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At least 703 records · Page 39Linked to original sources
PubMed2006

High-density single nucleotide polymorphism screen in a large multiplex neural tube defect family refines linkage to loci at 7p21.1-pter and 2q33.1-q35.

BACKGROUND: Neural tube defects (NTDs) are considered complex, with both genetic and environmental factors implicated. To date, no major causative genes have been identified in humans despite several investigations. The first genomewide screen in NTDs demonstrated evidence of linkage to chromosomes 7 and 10. This screen included 44 multiplex families and consisted of 402 microsatellite markers spaced approximately 10 cM apart. Further investigation of the genomic screen data identified a single large multiplex family, pedigree 8776, as primarily driving the linkage results on chromosome 7. METHODS: To investigate this family more thoroughly, a high-density single nucleotide polymorphism (SNP) screen was performed. Two-point and multipoint linkage analyses were performed using both parametric and nonparametric methods. RESULTS: For both the microsatellite and SNP markers, linkage analysis suggested the involvement of a locus or loci proximal to the telomeric regions of chromosomes 2q and 7p, with both regions generating a LOD* score of 3.0 using a nonparametric identity by descent relative sharing method. CONCLUSIONS: The regions with the strongest evidence for linkage map proximal to the telomeres on these two chromosomes. In addition to mutations and/or variants in a major gene, these loci may harbor a microdeletion and/or translocation; potentially, polygenic factors may also be involved. This single family may be promising for narrowing the search for NTD susceptibility genes.

Chromosomes, Human, Pair 2↗
PubMed2006

Multiplex analysis of cytokines in exhaled breath condensate.

BACKGROUND: To improve monitoring of lung diseases, we analyzed cytokines in exhaled breath condensate (EBC). The main challenge in measurement of cytokines in EBC is the low protein content, which requires concentration steps that conflict with the need for excessive fluid required by most commonly used kits. METHODS: Here, a multiplex bead array for the detection of interleukins (IL) -1beta, -6, -8, -10, TNF-alpha, and IL-12p70 was modified and validated for analysis in EBC samples. Furthermore, 33 healthy volunteers and 11 patients with acute lung injury were investigated. RESULTS: In patients with inflammatory lung diseases, cytokine levels for all investigated cytokines were higher in comparison to healthy smokers or healthy volunteers. DISCUSSION: Multiplexed immunoassays in highly sensitive approaches allow for cytokine detection in EBC. We found significant differences between patients and controls for all investigated cytokines.

Adult↗
PubMed2006

Multiplex analysis of circulating cytokines in the sera of patients with different clinical forms of visceral leishmaniasis.

BACKGROUND: The clinical spectrum of visceral leishmaniasis (VL), a chronic intracellular parasitic disease, ranges from a subclinical, asymptomatic infection to severe clinical disease (kala-azar). In experimental leishmaniasis, mice that have a Th1 response to infection tend to have limited disease while a Th2 response is associated with disease progression. Humans with VL most often have mixed rather than polarized responses. However, most clinical studies have used methods that require a relatively large sample volume, thus limiting their scope. Measuring multiple cytokine levels in blood samples using a multiplexed microsphere assay (MMA) may be useful to further evaluate the Th1/Th2 paradigm in humans. METHODS: Bangladeshi individuals (n=120) living in an area endemic for VL were categorized into one of the five clinical categories. Sera from these individuals were measured for levels of IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IFN-gamma, and TNF-alpha by multiplexed microsphere cytokine immunoassay. RESULTS: Circulating IL-8, IL-10, and IL-12 differed significantly among the clinical groups. Persons with kala-azar demonstrated the highest median levels of IL-8 and IL-10 but lower median levels of IL-12. CONCLUSIONS: The MMA for cytokines is an extremely time-and sample-efficient method for characterizing circulating cytokine levels in visceral leishmaniasis patients.

Adult↗
PubMed2006

Simultaneous detection of porcine proinflammatory cytokines using multiplex flow cytometry by the xMAP technology.

