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W Edward Highsmith

Publications and source records attributed to W Edward Highsmith.

17 recordsLinked to original sources
PubMed2006

Diagnosis of alpha-1-antitrypsin deficiency: An algorithm of quantification, genotyping, and phenotyping.

BACKGROUND: Laboratory testing in suspected alpha-1-antitrypsin (A1AT) deficiency involves analysis of A1AT concentrations and identification of specific alleles by genotyping or phenotyping. The purpose of this study was to define and evaluate a strategy that provides reliable laboratory evaluation of A1AT deficiency. METHODS: Samples from 512 individuals referred for A1AT phenotype analysis were analyzed by quantification, phenotype, and genotype. A1AT concentrations were measured by nephelometry. Phenotype analysis was performed by isoelectric focusing electrophoresis. The genotype assay detected the S and Z deficiency alleles by a melting curve analysis. RESULTS: Of the 512 samples analyzed, 2% of the phenotype and genotype results were discordant. Among these 10 discordant results, 7 were attributed to phenotyping errors. On the basis of these data we formulated an algorithm, according to which we analyzed samples by genotyping and quantification assays, with a reflex to phenotyping when the genotype and quantification results were not concordant. Retrospective analyses demonstrated that 4% of samples submitted for genotype and quantitative analysis were reflexed to phenotyping. Of the reflexed samples, phenotyping confirmed the genotype result in 85% of cases. In the remaining 15%, phenotyping provided further information, including identifying rare deficiency alleles and suggesting the presence of a null allele, and allowed for a more definitive interpretation of the genotype result. CONCLUSIONS: The combination of genotyping and quantification, with a reflex to phenotyping, is the optimal strategy for the laboratory evaluation of A1AT deficiency.

Algorithms↗
PubMed2006

A splice-site mutation in CCM1/KRIT1 is associated with retinal and cerebral cavernous hemangioma.

PURPOSE: To report a case of a unilateral retinal cavernous hemangioma associated with a novel splice-site mutation in CCM1/KRIT1. METHODS: An 11-year-old girl was noted to have an asymptomatic retinal cavernous hemangioma in the left eye. CCM1/KRIT1 was screened for mutations. RESULTS: Genetic evaluation of CCM1/KRIT1 revealed a single guanine-to-cytosine transversion in the invariant splice acceptor consensus sequence of intron 8 (c.1146-1G-->C), which is predicted to result in abnormal protein splicing. CONCLUSIONS: Mutations in CCM1/KRIT1 may be found in asymptomatic patients with retinal cavernous hemangioma.

Ankyrin Repeat↗
PubMed2006

Mutation scanning of the RET protooncogene using high-resolution melting analysis.

BACKGROUND: Single-base pair missense mutations in exons 10, 11, 13, 14, 15, and 16 of the RET protooncogene are associated with the autosomal dominant multiple endocrine neoplasia type 2 (MEN2) syndromes: MEN2A, MEN2B, and familial medullary thyroid carcinoma. The current widely used approach for RET mutation detection is sequencing of the exons. METHODS: Because RET mutations are rare and the majority are heterozygous mutations, we investigated RET mutation detection by high-resolution amplicon melting analysis. This mutation scanning technique uses a saturating double-stranded nucleic acid binding dye, LCGreen, and the high-resolution melter, HR-1, to detect heterozygous and homozygous sequence variations. Mutant genotypes are distinguished from the wild-type genotype by an altered amplicon melting curve shape or position. RESULTS: Samples of 26 unique RET mutations, 4 nonpathogenic polymorphisms, or the wild-type genotype were available for this study. The developed RET mutation-scanning assay differentiated RET sequence variations from the wild-type genotype by altered derivative melting curve shape or position. A blinded study of 80 samples (derived from the 35 mutant, polymorphism, or wild-type samples) demonstrated that 100% of RET sequence variations were differentiated from wild-type samples. For exons 11 and 13, the nonpathogenic polymorphisms could be distinguished from the pathogenic RET mutations. Some RET mutations could be directly genotyped by the mutation scanning assay because of unique derivative melting curve shapes. CONCLUSION: RET high-resolution amplicon melting analysis is a sensitive, closed-tube assay that can detect RET protooncogene sequence variations.

Genotype↗
PubMed2006

Introduction to molecular cystic fibrosis testing.

Technology improvements are rapidly bringing molecular diagnostics into routine laboratories. Recent recommendations for cystic fibrosis carrier testing by the American College of Medical Genetics (ACMG) have led to commercial test kit development and increased testing volumes. Molecular testing of genetic diseases presents a variety of challenges and situations that may be unfamiliar to laboratories with limited molecular genetic experience. We will briefly review the disease and discuss mutation testing indications, methodologies, quality assurance, and reporting issues associated with cystic fibrosis testing.

