PCR Capillary Electrophoresis Research Articles

A novel genotyping scheme for Vibrio parahaemolyticus with combined use of large variably-presented gene clusters (LVPCs) and variable-number tandem repeats (VNTRs)

A total of 18 variably-presented gene clusters (LVPCs) and nine previously characterized variable-number tandem repeats (VNTRs), and all known virulence markers were screened for their frequency and/or copy number in 251 global strains of Vibrio parahaemolyticus using PCR and gel or capillary electrophoresis. A two-step genotyping approach combining the use of LVPCs and VNTRs was established accordingly. The frequency profiles of LVPCs and virulence markers were primarily used to group the strains into six distinct complexes with different potential pathogenicity natures. The strains from each of these complexes were further analyzed with VNTRs to give a much more detailed discrimination of the strains. A genetic fingerprint-like database of a large collection of strains established with this two-stage approach would be very useful for identification, genotyping, origin tracing, and risk estimation of V. parahaemolyticus.

Gastric cancer and concomitant renal cancer: A systematic immunohistochemical and molecular analysis

The frequency of gastric cancer in patients with renal cell carcinoma (RCC) is exceptionally high in our region suggesting a common molecular basis. Our study aimed to characterize tumors and to analyze possible underlying molecular features in 12 patients with gastric cancer and concomitant RCC. We performed an immunohistochemical analysis including p53 protein expression, proliferative activity (MIB-1), mismatch repair status (hMLH1, hMSH2, hMSH6, PMS2) and E-cadherin expression in gastric cancers, which were additionally analyzed for Epstein-Barr-Encoded-RNA (EBER) by insitu hybridization. Microsatellite instability was analyzed with a PCR multiplex system and capillary electrophoresis. KRAS mutations in codons 12 and 13 were tested by pyrosequencing. All patients had clear cell RCCs, 10 of which were well differentiated and diagnosed in an early stage, while the gastric cancers of these patients were generally poorly or undifferentiated and diagnosed in an advanced stage. Gastric cancers showed reduced E‑cadherin staining in 10 out of 12 cases. Two gastric cancers demonstrated loss of hMLH1 and PMS2, which was confirmed by molecular analysis showing a high degree of microsatellite instability. All RCCs were microsatellite stable. KRAS mutation was detected in one of the two instable gastric cancers, while none of the RCCs had KRAS mutations. Another gastric cancer was positive for EBV. In conclusion, a coherent cause for gastric cancer and concomitant RCC, such as Lynch syndrome, a prominent role of KRAS mutation or EBV infection, was not found in our series. Other factors leading to a higher susceptibility for cancer must be explored to explain why individuals with RCC have a higher risk of developing gastric cancer in our region.

Fragment length analysis screening for detection of CEBPA mutations in intermediate-risk karyotype acute myeloid leukemia

During last years, molecular markers have been increased as prognostic factors routinely screened in acute myeloid leukemia (AML). Recently, an increasing interest has been reported in introducing to clinical practice screening for mutations in the CCAAT/enhancer-binding protein α (CEBPA) gene in AML, as it seems to be a good prognostic factor. However, there is no reliable established method for assessing CEBPA mutations during the diagnostic work-up of AMLs. We describe here a straightforward and reliable fragment analysis method based in PCR capillary electrophoresis (PCR-CE) for screening of CEBPA mutations; moreover, we present the results obtained in 151 intermediate-risk karyotype AML patients (aged 16-80 years). The method gave a specificity of 100% and sensitivity of 93% with a lower detection limit of 1-5% for CEBPA mutations. The series found 19 mutations and four polymorphisms in 12 patients, seven of whom (58%) presented two mutations. The overall frequency of CEBPA mutations in AML was 8% (n = 12). CEBPA mutations showed no coincidence with FLT3-ITD or NPM1 mutations. CEBPA mutation predicted better disease-free survival in the group of patients without FLT3-ITD, NPM, or both genes mutated (HR 3.6, IC 95%; 1.0-13.2, p = 0.05) and better overall survival in patients younger than 65 of this group without molecular markers (HR 4.0, IC 95%; 1.0-17.4, p = 0.05). In conclusion, the fragment analysis method based in PCR-CE is a rapid, specific, and sensitive method for CEBPA mutation screening and our results confirm that CEBPA mutations can identify a subgroup of patients with favorable prognosis in AML with intermediate-risk karyotype.