BACKGROUND: Multiplex flow cytometry is in widespread use for detection of cytokines in human samples. However, no report on the measurement of porcine cytokines using this method has previously been published. We report on the detection of the porcine proinflammatory cytokines TNF-alpha, IL-8, and IL-1beta by the xMap-assay for multiplex flow cytometry. METHODS: Commercially available antibodies to porcine cytokines were used as capture antibodies by attaching them to goat anti-mouse IgG coated microspheres with different fluorescent signatures. By the use of biotinylated detection antibodies and SAv-PE the amount of cytokines bound to the spheres were measured. Experiments were performed to determine the limits of detection and the amount of crossreactivity in buffer, serum, and plasma, using spiking with recombinant porcine cytokines. RESULTS: The limit of detection ranged from 0.18 to 12 ng/ml. Generally, the detection limit was higher in serum and plasma, than in buffer. No crossreactivity between reagents was found. CONCLUSIONS: Porcine proinflammatory cytokines can be detected utilizing this method with satisfactory detection limits, and no crossreaction between the reagents involved.

Animals↗
PubMed2004

Multiplex bead array assays for detection of soluble cytokines: comparisons of sensitivity and quantitative values among kits from multiple manufacturers.

BACKGROUND: Multiplex bead array assays permit simultaneous cytometric quantitation of multiple cytokines in solution by capturing these to spectrally distinct beads. Because several manufacturers offer reagents to quantitate the same cytokines on a single instrument, a comparison should be made to determine whether these kits yield similar data and whether these data are comparable to enzyme-linked immunosorbent assay (ELISA). METHODS: This study compared cytokine detection kits by using Luminex 100. Twenty-six serum samples from seven subjects were analyzed for interferon-gamma, interleukins 1beta, 6, and 8, and tumor necrosis factor-alpha by using multiplex kits from LINCO Research, Bio-Rad Laboratories, R&D Systems, and BioSource International. Each assay was performed according to the manufacturers' specifications. Standard curves were generated by using reference concentrations supplied by each manufacturer. ELISAs for interleukin-8 were performed by using kits from R&D and BioSource. RESULTS: Cytokine levels followed similar patterns, although absolute concentrations differed among kits. ELISA and Luminex values for interleukin-8 were similar in kits from the same manufacturer. CONCLUSIONS: Because relative cytokine measurements are often valuable when performed serially, it may be possible to make interlaboratory comparisons by using different kits. When comparison of absolute values is crucial, kits from the same supplier should be used. Within-vendor, bead array, and ELISA values appear comparable.

Cytokines↗
PubMed1998

Multiplex polymerase chain reaction of Alu polymorphic insertions.

Alu sequences are present in humans in excess of 500,000 copies per haploid genome and represent the largest family of short interspersed repetitive elements (SINEs). These mobile genetic elements are ancestrally derived from the 7SL RNA gene and move throughout the genomes of primates by retroposition. Polymorphic Alu insertions have proven to be useful for population studies, paternity determinations and forensic applications. Additionally, a simple polymerase chain reaction (PCR)-based assay has been established to examine these polymorphisms. In the present study, we have applied the technique of multiplex polymerase chain reaction to the Alu polymorphic system. Duplex and triplex PCR reactions were performed for the analysis of five different Alu polymorphic loci in different combinations. This study represents a starting point for further experimentation to improve and eventually optimize Alu multiplex PCR.

Alu Elements↗
PubMed2003

High-throughput screening of kinase inhibitors by multiplex capillary electrophoresis with UV absorption detection.

Protein kinases play a major role in the transformation of cells and are often used as molecular targets for the new generation of anticancer drugs. We present a novel technique for high-throughput screening of inhibitors of protein kinases. The technique involves the use of multiplexed capillary electrophoresis (CE) for the rapid separation of the peptides, phosphopeptides, and various inhibitors. By means of UV detection, diversified peptides with native amino acid sequences and their phosphorylated counterparts can be directly analyzed without the need for radioactive or fluorescence labeling. The effects of different inhibitors and their IC(50) value were determined using three different situations involving the use of a single purified kinase, two purified kinases, and crude cell extracts, respectively. The results suggest that multiplexed CE/UV may prove to be a straightforward and general approach for high-throughput screening of compound libraries to find potent and selective inhibitors of the various protein kinases.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed2004

Systematic multiplex polymerase chain reaction and reverse transcription-polymerase chain reaction analyses of changes in copy number and expression of proto-oncogenes and tumor suppressor genes in cancer tissues and cell lines.

