Detection of ultra-low levels of pathologic prion protein in scrapie infected hamster brain homogenates using real-time immuno-PCR.

Pathologic prion protein (PrP(Sc)), implicated in transmissible spongiform encephalopathies, is detected by antibody-based tests or bioassays to confirm the diagnosis of prion diseases. Presently, the Western blot or an ELISA is officially used to screen the brain stem in cattle for the presence of PrP(Sc). The immuno-polymerase chain reaction (IPCR), a technique whereby the exponential amplification ability of PCR is coupled to the detection of proteins by antibodies in an ELISA format, was applied in a modified real-time IPCR method to detect ultra-low levels of prion protein. Using IPCR, recombinant hamster PrP(C) was consistently detected at 1 fg/mL and proteinase K (PK)-digested scrapie infected hamster brain homogenates diluted to 10(-8) (approximately 10-100 infectious units) was detected with a semi-quantitative dose response. This level of detection is 1 million-fold more sensitive than the levels detected by Western blot or ELISA and poises IPCR as a method capable of detecting PrP(Sc) in the pre-clinical phase of infection. Further, the data indicate that unless complete PK digestion of PrP(C) in biological materials is verified, ultrasensitive assays such as IPCR may inaccurately classify a sample as positive.

Genotype-phenotype correlation and frequency of the 3199del6 cystic fibrosis mutation among I148T carriers: results from a collaborative study.

PURPOSE: We expect that the mutation panel currently recommended for preconception/prenatal CF carrier screening will be modified as new information is learned regarding the phenotype associated with specific mutations and allele frequencies in various populations. One such example is the I148T mutation, originally described as a severe CF mutation. After implementation of CF population-based carrier screening, we learned that I148T exists as a complex allele with 3199del6 in patients with clinical CF, whereas asymptomatic compound heterozygotes for I148T and a second severe CF mutation were negative for 3199del6. METHODS: We performed reflex testing for 3199del6 on 663 unrelated specimens, including I148T heterozygotes, compound heterozygotes, and a homozygous individual. RESULTS: Less than 1% of I148T carriers were also positive for 3199del6. Excluding subjects tested because of a suspected or known CF diagnosis or positive family history, 0.6% of I148T-positive individuals were also positive for 3199del6. We identified 1 I148T homozygote and 6 unrelated compound heterozygous individuals with I148T and a second CF variant (2 of whom also carried 3199del6). In addition, one fetus with echogenic bowel and one infertile male were heterozygous for I148T (3199del6 negative). CONCLUSIONS: Reflex testing for 3199del6 should be considered whenever I148T is identified. Reflex testing is of particular importance for any symptomatic patient or whenever one member of a couple carries a deleterious CF mutation and the other member is an I148T heterozygote. Further population data are required to determine if I148T, in the absence of 3199del6, is associated with mild or atypical CF or male infertility.

Differences in gene expression between B-cell chronic lymphocytic leukemia and normal B cells: a meta-analysis of three microarray studies.

MOTIVATION: A major focus of current cancer research is to identify genes that can be used as markers for prognosis and diagnosis, and as targets for therapy. Microarray technology has been applied extensively for this purpose, even though it has been reported that the agreement between microarray platforms is poor. A critical question is: how can we best combine the measurements of matched genes across microarray platforms to develop diagnostic and prognostic tools related to the underlying biology? RESULTS: We introduce a statistical approach within a Bayesian framework to combine the microarray data on matched genes from three investigations of gene expression profiling of B-cell chronic lymphocytic leukemia (CLL) and normal B cells (NBC) using three different microarray platforms, oligonucleotide arrays, cDNA arrays printed on glass slides and cDNA arrays printed on nylon membranes. Using this approach, we identified a number of genes that were consistently differentially expressed between CLL and NBC samples.

Sources of nonlinearity in cDNA microarray expression measurements.

BACKGROUND: A key assumption in the analysis of microarray data is that the quantified signal intensities are linearly related to the expression levels of the corresponding genes. To test this assumption, we experimentally examined the relationship between signal and expression for the two types of microarrays we most commonly encounter: radioactively labeled cDNAs on nylon membranes and fluorescently labeled cDNAs on glass slides. RESULTS: We uncovered two sources of nonlinearity. The first, which led to discrepancies in analysis affecting the fluorescent signals, was signal quenching associated with excessive dye concentrations. The second, affecting the radioactive signals, was a nonlinear transformation of the raw data introduced by the scanner. Correction for this transformation was made by some, but not all, image-quantification software packages. CONCLUSIONS: The second type of nonlinearity is more troublesome, because it could not have been predicted a priori. Both types of nonlinearities were detected by simple dilution series, which we recommend as a quality-control step.