Augmentation of Sensitivity of FLT3/ITD Assay Allows Detection of Minimal Residual Disease In Stem Cell Transplant Recipients-Correlation with Flow Cytometric MRD Assessment

AbstractAbstract 1717FLT3 internal tandem duplication (FLT3/ITD) is a somatic mutation that is associated with therapy resistance in acute myeloid leukemia (AML). Early data demonstrated low sensitivity for this assay, thus limiting its utility to the evaluation of diagnostic specimens, and precluding its utility in remission samples. We inquired whether the standard FLT3/ITD assay can be modified to enable its utility to detect presence of residual disease in remission specimens. Enhanced FLT3/ITD assay sensitivity was accomplished by altering annealing temperature, increasing the number of cycles as well as amount and concentration of the product that was subjected to capillary electropheresis. To assess the sensitivity of the enhanced assay, FLT3/ITD positive cells M4V11 were serially diluted in a population of ITD negative cells (HL60). The concentration of M4V11 cells in each sample ranged from 10% to 0.0001%. PCR product was subjected to capillary electropheresis and the appropriate region of the electropherogram was examined for the presence of the appropriate mutant product length. Appropriate FLT3/ITD signal was detected in dilutions down to 0.01%, validating our ability to detect extremely low levels of FLT3/ITD. We subsequently examined the remission marrows from patients with a history of FLT3/ITD who had undergone stem cell transplantation. Available bone marrow specimens (N = 51) from patients who underwent stem cell transplantation for FLT3/ITD-positive AML were analyzed and the result was correlated with the available standard PCR as well as the available MRD assessment by muti-dimensional flow cytometry; samples negative for FLT3/ITD by standard assay (N=11) were then subjected to the enhanced PCR methodology. Available ITD length for each patient was used for examination of the appropriate region of the electropherogram in each case. Of the available 51 bone marrow specimens analyzed, 23 specimens had FLT3/ITD detectable by standard PCR protocol. Using our modified PCR method and capillary electrophoresis, an additional 13 specimens had identifiable FLT3/ITD. In 6/11 patients, where initial FLT3/ITD was negative by standard methodology, enhanced assay identified FLT3/ITD signal. In each case, detection of FLT3/ITD by the enhanced assay was followed by morphologic or immunophenotypic emergence of disease, prompting therapeutic intervention. We further evaluated the ability to detect FLT3/ITD in patients with minimal residual disease by flow cytometry. 33 of the bone marrow specimens analyzed had a less than 5% abnormal blast population as detectable via flow cytometry. Among these samples, 7 had FLT3/ITD detectable using standard detection techniques. An additional 11 samples had detectable FLT3/ITD when our modified protocol was employed. Of the specimens that had less than 1% abnormal blast population as detectable via flow cytometry (N = 27), 4 had FLT3/ITD detectable using the standard detection assay; when our modified protocol was employed, an additional 6 samples had detectable FLT3/ITD. 17 bone marrow specimens had no abnormal blast cells detectable via flow cytometry; of these samples 1 had detectable FLT3/ITD using the standard detection assay, while an additional 3 had detectable FLT3/ITD using our modified assay. In four patients, FLT3/ITD was detected in bone marrow specimens found to have flow cytometric MRD of 0% (N=2), 0.1% (N=1) and 0.4% (N=1). In two patients with no detectable disease by MDF, both had emergence of morphologic (60% blast) or immunophenotypic disease by MDF (1.1%) within 4–6 weeks of detection of FLT3/ITD by enhanced assay. In this study, we demonstrate that simple modifications to the FLT3/ITD genotyping assay significantly increases its sensitivity and provides a highly sensitive and very specific assay for identifying this disease associated mutation in remission specimens. The enhanced assay can be incorporated into the standard evaluation of remission status for patients with FLT3/ITD. Disclosures:No relevant conflicts of interest to declare.

Non-Invasive Prenatal Detection of Trisomy 21 Using Tandem Single Nucleotide Polymorphisms