Systematic multiplex reverse transcription-polymerase chain reaction (SM RT-PCR) is distinguishable from other multiplex RT-PCR methods by (i) utilization of primers that amplify sequences that fall within a single exon of the genes, (ii) utilization of genomic DNA as a calibration standard, and (iii) optimized PCR conditions that allow amplification of bands of similar intensity using genomic DNA template. We previously developed the human experimental systems of 68 glycosyltransferase genes, 39 Hox genes, and 26 integrin subunit genes, and analyzed the expression of those genes in human adult tissues. Here we report the establishment of an SM RT-PCR system of proto-oncogenes and tumor suppressor genes and the analysis of gene expression in human cancer tissues and cell lines. We also demonstrate that the SM RT-PCR system, which was developed for cDNA expression analysis, could also be used successfully for more exquisite analysis of copy number changes in genomic DNA. We observed a decrease in band intensity of HRAS, TP73, CDKN2A, and CDKN2B genes in most of the breast and prostate cancer cell lines examined. The decrease in copy number of HRAS proto-oncogene leads us to suspect the presence of tumor suppressor genes in the vicinity of this gene on chromosome 11p15.5.

Cell Line, Tumor↗
PubMed2005

Multiplex multidimensional nanoLC-MS system for targeted proteomic analyses.

The present work describes a dual-column and dual-sprayer LC-MS system for high-throughput proteomic analyses. This system consists of two precolumns for sample desalting and two analytical columns. Each column is terminated by a nanoelectrospray emitter mounted on a robotic arm enabling their sequential positioning in front of the sampling cone of the mass spectrometer. The effluent from each emitter is recorded in separate acquisition channels without detectable crosstalk. Gradient elution to both nanoLC columns is delivered by a single HPLC system via a flow splitter. The reproducibility of retention time and peak intensity of the present multiplex system were comparable to those obtainable using a single emitter configuration. Replicate injections of complex tryptic digests (n = 10) indicated that this system provided good reproducibility of retention time and peak intensity on both columns with RSD values of less than 0.9 and 18.6%, respectively. The application of this system is demonstrated for the monitoring of protein expression changes in U937 human monocyte cells with and without phorbol ester administration. Furthermore, we also demonstrated the use of this multiplex system in a 2-D LC configuration to increase sample loading and throughput for the analysis of biomarker samples of higher complexity. Variations in peptide abundance down to two-fold change were identified across salt fractions for spiked tryptic digests present at a level of 50 fmol in 1.5 microg of plasma samples.

Animals↗
PubMed2006

Scanning copy number and gene expression on the 16p13.3-13.2 chromosomal region by the systematic multiplex polymerase chain reaction and reverse transcription-polymerase chain reaction methods.

We developed the systematic multiplex reverse transcription-PCR (SM RT-PCR) method that is distinguishable from other multiplex RT-PCR methods by optimized PCR conditions allowing amplification of sequences that fall within a single exon of genes of similar band intensity using genomic DNA template as a calibration standard. Using an SM RT-PCR system of proto-oncogenes and tumor suppressor genes, we previously showed that the SM RT-PCR system, which was developed for cDNA expression analysis, could also be used for a more exquisite analysis of copy number changes in genomic DNA. Here we report that the SM PCR method semiquantitatively detected less than a two-fold difference in copy number. Furthermore, we also report the results of subchromosomal scanning of copy number and expression using the SM PCR and SM RT-PCR methods. We identified and characterized the novel homozygous deletion that spans over 12-plus genes on 16p13.3-13.2 in the MDA-MB-468 breast cancer cell line.

Chromosomes, Human, Pair 16↗
PubMed2006

Multiplex primer extension analysis for rapid detection of major European mitochondrial haplogroups.

The evolution of the human mitochondrial genome is reflected in the existence of ethnically distinct lineages or haplogroups. Alterations of mitochondrial DNA (mtDNA) have been instrumental in studies of human phylogeny, in population genetics, and in molecular medicine to link pathological mutations to a variety of human diseases of complex etiology. For each of these applications, rapid and cost effective assays for mtDNA haplogrouping are invaluable. Here we describe a hierarchical system for mtDNA haplogrouping that combines multiplex PCR amplifications, multiplex single-base primer extensions, and CE for analyzing ten haplogroup-diagnostic mitochondrial single nucleotide polymorphisms. Using this rapid and cost-effective mtDNA genotyping method, we were able to show that within a large, randomly selected cohort of healthy Austrians (n = 1172), mtDNAs could be assigned to all nine major European haplogroups. Forty-four percent belonged to haplogroup H, the most frequent haplogroup in European Caucasian populations. The other major haplogroups identified were U (15.4%), J (11.8%), T (8.2%) and K (5.1%). The frequencies of haplogroups in Austria is within the range observed for other European countries. Our method may be suitable for mitochondrial genotyping of samples from large-scale epidemiology studies and for identifying markers of genetic susceptibility.

