Pyrosequencing PCR Research Articles

The frequency of methylation of the NT5E gene in metastatic breast cancer.

e11553 Background: Ecto-5-prime-nucleotidase (NT5E; CD73) catalyzes the conversion of purine 5-prime mononucleotides to nucleosides, the preferred substrate being AMP. Deficiency of NT5 occurs in a variety of immunodeficiency diseases and some studies have associated over-expression of NT5E with clinically aggressive neoplastic disease. Here we describe transcriptional down-regulation of the NT5E gene in breast cancer and show that this is associated with aberrant methylation in the CpG island located in its 5’ regulatory sequences. Methods: Methylation in the NT5E CpG island was analysed by methylation specific PCR (MSP) and quantitative pyrosequencing in a panel of 10 breast carcinoma cell lines and two independent clinical series of breast carcinomas, comprising 80 and 140 cases respectively and 17 brain metastatic breast cancer lesions. The breast carcinomas were predominantly invasive ductal carcinomas, untreated at the time of surgery and randomly selected from our clinical practice. Expression was assessed by qRT-PCR. Results: NT5E mRNA was down-regulated in 4/10 breast carcinoma cell lines analysed. Down-regulation was associated with aberrant methylation in the CpG island located in the 5’ regulatory sequences of the NT5E gene: methylation was present only in cell lines with down-regulation and the CpG island was unmethylated in cell lines which express the gene. Moreover, expression of NT5E was up-regulated by azacytidine. In clinical cases, methylation (assessed by pyrosequencing and/or MSP) was present in 28% and 41% of cases in independent clinical series of primary carcinomas. Methylation was more common in primary cases which ultimately relapsed at distant metastatic sites. Strikingly, methylation was particularly common in 9/16 brain metastatic lesions. Conclusions: In contrast to previous reports, we show that methylation of NT5E is common in metastatic breast cancer, particularly in intra-cranial metastases. Our data implicate transcriptional silencing of NT5E as a common epigenetic event in metastatic breast cancer and imply that detection of methylated DNA may have utility as a prognostic and/or predictive biomarker in breast cancer.

The Microbial Communities in Male First Catch Urine Are Highly Similar to Those in Paired Urethral Swab Specimens

Urine is the CDC-recommended specimen for STI testing. It was unknown if the bacterial communities (microbiomes) in urine reflected those in the distal male urethra. We compared microbiomes of 32 paired urine and urethral swab specimens obtained from adult men attending an STD clinic, by 16S rRNA PCR and deep pyrosequencing. Microbiomes of urine and swabs were remarkably similar, regardless of STI status of the subjects. Thus, urine can be used to characterize urethral microbiomes when swabs are undesirable, such as in population-based studies of the urethral microbiome or where multiple sampling of participants is required.

Abstract 3954: Identification of microRNAs epigenetically silenced in bladder cancer

Abstract Purpose: Bladder cancer is the one of the most common malignancies in the urothelial tract. MicroRNAs (miRNAs) are small, non-coding RNAs that control gene expression post-transcriptionally, either by degradation of target mRNAs or by inhibition of protein translation. MiRNAs are involved in cancer development and progression, acting as tumor suppressors or oncogenes. Recent studies have demonstrated a role of DNA methylation in silencing miRNAs in bladder cancer. The aim of current study is to identify bladder cancer-related miRNAs by screening epigenetically silenced miRNAs in bladder cancer. Materials & Methods: Two bladder cancer cell lines (T-24, UM-UC3) were treated with or without 5-aza-2’deoxycytidine (5-aza-dC) and 4-phenylbutyric acid (4-PBA), and expression of mature miRNAs was examined using a TaqMan Low Density Array System. We used UCSC genome browser to examine if CpG islands are located in the proximal upstream of the miRNAs upregulated after drug treatment. Methylation-specific PCR (MSP) and bisulfite pyrosequencing were carried out to analyze methylation status of the miRNA genes in bladder cancer cell lines and primary bladder cancer specimens. Results: Of 664 miRNAs examined, 208 miRNAs were up-regulated (>5-fold) in T24 cells, and 200 miRNAs were up-regulated in UM-UC3 cells, respectively. Among the miRNAs, 146 were commonly up-regulated in both T-24 and UM-UC3 cells. Among those 146 miRNAs, 36 miRNAs contain CpG islands in their 5’ end of the genes, and further analyzed. Methylation-specific PCR (MSP) was carried out, and 12 miRNAs showed aberrant methylation in bladder cancer cell lines. Bisulfite pyrosequencing of these 12 miRNAs was carried out, and we identified hypermethylation of 12 miRNAs in cultured and primary bladder cancer specimens. Conclusions: Our results suggest that DNA methylation is associated with miRNAs silencing in bladder cancer, and that these miRNAs may have a role in the development and progression of bladder cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3954. doi:10.1158/1538-7445.AM2011-3954