BackgroundScreening tests for Trisomy 21 (T21), also known as Down syndrome, are routinely performed for the majority of pregnant women. However, current tests rely on either evaluating non-specific markers, which lead to false negative and false positive results, or on invasive tests, which while highly accurate, are expensive and carry a risk of fetal loss. We outline a novel, rapid, highly sensitive, and targeted approach to non-invasively detect fetal T21 using maternal plasma DNA.Methods and FindingsHighly heterozygous tandem Single Nucleotide Polymorphism (SNP) sequences on chromosome 21 were analyzed using High-Fidelity PCR and Cycling Temperature Capillary Electrophoresis (CTCE). This approach was used to blindly analyze plasma DNA obtained from peripheral blood from 40 high risk pregnant women, in adherence to a Medical College of Wisconsin Institutional Review Board approved protocol. Tandem SNP sequences were informative when the mother was heterozygous and a third paternal haplotype was present, permitting a quantitative comparison between the maternally inherited haplotype and the paternally inherited haplotype to infer fetal chromosomal dosage by calculating a Haplotype Ratio (HR). 27 subjects were assessable; 13 subjects were not informative due to either low DNA yield or were not informative at the tandem SNP sequences examined. All results were confirmed by a procedure (amniocentesis/CVS) or at postnatal follow-up. Twenty subjects were identified as carrying a disomy 21 fetus (with two copies of chromosome 21) and seven subjects were identified as carrying a T21 fetus. The sensitivity and the specificity of the assay was 100% when HR values lying between 3/5 and 5/3 were used as a threshold for normal subjects.ConclusionsIn summary, a targeted approach, based on calculation of Haplotype Ratios from tandem SNP sequences combined with a sensitive and quantitative DNA measurement technology can be used to accurately detect fetal T21 in maternal plasma when sufficient fetal DNA is present in maternal plasma.

Copy number variation of trinucleotide repeat in dynamic mutation sites of autosomal dominant cerebellar ataxias related genes

To standardize the experimental procedure of the gene test for autosomal dominant cerebellar ataxias (ADCA), and provide the basis for quantitative criteria of the dynamic mutation of spinocerebellar ataxia (SCA) genes in Chinese population. Genotyping of the dynamic mutation loci of the SCA1, SCA2, SCA3, SCA6 and SCA7 genes was performed, using florescence PCR-capillary electrophoresis followed by DNA sequencing, to investigate the variation range of copy number of CAG tandem repeat of the genes in 263 probands of ADCA pedigrees and 261 non-related normal controls. Based on the sequencing result, the bias of the CAG copy number estimation using capillary electrophoresis with different DNA controls was compared to analyze the technical detailes of the electrophresis method in testing the dynamic mutation sites. PCR products containing dynamic mutation loci of the SCA genes showed significantly higher mobility than that of molecular weigh marker with relatively balanced GC content. This was particularly obvious in the SCA2, SCA 6 and SCA7 genes whereas the deviation of copy number could be corrected to +/-1 when known CAG copy number fragments were used as controls. The mobility of PCR products was primarily related to the copy number of CAG repeat when the fragments contained normal CAG repeat. In the 263 ADCA pedigrees, 6 (2.28%) carried SCA1 gene mutation, 8 (3.04%) had SCA2 mutation and 81 (30.80%) harbored SCA3 mutation. The gene mutation of SCA6 and SCA7 was not found. The normal variation range of the CAG repeat was 17-36 copies in SCA1 gene, 13-30 copies in SCA2, 14-39 copies in SCA3, 6-16 copies in SCA6 and 6-13 copies in SCA7. The heterozygosity was 76.1%, 17.7%, 74.4%, 72.1% and 41.3%, respectively. The mutation range of the CAG repeat was 49-56 copies in SCA1 gene, 36-41 copies in SCA2, 59-81 copies in SCA3. Neither homozygous mutation of an SCA gene nor double heterozygous mutation of the SCA genes was observed in the study. The copy number of the CAG repeat in SCA genes could be calculated accurately based on the result of florescence PCR-capillary electrophoresis when limited amount of known repeat copy number controls were used. Our result supported that the notion that SCA3 gene mutation was the most common cause for ADCA, and the obtained data would be helpful for establishing quantitative criteria of the dynamic mutation of the SCA genes in Chinese.

Adverse Prognostic Impact of FLT3 Internal Tandem Duplication (ITD) Is Age-Associated in Older [≥60 Years (Y)] De Novo cytogenetically Normal Acute Myeloid Leukemia (CN-AML) Patients (Pts): a Cancer and Leukemia Group B (CALGB) Study.