Austria↗
PubMed1989

Genetic analysis of IDDM: the GAW5 multiplex family dataset.

In a collaborative effort by 12 centers from Europe and North America, data were assembled from 94 multiplex families with insulin-dependent diabetes mellitus (IDDM) for analysis of genetic and other factors of possible etiological importance. The dataset contains information on the following genetic markers: HLA-DR beta and -DQ beta restriction fragment length polymorphisms (RFLPs), three RFLPs detected with two probes that map 5' to the insulin gene, the serologically defined HLA loci, and the immunoglobulin allotypes. Data also were included for auto-antibodies to insulin and pancreatic islet cells as possible indicators of pathogenesis and for antibodies to certain viruses that have been implicated as "triggering" agents in IDDM. Medical history of family members was obtained by means of a uniform questionnaire. Identical copies of the dataset were distributed to anyone wishing to participate in the analysis for the IDDM component of GAW5. The multiplex IDDM family dataset is now available on request for further analysis.

Adolescent↗
PubMed2002

A modified multiplex PCR assay for detection of large deletions in MSH2 and MLH1.

A method for detection of large genomic deletions in the MSH2 and MLH1 genes based on multiplex PCR and quantitative evaluation of PCR products is presented. All 35 exons of MSH2 and MLH1 were screened simultaneously in seven PCR reactions, each of them including primers for both genes. The method is reliable for uncovering large genomic deletions in patients suspected of HNPCC. With this method, six novel deletions were identified, two in MSH2: EX1_10del and EX1_16del (representing deletion of the entire MSH2 gene); and four in MLH1: EX1_10del in two unrelated patients, EX3_5del, and EX4del. The deletions were detected in 18 unrelated patients in whom no germline mutation had been identified by SSCP and DHPLC. These results indicate that our modified multiplex PCR assay is suited for the detection of large deletions both in the MSH2 and MLH1 gene and therefore represents an additional valuable tool for mutation screening in HNPCC families.

Adaptor Proteins, Signal Transducing↗
PubMed2003

A multiplex methylation PCR assay for identification of uniparental disomy of chromosome 7.

Uniparental disomy of chromosome 7 (UPD7) is associated with abnormal phenotypic effects because of inappropriate expression of imprinted genes on chromosome 7. Based on the differential methylation of the promoter region of the imprinted PEG1/MEST locus at 7q32, we designed a multiplex methylation PCR (mPCR) assay to rapidly distinguish UPD7 from biparental inheritance of chromosome 7. Primers were designed to produce different sized PCR amplicons based on the parent of origin-specific methylation at this locus; electrophoresis of PCR amplicons showed a 189-bp product from the methylated maternal allele and a 109-bp product from the unmethylated paternal allele. This mPCR assay correctly predicted the chromosome 7 imprinting status in normal control and UPD7 samples. Previous assays for UPD7 required genotyping of the proband and parents, or separate maternal- and paternal-specific mPCR reactions. The advantage of this assay is that parental samples are not required and that amplification of both alleles in the same reaction is simpler and provides an internal control. This multiplex mPCR assay will be useful in screening for UPD7 in patients with Silver-Russell syndrome (SRS; also Russell-Sliver syndrome, RSS), primordial growth retardation, and in patients with supernumerary marker chromosomes or chromosome rearrangements of chromosome 7 origin.

Alleles↗
PubMed2003

Evaluation of multiplex capillary heteroduplex analysis: a rapid and sensitive mutation screening technique.

Bardet-Biedl syndrome (BBS) is a heterogeneous disease; to date seven loci have been mapped and five identified (BBS1, BBS2, BBS4, BBS6, and BBS7). Inheritance in some families is complex with multiallelic participation making linkage analysis difficult. Previous mutation screens have been carried out by direct sequencing but with an increasing number of patients to be screened for five relatively large genes, a more rapid and cost-effective mutation assay for BBS was required. We have adapted the technique of heteroduplex analysis for use on the MegaBACE 1000, a capillary-based DNA fragment analyser, to improve the resolution and sensitivity of the system. Twelve known alterations (insertions, deletions, missenses, and SNPs) in BBS1, BBS2, BBS4, and BBS6 were used to test the sensitivity of the assay and subsequently used to screen new patients for mutations. We achieved a 100% detection rate while dramatically increasing the sample throughput by virtue of multiplexing up to six PCR products in each capillary. In addition, four novel variants were identified: two in BBS2 [c.522T>A (p.D174E) and c.805-20A>G] and two in BBS4 [c.332+27_28insA and c.1414A>G (p.M472V)]. Compared with sequencing and alternative screening methods, multiplex capillary heteroduplex analysis (MCHA) is extremely cost effective. Hum Mutat 22:151-157, 2003.