Abstract LB-260: Transcriptional silencing disrupts two levels of arginine biosynthesis in glioblastoma multiforme: a novel, targeted therapeutic strategy for high grade gliomas

Abstract Background: Changes in the structure and expression of enzymes involved in key metabolic pathways (such as isocitrate dehydrogenase) have been identified in glial brain tumours. To seek novel candidate brain tumour suppressors, we have performed pharmacological methylation reversal. Methods: Methylation reversal was performed in human GBM cell lines and primary explants using 5′ azacytidine (5′AZA), coupled with micro-array analysis, in GBM. Gene expression was analysed using qPCR and western blotting and by immunohistochemistry in clinical cases of supra-tentorial adult GBM. Methylation was analysed using methylation specific PCR and quantitative pyrosequencing. Results: Both ASS1, which encodes arginino succinate synthetase, and arginino succinate lyase (ASL) are down-regulated in GBM. Down-regulation correlates with aberrant, neoplasia-specific methylation in the CpG island of each gene and expression is restored by demethylation. Arginine starvation produced by exposure to the arginine-depleting agent pegylated arginine deiminase induces an adaptive transcriptional up-regulation of both ASS1 and ASL which is blocked in GBM cells by methylation. Cells lacking ASS1 or ASL are sensitive to arginine deprivation and die by apoptosis when treated with arginine deiminsase. In clinical cases of de novo GBM, treated by surgical debulking in our institution, ASS1 was silenced in 13/60 cases and ASL in 3/60. These cases had no expression of the proteins, as seen by immunohistochemistry, together with aberrant CpG methylation. Conclusions: Taken together, these results identify a novel treatment strategy and suggest that ADI-PEG20 warrants evaluation in phase 2 clinical trial in ASS1 negative GBM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-260. doi:10.1158/1538-7445.AM2011-LB-260

Use of Pyromark Q96 ID pyrosequencing system in identifying bacterial pathogen directly with urine specimens for diagnosis of urinary tract infections

Detection and identification of bacterial etiology in urine is critical for accurate diagnosis and subsequent rational treatment of urinary tract infections (UTIs). Urine culture followed by a series of biochemical reactions is currently the standard method for detecting and distinguishing microorganisms associated with UTIs. The whole procedure commonly takes more than 24h. Here we developed a new system combining 16S rRNA gene broad-range PCR with pyrosequencing technology that allows for bacteria detection and identification in urine in 5h. To evaluate this system for rapid diagnosis of bacteriuria, 768 urine specimens were collected from patients with suspected UTIs and were tested side-by-side using standard urine culture-based identification method and the pyrosequencing method. The results from pyrosequencing correlated well with those from traditional culture-based identification method. The overall agreement between these two methods reached 98.0% (753/768). In addition, we tested the sensitivity of pyrosequencing method and determined that urine bacterial numbers as low as 104cfu/ml could be accurately detected and identified. In conclusion, compared with traditional biochemical method, the PCR–pyrosequencing system significantly improved the detection and identification of bacteriuria with shorter time, higher accuracy, and higher throughput, thus allowing earlier pathogen-adapted antibiotic therapy for patients.

