Single Nucleotide Extension Research Articles

A Single Multiplex PCR and Single-Nucleotide Extension Assay for the Detection of Common Thanatophoric Dysplasia I and II Mutations

Mutation analysis is used to provide confirmation of a clinical and radiological diagnosis of Thanatophoric dysplasia types I and II (TDI & II). We developed a single multiplexed PCR and a single nucleotide extension (SNE) assay to identify 14 common mutations causing 99% of TD I and TD II, including the challenging three adjacent mutations in the stop codon of exon 18 of the FGFR3 gene. The assay design also provides a solution for resolving SNE PCR product sizing using performance optimized polymer-7 (POP-7).The assay was validated using thirty-seven previously characterized, de-identified patient samples representing the nine wild-types and 10/14 mutant genotypes. Four artificial templates were synthesized to mimic four TD I mutations not represented in the available patient samples. Fragment size and fluorophore channel for each SNE product from 10 samples and the four artificial templates were used to define bins and panels for analysis with GeneMarker v3.0 and GeneMapper v6.0 software. Allele calls (bin placement within the panels) were verified using the remaining 27 previously characterized samples.This TD I and II PCR and SNE assay is a robust multiplexed assay, streamlined, to identify 14 mutations in one single reaction. Turnaround time required for this assay performance and analysis is decreased in comparison to traditional Sanger or next generation sequencing.

P613: A multiplexed targeted PCR and single nucleotide extension assay for the 14 most common thanatophoric dysplasia I and II mutations

P613: A multiplexed targeted PCR and single nucleotide extension assay for the 14 most common thanatophoric dysplasia I and II mutations

Multiplex Snapshot minisequencing for detection of common PAH gene mutations in Iranian patients with Phenylketonuria.

Phenylketonuria (PKU) is one of the most common inborn faults of amino acid metabolism in the world. It is caused by an autosomal recessive insufficiency of the hepatic enzyme phenylalanine hydroxylase (PAH) that catalysis the irreversible hydroxylation of phenylalanine to tyrosine. More than 950 different disease-causing mutations in the PAH gene have been identified. The most prominent complication of PKU if not diagnosed and treated is severe mental retardation. Early diagnosis and initiation of nutritional therapy are the most important measures in preventing this mental retardation. For this reason, we developed a simple and rapid molecular test for detection of the most frequent phenylalanine hydroxylase mutations. We have developed a PCR multiplex SNaPshot reaction that targets all 10 common mutations identified in PAH gene. Multiplex assays based on the SNaPshot minisequencing approach were developed to simultaneous genotype ten mutations at the PAH gene. We have optimized the detection of this mutations in one multiplex polymerase chain reaction (PCR) followed by ten single-nucleotide extension reactions. The different genotypes were determined based on the position and the fluorescent color of the peaks in a single electropherogram. DNA sequencing assay also were used to verify genotyping results obtained by SNaPshot minisequencing, all mutations were concordant. This method can be useful as secondary test in neonatal screening for hyperphenylalaninemia in neonates with a positive screening test and also be suitable for carrier screening. The assay can be easily applied for accurate, time and cost efficient genotyping of the selected SNPs in various populations.

Epigenetic Pyrimidine Nucleotides in Competition with Natural dNTPs as Substrates for Diverse DNA Polymerases.

Five 2′-deoxyribonucleoside triphosphates (dNTPs) derived from epigenetic pyrimidines (5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine, 5-hydroxymethyluracil, and 5-formyluracil) were prepared and systematically studied as substrates for nine DNA polymerases in competition with natural dNTPs by primer extension experiments. The incorporation of these substrates was evaluated by a restriction endonucleases cleavage-based assay and by a kinetic study of single nucleotide extension. All of the modified pyrimidine dNTPs were good substrates for the studied DNA polymerases that incorporated a significant percentage of the modified nucleotides into DNA even in the presence of natural nucleotides. 5-Methylcytosine dNTP was an even better substrate for most polymerases than natural dCTP. On the other hand, 5-hydroxymethyl-2′-deoxyuridine triphosphate was not the best substrate for SPO1 DNA polymerase, which naturally synthesizes 5hmU-rich genomes of the SPO1 bacteriophage. The results shed light onto the possibility of gene silencing through recycling and random incorporation of epigenetic nucleotides and into the replication of modified bacteriophage genomes.

