High-resolution Agarose Gel Electrophoresis Research Articles

UGT1A1*28 detection using high-resolution agarose gel electrophoresis

A new UGT1A1*28 detection method combining PCR and high-resolution agarose gel electrophoresis was developed. The viability of this method was demonstrated on 15 healthy adult volunteers. Subjects included 13 wild type homozygotes (86.7 %), 2 heterozygotes (13.3 %), and no mutant type homozygotes (0 %). The new UGT1A1*28 detection method results were fully consistent with DNA sequencing. PCR and agarose gel electrophoresis are common techniques with high-resolution agarose gels available commercially. These results support the clinical viability of this method potentially reducing UGT1A1*28 diagnosis complexity and cost.

Monoclonal Gammopathy of Undetermined Significance with Three Bands on Serum Protein Electrophoresis: An Atypical Presentation

Monoclonal Gammopathy (MGM) is a group of B-cell disorders characterised by secretion of abnormal monoclonal Immunoglobulin (Ig) that can be detected by presence of M-protein in serum and urine electrophoresis. Monoclonal protein (M-protein) can be best identified by high resolution agarose gel electrophoresis where, it is usually perceived as a discrete band in the γ or β globulin region. Sometimes atypical banding patterns are seen in serum capillary electrophoresis that pose diagnostic dilemmas and may require repeat testing and further confirmation with a diagnostic test. In the present case, A 60-year-old female patient was advised assessment for gammopathies owing to the presence of skeletal lesions and hyperproteinemia clinically. Serum protein capillary electrophoresis revealed an unusual pattern with three prominent bands in the beta/early gamma region, indicative of triclonal gammopathy, although, it was monoclonal in nature which was depicted during confirmatory diagnostic testing. The authors further discuss the multiple aspects that may be attributed as a cause for such peculiar presentations which should be considered to avoid misdiagnosis.

Efficient synthesis of amino acids capped gold nanoparticles from easily reducible aryldiazonium tetrachloroaurate(III) salts for cellular uptake study.

Biomimetic synthesis of gold nanoparticles (GNPs) is critical in biomedical applications. Gold bioconjugates were fabricated by capping the water-dispersible gold-carbon nanoparticles with tyrosine, tryptophan and cysteine amino acids. Incubation of the water-soluble and easily reducible aryldiazonium gold(III) salt [HOOC-4-C6H4N≡N]AuCl4 with amino acids at room temperature formed a purple color over a few minutes with tryptophan and tyrosine and over two hours with cysteine. Rarely that cysteine is capable of reducing gold(III) precursors; however, a cysteine capped gold bioconjugate was synthesized and characterized in this study. Capping GNPs with amino acids was confirmed by high resolution transmission electron microscopy (TEM) and agarose gel electrophoresis. Depending on the amino acid, synthesized particles size was 27.2 ± 5.4nm, 14.6 ± 7.7nm and 8.6 ± 2.6nm for tyrosine, tryptophan and cysteine, respectively. The amino acids capped GNPs showed negligible cytotoxicity to human dermal normal fibroblast cell lines. The highly water dispersible bioconjugates were studied for in vitro cellular uptake by HeLa cancer cells using confocal laser scan microscopy (CLSM) after being labelled with FITC (GNPs-COOH-FITC) and the nuclei were counter stained with DAPI fluorescent dyes. The biomimetic route for the synthesis of the amino acids reduced gold-carbon nanoparticles will benefit the applications in biomedical devices and biosensors.

Analysis of DNA topology of EBV minichromosomes in HEK 293 cells.

Simian Virus 40 (SV40) and Epstein-Barr Virus (EBV) are frequently used as model systems to study DNA replication. Their genomes are both circular duplex DNAs organized in a single replicon where replication initiates at a precise site upon binding of a specific protein: the large tumor (T) antigen for SV40 and the Epstein-Barr Nuclear Antigen 1 (EBNA-1) for EBV. Despite the abundant information available on the genetics and biochemistry of the replication process in these systems, little is known about the changes in DNA topology that take place as molecules are transfected into eukaryotic cells, assembled into chromatin and bind initiator proteins to start replication. Here we used high-resolution two-dimensional agarose gel electrophoresis to demonstrate that in Human Embryonic Kidney (HEK) 293 cells, minichromosomes of almost the same mass carrying either the SV40 or the EBV replication origin showed similar topological features. The patterns were very similar regardless of the initiator proteins. We also showed that in a hybrid minichromosome, pEco3’Δ, that initiates replication from the SV40 origin, the presence of EBNA-1 and its putative binding to the EBV “family of repeats” induces no significant topological change. These observations challenge the idea that binding of EBNA-1 to oriP could induce negative supercoiling and favor a model suggesting that it binds to oriP in a two-step process where only the second step causes structural changes in a transient cell cycle specific manner.

