Quantitative trait loci for electrical seizure threshold mapped in C57BLKS/J and C57BL/10SnJ mice.

We mapped the quantitative trait loci (QTL) that contribute to the robust difference in maximal electroshock seizure threshold (MEST) between C57BLKS/J (BKS) and C57BL10S/J (B10S) mice. BKS, B10S, BKS × B10S F1 and BKS × B10S F2 intercross mice were tested for MEST at 8-9 weeks of age. Results of F2 testing showed that, in this cross, MEST is a continuously distributed trait determined by polygenic inheritance. Mice from the extremes of the trait distribution were genotyped using microarray technology. MEST correlated significantly with body weight and sex; however, because of the high correlation between these factors, the QTL mapping was conditioned on sex alone. A sequential series of statistical analyses was used to map QTLs including single-point, multipoint and multilocus methods. Two QTLs reached genome-wide levels of significance based upon an empirically determined permutation threshold: chromosome 6 (LOD = 6.0 at ∼69 cM) and chromosome 8 (LOD = 5.7 at ∼27 cM). Two additional QTLs were retained in a multilocus regression model: chromosome 3 (LOD = 2.1 at ∼68 cM) and chromosome 5 (LOD = 2.7 at ∼73 cM). Together the four QTLs explain one third of the total phenotypic variance in the mapping population. Lack of overlap between the major MEST QTLs mapped here in BKS and B10S mice and those mapped previously in C57BL/6J and DBA/2J mice (strains that are closely related to BKS and B10S) suggest that BKS and B10S represent a new polygenic mouse model for investigating susceptibility to seizures.

Rare structural variants found in attention-deficit hyperactivity disorder are preferentially associated with neurodevelopmental genes.

Attention-deficit/hyperactivity disorder (ADHD) is a common and highly heritable disorder, but specific genetic factors underlying risk remain elusive. To assess the role of structural variation in ADHD, we identified 222 inherited copy number variations (CNVs) within 335 ADHD patients and their parents that were not detected in 2026 unrelated healthy individuals. Although no excess CNVs, either deletions or duplications, were found in the ADHD cohort relative to controls, the inherited rare CNV-associated gene set was significantly enriched for genes reported as candidates in studies of autism, schizophrenia and Tourette syndrome, including A2BP1, AUTS2, CNTNAP2 and IMMP2L. The ADHD CNV gene set was also significantly enriched for genes known to be important for psychological and neurological functions, including learning, behavior, synaptic transmission and central nervous system development. Four independent deletions were located within the protein tyrosine phosphatase gene, PTPRD, recently implicated as a candidate gene for restless legs syndrome, which frequently presents with ADHD. A deletion within the glutamate receptor gene, GRM5, was found in an affected parent and all three affected offspring whose ADHD phenotypes closely resembled those of the GRM5 null mouse. Together, these results suggest that rare inherited structural variations play an important role in ADHD development and indicate a set of putative candidate genes for further study in the etiology of ADHD.

MLPA: a rapid, reliable, and sensitive method for detection and analysis of abnormalities of 22q.

In this study, essential test characteristics of the recently described multiplex ligation-dependent probe amplification (MLPA) method are presented, using chromosome 22 as a model. This novel method allows the relative quantification of approximately 40-45 different target DNA sequences in a single reaction. For the purpose of this study, MLPA was performed in a blinded manner on a training set containing over 50 samples, including typical 22q11.2 deletions, various atypical deletions, duplications (trisomy and tetrasomy), and unbalanced translocations. All samples in the training set have been previously characterized by fluorescence in situ hybridization (FISH) with cosmid or BAC clones and/or cytogenetic studies. MLPA findings were consistent with cytogenetic and FISH studies, no rearrangement went undetected and repeated tests gave consistent results. At a relative change in comparative signal strength of 30% or more, sensitivity and specificity values were 0.95 and 0.99, respectively. Given that MLPA is likely to be used as an initial screening method, a higher sensitivity, at the cost of a lower specificity, was deemed more appropriate. A receiver operator characteristic (ROC) curve analysis was performed to calculate the most optimal threshold range, with associated sensitivity and specificity values of 0.99 and 0.97, respectively. Finally, performance of each individual probe was analyzed, providing further useful information for the interpretation of MLPA results. In conclusion, MLPA has proven to be a highly sensitive and accurate tool for detecting copy number changes in the 22q11.2 region, making it a fast and economic alternative to currently used methods. The current study provides valuable and detailed information on the characteristics of this novel method.