Enzymatic DNA Synthesis by Engineering Terminal Deoxynucleotidyl Transferase

Template-free enzymatic approaches are considered the most promising solution for next-generation artificial DNA synthesis. However, the development of these technologies has been hampered by the lack of efficient enzymes specialized for stepwise nucleotide addition. By combining evolutionary analysis, high-throughput mutagenesis scanning, and rational design, we identified a terminal deoxynucleotidyl transferase from Zonotrichia albicollis (ZaTdT) and reshaped its catalytic cavity to better accommodate 3′-ONH2-modified nucleotides. The catalytic activity of the engineered ZaTdT for 3′-ONH2-dNTPs is 3 orders of magnitude higher than that of the commonly used mammalian TdT. The engineered ZaTdT enables highly efficient single-nucleotide extension of the growing oligonucleotide chain with an average stepwise yield of 98.7%, which makes it practical for de novo enzymatic DNA synthesis.

2-Formyl-dATP as Substrate for Polymerase Synthesis of Reactive DNA Bearing an Aldehyde Group in the Minor Groove.

2-Formyl-2'-deoxyadenosine triphosphate (dCHO ATP) was synthesized and tested as a substrate in enzymatic synthesis of DNA modified in the minor groove with a reactive aldehyde group. The multistep synthesis of dCHO ATP was based on the preparation of protected 2-dihydroxyethyl-2'-deoxyadenosine intemediate, which was triphosphorylated and converted to aldehyde through oxidative cleavage. The dCHO ATP triphosphate was a moderate substrate for KOD XL DNA polymerase, and was used for enzymatic synthesis of some sequences using primer extension (PEX). On the other hand, longer sequences (31-mer) with higher number of modifications, or sequences with modifications at adjacent positions did not give full extension. Single-nucleotide extension followed by PEX was used for site-specific incorporation of one aldehyde-linked adenosine into a longer 49-mer sequence. The reactive formyl group was used for cross-linking with peptides and proteins using reductive amination and for fluorescent labelling through oxime formation with an AlexaFluor647-linked hydroxylamine.

Assessment for familial pattern and association of polymorphisms in KIAA0319 gene with specific reading disorder in children from North India visiting a tertiary care centre: A case-control study.

Genetic association studies have identified KIAA0319 gene as a possible susceptibility locus for reading disorder; however, very few studies are available from India. The study was planned to investigate the familial pattern and association of KIAA0319 polymorphisms among children with reading disorder visiting a tertiary centre in North India. This is a case-control, familial, and genetic association study on 30 children diagnosed with reading disorder (ICD-10) and 30 matched healthy controls and their families. The Aggregate Neurobehavioral Student Health and Educational Review System was administered on parents of probands and controls for reading problems in their siblings, and Adult Reading Questionnaire was administered for parents of both groups. The blood sample was taken from probands, and DNA was isolated. Four KIAA0319 coding sequence single nucleotide polymorphisms (SNPs; rs4504469, rs6935076, rs2038137, and rs2179515) were genotyped using SNaPshot single nucleotide extension. The incidence of reading problem was significantly higher in families of probands as compared with families of controls. There were no significant differences in both groups regarding the frequency of alleles of four SNPs. The reading disorder showed a significant familial pattern, but KIAA0319 gene did not appear to be a susceptibility factor. Future replications with larger samples and whole genome studies are warranted.

Correlation between gene polymorphism of mannose-binding lectin 2 and posttraumatic susceptibility to sepsis in Hainan Province