The abundance of Fob1 modulates the efficiency of rRFBs to stall replication forks.

In eukaryotes, ribosomal genes (rDNA) are organized in tandem repeats localized in one or a few clusters. Each repeat encompasses a transcription unit and a non-transcribed spacer. Replication forks moving in the direction opposite to transcription are blocked at specific sites called replication fork barriers (rRFBs) in the non-transcribed spacer close to the 3′ end of the transcription unit. Here, we investigated and quantified the efficiency of rRFBs in Saccharomyces cerevisiae and to this end transfected budding yeast cells that express dissimilar quantities of Fob1 with circular minichromosomes containing different copies of the minimal 20-bp DNA segment that bind Fob1. To identify fork stalling we used high-resolution 2D agarose gel electrophoresis. The results obtained indicated that neighbor DNA sequences and the relative abundance of Fob1 modulate the efficiency of rRFBs to stall replication forks.

Increased Production Rate of Apolipoprotein(a) is the Primary Mechanism for Increased Lipoprotein(a) Concentration in Patients with Hypercholesterolaemia on Statin Therapy

Background: Elevated plasma concentration of lipoprotein(a) [Lp(a)] are associated with cardiovascular disease (CVD). Understanding the mechanism for the elevation in Lp(a), may be of clinical significance, especially in individuals with elevated Lp(a) concentrations. Aim: To determine the kinetic parameters of Lp(a)–apo(a) in individuals with high and low plasma Lp(a) concentrations (HLPA and LLPA, respectively). Methods: In a cross-sectional study, we recruited 14 subjects with high Lp(a) and 14 subjects with low Lp(a). Deuterated leucine (5 mg/kg) was administered to the subject. Lp(a) was isolated using magnetic beads/antibody, apo(a) was isolated using gel electrophoresis and enrichment measured using gas chromatography mass spectrometry. Lp(a) concentration in plasma was determined by a monoclonal antibody based ELISA and apo(a) isoform sizes were determined by high resolution agarose gel electrophoresis followed by immunoblotting. Results: Lp(a) concentration was significantly higher in HLPA subjects compared with LLPA (319.13 ± 28.00 vs 24.62 ± 8.59 nmol/L, p < 0.001). Apo(a) production rate (PR) was also significantly greater in HLPA subjects (195.89 ± 33.00 vs 6.93 ± 2.27 nmol/kg/day, p < 0.001). Apo(a) isoform size, expressed as number of apo(a) kringle 4 domains, was significantly larger in LLPA, compared with HLPA subjects (23.00 ± 2.54 vs 17.00 ± 0.29, p < 0.02). A high correlation was found between Lp(a) concentration and apo(a) PR (r = 0.95, p < 0.001). Conclusion: Apo(a) PR is the primary determinant of Lp(a) concentration in subjects with hypercholesterolaemia. This observation is consistent with the concept that Lp(a) isoform, which is determined genetically, determines apo(a) PR and hence Lp(a) concentration.

APOE Variants E2, E3, and E4 Can Be Miscalled By Classical PCR-RFLP When The Christchurch Variant Is Also Present.

The APOE Christchurch (APOECh) is a rare variant (c.543C>A) in codon 154. It was first described in an E2 patient with type III dyslipidemia, and thus initially called E2Ch. Its prevalence and the lipid profile of carriers remain unclear. E2, E3, and E4 screening for the APOE gene was performed by PCR-RFLP. The rare APOECh variant was firstly found after detecting an unexpected 109 base-pair band in the high-resolution agarose gel electrophoresis leading to a genotype misinterpretation: the presence of APOECh alters the restriction-bands pattern. To confirm the Ch variant, a second PCR-RFLP method was specifically designed to detect this variant and Sanger sequencing was also performed for all positive samples. We identified 12 unrelated subjects for the APOECh among a cohort of 2,560 patients: nine E3/E3Ch, two E3Ch/E4, and one E2/E3Ch or E2Ch/E3. The frequency of the variant is 0.4% in our study population, which represents the highest percentage published so far. If there is a 109 bp band, it is easy to recognize the presence of the variant. However, in APOE routine genotyping, an E4Ch allele is indistinguishable from a standard E3. Therefore, E4Ch alleles might be underrepresented in the results. We recommend APOE exon 4 sequencing to unequivocally detect the common three variants E2, E3, and E4 and the rare variants as well, to find out the real role they play in atherosclerosis and to estimate its real frequency which is nowadays unclear, in part by the small number of cases identified.