Integration profile of retroviral vector in gene therapy treated patients is cell‐specific according to gene expression and chromatin conformation of target cell

The analysis of genomic distribution of retroviral vectors is a powerful tool to monitor ‘vector-on-host’ effects in gene therapy (GT) trials but also provides crucial information about ‘host-on-vector’ influences based on the target cell genetic and epigenetic state. We had the unique occasion to compare the insertional profile of the same therapeutic moloney murine leukemia virus (MLV) vector in the context of the adenosine deaminase-severe combined immunodeficiency (ADA-SCID) genetic background in two GT trials based on infusions of transduced mature lymphocytes (peripheral blood lymphocytes, PBL) or a single infusion of haematopoietic stem/progenitor cells (HSC). We found that vector insertions are cell-specific according to the differential expression profile of target cells, favouring, in PBL-GT, genes involved in immune system and T-cell functions/pathways as well as T-cell DNase hypersensitive sites, differently from HSC-GT. Chromatin conformations and histone modifications influenced integration preferences but we discovered that only H3K27me3 was cell-specifically disfavoured, thus representing a key epigenetic determinant of cell-type dependent insertion distribution. Our study shows that MLV vector insertional profile is cell-specific according to the genetic/chromatin state of the target cell both in vitro and in vivo in patients several years after GT.

Identification of a Severe Acute Respiratory Syndrome Coronavirus-Like Virus in a Leaf-Nosed Bat in Nigeria

Bats are reservoirs for emerging zoonotic viruses that can have a profound impact on human and animal health, including lyssaviruses, filoviruses, paramyxoviruses, and severe acute respiratory syndrome coronaviruses (SARS-CoVs). In the course of a project focused on pathogen discovery in contexts where human-bat contact might facilitate more efficient interspecies transmission of viruses, we surveyed gastrointestinal tissue obtained from bats collected in caves in Nigeria that are frequented by humans. Coronavirus consensus PCR and unbiased high-throughput pyrosequencing revealed the presence of coronavirus sequences related to those of SARS-CoV in a Commerson’s leaf-nosed bat (Hipposideros commersoni). Additional genomic sequencing indicated that this virus, unlike subgroup 2b CoVs, which includes SARS-CoV, is unique, comprising three overlapping open reading frames between the M and N genes and two conserved stem-loop II motifs. Phylogenetic analyses in conjunction with these features suggest that this virus represents a new subgroup within group 2 CoVs.

Epigenetic Silencing of Somatostatin in Gastric Cancer

Somatostatin (SST), a primary inhibitor of gastrin-stimulated gastric acid secretion, has potent antitumor and anti-secretory activity in several human cancers. This study was performed to investigate the SST gene expression levels and possible epigenetic mechanisms that regulate expression of SST in gastric adenocarcinomas. Quantitative real-time (RT)-PCR and quantitative bisulfite pyrosequencing were used to study primary gastric cancer tissue samples and cell lines. Quantitative RT-PCR analysis revealed down-regulation of the SST transcript in 93% of gastric carcinoma samples (30/32), compared with 21 normal samples (P<0.001). Because of the presence of a large CpG island in the SST promoter, we next examined its promoter DNA methylation levels by use of quantitative bisulfite pyrosequencing. The results revealed a significant increase in SST promoter DNA methylation in tumor samples compared with normal samples (P<0.05). Promoter DNA hypermethylation and silencing of SST was also detected in seven gastric cancer cell lines that we tested. To confirm the role of promoter DNA methylation as an epigenetic mechanism regulating SST expression, AGS gastric cancer cells were treated with 5-Aza-dc. This treatment led to reduction of promoter DNA methylation levels of SST accompanied by restoration of its mRNA expression. Our results indicate that promoter DNA methylation levels play a critical role in regulating SST expression in gastric cancer. This finding provides a foundation for further studies on the role of SST in gastric carcinogenesis and its potential as a biomarker for gastric cancers.

Molecular identification of Helicobacter DNA in human gastric adenocarcinoma tissues using Helicobacter species-specific 16S rRNA PCR amplification and pyrosequencing analysis