Abstract 1579▪▪This icon denotes an abstract that is clinically relevant.Poster Board I-605While the prognostic role of FLT3 ITD has been validated in younger CN-AML adults (<60 y), its association with outcome has not been fully investigated in older pts. We studied the frequency and the clinical impact of FLT3 ITD in a cohort of older pts ≥60 y ([n=243; 111 (46%) aged ≥70 y] with de novo CN-AML. Pts were treated on CALGB protocols [10201, 9720, 9420, 8923 and 8525] with cytarabine/anthracycline-based induction and cytarabine-based consolidation. Diagnostic samples were assessed by quantitative fluorescence-based PCR capillary electrophoresis for FLT3 ITD and tyrosine kinase domain (TKD) mutations, and by DHPLC/sequencing analysis for NPM1 and WT1 mutations. Of the 243 pts, 78 (32%) presented with FLT3 ITD, 24 (10%) with FLT3 TKD and 6 (2%) had both. Outcome analyses were restricted to comparison of FLT3 wild-type (WT) pts (n=147) with those with FLT3 ITD alone (n=72). Of these pts, 121 (55%) had NPM1 mutations and 15 (7%) had WT1 mutations. Unfortunately, only a subset of these pts also had available material for BAALC and ERG measurements, thereby preventing analysis of the prognostic significance of FLT3 ITD in the context of a panel of variables that included these markers. Compared with FLT3 WT pts, FLT3 ITD pts had higher WBC counts and % bone marrow and blood blasts (P<.001, all), and higher frequencies of NPM1 (74% v 46%; P<.001) and WT1 mutations (15% v 3%; P=.001). Complete remission (CR) rates were similar, but disease-free (DFS) and overall survival (OS) were shorter in FLT3 ITD v FLT3 WT pts (Table 1). In multivariable analyses (MVA), FLT3 ITD associated with shorter DFS and OS (Table 2). The FLT3 ITD prognostic impact was associated with age. FLT3 ITD pts aged 60-69 y had shorter DFS and OS than FLT3 WT pts, whereas clinical outcomes were not different for ≥70 y FLT3 ITD v FLT3 WT pts (Table 1). In MVA for the 60-69 y subgroup, pts with FLT3 ITD had shorter DFS and OS (Table 2). The reasons for this age-associated effect remain to be explained. In previous studies of younger CN-AML, a higher (≥ median) FLT3 ITD:WT allelic ratio (AR) was associated with worse clinical outcome. In the current study, FLT3 ITD had an adverse prognostic impact on the 60-69 y pts and no significant impact on the ≥70 y pts regardless of the AR levels. However, a 27-microRNA (miR) signature differentiating between FLT3 ITD and FLT3 WT pts and characterized by >2-fold higher miR-155 expression in FLT3 ITD pts, was associated with shorter DFS and OS in the 60-69 y pt subgroup (P=.001, each) but not in the ≥70 y subgroup (P=.26 and P=.89, respectively), suggesting an age-associated prognostic role of the miRs. In summary, our data show FLT3 ITD is an independent marker for poor outcome in CN-AML pts aged 60-69 y but not in those aged ≥70 y. Although the ≥70 y pts with FLT3 ITD had a seemingly better prognosis than the corresponding 60-69 y pts, the outcome for both groups is poor and novel treatment approaches are needed in older pts.Table 1Outcomes in older CN-AML pts with and without FLT3ITDOverall60-69 y Pts≥70 y PtsFLT3 ITD (n=72)FLT3 WT (n=147)PFLT3 ITD (n=41)FLT3 WT (n=78)PFLT3 ITD (n=31)FLT3 WT (n=69)P% achieving CR67%70%.6471%75%.6761%65%.82DFS % disease-free at 3 y10%18%.0077%19%<.00116%18%.94OS % alive at 3 y14%23%<.00110%26%<.00119%20%.71Table 2Variables in Final MVA Models for DFS and OSOverall60-69 y PtsDFSOSDFSOSHR*PHR*PHR*PHR*PFLT3 ITD, positive v negative2.10<.001†1.97<.0012.94<.001†2.79<.001NPM1, mutated v wild-type0.59.0050.54<.001––0.62.021WBC, continuous, 50 unit increase1.44.028†––––––Hemoglobin, continuous1.27.045†––1.50.018†––*HRs <1 (>1) indicate lower (higher) risk for an event for the first category listed for the dichotomous variables and for the higher values of the continuous variables. Variables considered in the models were those significant at á=0.20 in univariable analyses.†Variable did not meet the proportional hazards assumption, a covariate was used to account for time dependence. DisclosuresStone:Cephalon: ad hoc consultancy; Novartis: Research Funding, ad hoc consultancy.