A Case of Multiple Myeloma with Unusual Serum Protein Electrophoresis

Monoclonal gammopathy is a group of B-cell disorders resulting in the secretion of a specific and unique monoclonal immunoglobulin (M-component); best detecting with high resolution agarose gel electrophoresis. An M-protein is usually visible as a localized band on agarose gel electrophoretic peak in the beta, gamma, or rarely in the alpha-2globulin region of the densitometer tracing. Here, we presented a multiple myeloma patient with IgA kappa paraprotein showing an M spike in the alpha-2 globulin region in agarose gel electrophoresis.Pulse Vol.8 January-December 2015 p.77-80

Recombinant VWF Promotes Platelet Adhesion Under Shear Stress Dependent on Its Multimer Size

Baxter has developed BAX 111, a recombinant von Willebrand factor (rVWF) currently being evaluated for the treatment of von Willebrand disease based on the results of a phase 3 clinical trial. BAX 111 contains ultra-large molecular weight multimers (ULMWM), which are the most hemostatically active type of multimer. ULMWM demonstrate higher functional activity than the multimers present in commercially available plasma-derived VWF (pdVWF) products in terms of VWF:RCo and VWF:CB activity. To investigate the contribution of VWF multimer size on BAX 111 hemostatic activity, fractions containing distinct portions of VWF multimers were generated and analyzed for their ability to mediate platelet adhesion under shear stress in vitro.rVWF fractions of varying VWF multimer size were purified by size-exclusion chromatography; size distribution was determined using low and high resolution agarose gel electrophoresis. A parallel-plate perfusion chamber system was used to investigate the rVWF-mediated adhesion of platelets from human blood to fibrillar collagen type I under shear stress. For this purpose, a VWF sample of 1.0 IU/mL VWF:Ag was added to healthy donor blood and the time course of surface coverage was determined by microscopy using fluorescent labeled platelets. Multiple pictures were taken every 5 seconds. Perfusion was performed for 3 minutes at +37°C and at a defined wall shear rate of 1500 s-1. Different batches of BAX 111, rVWF fractions of distinct multimer size, and three commercially available pdVWF products with low and high FVIII content were investigated.The endogenous VWF present in human whole blood mediated initial platelet adhesion with approximately 5% surface coverage after 120 seconds of perfusion. Additionally spiking human blood with 1.0 IU/mL of BAX 111 or rVWF fractions of different multimeric size resulted in increased, time-dependent platelet binding. The amount of platelets that became attached to collagen was clearly dependent on the size of rVWF. While highly multimerized BAX 111 effectively promoted platelet binding, lower-sized rVWF fractions containing VWF multimers of lower molecular weight showed decreased platelet adhesion properties. pdVWF products lacking ultra-large molecular weight multimers were less effective in mediating platelet adhesion to collagen, as observed by delayed platelet binding kinetics. After 60 seconds of perfusion, approximately twice the surface coverage was measured for BAX 111 compared to pdVWF products. The amount of FVIII contained in the pdVWF products had no influence on platelet adhesion.These results demonstrate that the improved hemostatic activity of BAX 111 over pdVWF is due to the presence of ULMWM and confirms their importance in mediating intact platelet binding. DisclosuresSchrenk:Baxter Innovations GmbH, Vienna, Austria: Employment. Schreiner:Baxter Innovations GmbH, Vienna, Austria: Employment. Gritsch:Baxter Innovations GmbH, Vienna, Austria: Employment. Turecek:Baxter Innovations GmbH, Vienna, Austria: Employment. Dockal:Baxter Innovations GmbH, Vienna, Austria: Employment. Scheiflinger:Baxter Innovations GmbH, Vienna, Austria: Employment.