Expression profiles of acute lymphoblastic and myeloblastic leukemias with ALL-1 rearrangements.

The ALL-1 gene is directly involved in 5-10% of acute lymphoblastic leukemias (ALLs) and acute myeloid leukemias (AMLs) by fusion to other genes or through internal rearrangements. DNA microarrays were used to determine expression profiles of ALLs and AMLs with ALL-1 rearrangements. These profiles distinguish those tumors from other ALLs and AMLs. The expression patterns of ALL-1-associated tumors, in particular ALLs, involve oncogenes, tumor suppressors, antiapoptotic genes, drug-resistance genes, etc., and correlate with the aggressive nature of the tumors. The genes whose expression differentiates between ALLs with and without ALL-1 rearrangement were further divided into several groups, enabling separation of ALL-1-associated ALLs into two subclasses. One of the groups included 43 genes that exhibited expression profiles closely linked to ALLs with ALL-1 rearrangements. Further, there were evident differences between the expression profiles of AMLs in which ALL-1 had undergone fusion to other genes and AMLs with partial duplication of ALL-1. The extensive analysis described here pinpointed genes that might have a direct role in pathogenesis.

Near-precise interchromosomal recombination and functional DNA topoisomerase II cleavage sites at MLL and AF-4 genomic breakpoints in treatment-related acute lymphoblastic leukemia with t(4;11) translocation.

We analyzed the der(11) and der(4) genomic breakpoint junctions of a t(4;11) in the leukemia of a patient previously administered etoposide and dactinomycin by molecular and biochemical approaches to gain insights about the translocation mechanism and the relevant drug exposure. The genomic breakpoint junctions were amplified by PCR. Cleavage of DNA substrates containing the normal homologues of the MLL and AF-4 translocation breakpoints was examined in vitro upon incubation with human DNA topoisomerase IIalpha and etoposide, etoposide catechol, etoposide quinone, or dactinomycin. The der(11) and der(4) genomic breakpoint junctions both involved MLL intron 6 and AF-4 intron 3. Recombination was precise at the sequence level except for the overall gain of a single templated nucleotide. The translocation breakpoints in MLL and AF-4 were DNA topoisomerase II cleavage sites. Etoposide and its metabolites, but not dactinomycin, enhanced cleavage at these sites. Assuming that DNA topoisomerase II was the mediator of the breakage, processing of the staggered nicks induced by DNA topoisomerase II, including exonucleolytic deletion and template-directed polymerization, would have been required before ligation of the ends to generate the observed genomic breakpoint junctions. These data are inconsistent with a translocation mechanism involving interchromosomal recombination by simple exchange of DNA topoisomerase II subunits and DNA-strand transfer; however, consistent with reciprocal DNA topoisomerase II cleavage events in MLL and AF-4 in which both breaks became stable, the DNA ends were processed and underwent ligation. Etoposide and/or its metabolites, but not dactinomycin, likely were the relevant exposures in this patient.

Etoposide metabolites enhance DNA topoisomerase II cleavage near leukemia-associated MLL translocation breakpoints.

Chromosomal breakage resulting from stabilization of DNA topoisomerase II covalent complexes by epipodophyllotoxins may play a role in the genesis of leukemia-associated MLL gene translocations. We investigated whether etoposide catechol and quinone metabolites can damage the MLL breakpoint cluster region in a DNA topoisomerase II-dependent manner like the parent drug and the nature of the damage. Cleavage of two DNA substrates containing the normal homologues of five MLL intron 6 translocation breakpoints was examined in vitro upon incubation with human DNA topoisomerase IIalpha, ATP, and either etoposide, etoposide catechol, or etoposide quinone. Many of the same cleavage sites were induced by etoposide and by its metabolites, but several unique sites were induced by the metabolites. There was a preference for G(-1) among the unique sites, which differs from the parent drug. Cleavage at most sites was greater and more heat-stable in the presence of the metabolites compared to etoposide. The MLL translocation breakpoints contained within the substrates were near strong and/or stable cleavage sites. The metabolites induced more cleavage than etoposide at the same sites within a 40 bp double-stranded oligonucleotide containing two of the translocation breakpoints, confirming the results at a subset of the sites. Cleavage assays using the same oligonucleotide substrate in which guanines at several positions were replaced with N7-deaza guanines indicated that the N7 position of guanine is important in metabolite-induced cleavage, possibly suggesting N7-guanine alkylation by etoposide quinone. Not only etoposide, but also its metabolites, enhance DNA topoisomerase II cleavage near MLL translocation breakpoints in in vitro assays. It is possible that etoposide metabolites may be relevant to translocations.

t(3;11) translocation in treatment-related acute myeloid leukemia fuses MLL with the GMPS (GUANOSINE 5' MONOPHOSPHATE SYNTHETASE) gene.