Genotype

Cutaneous melanoma is characterized by poor patient outcome in its later stages. The search for genetic markers is therefore crucial for the identification of populations at risk for melanoma. Highly polymorphic CA repeats in 3' proximity in the MANBA gene were examined by PCR-capillary electrophoresis in 185 Swedish melanoma patients and 441 tumor-free age- and gender-matched individuals. The associations of the polymorphisms with melanoma risk, the pigment phenotypes of the patients and tumor characteristics were analyzed. A significant difference in allelic distribution between melanoma patients and tumor-free individuals was observed. The frequency of the MANBA genotype <21CAs/≥21CAs was significantly higher in melanoma patients than in the controls. When comparing allele distribution in patients and their matched controls, the allele <21CAs was found to be associated with the female gender (39.8 vs. 31.2%, P=0.041, OR=1.46, 95% CI 1.02-2.10), but not with male gender (34.4 vs. 30.9%, P%0.39). Within the melanoma group, there were no differences in the distribution of the MANBA alleles associated with patient gender or age before or after 55 years at diagnosis, nor was there any association between the MANBA genotype and pigment phenotype or tumor sites. The MANBA allele <21CAs was, however, associated with thin melanomas at diagnosis (Breslow thickness ≤1.5 mm and Clark levels I and II). In conclusion, these data suggest that MANBA polymorphisms might be an indicator of tumor growth and progression and, together with other markers, could be used to identify individuals at increased risk of melanoma.

Multiplex PCR–capillary electrophoresis–SSCP used to identify foodborne pathogens

Multiplex PCR–CE–SSCP approach was used to identify foodborne pathogens. 16srRNA system including three target fragments localized at different position of 16srRNA gene and gyrB system including two target fragments at gyrB gene were established. SSCP profile was analyzed in a similar way as RAPD and the identity of each peak was determined by relative position to inner standard. Twenty-five reference bacteria strains representing 2 classes, 12 genus and 19 species were tested. All bacteria could be distinctly determined at genus level by 16srRNA system. GyrB system improved discriminating resolution of some bacterial at species level. Phylogenetic analysis showed that 16srRNA based phylogenetic relationship was consistent with traditional classification of these organisms. Good reproducibility and resolution make multiplex PCR–CE–SSCP protocol a promising approach for fast screening of foodborne pathogens.

Microsatellite instability analysis using BAT-26 marker in patients with gastric or colorectal cancer diagnosed at Hospital Universitario de Santander.

Introduction: The path of microsatellite instability has mainly been found in cancers of digestive tracts, of which 90% of cases are hereditary cancer patients and 15% are patients with sporadic cancer. That instability produces a phenotype called Microsatellite instability (IMI+) or phenotype by replication mistake (RER+) that leads to the accumulation of mutations in higher rates to normal. Objectives: To identify the presence of IMI+ phenotype in patients analyzed and evaluated its relationship with the differentiation histopathologic tumor, because which together have been associated with a better prognosis and improved response to treatment given to the patient. Methodology: DNA was extracted from blood samples (normal cells) and biopsy of the tumor (tumor cells) of the 23 patients who were included in the study and through the amplification by PCR and subsequent capillary electrophoresis was typified the microsatellite mononucleotidic BAT-26, who was employed by his sensitivity to detect IMI+ phenotype (95%). Finally, we made a statistical correlation between the microsatellite instability phenotype, the presence (IMI+) or the absence (IMI-), with histopathological findings using Fisher’s exact test. Results: In 17% of cases (4 patients) had IMI+: 3 of them with colorectal cancer (CRC) -2 cases possibly with hereditary cancer- and one with gastric cancer (GC). There was no association between phenotype IMI+ with the pathological diagnosis, age, sex and differentiation histopathologic tumor. Conclusion: We found the phenotype IMI + in 17% of cases without association with the degree of differentiation histopathologic tumor, possibly by the reduced number of samples analyzed, making it essential to review the methods of fixation, paraffin and storage tissue because current techniques hinder digestion of proteinase k and PCR.

Population-based case-control study of alpha 1-antitrypsin and SLC11A1 in Crohnʼs disease and ulcerative colitis

Crohn's disease (CD) and ulcerative colitis (UC) are chronic inflammatory diseases of the digestive tract. Genetic factors and an abnormal immune response to infections are suspected to be involved in inflammatory bowel diseases. In the present study 300 blood samples from CD patients (n = 100), UC patients (n = 100), and healthy controls (n = 100) were taken from a population-based case-control study. PCR assays and capillary electrophoresis were used to detect alpha 1 antitrypsin M, S, and Z alleles and the C-to-T transition at the -237 nucleotide position of the SLC11A1 promoter. Additionally, length polymorphism of (gt)n alleles in the promoter region and TGTG and CAAA insertion/deletion in the untranslated region (3' UTR) of the SLC11A1 gene were evaluated. The Z allele only for AAT was associated (P < 0.05) with CD. No other significant results were detected for AAT alleles. For SLC11A1, alleles 1 and 2 were significant (P < 0.05) for UC, but only allele 3 was significant (P < 0.05) for CD. There was a significant (P < 0.05) association of a CAAA insertion with CD but not for deletion in the 3' UTR. No differences (P < 0.05) were detected for TAAA. Because AAT and SLC11A1 proteins directly or indirectly function as inhibitors of human leukocyte elastase, mutations in the AAT and SLC11A1 genes may change the balance between elastase produced by leukocytes during phagocytosis.