Telomerase activity in the various regions of mouse brain: non-radioactive telomerase repeat amplification protocol (TRAP) assay.

Telomerase, a ribonucleoprotein, is responsible for maintaining the telomere length and therefore promoting genomic integrity, proliferation, and lifespan. In addition, telomerase protects the mitochondria from oxidative stress and confers resistance to apoptosis, suggesting its possible importance for the surviving of non-mitotic, highly active cells such as neurons. We previously demonstrated the ability of novel telomerase activators to increase telomerase activity and expression in the various mouse brain regions and to protect motor neurons cells from oxidative stress. These results strengthen the notion that telomerase is involved in the protection of neurons from various lesions. To underline the role of telomerase in the brain, we here compare the activity of telomerase in male and female mouse brain and its dependence on age. TRAP assay is a standard method for detecting telomerase activity in various tissues or cell lines. Here we demonstrate the analysis of telomerase activity in three regions of the mouse brain by non-denaturing protein extraction using CHAPS lysis buffer followed by modification of the standard TRAP assay. In this 2-step assay, endogenous telomerase elongates a specific telomerase substrate (TS primer) by adding TTAGGG 6 bp repeats (telomerase reaction). The telomerase reaction products are amplified by PCR reaction creating a DNA ladder of 6 bp increments. The analysis of the DNA ladder is made by 4.5% high resolution agarose gel electrophoresis followed by staining with highly sensitive nucleic acid stain. Compared to the traditional TRAP assay that utilize (32)P labeled radioactive dCTP's for DNA detection and polyacrylamide gel electrophoresis for resolving the DNA ladder, this protocol offers a non-toxic time saving TRAP assay for evaluating telomerase activity in the mouse brain, demonstrating the ability to detect differences in telomerase activity in the various female and male mouse brain region.

Topoisomerase 2 is dispensable for the replication and segregation of small yeast artificial chromosomes (YACs).

DNA topoisomerases are thought to play a critical role in transcription, replication and recombination as well as in the condensation and segregation of sister duplexes during cell division. Here, we used high-resolution two-dimensional agarose gel electrophoresis to study the replication intermediates and final products of small circular and linear minichromosomes of Saccharomyces cerevisiae in the presence and absence of DNA topoisomerase 2. The results obtained confirmed that whereas for circular minichromosomes, catenated sister duplexes accumulated in the absence of topoisomerase 2, linear YACs were able to replicate and segregate regardless of this topoisomerase. The patterns of replication intermediates for circular and linear YACs displayed significant differences suggesting that DNA supercoiling might play a key role in the modulation of replication fork progression. Altogether, this data supports the notion that for linear chromosomes the torsional tension generated by transcription and replication dissipates freely throughout the telomeres.

Evaluation of high resolution gel β2-transferrin for detection of cerebrospinal fluid leak

Cerebrospinal fluid (CSF) leaks are potentially life-threatening conditions that can be diagnosed by detection of β(2)-transferrin using protein electrophoresis. Another less commonly available test is β-trace protein quantitation using immunoassay. The objectives of this study were to evaluate a new immunofixation-based β(2)-transferrin test for detection of CSF leaks and to compare it to an existing agarose gel electrophoresis test and β-trace protein immunoassay. For method comparison, 63 consecutive samples from physician-ordered β(2)-transferrin tests were analyzed using two different electrophoresis methods, agarose gel fractionation followed by acid-violet staining, and high resolution agarose gel electrophoresis followed by β(2)-transferrin immunofixation. A subset of samples (16/63) were analyzed for β-trace protein. Results were compared against patient chart data for the presence of a CSF leak. Additional studies were performed to assess the stability, detection limit, and analytical specificity of the β(2)-transferrin immunofixation test. The β(2)-transferrin immunofixation test had a sensitivity of 100 % (40/40) and specificity of 71 % (12/17) for detection of CSF leaks. By comparison, the agarose gel test had a sensitivity of 87 % (35/40) and specificity of 94 % (16/17). β-trace protein had a sensitivity of 100 % (10/10) and specificity of 86 % (5/6). Serum and saliva could be differentiated from CSF by the β(2)-transferrin immunofixation test based on their migration patterns. However, whole blood samples appeared positive for β(2)-transferrin at a threshold of ~ 4 g/L hemoglobin. At a cut-off of 3 mg/L, β-trace protein was increased in 10/10 cases with documented CSF leak and in 1/6 patients without CSF leak. Both the new immunofixation test for β(2)-transferrin and the β-trace protein were effective at detecting CSF leaks. Users of the β(2)-transferrin immunofixation test should be cautioned against interpreting samples with blood contamination.