Clinical and biologic activity of the farnesyltransferase inhibitor R115777 in adults with refractory and relapsed acute leukemias: a phase 1 clinical-laboratory correlative trial.

R115777 is a nonpeptidomimetic enzyme-specific inhibitor of farnesyl protein transferase (FT) that was developed as a potential inhibitor of Ras protein signaling, with antitumor activity in preclinical models. This study was a phase 1 trial of orally administered R115777 in 35 adults with poor-risk acute leukemias. Cohorts of patients received R115777 at doses ranging from 100 mg twice daily (bid) to 1200 mg bid for up to 21 days. Dose-limiting toxicity occurred at 1200 mg bid, with central neurotoxicity evidenced by ataxia, confusion, and dysarthria. Non-dose-limiting toxicities included reversible nausea, renal insufficiency, polydipsia, paresthesias, and myelosuppression. R115777 inhibited FT activity at 300 mg bid and farnesylation of FT substrates lamin A and HDJ-2 at 600 mg bid. Extracellular signal-regulated kinase (ERK), an effector enzyme of Ras-mediated signaling, was detected in its phosphorylated (activated) form in 8 (36.4%) of 22 pretreatment marrows and became undetectable in 4 of those 8 after one cycle of treatment. Pharmacokinetics revealed a linear relationship between dose and maximum plasma concentration or area under the curve over 12 hours at all dose levels. Weekly marrow samples demonstrated that R115777 accumulated in bone marrow in a dose-dependent fashion, with large increases in marrow drug levels beginning at 600 mg bid and with sustained levels throughout drug administration. Clinical responses occurred in 10 (29%) of the 34 evaluable patients, including 2 complete remissions. Genomic analyses failed to detect N-ras gene mutations in any of the 35 leukemias. The results of this first clinical trial of a signal transduction inhibitor in patients with acute leukemias suggest that inhibitors of FT may have important clinical antileukemic activity. (Blood. 2001;97:3361-3369)

Comparison of the safety, vaccine virus shedding, and immunogenicity of influenza virus vaccine, trivalent, types A and B, live cold-adapted, administered to human immunodeficiency virus (HIV)-infected and non-HIV-infected adults.

Fifty-seven human immunodeficiency virus (HIV)-infected (CDC class A1-2) and 54 non-HIV-infected adults, not prescreened for influenza susceptibility, were randomized to receive trivalent live attenuated influenza vaccine (LAIV) or placebo intranasally. LAIV was safe and well tolerated with no serious adverse events attributable to vaccine. Reactogenicity rates were similar in LAIV and placebo recipients except that runny nose/nasal congestion was significantly more common in LAIV recipients regardless of HIV status. No prolonged shedding of LAIV was observed in HIV-infected participants. HIV RNA levels were not increased and CD4 counts were not decreased in HIV-infected LAIV recipients compared with placebo recipients after immunization. Shedding of LAIV and increases in antibody titers were infrequent, consistent with prior experience in unscreened adults. The data suggest that inadvertent vaccination with LAIV in relatively asymptomatic HIV-infected adults would not be associated with frequent significant adverse events.

Single-strand conformation polymorphism and heteroduplex analysis for gel-based mutation detection.

Single-strand conformation polymorphism (SSCP) and heteroduplex analysis (HA) are popular electrophoretic methods for the identification of sequences. The principle reasons for the popularity of these two methods are their technical simplicity and their relatively high sensitivity for the detection of mutations. Here we review the theory and practice of SSCP and HA, including the factors contributing to the sensitivity of mutation detection. For SSCP analysis, these factors include: choice of gel matrix, electrophoretic conditions, presence of neutral additives, fragment size, and G+C content For HA, the principle factors influencing sensitivity are the gel matrix and the identity of the base mismatch.

Use of a DNA toolbox for the characterization of mutation scanning methods. I: construction of the toolbox and evaluation of heteroduplex analysis.