Cystic Fibrosis↗
PubMed2005

MYH Y165C and G382D mutations in hepatocellular carcinoma and cholangiocarcinoma patients.

PURPOSE: Production of reactive oxygen species (ROS) during chronic inflammation has been implicated in the progression of liver diseases and carcinogenesis. Subjects with inflammatory liver disease and one non-functional allele of the base excision repair gene, MYH, may be more susceptible to progression to cancer due to MYH haploinsufficiency in repairing oxidative damage caused by ROS. Here, we investigated the association of two common germline MYH mutations in patients with hepatocellular carcinoma (HCC) and cholangiocarcinoma. METHODS: DNA from patients with HCC (n=48) or cholangiocarcinoma (n=84) compared to non-cancerous controls (n=308) were genotyped for the Y165C and G382D mutations in MYH. RESULTS: There was no significant difference in MYH mutation carrier status between patients with HCC (1/48), cholangiocarcinoma (3/84), and non-cancerous controls (4/308). CONCLUSIONS: Patients with HCC or cholangiocarcinoma do not have an increased incidence of monoallelic MYH mutations pre-disposing them to disease.

Bile Duct Neoplasms↗
PubMed2005

Denaturing gradient-based two-dimensional gene mutation scanning in a polymer microfluidic network.

An integrated two-dimensional (2-D) DNA separation platform, combining standard gel electrophoresis with temperature gradient gel electrophoresis (TGGE) on a polymer microfluidic chip, is reported. Rather than sequentially sampling DNA fragments eluted from standard gel electrophoresis, size-resolved fragments are simultaneously electrokinetically transferred into an array of orthogonal microchannels and screened for the presence of sequence heterogeneity by TGGE in a parallel and high throughput format. A bulk heater assembly is designed and employed to externally generate a temporal temperature gradient along an array of TGGE channels. Extensive finite element modeling is performed to determine the optimal geometries of the microfluidic network for minimizing analyte band dispersion caused by interconnected channels in the network. A pH-mediated on-chip analyte stacking strategy is employed prior to the parallel TGGE separations to further reduce additional band broadening acquired during the electrokinetic transfer of DNA fragments between the first and second separation dimensions. A comprehensive 2-D DNA separation is completed in less than 5 min for positive detection of single-nucleotide polymorphisms in multiplex PCR products that vary in size and sequence.

DNA↗
PubMed2005

Nine novel germline gene variants in the RET proto-oncogene identified in twelve unrelated cases.

We report nine novel DNA alterations in the RET proto-oncogene in 12 unrelated cases identified by DNA sequencing of exons 10 and 11 of the gene. The novel variants K666E, IVS9-11G-->A, D631V in cis with H665Q, D631E (with C634Y), E623K (in trans with C618S), 616delGAG (in trans with C609Y), Y606C, C630R, and R635-T636insELCR;T636P were detected in patients with various clinical presentations ranging from thyroid goiter, medullary thyroid carcinoma, and pheochromocytoma to classic multiple endocrine neoplasia type 2A. When novel DNA alterations are found, extended family studies can be helpful in determining the clinical significance of such findings. Segregation within families suggests that K666E and T636insELCR;T636P are likely to be disease-causing mutations. However, the mechanism by which they affect the normal activity of the RET receptor is unclear. Absence of segregation with disease was observed for E623K and 616delGAG. For the remainder of the DNA alterations, family studies were not possible, and the clinical significance of these novel variants needs further assessment. Additional case reports, animal models, and/or functional studies are needed to determine the clinical significance of these newly identified variants.

Adult↗
PubMed2005

Biological validation of differentially expressed genes in chronic lymphocytic leukemia identified by applying multiple statistical methods to oligonucleotide microarrays.

Oligonucleotide microarrays are a powerful tool for profiling the expression levels of thousands of genes. Different statistical methods for identifying differentially expressed genes can yield different results. To our knowledge, no experimental test has been performed to decide which method best identifies genes that are truly differentially expressed. We applied three statistical methods (dChip, t-test on log-transformed data, and Wilcoxon test) to identify differentially expressed genes in previously untreated patients with chronic lymphocytic leukemia (CLL). We used a training set of Affymetrix Hu133A microarray data from 11 patients with unmutated immunoglobulin (Ig) heavy chain variable region (VH) genes and 8 patients with mutated Ig VH genes. Differential expression was validated using semiquantitative real-time polymerase chain reaction assays and by validating models to predict the somatic mutation status of an independent test set of nine CLL samples. The methods identified 144 genes that were differentially expressed between cases of CLL with unmutated compared with mutated Ig VH genes. Eighty genes were identified by Wilcoxon test, 60 by t-test, and 65 by dChip, but only 11 were identified by all three methods. Greater agreement was found between the t-test and the Wilcoxon test. Differential expression was validated by semiquantitative real-time polymerase chain reaction assays for 83% of individual genes, regardless of the statistical method. However, the Wilcoxon test gave the most accurate predictions on new samples, and dChip, the least accurate. We found that all three methods were equally good for finding differentially expressed genes, but they found different genes. The genes selected by the nonparametric Wilcoxon test are the most robust for predicting the status of new cases. A comprehensive list of all differentially expressed genes can only be obtained by combining the results of multiple statistical tests.