Helicobacter pylori (H. pylori) is a microaerophilic gram-negative bacterium known to be associated with chronic gastritis, peptic ulcer and gastric adenocarcinoma. In the present study, the presence of Helicobacter DNA was investigated using a Helicobacter species-specific 16S rRNA PCR amplification and pyrosequencing analysis in 51 resected gastric adenocarcinomas. DNA was extracted from paraffin-embedded tissues of resected gastric adenocarcinomas. PCR primers were designed to amplify the 133-bp PCR fragment in highly conserved regions of the 16S rRNA gene. The sequence of the PCR products was analyzed using a PSQ 96 system with SQA software. The pyrosequencing analysis of 16S rRNA showed that H. pylori was present in 47 (92.2%) of the 51 gastric adenocarcinomas. In the 4 H. pylori-negative cases, Helicobacter cinaedi (2 cases), Helicobacter mustelae (1 case) and Campylobacter hyointestinalis (1 case) were detected. Pyrosequencing technology was useful in the identification and differentiation of H. pylori from other species by analyzing the gene encoding 16S rRNA. Gastric adenocarcinoma tissues contain bacteria, and the majority are H. pylori. Helicobacter cinaedi, Helicobacter mustelae and Campylobacter hyointestinalis rarely occur. The roles of these organisms in the pathogenesis of gastric adenocarcinoma remain unclear.

Abstract 2707: Impact of accuracy in O6-methylguanine-DNA methyltransferase methylation assays for temozolomide efficacy on glioblastoma

Abstract Background: O6-methylguanine-DNA methyltransferase (MGMT) is implicated in resistance to temozolomide (TMZ). MGMT status has been shown to correlate with survival of patients with glioblastoma (GBM). We evaluated the relationship between promoter methylation status and protein expression of the MGMT gene in patients with GBM treated with TMZ, and also analyzed their prognostic value regarding response to TMZ and survival. Methods: We analyzed 47 GBM tissues from 43 patients treated with TMZ from September 2003 to January 2009. Among them, 22 patients with recurrent GBM treated with standard TMZ single therapy were subjected to survival analyses. TMZ was given daily orally 75mg/m2 during radiotherapy, or as standard 5/28-day regimen. MGMT protein expression was quantified by Western blotting, and promoter methylation status was determined by both methylation-specific PCR (MSP) and quantitative pyrosequencing. Results: MGMT promoter methylation was detected in 19/47 cases (40.4%) by MSP, 16/47 (34%) by pyrosequencing, and was significantly correlated with each other (p&amp;lt;0.001). The methylation status correlated significantly with MGMT protein expression as well. Upon TMZ treatment for recurrent GBM, patients with both methylated MGMT promoter by MSP and low MGMT protein expression had a significantly improved progression-free survival (PFS) and overall survival (OS), whereas methylation status by pyrosequencing data did not. MGMT status was the only significant prognostic factor for PFS and OS. Conclusions: The three MGMT assays revealed similar methylation/expression patterns and were suggested to be important prognostic factors for patients treated with TMZ. Whether precise quantification of the methylation status provides any clinically relevant information on the treatment strategy for GBM needs to be further investigated. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2707.

Abstract 4088: Effects of exposure to particulate matter on methylation of miRNA genes coding for miR-21 and miR-222

Abstract BACKGROUND: Inhalation of Particulate Matter (PM) and PM metal components has been associated with increased risk of lung cancer. MicroRNAs (miRNAs) are small, non-coding RNAs with an important role in biological activities and are linked to human diseases such as cancer. miRNA expression needs tight regulation, which also takes place at the epigenetic level. miRNAs undergo regulation by DNA methylation in miRNA coding genes, associate with silencing of miRNA expression. Increased expression of miR-21 has been implicated in various processes involved in carcinogenesis, including inhibition of apoptosis, promotion of cell proliferation and stimulation of tumor growth by targeting multiple tumor/metastasis suppressor genes. In cancer cells miR-222 promotes cell proliferation by targetting the cell cycle inhibitor and tumor suppressor p27. AIMS: We evaluated the effects of exposure to PM and PM metal components on candidate miRNAs methylation (miR-222 and miR-21) in 63 workers of an electric-furnace steel plant with well-characterized exposure. METHODS: We measured miR-222 and miR-21 methylation in blood leukocyte RNA obtained from 63 workers on the first day of a workweek (baseline) and after three days of work (post-exposure). Quantitative miRNA methylation analysis was performed through bisulfite PCR Pyrosequencing. We estimated individual exposures to PM1, PM10, coarse particles and PM metal components (chromium, lead, cadmium, arsenic, nickel, manganese) between the baseline and post-exposure measurements. RESULTS: miR-222 and miR-21 methylation was significantly decreased in post-exposure samples, compared with baseline (post-exposure=60.07±9.38, baseline=63.05±6.38; p=0.013 for miR-222; post-exposure=62.47±6.87, baseline=66.24±6.32; p=0.004 for miR-21). In post-exposure samples, miR-222 methylation was negatively associated with the average exposure to the levels of PM (βstd=-0.012, p=0.03 for PM10, βstd=-0.579, p=0.005 for PM1 and βstd=-0.013, p=0.032 for Coarse) and to the levels of PM metal components (βstd=-111.345, p=0.008 for Chromium and βstd=17.195, p=0.023 for Arsenic). CONCLUSIONS: Changes in miRNA methylation may represent a novel epigenetic mechanism mediating responses to PM and its metal components. Whether these modifications have a role in the initiation and progression of cancer needs to be evaluated in future studies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4088.