The partner gene of MLL was identified in a patient with treatment-related acute myeloid leukemia in which the karyotype suggested t(3;11)(q25;q23). Prior therapy included the DNA topoisomerase II inhibitors, teniposide and doxorubicin. Southern blot analysis indicated that the MLL gene was involved in the translocation. cDNA panhandle polymerase chain reaction (PCR) was used, which does not require partner gene-specific primers, to identify the chimeric transcript. Reverse-transcription of first-strand cDNAs with oligonucleotides containing known MLL sequence at the 5' ends and random hexamers at the 3' ends generated templates with an intra-strand loop for PCR. In-frame fusions of either MLL exon 7 or exon 8 with the GMPS (GUANOSINE 5'-MONOPHOSPHATE SYNTHETASE) gene from chromosome band 3q24 were detected. The fusion transcript was alternatively spliced. Guanosine monophosphate synthetase is essential for de novo purine synthesis. GMPS is the first partner gene of MLL on chromosome 3q and the first gene of this type in leukemia-associated translocations. (Blood. 2000;96:4360-4362)

Panhandle PCR for cDNA: a rapid method for isolation of MLL fusion transcripts involving unknown partner genes.

Identifying translocations of the MLL gene at chromosome band 11q23 is important for the characterization and treatment of leukemia. However, cytogenetic analysis does not always find the translocations and the many partner genes of MLL make molecular detection difficult. We developed cDNA panhandle PCR to identify der(11) transcripts regardless of the partner gene. By reverse transcribing first-strand cDNAs with oligonucleotides containing coding sequence from the 5' MLL breakpoint cluster region at the 5' ends and random hexamers at the 3' ends, known MLL sequence was attached to the unknown partner sequence. This enabled the formation of stem-loop templates with the fusion point of the chimeric transcript in the loop and the use of MLL primers in two-sided PCR. The assay was validated by detection of the known fusion transcript and the transcript from the normal MLL allele in the cell line MV4-11. cDNA panhandle PCR then was used to identify the fusion transcripts in two cases of treatment-related acute myeloid leukemia where the karyotypes were normal and the partner genes unknown. cDNA panhandle PCR revealed a fusion of MLL with AF-10 in one case and a fusion of MLL with ELL in the other. Alternatively spliced transcripts and exon scrambling were detectable by the method. Leukemias with normal karyotypes may contain cryptic translocations of MLL with a variety of partner genes. cDNA panhandle PCR is useful for identifying MLL translocations and determining unknown partner sequences in the fusion transcripts.

Detection of leukemia-associated MLL-GAS7 translocation early during chemotherapy with DNA topoisomerase II inhibitors.

Leukemias with MLL gene translocations are a complication of primary cancer treatment with DNA topoisomerase II inhibitors. How early translocations appear during primary cancer treatment has not been investigated. We tracked the leukemic clone with an MLL gene translocation during neuroblastoma therapy in a child who developed acute myeloid leukemia. The karyotype of the leukemic clone showed del(11)(q23). We used panhandle PCR-based methods to isolate the breakpoint junction involving MLL and an unknown partner gene. Marrow DNA from neuroblastoma diagnosis and DNA and RNA from serial preleukemic marrows were examined for the translocation. The karyotypic del(11)(q23) was a cryptic t(11;17). GAS7, a growth arrest-specific gene at chromosome band 17p13, was the partner gene of MLL. Two different MLL-GAS7 fusion transcripts were expressed. The translocation was already detectable by 1.5 months after the start of neuroblastoma treatment. The translocation was not detectable in the marrow at neuroblastoma diagnosis or in peripheral blood lymphocyte DNAs of six normal subjects. GAS7 is a new partner gene of MLL in treatment-related acute myeloid leukemia. MLL gene translocations can be present early during anticancer treatment at low cumulative doses of DNA topoisomerase II inhibitors. Although MLL has many partner genes and most have not been characterized, panhandle PCR strategies afford new means for detecting MLL gene translocations early during therapy when the partner gene is unknown.

Duplicated regions of AF-4 intron 4 at t(4;11) translocation breakpoints.

