun Sood ◽

Titto A Augustine ◽

...

Keyword(s):

High Throughput ◽

Direct Sequencing ◽

Cost Effective ◽

Rapid Screening ◽

Accurate Identification ◽

Multiple Snps ◽

Fast Screening ◽

Effective Manner ◽

Multiplex Sequencing ◽

Multiple Samples

418 Background: Accurate and fast screening of mutations is essential for designing individualized therapy necessary and critical for efficient disease management and better patient outcome in mCRC. Detection of hotspots by gold standard direct sequencing (DS) is time consuming and cost ineffective. Pyrosequencing (PS) technique is rapid and precisely committed towards SNP detection. Recent introduction of high throughput multiplex PCR based extension on microarray (Sequenom, SEQ) offers a robust platform capable of detecting multiple SNPs simultaneously in a rapid and cost effective manner. The current study analyzes the concordance and efficacy of the cutting edge SEQ technique to the well established DS and PS methods. Methods: DNA isolated from 122 specimens from 76 mCRC patients were sequenced by all three methods. DS and PS were performed on 4 genes at 10 hotspots. SEQ multiplexing was performed on 31 hotspots in 19 genes by 4 multiplex reactions. Results: We were able to make "calls" for all samples by DS and PS. With the multiplex system, the “calls” rate was 97.8% of successful reactions. Using PS data as our standard in the assay we calculated the percent concordance of DS and SEQ. Futhermore SEQ offered a more accurate identification of the substituted nucleotide in Kras codon 12 as compared to PS. Conclusions: The multiplexing of PCR reactions offers an excellent advantage of high throughput with strong feasibility of analyzing several samples for multiple SNPs simultaneously. The concordance rate of > 90% when compared to PS along with the ability to analyze multiple samples/ hotspots plexed together in a time effective rapid mode provided a trifold advantage of the sequenom technology. It is therefore the next generation technology for rapid genetic evaluation of cancer patients. [Table: see text]

Download Full-text