Algorithms↗
PubMed2004

DNA mutation detection in a polymer microfluidic network using temperature gradient gel electrophoresis.

A miniaturized system for DNA mutation analysis, utilizing temperature gradient gel electrophoresis (TGGE) in a polycarbonate (PC) microfluidic device, is reported. TGGE reveals the presence of sequence heterogeneity in a given heteroduplex sample by introducing a thermal denaturing gradient that results in differences between the average electrophoretic mobilities of DNA sequence variants. Bulk heater assemblies are designed and employed to externally generate temperature gradients in spatial and temporal formats along the separation channels. TGGE analyses of model mutant DNA fragments, each containing a single base substitution, are achieved using both single- and 10-channel parallel measurements in a microfluidic platform. Additionally, a comprehensive polymer microfluidic device containing an integrated microheater and sensor array is developed and demonstrated for performing spatial TGGE for DNA mutation analysis. The device consists of two PC modular substrates mechanically bonded together. One substrate is embossed with microchannels, and the other contains a tapered microheater, lithographically patterned along with an array of temperature sensors. Compared with the external heating approaches, the integrated platform provides significant reduction in power requirement and thermal response time while establishing more accurate and highly effective control of the temperature gradient for achieving improved separation resolution.

DNA Mutational Analysis↗
PubMed2004

Telomerase activity measurement in magnetically captured epithelial cells: comparison of slab-gel and capillary electrophoresis.

We have compared telomerase activity measurements by slab-gel and capillary electrophoresis in cultured cells (A549 and H125 human cancer cell lines) and in cells isolated from clinical peripheral blood specimens epithelial cells of patients with lung and esophageal cancer. Telomerase activity was determined using the telomerase repeat amplification protocol (TRAP) assay with phosphoimager scanning of slab-gels and by laser-induced fluorescence capillary electrophoresis (LIF-CE). Experiments using A549 and H125 cells were performed to determine the reproducibility of each method and to identify the contribution of each stage of the TRAP/polymerase chain reaction (PCR) assay to the variability. In these experiments, it was found that more than half of the overall variability (coefficient of variation, CV = 35%) of the slab-gel method and almost all of the overall variability (CV = 20%) of the CE method was due to the PCR stage of the TRAP assay. In the clinical samples, classification as positive or negative was by visual inspection of the slab-gel and CE electropherograms for the presence of the characteristic 6 base-pair TRAP ladder and by GeneScan analysis of the CE. We examined several criteria including the use of 3, 4, or 5 TRAP bands as the definition of a positive test. Using the slab-gel method, the 5-band criterion gave 40% sensitivity with 100% specificity (no false positives in inactive controls). The CE method yielded a comparable 38% sensitivity and 100% specificity using this criterion. These data indicate that detection of telomerase activity in epithelial cells isolated from peripheral blood has a useful level of sensitivity and specificity and may be useful in the detection and monitoring of aerodigestive cancers. However, analysis by slab-gel is cumbersome and the precision is poor (inter-replicate CV = 20%) compared to LIF-CE (CV = 5%). A high-throughput CE-LIF detection platform will be indispensable for validation studies of telomerase activity measurements.

Electrophoresis, Capillary↗
PubMed2003

High-throughput analysis of telomerase by capillary electrophoresis.