Objective To investigate the clinical relevance of mannose-binding lectin 2 (MBL2) gene polymorphism with traumatic sepsis in Hainan Province. Methods A retrospective case control study was conducted to analyze the clinical data of 112 severe trauma patients admitted to the First Affiliated Hospital of Hainan Medical College and Haikou People's Hospital from June 2017 to June 2018. There were 73 males and 39 females, aged 17-83 years [(41.8±8.9)years]. There were 48 patients in the sepsis group and 64 patients in the non-sepsis group. Multiplex single nucleotide extension polymorphism (SNaPshot) typing technique was used to detect the MBL2 gene polymorphism. The correlation between different genotypes and the risk of sepsis was analyzed. ELISA method was used to detect the level of MBL2 in plasma of each group. Results Among the three polymorphic loci of MBL2 gene (rs5030737, rs1800450 and rs1800451), the mutation frequency of rs1800450 was 27.7%, while the mutation frequency of rs5030737 and of rs1800451 was 0. The genotype distribution in two groups was in accordance with Hardy-Weinberg equilibrium. The frequency of GA genotype in sepsis group was significantly higher than that in non-sepsis group (P<0.05). A allele frequency in sepsis group was also much higher than that in non-sepsis group (P<0.05). Patients with GA genotype had increased risk of traumatic sepsis when compared to GG genotype(OR=3.442, 95%CI 1.447-8.187). Allele A increased the prevalence of sepsis significantly as well when compared to allele G(OR=2.799, 95%CI 1.270-6.170). The MBL2 level in serum in sepsis patients with genotype GG and GA was significantly lower than that in non-sepsis group (P<0.05). In sepsis group, the MBL2 serum level of patients with genotype GA was obviously lower than that in patients with genotype GG (P<0.05). Conclusion MBL2 rs1800450G/A polymorphism is closely related to the occurrence of sepsis in Hainan province, and may be related to the decrease of serum MBL2 level in patients with mutant type. Key words: Wounds and injuries; Sepsis; Mannose-binding lectin 2

Candidate Gene Testing in Clinical Cohort Studies with Multiplexed Genotyping and Mass Spectrometry.

Complex diseases are often underpinned by multiple common genetic variants that contribute to disease susceptibility. Here, we describe a cost-effective tag single nucleotide polymorphism (SNP) approach using a multiplexed genotyping assay with mass spectrometry, to investigate gene pathway associations in clinical cohorts. We investigate the food allergy candidate locus Interleukin13 (IL13) as an example. This method efficiently maximizes the coverage by taking advantage of shared linkage disequilibrium (LD) within a region. Selected LD SNPs are then designed into a multiplexed assay, enabling up to 40 different SNPs to be analyzed simultaneously, boosting cost-effectiveness. Polymerase chain reaction (PCR) is used to amplify the target loci, followed by single nucleotide extension, and the amplicons are then measured using matrix-assisted laser desorption/ionization-time of flight(MALDI-TOF) mass spectrometry. The raw output is analyzed with the genotype calling software, using stringent quality control definitions and cut-offs, and high probability genotypes are determined and output for data analysis.

Japaneseplex: A forensic SNP assay for identification of Japanese people using Japanese-specific alleles.

It is sometimes necessary to determine whether a forensic biological sample came from a Japanese person. In this study, we developed a 60-locus SNP assay designed for the differentiation of Japanese people from other East Asians using entirely and nearly Japanese-specific alleles. This multiplex assay consisted of 6 independent PCR reactions followed by single nucleotide extension. The average number and standard deviation of Japanese-specific alleles possessed by an individual were 0.81 ± 0.93 in 108 Koreans from Seoul, 8.87 ± 2.89 in 103 Japanese from Tottori, 17.20 ± 3.80 in 88 Japanese from Okinawa, and 0 in 220 Han Chinese from Wuxi and Changsha. The Koreans had 0-4 Japanese-specific alleles per individual, whereas the Japanese had 4-26 Japanese-specific alleles. Almost all Japanese were distinguished from the Koreans and other people by the factorial correspondence and principal component analyses. The Snipper program was also useful to estimate the degree of Japaneseness. The method described here was successfully applied to the differentiation of Japanese from non-Japanese people in forensic cases. This Japanese-specific SNP assay was named Japaneseplex.

DNA polymerase I proofreading exonuclease activity is required for endonuclease V repair pathway both in vitro and in vivo