A systematic characterization of the effects of important physical parameters on the sensitivity and specificity of methods in searching for unknown base changes (mutations or single nucleotide polymorphisms) over a relatively long DNA segment has not been previously reported. To this end, we have constructed a set of molecules of varying G+C content (40, 50, and 60% GC) having all possible base changes at a particular location - the "DNA toolbox". Exhaustive confirmatory sequencing demonstrated that there were no other base changes in any of the clones. Using this set of clones as polymerase chain reaction (PCR) templates, amplicons of various lengths with the same base mutated to all other bases were generated. The behavior of these constructs in manual and automated heteroduplex analysis was analyzed as a function of the size and overall base content of the fragment, the nature and location of the base change. Our results show that in heteroduplex analysis, the nature of the mismatched base pair is the overriding determinant for the ability to detect the mutation, regardless of fragment length, GC content, or the location of the mutation.

Use of DNA toolbox for the characterization of mutation scanning methods. II: evaluation of single-strand conformation polymorphism analysis.

Single-strand conformation polymorphism (SSCP) is one of the most commonly used methods for searching for unknown base changes (mutations). In order to characterize systematically the effects of important physical parameters on the sensitivity and specificity of SSCP, we used the DNA toolbox constructed as described in the companion paper [2]. Using this set of DNA molecules as polymerase chain reaction (PCR) templates, amplicons of various lengths with the same base, mutated to all other bases, were generated. The behavior of these constructs in manual and automated SSCP was analyzed as a function of the size, overall base content of the fragment, nature and location of the base change, and the temperature and pH of electrophoresis. Our results demonstrate that all of these variables interact to determine the rate of detection of single-base changes, with the GC content being the predominant determinant of detection sensitivity.

Quantitative measurement of telomerase activity in lymphadenopathy: correlation with histologic features and human immunodeficiency virus-1 infection.

Telomerase is a ribonucleoprotein that uses its internal RNA component as a template for synthesis of telomeric DNA on the ends of chromosomes after each round of cell division. It is expressed in approximately 90% of all human cancers tested to date, as well as in most immortal cell lines. Recently, telomerase activity was detected in normal proliferating lymphoid tissue and in non-Hodgkin's lymphomas (NHLs) by use of the telomeric repeat amplification protocol assay, a qualitative measure of telomerase activity. In this study, we modified the assay to measure quantitatively the telomerase activity in lymph node biopsy specimens obtained from patients with lymphadenopathy. The lymph nodes either contained benign reactive changes, were involved by NHL of B-cell lineage, or were involved by Hodgkin's disease. Telomerase activity was detected in all of our samples, benign as well as malignant. The levels of activity were unaffected by the patient's human immunodeficiency virus-1 status. Although the specimens involved by NHLs showed a range in telomerase activity from low to high, the levels did not correlate strictly with the histologic grade according to the Working Formulation. All of the cases of Hodgkin's disease also expressed telomerase activity, and the levels were similar regardless of histologic subtype. Our results showed that telomerase activity was expressed in both benign and malignant lymphoproliferative processes.

Mismatch cleavage detects base deletion in cystic fibrosis gene.

The delta F508 is the most common defect in the cystic fibrosis (CF) gene; it involves in a 3-base deletion in codon 508 and results in the loss of a phenylalanine residue at amino acid position 508. Our previous results have shown the mismatch enzyme cleavage at the mismatch of a DNA duplex in identifying a specific DNA sequence or a point mutation. The assay is simple and reliable. By manipulating the melting temperature (Tm) for the hybrids of the DNA targets and the deoxynucleotide probes, the mismatch cleavage assays are able to detect the most common defective CF gene, delta F508. The assays with a delta F508 and a normal wild-type probe can differentiate the three genotypes, i.e., delta F508/delta F508, delta F508/normal and normal/normal. Furthermore, the addition of ammonium acetate amplifier to the assay for recycling the target DNA can increase the sensitivity to a level that is sufficient to detect the mutated target in a few micrograms of genomic DNA without the aid of PCR amplification. The detection of the base deletion, the amplification of sensitivity and the differentiation among the genotypes of normal, carrier delta F508 and mutant delta F508 suggest the useful application of mismatch cleavage in genetic diagnosis at the DNA level.

Identification of a splice site mutation (2789 +5 G > A) associated with small amounts of normal CFTR mRNA and mild cystic fibrosis.