Topo IV is the topoisomerase that knots and unknots sister duplexes during DNA replication

DNA topology plays a crucial role in all living cells. In prokaryotes, negative supercoiling is required to initiate replication and either negative or positive supercoiling assists decatenation. The role of DNA knots, however, remains a mystery. Knots are very harmful for cells if not removed efficiently, but DNA molecules become knotted in vivo. If knots are deleterious, why then does DNA become knotted? Here, we used classical genetics, high-resolution 2D agarose gel electrophoresis and atomic force microscopy to show that topoisomerase IV (Topo IV), one of the two type-II DNA topoisomerases in bacteria, is responsible for the knotting and unknotting of sister duplexes during DNA replication. We propose that when progression of the replication forks is impaired, sister duplexes become loosely intertwined. Under these conditions, Topo IV inadvertently makes the strand passages that lead to the formation of knots and removes them later on to allow their correct segregation.

Association of Apo(a)isoform size with dyslipoproteinemia in male Venous Thrombosis patients

Background Lp(a) is a proatherogenic lipoprotein that may also be prothrombotic. Apo(a) size isoforms have differential effects on fibrinolysis. Whereas Lp(a) concentrations have been linked to venous thromboembolic disease (VTE) risk, apo(a) polymorphisms in VTE have not been studied. Methods We used a standardized high resolution agarose gel electrophoresis technique to determine apo(a) isoform size, and a Lp(a) immunoassay insensitive to apo(a) size to measure Lp(a) concentration in 46 men with VTE and 46 age-matched healthy controls. Results Apo(a) isoform distribution in VTE cases and controls was bimodal and VTE patients tended to have more medium-sized isoforms K 4-(19-27) (54.3% vs. 34.8%, p = 0.06). Cases and controls had the same median predominant apo(a) size isoform (23.5 K 4 repeats) and comparable Lp(a) concentrations. However, subgroup analysis based on apo(a) isoform size (K 4 ≤ 23 or K 4 ≥ 24) revealed that cases in the K 4 ≥ 24 subgroup had higher Lp(a) concentrations than the controls in this isofrom subgroup (14.5 mmol vs. 6.6 mmol, p = 0.029). Also, dyslipoproteinemia (smaller LDL and HDL particles, higher LDL and lower HDL parameters) was strongly associated with VTE only in this larger apo(a) isoform group. Conclusions These observations provide the first evidence that determination of apo(a) isoforms may provide useful novel insights into VTE risk.

Fast short-fragment PCR for rapid and sensitive detection of shrimp viruses

This article describes a fast short-fragment PCR method for the detection of white spot syndrome virus (WSSV), infectious hypodermal and hematopoietic necrosis virus (IHHNV), and monodon baculovirus (MBV). Fast two-temperature (95 °C denaturation and 60 °C annealing/extension) PCRs were performed in 5–10 μl volume samples in miniaturized microplates using a fast Peltier thermal cycler. 40 cycles were completed in 25–30 min. Rapid high-resolution agarose gel electrophoresis of 70–150 bp PCR fragments was performed in 10 min. High sensitivity of PCR product detection (50–100 pg) was obtained using ultra sensitive dyes such as GelStar ® and a gel documentation system equipped with a blue-light transilluminator. This novel method is faster and more sensitive than its TaqMan ® real-time PCR counterparts.

Interplay of DNA supercoiling and catenation during the segregation of sister duplexes

The discrete regulation of supercoiling, catenation and knotting by DNA topoisomerases is well documented both in vivo and in vitro, but the interplay between them is still poorly understood. Here we studied DNA catenanes of bacterial plasmids arising as a result of DNA replication in Escherichia coli cells whose topoisomerase IV activity was inhibited. We combined high-resolution two-dimensional agarose gel electrophoresis with numerical simulations in order to better understand the relationship between the negative supercoiling of DNA generated by DNA gyrase and the DNA interlinking resulting from replication of circular DNA molecules. We showed that in those replication intermediates formed in vivo, catenation and negative supercoiling compete with each other. In interlinked molecules with high catenation numbers negative supercoiling is greatly limited. However, when interlinking decreases, as required for the segregation of newly replicated sister duplexes, their negative supercoiling increases. This observation indicates that negative supercoiling plays an active role during progressive decatenation of newly replicated DNA molecules in vivo.