Prenatal diagnosis of β‐thalassemia by chip‐based capillary electrophoresis

To explore the potential of chip-based capillary electrophoresis for rapid prenatal diagnosis of genetic diseases, we developed a prenatal diagnosis method for beta-thalassemia, one of the most common recessive inherited disorders. DNA samples containing known mutations of beta-thalassemia were used to optimize multiplex allele-specific PCR and chip-based capillary electrophoresis was used to simultaneously detect Cy3-labeled PCR products and Cy5-labeled ladder. This system was validated by detecting 40 unknown cases, 4 of which were processed further for prenatal diagnosis. We analyzed previously genotyped samples and unknown samples by this method and the results were concordant with those from reverse dot blot and direct sequencing. Prenatal diagnosis was carried out successfully and rapidly on 1 microL (1 ng/microL) sample in 4 pregnancy cases carrying potential beta-thalassemia risk. This chip-based capillary electrophoresis detecting system could be used for prenatal diagnosis of beta-thalassemia and will facilitate the rapid and sensitive prenatal diagnosis of genetic disorder.

Large Granular Lymphocyte Leukemia: Clonality Reconsidered.

Background: T-cell large granular lymphocyte (LGL) leukemia is widely considered to represent a monoclonal proliferation of lymphocytes. Clonality assessment methods have evolved from Southern blots (first-generation) to polymerase chain reaction with heteroduplex electrophoresis (second-generation) to high-resolution capillary electrophoresis (third-generation) testing.Aims: To determine if third-generation T-cell clonality assays result in a higher frequency of oligoclonal results, to compare the concordance for testing at the T-cell receptor (TCR) gamma (TCRG) and TCR beta (TCRB) loci, and to compare the clinical characteristics of patients with monoclonal vs. oligoclonal TCRs.Methods: The study population consisted of patients from August 1999-April 2007 with elevated circulating LGLs and cytopenia(s). TCRG locus clonality was determined by both the heteroduplex method and capillary electrophoresis in 35 patients. 89 samples were tested for TCRG and TCRB clonality using the Biomed II PCR primer sets and capillary electrophoresis on an ABI 3100 automated DNA sequencer. Determinations of clonality were made independently by three pathologists blinded to the clinical characteristics of the patients.Results: A total of 93 patients (median age 50 years, 53% female) were evaluated. Median absolute neutrophil count was 1.56 × 109/L (range 0.2–7.8 × 109/L), median lymphocyte count was 1.81 × 109/L (range 0.6–13 × 109/L), and median hemoglobin was 13 g/dL (range 6.3–17.4 g/dL). The concordance rate for TCRG clonality testing by the heteroduplex and capillary electrophoresis methods was only 40%. The primary difference was a striking increase in the frequency of oligoclonal results by the capillary electrophoresis method (p= 0.00007). All of these samples appeared monoclonal by the lower resolution heteroduplex assay (Table 1). Concordance for clonality for TCRG vs. TCRB was 54% (Table 2). All samples had monoclonality or oligoclonality demonstrated at TCRG or TCRB, but only 26% were monoclonal at both loci. The clinical characteristics for the 23 patients with monoclonal TCRG and TCRB appeared similar to the 23 patients with oligoclonal TCRG and TCRB. The median age in both groups was 53 years, with 61% of patients in each group requiring treatment after a median of 36.8 and 38.6 months of follow-up, respectively.Discussion: The high resolution of capillary electrophoresis appears to result in a much greater proportion of oligoclonal TCRG results, which by the older heteroduplex method would have been considered monoclonal. Furthermore, the concordance rate at TCRG and TCRB appears to be remarkably low. Though oligoclonal T-cell populations are generally believed to be transient and reactive processes, the clinical characteristics of our oligoclonal and monoclonal cohorts did not differ significantly.Conclusion: Capillary electrophoresis frequently identifies patients with oligoclonal TCR whose clinical features are indistinguishable from those of patients with classic monoclonal LGL leukemia.HeteroduplexMonoclonalNegativeOligoclonalTotalMonoclonal121114Capillary ElectrophoresisNegative4206Oligoclonal150015Total313135TCRGMonoclonalNegativeOligoclonalTotalMonoclonal2331238TCRBNegative7018Oligoclonal1532543Total4563889