Bardet-Biedl Syndrome↗
PubMed2004

Subtelomeric deletions detected in patients with idiopathic mental retardation using multiplex ligation-dependent probe amplification (MLPA).

Subtelomeric rearrangements are responsible for 5% to 10% of cases of unexplained mental retardation. Despite their clinical relevance, methods to screen for these cytogenetically invisible abnormalities on a routine base are scarce. We screened patients with idiopathic mental retardation for subtelomeric aberrations using multiplex ligation-dependent probe amplification (MLPA). This recently developed technique is based on PCR amplification of ligated probes hybridized to chromosome ends. Currently, 41 telomeres can be screened in just two multiplex reactions. Four subtelomeric rearrangements (5.3%) were detected in a group of 75 patients with mild to severe mental retardation in combination with dysmorphic features and/or a familial history of mental retardation: two terminal 1p deletions, a terminal 1q deletion, and a terminal 3p deletion. Deletions could be verified by FISH and marker analysis. In one case the MLPA indicated a terminal 21q deletion due to a 3-bp deletion at the site of the probe, giving a false-positive rate of 1.3%. This study demonstrates that MLPA is a fast and reliable screening method, potentially suitable for use in routine diagnostics.

Adolescent↗
PubMed1995

Multiplex PCR analysis and genotype-phenotype correlations of frequent APC mutations.

Germline mutations of the adenomatous polyposis coli (APC) gene tend to cluster in discrete regions. Some of these mutations occur frequently in familial adenomatous polyposis coli (FAP) patients, and strategies for genetic diagnosis of the disease should include simple methods for their detection. We studied a total of 48 FAP-affected or "at-risk" members from 31 unrelated FAP pedigrees. Unrelated patients were analyzed using heteroduplex analysis on agarose minigels (HAAM) and multiplex allele-specific PCR. This novel strategy readily and reliably detected the three frequently occurring APC deletions at codons 1061, 1068, and 1309, allowing identification of mutant alleles in nine unrelated patients. A targeted mutational analysis, based on HAAM and amplification refractory mutation system (ARMS), allowed the rapid identification of 11 additional subjects with germline deletions, among relatives of the patients in whom mutations had been detected by multiplex PCR and HAAM. The use of two independent PCR-based tests, employing distinct sets of primers, reduces the possibility that artifacts occurring during DNA amplification may interfere with the diagnostic evaluation. The analysis of genotype-phenotype correlations provided evidence for heterogeneity with regard to the extent of colonic and extracolonic manifestations of the disease in subjects bearing identical mutations. However, the consistent association of the deletion at codon 1309 with more severe colonic disease than that observed in patients with mutations at codons 1061 and 1068, supports a correlation between mutation site and penetrance of FAP.

Adenomatous Polyposis Coli↗
PubMed2004

RT-PCR permits simultaneous genotyping of thiopurine S-methyltransferase allelic variants by multiplex induced heteroduplex analysis.

Thiopurine-based drugs are a widely prescribed group of medications. Their tolerance and effectiveness is dependent on an individual's ability to metabolize these compounds. An essential enzyme for the metabolism of these drugs is thiopurine S-methyltransferase (TPMT), whose activity is subject to genetic variation. Genotyping of the most frequent allelic variants in TPMT affords an extremely accurate prediction of the three clinical phenotypes: high, intermediate, and low enzyme activity. One constraint of most genotyping methods is the inability to demonstrate physical linkage between two sequence variants that occur in different exons, e.g., c.460G>A and c.719A>G, which give rise to TPMT*3, the most common defective allele in Caucasian populations. Using mRNA/cDNA as a template enables analysis of both sequence variants in a single assay. This approach could be applicable to other genes where allelic variation (in-cis and in-trans) is due to alterations in different exons. Induced heteroduplex generator analysis has previously been shown to discriminate in-cis and has also been suitable for multiplexing. In this method we have exploited both these features and for the first time have applied them to a RT-PCR analysis. The primary reagent developed allows unequivocal resolution of TPMT*3A and the alleles carrying the c.719A>G allelic variant, TPMT*3C, as well as the silent alteration c.474T>C. The TPMT*3B variant has not been observed. A secondary reagent, which can be multiplexed, identifies the TPMT*2 allele.

Alleles↗