Deamination of adenine can occur spontaneously under physiological conditions to generate the highly mutagenic lesion, deoxyinosine (hypoxanthine deoxyribonucleotide, dI). In DNA, dI preferably pairs with cytosine rather than thymine and results in A:T to G:C transition mutations after DNA replication. The deamination of adenine is enhanced by ROS from exposure of DNA to ionizing radiation, UV light, nitrous acid, or heat. In Escherichia coli, dI repair is initiated by endonuclease V (endo V; nfi gene product) nicking but a complete repair mechanism has yet to be elucidated. Using in vitro minimum component reconstitution assays, we previously showed that endo V, DNA polymerase I (pol I), and E. coli DNA ligase were sufficient to repair this dI lesions efficiently and that the 3′-5′ exonuclease of pol I is essential. Here we employed a phagemid-based T-I substrate mimicking adenine deamination product to demonstrate pol I proofreading exonuclease is required by the endo V repair pathway both in vitro and in vivo. In vivo we found that the repair level of an nfi mutant (11%) was almost 8-fold lower than the wild type (87%). while the polA-D424A strain, a pol I mutant defective in 3′-5′ exonuclease, showed a high repair level similar to wild type (both more than 80%). Using additional C–C mismatch as strand discrimination marker we found that the high level of dI removal in polA-D424A was due to strand loss (more than 60%) associated with incomplete repair. Thus, pol I proofreading exonuclease is the major function responsible for dI lesion removal after endoV nicking both in vitro and in vivo. Finally, using MALDI-TOF to analyze single-nucleotide extension product we show that the pol I proofreading exonuclease excises only 2-nt 5′ upstream of endo V incision site further honing the role of pol I in the endoV dI dependent repair pathway.

Application of single nucleotide extension and MALDI-TOF mass spectrometry in proofreading and DNA repair assay

Proofreading and DNA repair are important factors in maintaining the high fidelity of genetic information during DNA replication. Herein, we designed a non-labeled and non-radio-isotopic simple method to measure proofreading. An oligonucleotide primer is annealed to a template DNA forming a mismatched site and is proofread by Klenow fragment of Escherichia coli DNA polymerase I (pol I) in the presence of all four dideoxyribonucleotide triphosphates. The proofreading excision products and re-synthesis products of single nucleotide extension are subjected to MALDI-TOF mass spectrometry (MS). The proofreading at the mismatched site is identified by the mass change of the primer. We examined proofreading of Klenow fragment with DNAs containing various base mismatches. Single mismatches at the primer terminus can be proofread efficiently. Internal single mismatches can also be proofread at different efficiencies, with the best correction for mismatches located 2–4-nucleotides from the primer terminus. For mismatches located 5-nucleotides from the primer terminus there was partial correction and extension. No significant proofreading was observed for mismatches located 6–9-nucleotides from the primer terminus. We also subjected primers containing 3′ penultimate deoxyinosine (dI) lesions, which mimic endonuclease V nicked repair intermediates, to pol I repair assay. The results showed that T-I was a better substrate than G-I and A-I, however C-I was refractory to repair. The high resolution of MS results clearly demonstrated that all the penultimate T-I, G-I and A-I substrates had been excised last 2 dI-containing nucleotides by pol I before adding a correct ddNMP, however, pol I proofreading exonuclease tolerated the penultimate C-I mismatch allowing the primer to be extended by polymerase activity.

Validation of an Extensive CYP2D6 Assay Panel Based on Invader and TaqMan Copy Number Assays.

CYP2D6 is involved in the oxidative metabolism of approximately 20% of all clinically used medications. Genotyping cytochrome P450, family 2, subfamily D, polypeptide 6 (CYP2D6), is a challenge because of the high complexity of the locus. Twenty-nine CYP2D6 sequence variants were genotyped in 50 deidentified patient samples and 29 Coriell DNAs by Invader assay, and results were compared with Infiniti assay and Sanger sequencing. To determine CYP2D6 copy number, 3 TaqMan real-time hydrolysis probes were used and results were compared with long-range PCR. Discrimination of the duplicated alleles was done on 17 DNA samples with 3 copies of CYP2D6 by long-range PCR followed by Invader genotyping and single nucleotide extension for the comparison. Complete concordance was observed for all samples between platforms except for 2 samples due to the lack of the *45 allele in the Infiniti panel. Reproducibility with the Invader assay and TaqMan copy number was 100%. Analytical sensitivity using DNA with 2 copies was determined to be 10 ng DNA for the Invader assay and 1 ng/μL DNA for the TaqMan assay, respectively. Complete concordance and reproducibility were observed for duplicated allele discrimination with the exception of 1 sample, determined to be *29/*43X2 by the Invader test and *1X2/*29 by the Infiniti method, which did not test for *43. This validation study showed that Invader and TaqMan assay combined panel provides an attractive, valid, highly accurate, and reproducible approach for CYP2D6 genotyping for clinical implementation.

Somatic DNA Mutation Analysis.