BACKGROUND: AF-4 is a common partner gene of MLL. AF-4 breakpoints occur in introns, but most AF-4 introns are uncharacterized. METHODS AND RESULTS: We cloned AF-4 intron 4 and examined the frequency of breakpoints in this intron. The 5.8-kb intron is rich in repeat sequences and was the site of translocation in 3 of 17 leukemias with t(4;11). We cloned the der (11) and der (4) breakpoints and isolated the fusion transcripts in the cell line MV4-11 and in a de novo acute lymphoblastic leukemia (ALL). Both translocations joined MLL intron 6 and AF-4 intron 4. In MV4-11, 249 bases from AF-4 were present in both derivative chromosomes, indicating duplication. In the de novo ALL, duplication of 446 bases from MLL and AF-4 occurred. Reciprocal fusion transcripts were expressed. CONCLUSIONS: Intronic sequence of AF-4 is useful for molecular diagnosis of t(4;11). Duplicated intronic regions suggest staggered chromosomal breakage.

t(11;22)(q23;q11.2) In acute myeloid leukemia of infant twins fuses MLL with hCDCrel, a cell division cycle gene in the genomic region of deletion in DiGeorge and velocardiofacial syndromes.

We examined the MLL genomic translocation breakpoint in acute myeloid leukemia of infant twins. Southern blot analysis in both cases showed two identical MLL gene rearrangements indicating chromosomal translocation. The rearrangements were detectable in the second twin before signs of clinical disease and the intensity relative to the normal fragment indicated that the translocation was not constitutional. Fluorescence in situ hybridization with an MLL-specific probe and karyotype analyses suggested t(11;22)(q23;q11. 2) disrupting MLL. Known 5' sequence from MLL but unknown 3' sequence from chromosome band 22q11.2 formed the breakpoint junction on the der(11) chromosome. We used panhandle variant PCR to clone the translocation breakpoint. By ligating a single-stranded oligonucleotide that was homologous to known 5' MLL genomic sequence to the 5' ends of BamHI-digested DNA through a bridging oligonucleotide, we formed the stem-loop template for panhandle variant PCR which yielded products of 3.9 kb. The MLL genomic breakpoint was in intron 7. The sequence of the partner DNA from band 22q11.2 was identical to the hCDCrel (human cell division cycle related) gene that maps to the region commonly deleted in DiGeorge and velocardiofacial syndromes. Both MLL and hCDCrel contained homologous CT, TTTGTG, and GAA sequences within a few base pairs of their respective breakpoints, which may have been important in uniting these two genes by translocation. Reverse transcriptase-PCR amplified an in-frame fusion of MLL exon 7 to hCDCrel exon 3, indicating that an MLL-hCDCrel chimeric mRNA had been transcribed. Panhandle variant PCR is a powerful strategy for cloning translocation breakpoints where the partner gene is undetermined. This application of the method identified a region of chromosome band 22q11.2 involved in both leukemia and a constitutional disorder.

Potential role for wild-type p53 in leukemias with MLL gene translocations.

We used single-strand conformation polymorphism (SSCP) analysis of p53 exons 4-8 to screen for possible mutations in 25 pediatric de novo leukemias with translocations of the MLL gene at chromosome band 11q23. Of the 25 patients, 21 were infants. Fifteen cases were acute myeloid leukemia (AML), eight were acute lymphoblastic leukemia (ALL), and two cases were biphenotypic. Nineteen cases were studied at diagnosis and six at time of relapse. p53 mutations were absent in all 19 cases studied at the time of diagnosis. The only mutation was a TGC-->TTC transversion (cys-->phe) at codon 141 in exon 5 in a case of infant ALL at relapse that occurred by subclone evolution after MLL gene translocation. We previously showed that p53 mutations are also absent in pediatric treatment-related leukemias with MLL gene translocations. The absence of p53 mutations at initial transformation may suggest that the anti-apoptotic effect of mutant p53 is not important in leukemias with MLL gene translocations. Alternatively, exogenous DNA damage may be the common feature in treatment-related and de novo cases. Since MLL gene translocations may occur through DNA repair and wild-type p53 is central to DNA repair, the absence of p53 mutations raises the possibility that wild-type p53, not mutant p53, may be important in the genesis of leukemias with these translocations.

Molecular analysis of a compound heterozygote for hypoprothrombinemia and dysprothrombinemia (-G 7248/7249 and ARG 340 TRP).