Genomic Screening for Genes Silenced by DNA Methylation Revealed an Association between RASD1 Inactivation and Dexamethasone Resistance in Multiple Myeloma

Epigenetic changes such as DNA methylation play a key role in the development and progression of multiple myeloma. Our aim in the present study was to use genomic screening to identify genes targeted for epigenetic inactivation in multiple myeloma and assess their role in the development of resistance to dexamethasone. Gene expression was examined using microarray screening, reverse transcription-PCR, and real-time quantitative PCR. DNA methylation was examined using bisulfite PCR, bisulfite sequencing, and bisulfite pyrosequencing in 14 multiple myeloma cell lines, 87 multiple myeloma specimens, and 12 control bone marrow samples. WST-8 assays were used to assess cell viability after treatment with 5-aza-2'-deoxycytidine and/or dexamethasone. Microarray analysis was done to screen for genes up-regulated by 5-aza-2'-deoxycytidine. In RPMI8226 cells, 128 genes were up-regulated, whereas 83 genes were up-regulated in KMS12PE cells. Methylation of 22 genes with CpG islands in their 5' regions, including RASD1, was confirmed. Methylation of RASD1 was associated with its inactivation, which correlated with resistance to dexamethasone. Treating multiple myeloma cells with 5-aza-2'-deoxycytidine restored sensitivity to dexamethasone. Methylation of RASD1 was also detected in a subset of primary multiple myeloma specimens, and the levels of methylation were increased after repeated antitumor treatments. Gene signature analysis revealed various genes to be synergistically induced by treatment with a combination of 5-aza-2'-deoxycytidine plus dexamethasone. Our findings indicate that epigenetic inactivation of genes, including RASD1, plays a key role in the development of dexamethasone resistance in multiple myeloma. Moreover, they show the utility of demethylation therapy in cases of advanced multiple myeloma.

Non-Random Lentiviral Vector Insertions in Bone Marrow Progenitors from Fanconi Anemia Patients

Improved protocols using lentiviral vectors have been established with minimal cytokine exposure and short transduction times proving more suitable for overcoming the disease-specific challenge in correcting functionally defective hematopoietic stem cells (HSCs) of Fanconi Anemia (FA) patients. Bone marrow (BM) cells from FA patients were transduced ex vivo with lentiviral vectors (LVs) expressing FANCA and/or EGFP using optimized conditions to preserve the repopulating properties of the primitive hematopoietic stem cells (manuscript submitted). In a forward preclinical screening of possible LV-induced side effects we analyzed the insertional inventory in colonies generated by FA BM cells previously transduced with the LVs. We have established and optimized DNA and RNA isolation procedures for minimal cell numbers, suitable for large scale screening of colony forming cell (CFC) derived colonies by linear amplification-mediated PCR (LAM-PCR) and massive parallel pyrosequencing (454 GS Flx system; Roche). This approach is applicable for detecting early indicators of clonal selection, and is based on the analysis of common integration sites (CIS) and non-random distribution of vector insertions in particular genomic loci. From a total of 180 CFC-derived colonies expressing the EGFP LV marker gene, 298 vector insertions could be sequenced and mapped to the human genome. The analysis of vector targeted gene coding regions showed a non-random genomic distribution of LV insertions, with a significant overrepresentation of RefSeq genes that are part of distinct functional categories. Accordingly vector associated genes are predominantly involved in cellular signal cascades regulated by the MAP Kinase family known to be involved in a wide variety of cellular processes such as proliferation, differentiation, transcription regulation and development. Apart from the observed high integration frequency in genes (>80%), partial loss of vector LTR nucleotides was detected in >10% of the integrants (3–25bp). Notably, >20% of the lentiviral insertions were found to be located in CIS of predominantly 2nd order. Further screening assays of LV transduced CFC-derived colonies will allow a deeper investigation in the functional consequences of such CIS targeting in gene therapy protocols of FA. However our results suggest that the LV transduction of FA BM progenitors leads to a relatively high frequency of insertions in CIS which may be indicative of an insertion based (specific) selection mechanism. We herby show that the ex vivo large scale integration site analyses of CFC-derived colonies from patients considered to undergo gene therapeutic treatments constitutes a robust approach, which combined with mouse preclinical biosafety studies will help to improve the safety of clinical gene therapy protocols. The non-random distribution of LV integrations in CIS associated genes and in genes involved in particular cellular pathways may be indicative for the altered biochemical pathways characteristic of FA stem cells, with reported defects in DNA repair and self-renewal.