Microfluidic Chips for Detecting the t(4;14) Translocation and Monitoring Disease during Treatment Using Reverse Transcriptase-Polymerase Chain Reaction Analysis of IgH-MMSET Hybrid Transcripts

Diagnosis platforms incorporating low-cost microfluidic chips enable sensitive, rapid, and accurate genetic analysis that could facilitate customized therapies tailored to match the vulnerabilities of any types of cancer. Using ex vivo cancer cells, we have detected the unique molecular signature and a chromosomal translocation in multiple myeloma. Multiple myeloma is characterized by IgH rearrangements and translocations that enable unequivocal identification of malignant cells, detected here with integrated microfluidic chips incorporating genetic amplification via reverse transcriptase-polymerase chain reaction and capillary electrophoresis. On microfluidic chips, we demonstrated accurate and versatile detection of molecular signatures in individual cancer cells, with value for monitoring response to therapy, detecting residual cancer cells that mediate relapse, and evaluating prognosis. Thus, testing for two clinically important molecular biomarkers, the IgH VDJ signature and hybrid transcripts signaling the t(4;14) chro-mosomal translocation, with predictive value in diagnosis, treatment decisions, and monitoring has been efficiently implemented on a miniaturized microfluidic system.

Specific isolation of disseminated cancer cells: a new method permitting sensitive detection of target molecules of diagnostic and therapeutic value

Molecular studies of rare cells, such as circulating cancer cells, require efficient pre-enrichment steps to obtain a pure population of target cells for further characterization. We have developed a two-step approach, starting with immunomagnetic enrichment, followed by specific isolation of individual, easily identifiable bead-rosetted target cells using a new semi-automated CellPick system. With this procedure, 1-50 live target cells can now be isolated. As a model system, we spiked a small number of tumor cells into millions of normal mononuclear cells (MNCs). Efficient isolation of pure target cells was obtained by use of the CellPick system, and the nature of isolated, bead-rosetted cells was verified by use of FISH. Single breast cancer cells were picked directly into an RNA preserving lysis buffer, reverse transcribed, and PCR amplified with two cDNA specific primer sets. With the isolated cells we consistently obtained both ubiquitously expressed and tumor cell specific PCR products. We also performed a successful mutation analysis of single cells using PCR and cycling temperature capillary electrophoresis (CTCE). This may have significant clinical implications in cancer and in other diseases, e.g. in characterizing micrometastatic cancer cells in blood and lymph nodes to help identifying patients who most likely will respond to therapies like tyrosine kinase inhibitors and compounds targeting specific mutations. By use of the CellPick system it is possible to specifically isolate bead-rosetted or otherwise labelled target cells from a heterogeneous cell population for further molecular characterization.

Macrophage migration inhibitory factor polymorphisms do not predict therapeutic response to glucocorticoids or to tumour necrosis factor α-neutralising treatments in rheumatoid arthritis

Background:Macrophage migration inhibitory factor (MIF) is an inflammatory mediator associated with RA severity. In various diseases, MIF polymorphisms are associated with clinical response glucocorticoid (GC) treatment. It is unclear whether...

Octaplex PCR and fluorescence-based capillary electrophoresis for identification of human diarrheagenic Escherichia coli and Shigella spp.

A multiplex PCR assay, amplifying seven specific virulence genes and one internal control gene in a single reaction, was developed to identify the five main pathotypes of diarrheagenic Escherichia coli and Shigella spp. The virulence genes selected for each category were Stx1, Stx2, and eaeA for enterohemorrhagic E. coli (EHEC), eaeA for enteropathogenic E. coli (EPEC), STIb and LTI for enterotoxigenic E. coli (ETEC), ipaH for enteroinvasive E. coli (EIEC) and Shigella spp., and aggR for enteroaggregative E. coli (EAEC). Each forward primer was labelled with a fluorochrome and the PCR products were separated by multicolour capillary electrophoresis on an ABI PRISM™310 Genetic Analyzer (Applied Biosystems). If present, several gene variants of each virulence gene were identified. The internal control gene rrs, encoding 16S rRNA, was amplified in all 110 clinical strains analyzed. Virulence genes were demonstrated in 103 (94%) of these strains. In the majority of the cases (98/103, 95%), classification obtained by the novel multiplex PCR assay was in agreement with that previously determined by phenotypic assays combined with other molecular genetic approaches. Numerous multiplex PCR assays have been published, but only a few of them detect all five E. coli pathotypes within a single reaction, and none of them has used multicolour capillary electrophoresis to separate the PCR products. The octaplex PCR assay followed by capillary electrophoresis presented in the present paper provides a simple, reliable, and rapid procedure that in a single reaction identifies the five main pathotypes of E. coli, and Shigella spp. This assay will replace the previous molecular genetic methods used in our laboratory and work as an important supplement to the more time-consuming phenotypic assays.

