Relapse of acute promyelocytic leukemia with PML-RARalpha mutant subclones independent of proximate all-trans retinoic acid selection pressure.

Relapse of acute promyelocytic leukemia (APL) following all-trans retinoic acid (ATRA) therapy has been associated with the acquisition of mutations in the high-affinity ATRA binding site in PML-RARalpha, but little information is available about the selection dynamics of the mutation-harboring subclones. In this study, 6/18 patients treated with sequential ATRA and chemotherapy on protocol INT0129 relapsed with complete replacement of the nonmutant pretreatment APL cell population by a PML-RARalpha mutant subclone. Two patients relapsed in proximity of ATRA treatment; however, in four patients there was a 6-48 month hiatus between the last ATRA treatment and relapse. The mutant subclones were not detectable in samples tested > or = 3 months before relapse at > or = 1 in 10(2) (10(-2)) sensitivity. In one patient, a functionally weak mutation was detected at 10(-4) sensitivity before therapy but only limited pre-relapse enrichment of the mutant subclone was observed on subsequent ATRA therapy. These results indicate that proximate ATRA selection pressure is frequently not the main determinant for the emergence of strongly dominant PML-RARalpha mutant subclones and suggest that APL subclones harboring PML-RARalpha mutations are predisposed to the acquisition of secondary genetic/epigenetic alterations that result in a growth/survival advantage.

Factors affecting the performance of 5' nuclease PCR assays for Listeria monocytogenes detection.

The design and operating parameters affecting the performance of 5' nuclease PCR (TaqMan) assays for the detection of Listeria monocytogenes was investigated. A system previously developed and based on the hlyA gene was used as a model [Appl. Environ. Microbiol. 61 (1995) 3724]. A series of fluorogenic probes labeled with a reporter and a quencher dye was synthesized to explore the effect of probe position and sequence content on the efficiency of probe hydrolysis. In addition, a series of PCR primer pairs that altered the distance between the upstream primer and the interceding probe was examined. The effects of various assay parameters were evaluated by measuring the ratio of the fluorescence intensity of the reporter dye over the quencher dye (deltaRQ). For a given probe sequence, the deltaRQ was typically lower if the 5' terminus was a G residue. Decreasing the probe concentration increased the deltaRQ, although this was at the expense of reproducibility in the assay readout. The distance between the upstream primer and the interceding probe has a significant effect on probe hydrolysis. Reducing the primer-probe distance from, for example, 127 to 4 nt increased the deltaRQ from 2.87 to 5.00. These general rules were used to develop a 5' nuclease PCR (TaqMan) assay with enhanced signal output, providing higher and more reproducible deltaRQ values for L. monocytogenes detection.

Pre-clinical validation of a novel, highly sensitive assay to detect PML-RARalpha mRNA using real-time reverse-transcription polymerase chain reaction.

We have developed a sensitive and quantitative reverse-transcription polymerase chain reaction (RT-PCR) assay for detection of PML-RARalpha, the fusion oncogene present as a specific marker in >99% of cases of acute promyelocytic leukemia (APL). The assay is linear over at least 5 orders of magnitude of input DNA or RNA, and detects as few as 4 copies of PML-RARalpha plasmid DNA. PML-RARalpha transcripts could be detected in mixtures containing 2 to 5 pg of RNA from fusion-containing cells in a background of 1 microg of RNA from PML-RARalpha-negative cells. Using 1.0 to 2.5 microg of input RNA, the sensitivity of the assay was between 10(-5) and 10(-6). Furthermore, determination of GAPDH copy number in each reaction allowed an accurate assessment of sample-to-sample variation in RNA quality and reaction efficiency, with consequent definition of a detection limit for each sample assayed. Using an internal calibrator, assay precision was high, with coefficients of variation between 10 and 20%. An interlaboratory study using coded samples demonstrated excellent reproducibility and high concordance between laboratories. This assay will be used to test the hypothesis that sensitive and quantitative measurement of leukemic burden, during or after therapy of APL, can stratify patients into discrete risk groups, and thereby serve as a basis for risk-adapted therapy in APL.

Fluorescence polarization in homogeneous nucleic acid analysis II: 5'-nuclease assay.