Real‐time PCR and subsequent pyrosequencing for screening of penA mosaic alleles and prediction of reduced susceptibility to expanded‐spectrum cephalosporins in Neisseria gonorrhoeae

Real-time PCR and subsequent pyrosequencing for screening of penA mosaic alleles and prediction of reduced susceptibility to expanded-spectrum cephalosporins in Neisseria gonorrhoeae

Effects of Particulate Matter on Genomic DNA Methylation Content and iNOS Promoter Methylation

BackgroundAltered patterns of gene expression mediate the effects of particulate matter (PM) on human health, but mechanisms through which PM modifies gene expression are largely undetermined.ObjectivesWe aimed at identifying short- and long-term effects of PM exposure on DNA methylation, a major genomic mechanism of gene expression control, in workers in an electric furnace steel plant with well-characterized exposure to PM with aerodynamic diameters < 10 μm (PM10).MethodsWe measured global genomic DNA methylation content estimated in Alu and long interspersed nuclear element-1 (LINE-1) repeated elements, and promoter DNA methylation of iNOS (inducible nitric oxide synthase), a gene suppressed by DNA methylation and induced by PM exposure in blood leukocytes. Quantitative DNA methylation analysis was performed through bisulfite PCR pyrosequencing on blood DNA obtained from 63 workers on the first day of a work week (baseline, after 2 days off work) and after 3 days of work (postexposure). Individual PM10 exposure was between 73.4 and 1,220 μg/m3.ResultsGlobal methylation content estimated in Alu and LINE-1 repeated elements did not show changes in postexposure measures compared with baseline. PM10 exposure levels were negatively associated with methylation in both Alu [β = −0.19 %5-methylcytosine (%5mC); p = 0.04] and LINE-1 [β = −0.34 %5mC; p = 0.04], likely reflecting long-term PM10 effects. iNOS promoter DNA methylation was significantly lower in postexposure blood samples compared with baseline (difference = −0.61 %5mC; p = 0.02).ConclusionsWe observed changes in global and gene specific methylation that should be further characterized in future investigations on the effects of PM.

Methylation Linear Discriminant Analysis (MLDA) for identifying differentially methylated CpG islands

BackgroundHypermethylation of promoter CpG islands is strongly correlated to transcriptional gene silencing and epigenetic maintenance of the silenced state. As well as its role in tumor development, CpG island methylation contributes to the acquisition of resistance to chemotherapy. Differential Methylation Hybridisation (DMH) is one technique used for genome-wide DNA methylation analysis. The study of such microarray data sets should ideally account for the specific biological features of DNA methylation and the non-symmetrical distribution of the ratios of unmethylated and methylated sequences hybridised on the array. We have therefore developed a novel algorithm tailored to this type of data, Methylation Linear Discriminant Analysis (MLDA).ResultsMLDA was programmed in R (version 2.7.0) and the package is available at CRAN [1]. This approach utilizes linear regression models of non-normalised hybridisation data to define methylation status. Log-transformed signal intensities of unmethylated controls on the microarray are used as a reference. The signal intensities of DNA samples digested with methylation sensitive restriction enzymes and mock digested are then transformed to the likelihood of a locus being methylated using this reference. We tested the ability of MLDA to identify loci differentially methylated as analysed by DMH between cisplatin sensitive and resistant ovarian cancer cell lines. MLDA identified 115 differentially methylated loci and 23 out of 26 of these loci have been independently validated by Methylation Specific PCR and/or bisulphite pyrosequencing.ConclusionMLDA has advantages for analyzing methylation data from CpG island microarrays, since there is a clear rational for the definition of methylation status, it uses DMH data without between-group normalisation and is less influenced by cross-hybridisation of loci. The MLDA algorithm successfully identified differentially methylated loci between two classes of samples analysed by DMH using CpG island microarrays.