Common Y-chromosomal STR database for three closely related European populations

Eighteen Y-chromosomal microsatellite loci (DYS19, DYS388, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS426, DYS437, DYS438, DYS439, DYS460, GATA H4.1, DYS385 a/b, YCAII a/b) were analyzed in 568 males from Belarus, Poland and Slovakia by means of a multiplex (octadecaplex) PCR reaction and capillary electrophoresis in order to compare their usefulness in forensic practice individually and in a haplotype format. Analysis of molecular variance (AMOVA) revealed significant differences between the three populations and excluded one common Y-STR database. Markers responsible for the differences between populations were identified using a chi-square test for homogeneity and locus-by-locus AMOVA. Selection of loci showing homogeneity enabled establishment of a common haplotype database for the studied populations. This approach may become an efficient method for Y-STR databasing for larger ethnogeographic areas as soon as consecutive homogeneity-showing markers are identified.

Minimal Disease Detection and Confirmation in Hematologic Malignancies: Combining Cell Sorting with Clonality Profiling

In this study we demonstrate the technical application of flow cytometry and cell sorting combined with gene-rearrangement clonality profiling to detect and confirm minimal disease in 2 leukemia and 2 lymphoma cases. Specimens with low percentages (0.05%-5%) of abnormal lymphoid populations were identified by flow cytometry. The abnormal lymphoid populations were sorted by flow cytometry, and the purified tumor populations along with unsorted fractions were subsequently analyzed for the presence of clonal gene rearrangements by PCR and fluorescence-based capillary electrophoresis fragment analysis. In 3 cases, distinct clonality profiles could be detected in the purified tumor cell fraction, and suspicious amplicons of identical sizes were detected among the polyclonal backgrounds in the unsorted specimens. For 1 patient, a monoclonal signal was detected in the sorted tumor cell fraction but not in the unseparated bone marrow specimen containing 0.05% abnormal lymphoblasts. A subsequent bone marrow specimen containing 4.8% recurring leukemia cells tested positive with a clonality profile that matched the previous profile in the sorted cell population. The described method integrating 2 technologies allows genotypic confirmation of an aberrant population detected by immunophenotype to increase diagnostic certainty. This strategy provides a sensitive tool for disease monitoring without the need for patient-specific primer design and assay optimization required for quantitative PCR analysis.

Extensive Variation in Intracellular Symbiont Community Composition among Members of a Single Population of the Wood-Boring Bivalve Lyrodus pedicellatus (Bivalvia: Teredinidae)

Shipworms (wood-boring bivalves of the family Teredinidae) harbor in their gills intracellular bacterial symbionts thought to produce enzymes that enable the host to consume cellulose as its primary carbon source. Recently, it was demonstrated that multiple genetically distinct symbiont populations coexist within one shipworm species, Lyrodus pedicellatus. Here we explore the extent to which symbiont communities vary among individuals of this species by quantitatively examining the diversity, abundance, and pattern of occurrence of symbiont ribotypes (unique 16S rRNA sequence types) among specimens drawn from a single laboratory-reared population. A total of 18 ribotypes were identified in two clone libraries generated from gill tissue of (i) a single specimen and (ii) four pooled specimens. Phylogenetic analysis assigned all of the ribotypes to a unique clade within the gamma subgroup of proteobacteria which contained at least five well-supported internal clades (phylotypes). By competitive quantitative PCR and constant denaturant capillary electrophoresis, we estimated the number and abundance of symbiont phylotypes in gill samples of 13 individual shipworm specimens. Phylotype composition varied greatly; however, in all specimens the numerically dominant symbiont belonged to one of two nearly mutually exclusive phylotypes, each of which was detected with similar frequencies among specimens. A third phylotype, containing the culturable symbiont Teredinibacter turnerae, was identified in nearly all specimens, and two additional phylotypes were observed more sporadically. Such extensive variation in ribotype and phylotype composition among host specimens adds to a growing body of evidence that microbial endosymbiont populations may be both complex and dynamic and suggests that such genetic variation should be evaluated with regard to physiological and ecological differentiation.