When the temperature and viscosity of the solvent is held constant, the degree of fluorescence polarization (FP) detected when a fluorescent dye is excited by plane polarized light depends mostly on the molecular weight of the dye molecule. By monitoring the FP of a fluorescent dye molecule, one can detect significant changes in the molecular weight of a fluorescent molecule without separation or purification. The 5'-nuclease (TaqMan) assay is a robust single nucleotide polymorphism genotyping method where an allele-specific probe that binds to a perfectly complementary target is cleaved by the 5'-nuclease activity of Taq DNA polymerase. Because the TaqMan probe is labeled with a fluorescent dye, it has high FP value when intact but a low FP value after cleavage. In this study, we compared the results of the 5'-nuclease assay based on standard fluorescence intensity readings and FP readings when genotyping 90 individuals with 20 single nucleotide polymorphisms. Our results show that FP is just as robust and reliable as the standard fluorescence detection method. Use of FP detection makes it possible to reduce the cost of TaqMan probes by abrogating the need for a fluorescence quencher.

Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.

The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-Delta Delta C(T)) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-Delta Delta C(T)) method. In addition, we present the derivation and applications of two variations of the 2(-Delta Delta C(T)) method that may be useful in the analysis of real-time, quantitative PCR data.

SNPing away at complex diseases: analysis of single-nucleotide polymorphisms around APOE in Alzheimer disease.

There has been great interest in the prospects of using single-nucleotide polymorphisms (SNPs) in the search for complex disease genes, and several initiatives devoted to the identification and mapping of SNPs throughout the human genome are currently underway. However, actual data investigating the use of SNPs for identification of complex disease genes are scarce. To begin to look at issues surrounding the use of SNPs in complex disease studies, we have initiated a collaborative SNP mapping study around APOE, the well-established susceptibility gene for late-onset Alzheimer disease (AD). Sixty SNPs in a 1.5-Mb region surrounding APOE were genotyped in samples of unrelated cases of AD, in controls, and in families with AD. Standard tests were conducted to look for association of SNP alleles with AD, in cases and controls. We also used family-based association analyses, including recently developed methods to look for haplotype association. Evidence of association (P

Seven-color, homogeneous detection of six PCR products.

We describe an extension of the fluorogenic PCR 5'-nuclease assay, or "Taq-Man" assay. Sequence-specific probes consisted of a novel nonfluorescent quencher, nitrothiazole blue (NTB), at the 3' terminus and six different reporter dyes at the 5' terminus. The six reporters were 6-FAM, dR110, dR6G, dTMR, dROX and JAZ dyes. The seventh color was from aluminum phthalocyanine tetrasulfonate and was utilized as a "passive reference" to calibrate concentration variations. Our test system was a set of three single-nucleotide polymorphisms (SNPs). Each SNP system consisted of two primers and two sequence-specific probes, each labeled with a different reporter dye and NTB. Following PCR, the reactions were diluted with water and measured in a microcuvette on a luminescence spectrometer in synchronous scanning mode. In this method, both the excitation and emission wavelengths were scanned, with a fixed wavelength difference (delta gamma) between excitation and emission wavelengths. The spectral overlap in the set was evaluated by calculation of the condition number of the 7 x 7 matrix (dye fluorescence vs. wavelength). The small value of the condition number (1.5) proved that the cross-talk between the dyes was minimal. SNP analyses of known, synthetic target sequences and genomic DNA were plotted both as normalized, subtracted spectra and as data points in three separate dot plots.

Allelic discrimination using fluorogenic probes and the 5' nuclease assay.

Large-scale screening for known polymorphisms will require techniques with few steps and the ability to automate each of these steps. In this regard, the 5' nuclease, or TaqMan, PCR assay is especially attractive. A fluorogenic probe, consisting of an oligonucleotide labeled with both a fluorescent reporter dye and a quencher dye, is included in a typical PCR. Amplification of the probe-specific product causes cleavage of the probe, generating an increase in reporter fluorescence. By using different reporter dyes, cleavage of allele-specific probes can be detected in a single PCR. The 5' nuclease assay has been successfully used to discriminate alleles that differ by a single base substitution. Guidelines have been developed so that an assay for any single nucleotide polymorphism (SNP) can be quickly designed and implemented. All assays are performed using a single reaction buffer and single thermocycling protocol. Furthermore, a standard method of analysis has been developed that enables automated genotype determination. Applications of this assay have included typing a number of polymorphisms in human drug metabolism genes.

Detection of minimal residual disease in patients with AML1/ETO-associated acute myeloid leukemia using a novel quantitative reverse transcription polymerase chain reaction assay.