JAK2 Mutations are present in all cases of polycythemia vera

The JAK2V617F point mutation has been described in 65 to 97% of patients with polycythemia vera (PV). Previously, 17 phenotypical cases of PV from a large group of patients from three institutions were initially reported as JAK2V617F negative. These cases were re-analyzed in detail to determine the cause, if any, of the JAK2V617F negativity. Reasons for this initial negativity included inaccurate clinical diagnosis, relatively insensitive laboratory techniques, insufficient material for re-testing, and possible treatment effect. It was predicted that when tested appropriately, a JAK2V617F mutation would be found in all new cases of PV. We developed a highly sensitive amplification refractory mutation system assay (ARMS) for the detection of JAK2V617F. The analytic sensitivity of the assay is 0.05-0.1% as defined by mixing JAK2V617F-containing HEL cells with normal peripheral blood mononuclear cells. At this level of sensitivity, the assay has a false positive rate of less than 1% (we found 1 positive case in 300 individuals without myeloid disorders). Pyrosequencing involves synthetic nucleotide extension by DNA polymerase. Unlike ARMS, the PCR pyrosequencing method reliably detects JAK2V617F only when the mutant allele is more than 5%. The sensitivity of the ARMS assay is therefore approximately 50 times greater than the pyrosequencing method. Of 105 PV cases diagnosed by Polycythemia Vera Study Group criteria, pyrosequencing detected the mutation in 96 cases. The ARMS technique detected JAK2V617F alleles in 8 of the remaining 9 cases. Of the 8, 2 had been treated by phlebotomy only, 4 had been on interferon for a median of 13 years, and 2 on imatinib for 3 years. In the last patient, who was negative by both assays, the JAK2 exon 12 deletion (E543-D544) was detected. This patient had been on imatinib for 3 years and remains in complete remission. We conclude that a sensitive assay is required to detect the JAK2 mutations in patients with polycythemia vera, especially in patients whose JAK2 mutation load might be affected by treatment.

JAK2 Mutations Are Present in All Cases of Polycythemia Vera.

The JAK2V617Fpoint mutation has been described in 65 to 97% of patients with polycythemia vera (PV). Previously, 17 phenotypical cases of PV from a large group of patients from three institutions were initially reported as JAK2V617Fnegative. These cases were re-analyzed in detail to determine the cause, if any, of the JAK2V617Fnegativity. Reasons for this initial negativity included inaccurate clinical diagnosis, relatively insensitive laboratory techniques, insufficient material for re-testing, and possible treatment effect. It was predicted that when tested appropriately, a JAK2V617Fmutation would be found in all new cases of PV. We developed a highly sensitive amplification refractory mutation system assay (ARMS) for the detection of JAK2V617F. The analytic sensitivity of the assay is 0.05-0.1% as defined by mixing JAK2V617F-containing HEL cells with normal peripheral blood mononuclear cells. At this level of sensitivity, the assay has a false positive rate of less than 1% (we found 1 positive case in 300 individuals without myeloid disorders). Pyrosequencing involves synthetic nucleotide extension by DNA polymerase. Unlike ARMS, the PCR pyrosequencing method reliably detects JAK2V617Fonly when the mutant allele is more than 5%. The sensitivity of the ARMS assay is therefore approximately 50 times greater than the pyrosequencing method. Of 105 PV cases diagnosed by Polycythemia Vera Study Group criteria, pyrosequencing detected the mutation in 96 cases. The ARMS technique detected JAK2V617Falleles in 8 of the remaining 9 cases. Of the 8, 2 had been treated by phlebotomy only, 4 had been on interferon for a median of 13 years, and 2 on imatinib for 3 years. In the last patient, who was negative by both assays, the JAK2 exon 12 deletion (E543-D544) was detected. This patient had been on imatinib for 3 years and remains in complete remission. We conclude that a sensitive assay is required to detect the JAK2 mutations in patients with polycythemia vera, especially in patients whose JAK2 mutation load might be affected by treatment.