Hypoprothrombinemia is an uncommon hereditary coagulation defect characterized by low levels of biologically active prothrombin. Automated fluorescence-based DNA sequence analysis of amplified genomic DNA was used to define prothrombin gene regions from a patient with severe functional hypoprothrombinemia and little detectable prothrombin antigen. Two changes that alter amino acid sequence were observed: a deletion of one nucleotide (-G, 7248/7249) in exon 8 of one allele, causing a frameshift at codon 249/250 that results in premature termination of translation; and a C --> T change resulting in the substitution of tryptophan (TGG) for arginine (CGG) at amino acid 340 in exon 10 of the prothrombin gene. Computer modeling of the thrombin molecule confirmed that arginine 340 is located at the surface of the thrombin molecule, which points to the aqueous solvent. As tryptophan is a highly hydrophobic amino acid, the Arg --> Trp change may be associated with instability of the thrombin molecule.

Panhandle PCR strategy to amplify MLL genomic breakpoints in treatment-related leukemias.

Panhandle PCR amplifies genomic DNA with known 5' and unknown 3' sequences from a template with an intrastrand loop schematically shaped like a pan with a handle. We used panhandle PCR to clone MLL genomic breakpoints in two pediatric treatment-related leukemias. The karyotype in a case of treatment-related acute lymphoblastic leukemia showed the t(4;11)(q21;q23). Panhandle PCR amplified the translocation breakpoint at position 2158 in intron 6 in the 5' MLL breakpoint cluster region (bcr). The karyotype in a case of treatment-related acute myeloid leukemia was normal, but Southern blot analysis showed a single MLL gene rearrangement. Panhandle PCR amplified the breakpoint at position 1493 in MLL intron 6. Screening of somatic cell hybrid and radiation hybrid DNAs by PCR and reverse transcriptase-PCR analysis of the leukemic cells indicated that panhandle PCR identified a fusion of MLL intron 6 with a previously uncharacterized sequence in MLL intron 1, consistent with a partial duplication. In both cases, the breakpoints in the MLL bcr were in Alu repeats, and there were Alu repeats in proximity to the breakpoints in the partner DNAs, suggesting that Alu sequences were relevant to these rearrangements. This study shows that panhandle PCR is an effective method for cloning MLL genomic breakpoints in treatment-related leukemias. Analysis of additional pediatric cases will determine whether breakpoint distribution deviates from the predilection for 3' distribution in the bcr that has been found in adult cases.

Panhandle polymerase chain reaction amplifies MLL genomic translocation breakpoint involving unknown partner gene.

We used a new approach called panhandle polymerase chain reaction (PCR) to clone an MLL genomic translocation breakpoint in a case of acute lymphoblastic leukemia of infancy in which karyotype analysis was technically unsuccessful and did not show the translocation partner. Panhandle PCR amplified known MLL sequence 5' of the breakpoint and 3' sequence from the unknown partner gene from a DNA template with an intrastrand loop schematically shaped like a pan with a handle. The 7-kb panhandle PCR product contained the translocation breakpoint in MLL intron 8. The partner DNA included unique nonrepetitive sequences, Alu and mammalian apparent LTR-retrotransposon (MaLR) repetitive sequences, and a region of homology to expressed sequence tags. MaLR sequences have not been found before near leukemia-associated translocation breakpoints. The nonrepetitive sequences were not homologous to known partner genes of MLL. Screening of somatic cell hybrid and radiation hybrid lines by PCR and fluorescence in situ hybridization analysis of normal metaphase chromosomes mapped the partner DNA to chromosome band 4q21. Reverse transcriptase-PCR identified an MLL-AF-4 chimeric mRNA, indicating that panhandle PCR identified a fusion of MLL with a previously uncharacterized AF-4 intronic sequence. Panhandle PCR facilitates cloning translocation breakpoints and identifying unknown partner genes.

Lineage-specific alternative splicing of the human Fc gamma RIIA transmembrane exon requires sequences near the 3' splice site.

The human Fc gamma RIIA gene produces multiple transcripts, including those with (Fc gamma RIIa1) and without (Fc gamma RIIa2) the single exon encoding the transmembrane domain (TM). Previously, a fluorescence-based RT-PCR assay showed lineage-specific differences in Fc gamma RIIA transcript ratios (Fc gamma RIIa2/Fc gamma RIIa1). The mechanism of this lineage-specific expression was investigated in this study. Differential transcript stability does not play a major role, because transcript ratios remained constant in cells with both low (K562) and high (Dami) ratios following actinomycin D treatment. Transient expression studies in K562 and Dami cells using a minigene construct containing a 5.0 kb genomic fragment including the TM exon and adjacent intron and exon sequences showed recapitulation of endogenous transcript ratios. The TM exon was efficiently spliced in by the constitutive splicing machinery in HeLa cells, an Fc gamma RIIA-negative cell line. Lineage-specific TM exon skipping was markedly diminished by two independent minigene mutations: a point mutation of the first nucleotide of the TM exon, and a five basepair intronic deletion near a putative branchpoint. These data demonstrate that cis-acting sequences in or near the TM exon 3' splice acceptor site contribute to lineage-specific differences in Fc gamma RIIA transcript ratios.