The AML1/ETO fusion transcript can be detected by reverse transcription polymerase chain reaction (RT-PCR) in patients with t(8;21)-associated acute myeloid leukemia (AML) in long-term complete remission (CR). Quantitation of the amount of the fusion transcript during CR may therefore be more predictive of cure or relapse than a simple qualitative assessment. Real Time PCR, a fluorometric-based technique, allows simple and rapid quantitation of a target sequence during the extension phase of PCR amplification, in contrast to end-point quantitative methods. Six patients with t(8;21)(q22;q22) AML, who achieved CR were studied by Real Time RT-PCR at different time intervals following diagnosis and high-dose cytarabine and anthracycline-based induction therapy. Five patients had a diagnostic bone marrow (BM) sample available for molecular analysis. Each patient showed > or = 10(3) copies of the AML1/ETO fusion transcript at diagnosis, and each showed a 2- to 4-log decrease in copy number following successful induction chemotherapy. This is comparable to the log-fold reduction in leukemic blasts that is thought to occur in patients successfully cytoreduced into CR by induction chemotherapy. The sixth patient showed a relatively high copy number immediately following successful remission induction chemotherapy, which continued to increase during early CR and was later followed by relapse. Real Time RT-PCR appears to offer advantages over previously used quantitative RT-PCR methods by providing absolute quantitation of the target sequence, expanding the dynamic range of quantitation to over six orders of magnitude, eliminating the post-PCR processing, and reducing labor and carryover contamination. These features make this an attractive method to prospectively evaluate the prognostic value of AML1/ETO fusion transcript quantitation in a larger patient population with t(8;21)(q22;q22) AML in CR.

A homogeneous, ligase-mediated DNA diagnostic test.

Single-nucleotide variations are the most widely distributed genetic markers in the human genome. A subset of these variations, the substitution mutations, are responsible for most genetic disorders. As single nucleotide polymorphism (SNP) markers are being developed for molecular diagnosis of genetic disorders and large-scale population studies for genetic analysis of complex traits, a simple, sensitive, and specific test for single nucleotide changes is highly desirable. In this report we describe the development of a homogeneous DNA detection method that requires no further manipulations after the initial reaction is set up. This assay, named dye-labeled oligonucleotide ligation (DOL), combines the PCR and the oligonucleotide ligation reaction in a two-stage thermal cycling sequence with fluorescence resonance energy transfer (FRET) detection monitored in real time. Because FRET occurs only when the donor and acceptor dyes are in close proximity, one can infer the genotype or mutational status of a DNA sample by monitoring the specific ligation of dye-labeled oligonucleotide probes. We have successfully applied the DOL assay to genotype 10 SNPs or mutations. By designing the PCR primers and ligation probes in a consistent manner, multiple assays can be done under the same thermal cycling conditions. The standardized design and execution of the DOL assay means that it can be automated for high-throughput genotyping in large-scale population studies.

Polymorphisms in the human DNA ligase I gene (LIG1) including a complex GT repeat.

Sequencing of a human DNA ligase I cDNA clone derived from HeLa cells revealed two unreported differences with the published sequence: a single base change and a three-base deletion. Both differences are in exon 6, and were analyzed by amplifying a segment containing exon 5, intron 6, and exon 6. The first finding was that intron 6 is approximately 2.6 kb in size, not the 1 kb reported in the literature. By sequence analysis of amplified segments, the single-base difference in exon 6 was shown to be polymorphic, with HeLa cells heterozygous for the A/C difference. Analysis of 60 unrelated individuals found a frequency of 0.5 for each allele. Primer extension reactions across the exon 5/exon 6 boundary were performed on cDNA obtained from HeLa cells and human thymus. The results show that the three-base deletion is due to a variation in splicing. For both HeLa and thymus, two-thirds of the transcripts are like the published cDNA sequence and one-third have the three-base deletion. Finally, sequencing of part of intron 6 revealed the presence of a complex GT repeat consisting of a 48-50 nucleotide polypurine tract followed by a variable number of GT residues. This entire unit of polypurine tract plus GTs is repeated three times. Detection of the repeated sequences required the development of specialized cloning and PCR conditions. Analysis of a pedigree showed that this complex repeat is polymorphic.

Structural analogues of TaqMan probes for real-time quantitative PCR.

We have prepared and evaluated a series of structural analogues of TaqMan PCR probes in an effort to identify second-generation probes with improved physical properties and performance. Modifications have included non-nucleosidic dye linkers, 2'-O-Me RNA substitutions, and pyrimidine C5-propyne substitutions.

Real time quantitative PCR.