Identification of Amyloidogenic Light Chains Requires the Combination of Serum-Free Light Chain Assay with Immunofixation of Serum and Urine

The diagnosis of systemic immunoglobulin light-chain (AL) amyloidosis requires demonstration of amyloid deposits in a tissue biopsy and amyloidogenic monoclonal light chains. The optimal strategy to identify the amyloidogenic clone has not been established. We prospectively assessed the diagnostic sensitivity of the serum free light chain (FLC) kappa/lambda ratio, a commercial serum and urine agarose gel electrophoresis immunofixation (IFE), and the high-resolution agarose gel electrophoresis immunofixation (HR-IFE) developed at our referral center in patients with AL amyloidosis, in whom the amyloidogenic light chain was unequivocally identified in the amyloid deposits. The amyloidogenic light chain was identified in 121 consecutive patients with AL amyloidosis by immunoelectron microscopy analysis of abdominal fat aspirates and/or organ biopsies. We characterized the monoclonal light chain by using IFE and HR-IFE in serum and urine and the FLC kappa/lambda ratio in serum. We then compared the diagnostic sensitivities of the 3 assays. The HR-IFE of serum and urine identified the amyloidogenic light chain in all 115 patients with a monoclonal gammopathy. Six patients with a biclonal gammopathy were omitted from the statistical analysis. The diagnostic sensitivity of commercial serum and urine IFE was greater than that of the FLC kappa/lambda ratio (96% vs 76%). The combination of serum IFE and the FLC assay detected the amyloidogenic light chain in 96% of patients. The combination of IFE of both serum and urine with the FLC kappa/lambda ratio had a 100% sensitivity. The identification of amyloidogenic light chains cannot rely on a single test and requires the combination of a commercially available FLC assay with immunofixation of both serum and urine.

Familial frontotemporal dementia with neuronal intranuclear inclusions is not a polyglutamine expansion disease

BackgroundMany cases of frontotemporal dementia (FTD) are familial, often with an autosomal dominant pattern of inheritance. Some are due to a mutation in the tau- encoding gene, on chromosome 17, and show an accumulation of abnormal tau in brain tissue (FTDP-17T). Most of the remaining familial cases do not exhibit tau pathology, but display neuropathology similar to patients with dementia and motor neuron disease, characterized by the presence of ubiquitin-immunoreactive (ub-ir), dystrophic neurites and neuronal cytoplasmic inclusions in the neocortex and hippocampus (FTLD-U). Recently, we described a subset of patients with familial FTD with autopsy-proven FTLD-U pathology and with the additional finding of ub-ir neuronal intranuclear inclusions (NII). NII are a characteristic feature of several other neurodegenerative conditions for which the genetic basis is abnormal expansion of a polyglutamine-encoding trinucleotide repeat region. The genetic basis of familial FTLD-U is currently not known, however the presence of NII suggests that a subset of cases may represent a polyglutamine expansion disease.MethodsWe studied DNA and post mortem brain tissue from 5 affected members of 4 different families with NII and one affected individual with familial FTLD-U without NII. Patient DNA was screened for CAA/CAG trinucleotide expansion in a set of candidate genes identified using a genome-wide computational approach. Genes containing CAA/CAG trinucleotide repeats encoding at least five glutamines were examined (n = 63), including the nine genes currently known to be associated with human disease. CAA/CAG tract sizes were compared with published normal values (where available) and with those of healthy controls (n = 94). High-resolution agarose gel electrophoresis was used to measure allele size (number of CAA/CAG repeats). For any alleles estimated to be equal to or larger than the maximum measured in the control population, the CAA/CAG tract length was confirmed by capillary electrophoresis. In addition, immunohistochemistry using a monoclonal antibody that recognizes proteins containing expanded polyglutamines (1C2) was performed on sections of post mortem brain tissue from subjects with NII.ResultsNo significant polyglutamine-encoding repeat expansions were identified in the DNA from any of our FTLD-U patients. NII in the FTLD-U cases showed no 1C2 immunoreactivity.ConclusionWe find no evidence to suggest that autosomal dominant FTLD-U with NII is a polyglutamine expansion disease.