Evaluation of a New Assay in Comparison with Reverse Hybridization and Sequencing Methods for Hepatitis C Virus Genotyping Targeting Both 5′ Noncoding and Nonstructural 5b Genomic Regions

We report the evaluation of a new real-time PCR assay for hepatitis C virus (HCV) genotyping. The assay design is such that genotype 1 isolates are typed by amplification targeting the nonstructural 5b (NS5b) subgenomic region. Non-genotype 1 isolates are typed by type-specific amplicon detection in the 5' noncoding region (5'NC) (method 1; HCV genotyping analyte-specific reagent assay). This method was compared with 5'NC reverse hybridization (method 2; InnoLiPA HCV II) and 5'NC sequencing (method 3; Trugene HCV 5'NC). Two hundred ninety-five sera were tested by method 1; 223 of them were also typed by method 2 and 89 by method 3. Sequencing and phylogenetic analysis of an NS5b fragment were used to resolve discrepant results. Suspected multiple-genotype infections were confirmed by PCR cloning and pyrosequencing. Even though a 2% rate of indeterminates was obtained with method 1, concordance at the genotype level with results with methods 2 and 3 was high. Among eight discordant results, five mixed infections were confirmed. Genotype 1 subtyping efficiencies were 100%, 77%, and 74% for methods 1, 2, and 3, respectively; there were 11/101 discordants between methods 1 and 2 (method 1 was predominantly correct) and 2/34 between methods 2 and 3. Regarding genotype 2, subtyping efficiencies were 100%, 45%, and 92% by methods 1, 2, and 3, respectively; NS5b sequencing of discordants (16/17) revealed a putative new subtype within genotype 2 and that most subtype calls were not correct. Although only sequencing-based methods provide the possibility of identifying new variants, the real-time PCR method is rapid, straightforward, and simple to interpret, thus providing a good single-step alternative to more-time-consuming assays.

Multiple strand displacement amplification of DNA isolated from human archival plasma/serum: Identification of cytokine polymorphism by pyrosequencing analysis

BackgroundDNA isolation from formalin-fixed paraffin-embedded tissue appears to be problematic due to degradation caused by fixative. Our aim was to investigate if the isolated genomic DNA from archival plasma/serum, combined with multiple strand displacement amplification (MDA) can be used for genotyping. MethodsNine archival plasma/serum samples and freshly frozen gastric biopsies from the same nine H. pylori-infected subjects were used for DNA isolation. Subsequently, MDA-DNA derived from the plasma/serum samples and DNA isolated from the antrum biopsies were analyzed by PCR amplification and pyrosequencing for the presence of interleukin-1beta gene (IL-1B) single nucleotide polymorphism (SNP). In addition, Southern blot and pyrosequencing analysis of H. pylori-specific PCR amplicons were performed. ResultsIL-1B SNP profiles obtained from the plasma/serum MDA-DNA and antrum biopsy DNA were identical. A C/C genotype was observed in 7 of 9 samples, and 2 of 9 revealed a C/T genotype for IL-1B −511. Similarly, 7 of 9 had a T/T, and 2 of 9 had a C/T genotype for IL-1B −31; 4 of 9 had a C/C, 4 of 9 had a C/T, and 1 of 9 had a T/T genotype, respectively, for IL-1B +3954. Moreover, pyrosequencing analysis revealed the presence of H. pylori 26695 and J99-like 16S rDNA variable V3 region sequence motifs in the antrum biopsies but not in the plasma/serum samples. ConclusionsWe conclude that MDA combined with pyrosequencing enables a rapid and accurate molecular typing of cytokine single nucleotide polymorphisms from archival plasma/serum samples.