A splicing mutation was identified at the +5 position of the splice donor site of exon 14b of CFTR in CF patients in a consanguineous family that is remarkable for unusually mild disease. Quantitative studies of nasal epithelial mRNA revealed that homozygotes for the spice site mutation produced approximately 4% of the normal amount of normally-spliced CFTR. We propose that this small amount of normally spliced mRNA is associated with synthesis of some normal CFTR protein, and accounts for the mild phenotype. Further characterization of epithelial function and clinical phenotype in patients bearing this form of mutation, termed a type V mutation, will be useful in determining the level of CFTR associated with amelioration of lung disease.

Optimization, assessment, and proposed use of a direct nested reverse transcription-polymerase chain reaction protocol for the detection of hepatitis C virus.

OBJECTIVES: To compare the performance of reverse transcription followed by the polymerase chain reaction (RT-PCR), without RNA purification, with the performance of classic protocols. STUDY DESIGN/METHODS: Direct and classic techniques were used to test three groups of samples: six hepatitis C virus (HCV) seroconversion panels (n = 90), a HCV RNA reference panel (n = 26), and serial dilutions of four HCV-positive sera (n = 24). These methods were then applied sequentially through a clinical diagnostic algorithm to test 268 samples from high-risk patients. RESULTS: For the three groups of samples, we found a 94% concordance between direct and purified RT-PCR methods. For the detection of HCV RNA in clinical samples, sensitivity was maximized and cost minimized using both protocols according to the proposed algorithm. CONCLUSIONS: The direct PCR method is reliable, sensitive, and can result in time and cost savings. The suggested testing algorithm can enhance sensitivity and time savings for populations with a high prevalence of infection.

Heteroduplex generator in analysis of Rh blood group alleles.

We describe the use of heteroduplex analysis to enhance the resolution of different rhesus-derived (Rh) isotypes. Heteroduplex analysis of different domains of the Rh D and Rh CE loci can be performed to diagnose a variety of blood group incompatibilities. One application of this technique is the ability to test for fetal-maternal blood group incompatibilities during pregnancy. Several new serotype-specific sequence variations were discovered in the mapping of the Rh locus, and used in the construction of artificial heteroduplex generators (HGs). HGs facilitate the resolution of the Rh isotypes by electrophoretic methods.

Screening Young syndrome patients for CFTR mutations.

Young syndrome is characterized by obstructive azoospermia associated with chronic sinobronchial disease of an infectious nature, but normal sweat-gland and pancreatic function as well as normal nasal potential differences. Congenital bilateral absence of the vas deferens (CBAVD) in some patients arises from mutations within the cystic fibrosis (CF) transmembrane regulator (CFTR) gene. Because of some similarities between Young syndrome, CF, and CBAVD, we evaluated 13 patients with Young syndrome, including screening for more than 30 different mutations within the CFTR gene. The mean age of the patients was 43 yr (range, 32 to 50 yr), and all were of northern European extraction. The sweat chloride concentration was normal in all patients (mean = 29 mEq/L; range, 8 to 43 mEq/L). Most had intermittent bronchial and sinus infections, but none was chronically colonized with Staphylococcus aureus or Pseudomonas aeruginosa. The FEV1 was normal or only mildly reduced in most patients (mean = 74%; range, 48 to 100% predicted). Of 26 Young syndrome chromosomes, we identified one with the recognized CF mutation delta F508. The incidence of CFTR mutations (1 in 26) did not differ significantly from the expected carrier frequency in this population. In summary, it is unlikely that the typical Young syndrome patient has a clinical disease associated with CFTR mutation on both alleles.

Rapid DNA haplotyping using a multiplex heteroduplex approach: application to Duchenne muscular dystrophy carrier testing.

A new strategy has been developed for rapid haplotype analysis based on an initial multiplex amplification of several polymorphic sites, followed by heteroduplex detection. Heteroduplexes formed between two different alleles are detected because they migrate differently than the corresponding homoduplexes in Hydrolink-MDE gel. This simple, rapid method does not depend on specific sequences such as restriction enzyme sites or CA boxes and does not require the use of isotope. This approach has been tested using commonly occurring polymorphisms spanning the dystrophin gene as a model. We describe the use of the method to assign the carrier status of females in Duchenne muscular dystrophy (DMD) pedigrees. The method may be used for other genetic diseases when mutations are unknown or there are few dinucleotide markers in the gene proximity, and for the identification of haplotype backgrounds of mutant alleles.