The enzyme telomerase is expressed in (85-90)% of all human cancers, but not in normal, non-stem cell somatic tissues. Clinical assays for telomerase in easily obtained body fluids would have great utility as noninvasive, cost-effective methods for the early detection of cancer. The most commonly used method for the detection and quantification of telomerase enzyme activity is the polymerase chain reaction (PCR)-based assay known as the telomerase repeat amplification protocol or TRAP assay. Most of the TRAP assay systems use a slab-gel based electrophoresis system to size and quantify the PCR-amplified extension products. We are developing high-throughput capillary electrophoresis (CE) methods for the analysis of TRAP/PCR products. The TRAP assay was conducted on lysates of the human lung cancer cell line A-549 in reactions containing 5-100 cells. TRAP/PCR products were generated using a fluorescent 4,7,2'4'5'7',-hexachloro-6-carboxyfluorescein(HEX)-labeled TS primer and analyzed on the Applied Biosystems Model 310 CE system using POP4 polymer. After analysis with GeneScan and Genotyper software, the total peak areas of the TRAP ladder extension products were computed using Microsoft Excel. Results were compared with unlabeled TRAP/PCR products analyzed on the Bio-Rad BioFocus 3000 CE system using 6% high molecular weight polyvinylpyrrolidone (HMW PVP) polymer and SYBR Green I dye. Both CE systems were able to resolve the TRAP ladder products with high reproducibility and sensitivity (5-15 cells). With the appropriate robotic sample handling system, these CE methods would enable performing the telomerase TRAP assay with increased sensitivity, reproducibility and automation over slab-gel methods.

Base Sequence↗
PubMed2002

Phage display technology: clinical applications and recent innovations.

Phage display is a molecular diversity technology that allows the presentation of large peptide and protein libraries on the surface of filamentous phage. Phage display libraries permit the selection of peptides and proteins, including antibodies, with high affinity and specificity for almost any target. A crucial advantage of this technology is the direct link that exists between the experimental phenotype and its encapsulated genotype, which allows the evolution of the selected binders into optimized molecules. Phage display facilitates engineering of antibodies with regard to their size, valency, affinity, and effector functions. The selection of antibodies and peptides from libraries displayed on the surface of filamentous phage has proven significant for routine isolation of peptides and antibodies for diagnostic and therapeutic applications. This review serves as an introduction to phage display, antibody engineering, the development of phage-displayed peptides and antibody fragments into viable diagnostic reagents, and recent trends in display technology.

Animals↗
PubMed2002

Identifying and quantifying sources of variation in microarray data using high-density cDNA membrane arrays.

Microarray experiments involve many steps, including spotting cDNA, extracting RNA, labeling targets, hybridizing, scanning, and analyzing images. Each step introduces variability, confounding our ability to obtain accurate estimates of the biological differences between samples. We ran repeated experiments using high-density cDNA microarray membranes (Research Genetics Human GeneFilters Microarrays Version I) and 33P-labeled targets. Total RNA was extracted from a Burkitt lymphoma cell line (GA-10). We estimated the components of variation coming from: (1) image analysis, (2) exposure time to PhosphorImager screens, (3) differences in membranes, (4) reuse of membranes, and (5) differences in targets prepared from two independent RNA extractions. Variation was assessed qualitatively using a clustering algorithm and quantitatively using a version of ANOVA adapted to multivariate microarray data. The largest contribution to variation came from reusing membranes, which contributed 38% of the total variation. Differences in membranes and in exposure time each contributed about 10%. Differences in target preparations contributed less than 5%. The effect of image quantification was negligible. Much of the effect from reusing membranes was attributable to increasing levels of background radiation and can be reduced by using membranes at most four times. The effects of exposure time, which were partly attributable to variation in the scanning process, can be minimized by using the same exposure time for all experiments.

Algorithms↗
PubMed2002

Telomerase as a diagnostic and therapeutic target for cancer.

Telomerase is a ribonucleoprotein enzyme responsible for the elongation of telomeres at the ends of chromosomes. It is widely expressed in most cancers, while absent from most normal somatic cells. Telomerase is partially responsible for the cellular immortalization that allows human cancers to progress indefinitely. Due to its widespread occurrence in cancer and its crucial role in the maintenance of the tumor, telomerase is an attractive target for cancer diagnosis and treatment.

Animals↗
PubMed2002

Telomerase detection in body fluids.

BACKGROUND: Telomerase is a ribonucleoprotein that maintains chromosomal telomere length. Telomerase is not active in nonmalignant somatic cells, but is activated in most human cancers. Telomerase activity in easily obtainable body fluids that bathe tumors may be a useful cancer marker, especially when used in conjunction with conventional cytology. APPROACH: Results from studies that assayed telomerase activity in easily obtainable body fluids are reviewed. CONTENT: The telomerase repeat amplification protocol (TRAP) assay has been used to measure telomerase activity in body fluids, including ascites, pleural effusions, pelvic washes, bronchial washings, bronchial lavage, urine, bladder washings, oral rinses, and plasma. Telomerase activity has sensitivities of 60-90% as a tumor marker with clinical specificities for cancer of approximately 90%. Telomerase activity is more sensitive than conventional cytology, the sensitivity of which was 40-65% in various studies. SUMMARY: Telomerase activity in body fluids, as measured by the TRAP assay, is a sensitive potential tumor marker that might help increase the cancer detection rate and the cancer treatment success rate when combined with conventional cytology.

Biomarkers, Tumor↗