Measurement of serum monoclonal components: comparison between densitometry and capillary zone electrophoresis

Quantitative measurement of serum monoclonal protein (M-protein) is one of the most important tools for monitoring disease activity in monoclonal gammopathies. The aims of this study were to evaluate serum M-protein quantification by capillary zone electrophoresis (CZE) and to compare results with those obtained by densitometric scanning of high-resolution agarose gel electrophoresis (HRE-AGE). The evaluation was carried out on 82 samples from patients with various monoclonal gammopathies. All the suspected M-proteins were confirmed and characterised by immunofixation on agarose gel (IFE). CZE was performed on a Paragon CZE 2000 system (Beckman Coulter). Passing-Bablok regression was: y (CZE)=1.27 x(HRE-AGE)-5.21 g/L. The correlation coefficient was 0.92. Bland-Altman analysis demonstrated a mean difference of -1.83 g/L (95% CI -0.76 to -2.90) with clear evidence of a concentration-related bias. Densitometry gave higher values at low M-spikes (<20 g/L), whereas CZE gave higher values at large M-spikes (>20 g/L). The concentration-related bias was found to be independent of the immunoglobulin isotype. In conclusion, to compare previous results obtained by M-protein densitometric scanning with those obtained by direct measurement of CZE peaks, the calculation of a univocal transforming factor appears to be unreliable.

Polymorphisms in Genes of Folate Metabolism and Response to High-Dose Methotrexate in Patients with Primary Central Nervous System Lymphoma.

The antifolate methotrexate (MTX) was shown to be the single most-effective agent in first-line treatment of patients with primary central nervous system lymphoma (PCNSL) when given intravenously in high doses. MTX inhibits 5,10-methylentetrahydrofolate reductase (MTHFR) as well as thymidylate synthase (TS) in target cells what eventually results in decreased DNA synthesis. Intracellular uptake of both folate and MTX is mediated by the reduced folate carrier (RFC). Genetic polymorphisms for all three proteins were described: a C to T base transition at nucleotide 667 (C677T) and an A to C transition at 1298 (A1298C) of MTHFR; a 28-base pair (bp) tandemly repeated sequence polymorphic in the numbers of the repeat (2R/2R; 3R/3R; and 2R/3R) in the 5′-UTR of TS; a 6bp insertion-(+6bp)/deletion(−6bp)-polymorphism in the 3′-UTR of TS; and a polymorphism at nucleotide 80 (G80A) of RFC. We hypothesized that these polymorphisms may be directly linked to response, survival and toxicity in patients with PCNSL who are treated with single high-dose MTX. We prospectively collected blood from 152 subjects who were enrolled into a German multicenter PCNSL trial. They were scheduled to receive HD-MTX 4 g/m2 every 2 weeks. Genomic DNA was extracted from mononuclear cells using QIAGEN DNA Blood Mini Kit®. Genotyping of the two MTHFR (C677T; A1298C) and the RFC (G80A) single nucleotide polymorphisms (SNPs) was performed by melting point analysis with Master Hybridization Probes® using the Light Cycler® technology. Genotyping of the TS 28bp tandem/triplet repeats in the 5′-UTR was performed by conventional PCR followed by high resolution agarose gel electrophoresis. The TS +6bp/−6bp polymorphism in the 3′-UTR was analyzed with PCR, followed by restriction enzyme digest with draI and agarose gel electrophoresis. The fragment sizes were 70 bp and 88 bp for the 6 bp insertion allele and 152 bp for the 6 bp deletion allele. Allele frequencies for heterozygosity and for homozygous mutations of the two MTHFR and the RFC SNPs as well as of the two TS polymorphisms were as expected for a Caucasian population. For the currently 120 evaluable patients logistic regression analysis, Armitage Trend test and Kaplan-Meier survival curves were performed to analyze the role of the polymorphisms as potential predictors for MTX response. Up to now, we only found a trend for an association between response to treatment and survival for the 2 MTHFR mutations. We will analyze haematological and non-haematological toxicities which will be presented at the meeting with updated results for survival and response in the patients who will have completed treatment until then.