Somatic mutations in patient tumor DNA samples can be readily detected based on mass spectrometry. The MassARRAY system is a high-throughput matrix-assisted laser desorption time-of-flight (MALDI) mass spectrometer for detection of nucleic acids. The technique is based on single-nucleotide base extension. A series of PCR assays amplify specific DNA regions of interest harboring mutations. A third primer is then introduced into the reaction which corresponds to the DNA template immediately in front of the mutation site. A final round of PCR is then performed using mass-modified nucleotides. These nucleotides are designed so that no additional bases can be added to the extension primer (terminating bases) after a single-base extension and are mass modified to exaggerate mass differences between nucleotides allowing easier identification by mass spectrometry.The sequences of the extension primer and possible extension products (wild type and mutations) are known; therefore, it is possible to calculate their mass. The mass spectrometer can identify the mass peaks for each assay and identify those with mutations (multiple peaks). The technique was originally designed to screen multiple single-nucleotide polymorphisms (SNPs) in a large number of specimens. A SNP in the coding region of DNA that alters the gene and subsequent protein expression is considered a mutation. Mutations often occur in genes whose protein product is in a key signaling pathway and/or drug target. Rationale treatment options can be designed based upon the presence or absence of these mutations. In this chapter, we describe the process for detection of somatic mutations in DNA extracted from formalin-fixed paraffin-embedded (FFPE) material.

Abstract 428: Hypermethylation of GATA2 is validated as a marker of progression in non-muscle invasive bladder cancer

Abstract Objective Hypermethylation patterns of a set of four genes (GATA2, TBX2, TBX3, and ZIC4) were previously identified to predict progression of non-muscle-invasive bladder cancer (NMIBC) to muscle-invasive bladder cancer (MIBC). In this prospective study we aimed to validate these four genes as progression markers in NMIBC. Materials and Methods A total of 856 patients in follow-up for stage Ta urothelial carcinoma of the bladder were included in hospitals in Denmark, Germany, Serbia, Spain, Sweden and the Netherlands. Available pathology sections were reviewed by a single expert uropathologist. According to the WHO 2003 grading, 633 patients were diagnosed with a Ta low-grade tumor and 223 with a Ta high-grade tumor. Progression to muscle-invasive disease was seen in 18 (3%) of the low-grade tumors and in 12 (5%) of the high-grade tumors. Mean follow up was 22 months (range 0 to 76 months). DNA was isolated from fresh frozen tumor tissue and analyzed for mutations in FGFR3, TERT, RAS and PIK3CA. Methylation assays were done using a single-nucleotide probe extension assay for GATA2, TBX2, TBX3 and ZIC4. Methylation percentages were calculated and cut-off values were determined for dichotomization. All results were combined and the optimal model for predicting progression was selected by logistic regression analysis. Survival analysis was done using the Kaplan-Meier method. Results Multivariate logistic regression analysis using backward selection resulted in an optimal prediction model including GATA2, TBX2, age and gender for Ta low-grade tumors. The predictive capacity of the model represented by the AUC was 0.896. For Ta high-grade tumors, multivariate regression analysis resulted in a predictive model that included only GATA2 and smoking status with an AUC of 0.783. Mutation analysis did not add to these models. KM curves were plotted of the best performing methylation marker GATA2 and progression-free survival. Progression-free survival was significantly worse in patients having a GATA2-positive Ta high-grade tumor (P = 0.025). Conclusion Hypermethylation of the GATA2 gene can predict progression of NMIBC to MIBC in low- as well as high-grade Ta tumors. Further, survival is significantly worse in Ta high grade patients with a GATA2 positive primary tumor. Hypermethylation of GATA2 is therefore a validated progression marker for NMIBC. Citation Format: Kim E.M. van Kessel, Kirstin A. van der Keur, Lars Dyrskjøt, Ferran Algaba, Naeromy Welvaart, Willemien Beukers, Ulrika Segersten, Bastian Keck, Tobias Maurer, Tatjana Simic, Marcus Horstmann, Joost L. Boormans, Francisco X. Real, Nuria Malats, Per-Uno Malmström, Torben F. Ørntoft, Ellen C. Zwarthoff. Hypermethylation of GATA2 is validated as a marker of progression in non-muscle invasive bladder cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 428.

Population Screening for Hemoglobinopathy Profiling: Is the Development of a Microarray Worthwhile?