Genetic diversity in human Fc receptor II for immunoglobulin G: Fc gamma receptor IIA ligand-binding polymorphism.

Fc gamma receptors, and in particular genetic variation in these receptors, are important in disorders of hose defense, immunohematologic disease, and systemic autoimmune diseases. We investigated the His-Arg (CAT/CGT) polymorphism at codon 131 of the Fc gamma receptor IIA gene, which influences ligand binding by the receptor. Previously, individuals had been classified phenotypically on the basis of differential binding of murine immunoglobulin G1, but the Fc gamma receptor IIA genotype distribution has not been reported. We used selective PCR-based sequence analysis of genomic DNA to determine the distribution in healthy individuals. For African-Americans, the genotype distribution was determined to be A/A (14%), A/G (60%), and G/G (26%); for Caucasian Americans, the distribution was A/A (30%), A/G (51%), and G/G (19%). These data correlate well with phenotypic data. We implemented a nonradioactive single-stranded conformational polymorphism analysis to rapidly identify all three genotypes. The PCR-single-stranded conformational polymorphism analysis method will facilitate studies of the genotype distribution in individuals with disorders of immune function.

Systematic use of automated fluorescence-based sequence analysis of amplified genomic DNA for rapid detection of point mutations.

Several approaches are now available for screening populations for known mutations in a given gene. However, for detection of multiple mutations in a population that has not been characterized or for detection of new mutations, the value and efficiency of these screening procedures decreases. Although more than 100 different beta-thalassemia mutations have so far been described, the spectrum of mutations in the Eastern Mediterranean and Israel has not been defined in detail. We have used automated fluorescence-based DNA sequence analysis of PCR-amplified genomic DNA employing a cycle-sequencing strategy coupled with advanced analysis software to rapidly detect beta-thalassemia mutations in Israeli patients. This method enabled rapid identification of eight different mutations in 10 patients, including two rare mutations, one of which has never been described in this geographic region. Our results show that automated fluorescence-based DNA sequence analysis of amplified genomic DNA is a rapid and reliable method for detection of point mutations and small deletions or insertions in both heterozygous and homozygous states. This approach is particularly effective for a relatively small gene such as beta-globin, but it can also be used for rapid detection of mutations in large genes by first sequencing clusters of exons and intron/exon borders.

Fluorescence-based RT PCR analysis: determination of the ratio of soluble to membrane-bound forms of Fc gamma RIIA transcripts in hematopoietic cell lines.

We have developed a fluorescence-based RT PCR assay for determination of the ratio of two alternatively spliced transcripts in different cell types. Fluorescence detection, by an automated DNA sequencer, allows enhanced sensitivity and ease of data processing. PCR products are fluorescently tagged using a dye-labeled oligonucleotide primer during the PCR reaction. Assay conditions were first defined so that fluorescence intensity of the PCR products was linear with respect to input RNA and exponential relative to PCR cycle number. Sensitivity and reproducibility of detection were evaluated with serial dilutions of RT PCR reactions. We have applied this assay to an analysis of the lineage-specific expression of two human Fc gamma RIIA transcripts, Fc gamma RIIa1 and Fc gamma RIIa2, in different hematopoietic cell lines. Previously, we noted that when standard RT PCR conditions are used with primers that bracket the TM exon, the pattern of expression of these transcripts as assessed by ethidium bromide staining of agarose gels varied in different hematopoietic cell lineages. Using the fluorescence-based RT PCR method, we now confirm our previous findings and quantitate transcript ratios (Fc gamma RIIa2/Fc gamma RIIa1) in several hematopoietic cell lines. The ratio varies from 0.70 (41% Fc gamma RIIa2) in the erythroleukemic cell line HEL, to 0.14 (12% Fc gamma RIIa2) in the monocytic cell line U937, to 0.07 (6% Fc gamma RIIa2) in the multipotential cell line K562. This fluorescent RT PCR method provides a general approach to quantitating mRNA levels and ratios of PCR products in other gene systems.