We have developed a novel "real time" quantitative PCR method. The method measures PCR product accumulation through a dual-labeled fluorogenic probe (i.e., TaqMan Probe). This method provides very accurate and reproducible quantitation of gene copies. Unlike other quantitative PCR methods, real-time PCR does not require post-PCR sample handling, preventing potential PCR product carry-over contamination and resulting in much faster and higher throughput assays. The real-time PCR method has a very large dynamic range of starting target molecule determination (at least five orders of magnitude). Real-time quantitative PCR is extremely accurate and less labor-intensive than current quantitative PCR methods.

The world-wide distribution of allele frequencies at the human dopamine D4 receptor locus.

The dopamine D4 receptor gene (DRD4) has an expressed polymorphism in the third exon that may have functional relevance. The polymorphism exists at two levels. At the higher level there is an imperfect tandem repeat of 48 base pairs (bp) coding for 16 amino acids; alleles have been identified with 2 (32 amino acids) to 10 (160 amino acids) repeats. The imperfect nature of the repeats is responsible for a more subtle level of variation since alleles with the same number of repeats can differ in the exact sequences or in the order of the variants of the 48-bp unit. We have undertaken a global survey of this expressed polymorphism as one approach to understanding the evolutionary significance and origins of the polymorphism as well as understanding what selective forces, if any, may be operating at this locus. As the first step, we have determined the repeat number genotype of the DRD4 repeat polymorphism in 1,327 individuals from 36 different populations. The allele frequencies differ considerably among the different populations. The 4-repeat allele was the most prevalent (global mean allele frequency = 64.3%) and appeared in every population with a frequency ranging from 0.16 to 0.96. The 7-repeat allele was the second most common (global mean = 20.6%), appearing quite frequently in the Americas (mean frequency = 48.3%) but only occasionally in East and South Asia (mean frequency = 1.9%). The 2-repeat allele was the third most common (global mean frequency = 8.2%) and was quite frequent in East and South Asia (mean frequency = 18.1%) while uncommon in the Americas (mean frequency = 2.9%) and Africa (mean frequency = 1.7%). The universality of the polymorphism with only three common repeat-number alleles (4, 7, and 2) indicates that the polymorphism is ancient and arose before the global dispersion of modern humans. The diversity of actual allele frequencies for this expressed polymorphism among different populations emphasizes the importance of population considerations in the design and interpretation of any association studies carried out with this polymorphism.

A PCR-based assay for the detection of Escherichia coli Shiga-like toxin genes in ground beef.

A detection system based on the PCR has been developed for Escherichia coli strains which harbor the Shiga-like toxin genes. This quantitative detection system involves the 5'-->3' nuclease activity of Thermus aquaticus DNA polymerase, which cleaves an internal oligonucleotide probe that has been labeled with both a fluorescent reporter dye (6-carboxy-fluorescein [FAM]) and a quencher dye (6-carboxytetramethyl-rhodamine [TAMRA]). Parameters which affected the performance of the assay included primer probe distance, probe concentration, and probe target sequence homology. The optimized assay format includes two PCR primers that generate a 497-bp amplicon specific for the sltI gene with the fluorogenic probe located 19 bp from the upstream PCR primer. When the distance between the upstream PCR primer and the probe was reduced from 190 to 19 bp, delta RQ values increased from approximately 1.5 to 3.0. The delta RQ for Shiga-like toxin I probe 102 reached a maximum of 4.15 at concentrations between 25 and 50 nM. The assay is sensitive and can detect approximately 10 +/- 5 CFU per PCR. As few as 0.5 CFU of Shiga-like toxin I-producing E. coli per g could be detected in ground beef with only 12 h of enrichment in modified E. coli broth.

Use of a fluorogenic probe in a PCR-based assay for the detection of Listeria monocytogenes.

A PCR-based assay for Listeria monocytogenes that uses the hydrolysis of an internal fluorogenic probe to monitor the amplification of the target has been formatted. The fluorogenic 5' nuclease PCR assay takes advantage of the endogenous 5' --> 3' nuclease activity of Taq DNA polymerase to digest a probe which is labelled with two fluorescent dyes and hybridizes to the amplicon during PCR. When the probe is intact, the two fluorophores interact such that the emission of the reporter dye is quenched. During amplification, the probe is hydrolyzed, relieving the quenching of the reporter and resulting in an increase in its fluorescence intensity. This change in reporter dye fluorescence is quantitative for the amount of PCR product and, under appropriate conditions, for the amount of template. We have applied the fluorogenic 5' nuclease PCR assay to detect L. monocytogenes, using an 858-bp amplicon of hemolysin (hlyA) as the target. Maximum sensitivity was achieved by evaluating various fluorogenic probes and then optimizing the assay components and cycling parameters. With crude cell lysates, the total assay could be completed in 3 h with a detection limit of approximately 50 CFU. Quantification was linear over a range of 5 x 10(1) to 5 x 10(5) CFU.

Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization.

The 5' nuclease PCR assay detects the accumulation of specific PCR product by hybridization and cleavage of a double-labeled fluorogenic probe during the amplification reaction. The probe is an oligonucleotide with both a reporter fluorescent dye and a quencher dye attached. An increase in reporter fluorescence intensity indicates that the probe has hybridized to the target PCR product and has been cleaved by the 5'-->3' nucleolytic activity of Taq DNA polymerase. In this study, probes with the quencher dye attached to an internal nucleotide were compared with probes with the quencher dye attached to the 3'-end nucleotide. In all cases, the reporter dye was attached to the 5' end. All intact probes showed quenching of the reporter fluorescence. In general, probes with the quencher dye attached to the 3'-end nucleotide exhibited a larger signal in the 5' nuclease PCR assay than the internally labeled probes. It is proposed that the larger signal is caused by increased likelihood of cleavage by Taq DNA polymerase when the probe is hybridized to a template strand during PCR. Probes with the quencher dye attached to the 3'-end nucleotide also exhibited an increase in reporter fluorescence intensity when hybridized to a complementary strand. Thus, oligonucleotides with reporter and quencher dyes attached at opposite ends can be used as homogeneous hybridization probes.

Variability of dopamine D4 receptor (DRD4) gene sequence within and among nonhuman primate species.

The dopamine D4 receptor is one of five receptors known to function in mammalian dopaminergic pathways. The DNA sequence of the human dopamine D4 receptor gene (DRD4) has previously been investigated in several populations and found to be highly polymorphic at both the DNA and amino acid levels, exhibiting at least 25 alleles. This variation results from differences in the number and DNA sequence of a 48-bp (16-amino acid) repeat unit in the coding region of DRD4. In the present study, DRD4 DNA sequence was examined in at least two individuals from each of five nonhuman primate species. All five species exhibit intraspecies variability, including both single nucleotide substitutions and variation in the number of 48-bp repeat units. No differences were found between the two alleles of one individual from a sixth nonhuman species. Within each species, all of the DRD4 alleles share species-specific features, indicating that while repeat-unit variation is nearly ubiquitous, ancestral variation has been lost and subsequently regenerated in each of the evolutionary lineages studied. Chimpanzees and gorillas share a unique 12-bp deletion in the coding region of DRD4, outside the repeat-unit segment of the gene. This suggest that the extant chimpanzee DRD4 is more closely related to the gorilla DRD4 than either is to the human DRD4.

Effect of the acyl-CoA:cholesterol acyltransferase inhibitor DuP 128 on cholesterol absorption and serum cholesterol in humans.

Intestinal cholesterol esterification by the enzyme acyl-CoA:cholesterol acyltransferase (ACAT) is a presumed prerequisite for cholesterol absorption. We evaluated the effect of a potent, poorly absorbed ACAT inhibitor (DuP 128: N'-(2,4-difluorophenyl)-N-[5-(4,5-diphenyl-1H-imidazol-2-ylthio)pe ntyl]- N-heptylurea) on cholesterol absorption in a randomized trial. Thirty subjects received DuP 128 for 7 weeks, 10 each at 900 mg per day, 1800 mg per day, and 3600 mg per day; six subjects received placebo; and nine subjects received 1 gm neomycin twice a day. Cholesterol absorption determinations used a continuous dual isotope 14C-cholesterol and 3H-beta sitosterol method. DuP 128 (pooled doses) induced at 14.4% +/- 11.4% reduction in cholesterol absorption (p < 0.05 versus placebo): 17.6% +/- 8.4% at 900 mg, 9.1% +/- 11.4% at 1800 mg, and 17.1% +/- 12.9% at 3600 mg. Neomycin induced a 26.4% +/- 10.7% reduction (p < 0.01). After 6 weeks, neomycin reduced serum total and low-density lipoprotein cholesterol by 22.4% +/- 9.2% and 24.0% +/- 11.6%, respectively (p < 0.01 versus placebo). DuP 128 induced reductions of 3.9% +/- 11% (difference not significant) and 4.95% +/- 14.3% (p = 0.05). ACAT inhibitors limit cholesterol absorption in humans; however, the magnitude of the effect, as exemplified by DuP 128, is small.