In order to perform affordable and expedient whole population scans for the single nucleotide polymorphisms (SNPs) involved in hemoglobinopathies, microarrays based on single nucleotide extension (SNE) might prove advantageous to whole genome/exome sequencing in terms of cost, speed, interpretation and discretion as they focus on a very small part of the tested genome. The development of a microarray assay entails most of the cost, to be deferred by the massive use of the end product. A microarray assay development project, involving multiplex polymerase chain reaction (PCR), labeling, hybridization and scanning/scoring steps is presented as a paradigm of objective bug ratios expected to such procedures and of ways to cope with them. Qualification of the microarray genotypes needs a reference method, which may still be restriction digestion or other, as sequencing remains an expensive commodity. Optimization of wet steps should also be followed by careful and perhaps individualized dye excitation and in silico scoring rules, taking into consideration decay and bleaching effects that perplex development. The strategy of successive elimination of problems, a top-bottom procedure, which had been used and is usually preferred by developing agencies, might have been erroneous; a bottom-up course to delineate issues in different levels, although more laborious, might be the correct choice, especially as software and robotic hardware, high throughput tools become more mature and available. The testing for interlocus compatibility, specificity and robustness is demanding and warranted only in the case of steady, high volume use of an assay for territorial, national or international use.

In the most popular type of next‐generation DNA sequencer, DNA fragments are PCR‐amplified in the slide's channel coated by a lawn of PCR primers to form myriads of clone DNA clusters. After that, the DNA sequence of each cluster is determined by taking photographs of fl uorescent signals on the slide with the image sensor again and again after each

Genes to CellsVolume 21, Issue 2 p. 117-117 Cover ImageFree Access In the most popular type of next-generation DNA sequencer, DNA fragments are PCR-amplified in the slide's channel coated by a lawn of PCR primers to form myriads of clone DNA clusters. After that, the DNA sequence of each cluster is determined by taking photographs of fl uorescent signals on the slide with the image sensor again and again after each single-nucleotide extension step of a novel dye-terminator method. Thus, the reaction proceeding in the next-generation sequencer is massively parallel sequencing. In this drawing, we schematically expressed this mechanism with a flying hawk (image sensor) viewing clusters of pine trees (DNA) on the channel-shaped reclaimed land divided by embankments. Designed by TRAIS Co., Ltd. (Kobe, Japan) First published: 02 February 2016 https://doi.org/10.1111/gtc.12347AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume21, Issue2February 2016Pages 117-117 RelatedInformation

Abstract 549: A urine based assay to select patients for initial cystoscopy

Abstract Objective Only 2.6 - 28% of the patients that present with haematuria will be diagnosed with bladder cancer. Unnecessary cystoscopies and upper tract imaging lead to high diagnostic costs and patient burden. In order to better select patients for diagnostic cystoscopy and thereby reduce cost and over-testing, we aimed to develop a diagnostic urine assay by combining mutation status of FGFR3, TERT and PIK3CA with methylation markers OTX1 and ONECUT2. Material and Methods A total of 258 patients undergoing cystoscopy for haematuria were included, 175 were diagnosed with bladder cancer and 83 had a non-malignant source of the haematuria. Voided urine samples were collected prior to cystoscopy. DNA was extracted and analyzed for mutations in FGFR3, TERT and PIK3CA. Methylation assays were done using a single-nucleotide probe extension assay with multiple probes for OTX1 (4 probes) and ONECUT2 (5 probes). Methylation percentages were calculated and cut-off values (sensitivity ≥ 85%) were determined for dichotomization. All results were combined and the optimal model was selected through logistic regression analysis. The Bootstrap procedure was used for internal validation. Results Combining the results of the dichotomized methylation markers OTX1 (probe 2), ONECUT2 (probe 1+4) with the mutation markers FGFR3, TERT, PIK3CA and the clinical parameters Age and Gender led to a prediction model with an AUC of 0.95 (95% CI 0.92-0.98) and an optimism-corrected AUC of 0.94. To further improve the model, analyses were repeated for continuous methylation marker variables. Now, adding ONECUT2 probe 2 enhanced the predictive capacity of the model even further (AUC 0.97, 95% CI 0.94 - 0.99 and an optimism-corrected AUC of 0.96). Overall material cost was estimated to be 20 euro per patient. A mutation-analysis first strategy would lead to a potentially larger reduction in cost. This strategy identified 120 patients with bladder cancer based on mutation assays alone. For the remaining 125 mutation-naive patients the prediction model containing the previously selected continuous methylation markers combined with Age and Gender was used. The overall combined strategy sensitivity was 92,1% and specificity was 89% (AUC of 0.96). Conclusion Analyzing haematuria patients for their risk of bladder cancer may lead to a reduction of unnecessary diagnostic cystoscopies and upper urinary tract imaging. This will present a major cost reduction in the urological practice. Combining continuous marker variables for OTX1 and three probes for ONECUT2 with mutation markers FGFR3, TERT, PIK3CA and Gender and Age results in an accurate prediction model with an AUC of 0.97 and an optimism-corrected AUC of 0.96. A mutation-analysis first strategy seems most practical and feasible (Sens 92%, Spec 89%). The assay must be further improved to reach a more satisfactory overall sensitivity (&amp;gt;95%), for instance by adding RAS mutation analysis. Citation Format: Kim E.M. van Kessel, Willemien Beukers, Irene Lurkin, Kirstin A. van der Keur, Lars Dyrskjot, Ulrika Segersten, Torben F. Ørntoft, Nuria Malats, Per-Uno Malmström, Francisco X. Real, Chris H. Bangma, Ellen C. Zwarthoff. A urine based assay to select patients for initial cystoscopy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 549. doi:10.1158/1538-7445.AM2015-549

Fluorescence polarization-based method with bisulfite conversion-specific one-label extension for quantification of single CpG dinucleotide methylation.

To quantify the methylation at individual CpG dinucleotide sites in large biological or clinical samples, we developed a bisulfite conversion-specific one-label extension (BS-OLE) method using visualization by fluorescence polarization (FP) measurement of methylation at single CpG sites in small amounts of genomic DNA. Genomic DNA was treated with sodium bisulfite to convert unmethylated cytosine to uracil leaving 5-methylcytosine unaltered, and BS-PCR was used to generate DNA template containing target CpG sites. BS-OLE uses a BS-primer hybridized immediately upstream of the target CpG site being examined and then fluorescent dCTP or dUTP is incorporated into the methylated (CpG) or unmethylated (TpG) form of the target site through single-nucleotide chain extension, yielding an FP ratio between the fluorescent dCTP- and dUTP-incorporated products as a measure of methylation. This provides stable estimates of the methylation level of human genomic DNA and of a 250-bp plasmid DNA segment containing a single TCGA TaqI cleavage site, in accordance with the results of a combined bisulfite restriction analysis method. We used BS-OLE to measure dose-dependent DNA hypomethylation in human embryonic kidney 293T cells treated with the DNA methyltransferase inhibitor 5-aza-dC. BS-OLE is well suited to high-throughput multi-sample applications in biological and medical studies.

Validation of DNA methylation biomarkers for diagnosis of acute lymphoblastic leukemia.

DNA methylation biomarkers capable of diagnosis and subtyping have been found for many cancers. Fifteen such markers have previously been identified for pediatric acute lymphoblastic leukemia (ALL). Validation of these markers is necessary to assess their clinical utility for molecular diagnostics. Substantial efficiencies could be achieved with these DNA methylation markers for disease tracking with potential to replace patient-specific genetic testing. We evaluated DNA methylation of promoter regions of TLX3 (T-cell leukemia homeobox) and FOXE3 (forkhead box E3) in bone marrow biopsies from 197 patients classified as leukemic (n = 95) or clear of the disease (n = 102) by MALDI-TOF. Using a single nucleotide extension assay (methylSABER), we tested 10 bone marrow biopsies collected throughout the course of patient chemotherapy. Using reference materials, diagnostic thresholds and limits of detection were characterized for both methods. Reliable detection of DNA methylation of TLX3 and FOXE3 segregated ALL from those clear of disease with minimal false-negative and false-positive results. The limit of detection with MALDI-TOF was 1000-5000 copies of methylated allele. For methylSABER, the limit of detection was 10 copies of methylated TLX3, which enabled monitoring of minimal residual disease in ALL patients. Mass spectrometry procedures can be used to regionally multiplex and detect rare DNA methylation events, establish DNA methylation loci as clinically applicable biomarkers for disease diagnosis, and track pediatric ALL.