Detection of clonal immunoglobulin heavy-chain gene rearrangements in cases of suspected lymphoproliferative disorders: comparison of polymerase chain reaction and Southern blot analysis.

Demonstration of clonality is supportive of a diagnosis of malignancy in cases of lymphoproliferative disorders. Determination of clonality at the molecular level is currently accomplished by Southern analysis; however, the polymerase chain reaction offers a potential alternative that is rapid, simple, and less expensive. To test its feasibility as a diagnostic test, we amplified the DNA from 121 suspected lymphoproliferative disorders submitted for gene rearrangement studies. In comparison to Southern analyses, a sensitivity of 70% and specificity of 96% were obtained. To test the effect of primer variability in the joining region of the heavy-chain gene, we substituted a more degenerate primer but found no changes in sensitivity or specificity. We conclude that the polymerase chain reaction has current application with minute or fixed specimens and may generally serve as a rapid, initial evaluation for B-cell clonality, followed by Southern analysis in negative cases. However, higher overall sensitivity must be achieved before this technique can replace Southern analysis as the method of choice in determining clonal gene rearrangements.

Update on the Human Genome Project.

The Human Genome Project is essentially on schedule in accomplishing its short-term goals. Extensive genetic maps and partial physical maps have been made of all the chromosomes, an achievement which has accelerated the rate at which genes are being cloned. Preliminary to the concerted sequencing efforts that will occupy the later years of the Project, extensive activity has been directed toward the development of conventional and novel sequencing methods. The impact that the Human Genome Project will have on society and the future of medicine is discussed.

The lymphoid proliferation-associated gene EPAG maps to human chromosome region Xq21-->q22.

Using Southern blot and fluorescence in situ hybridization analyses, we have shown that the novel human gene EPAG, expressed in association with the mitogenic activation of T and B cells in peripheral blood, maps to chromosome region Xq21-->q22. These data establish EPAG as a candidate gene in X-linked disorders.

Properties of a novel gene isolated from a Hodgkin's disease cell line that is expressed early during lymphoid cell activation.

We have isolated a novel 667-bp cDNA clone, designated epag, from a Hodgkin's-disease cell line-derived library that is expressed in association with T cell activation and which is not related to any known gene family. By using reverse transcription/PCR, we have demonstrated that epag mRNA is expressed as early as 1 h after stimulation of normal PBMCs with anti-CD3. The levels of mRNA peaked by 4 h, and no expression was detectable by 12 h postactivation or in resting cells incubated in culture without activation. Expression of epag was also detected in PMA- and PHA-stimulated, but not in nonstimulated Jurkat cells, and overall its expression in transformed cell lines of hemopoietic origin is highly restricted. Sequence analysis of multiple independent cDNA clones showed that epag expressed in the Hodgkin's-disease cell line L428 is identical to the gene expressed in normal activated PBMC. Epag expression was detected by reverse transcription/PCR in RNA preparations made from various normal nonlymphoid tissues. Computer analysis of the sequence identified an open reading frame encoding a putative protein of 13.2 kDa initiating at a CUG translational codon. In vitro translation and Western blot analysis with anti-peptide serum supported this analysis. We hypothesize that epag functions as an early signal that helps mediate the activation of T cells.

Increased incidence of lymphoproliferative disorder after immunosuppression with the monoclonal antibody OKT3 in cardiac-transplant recipients.

BACKGROUND: A sudden increase in the incidence of post-transplantation lymphoproliferative disorder among the patients in our cardiac-transplantation program was temporally related to introduction of the immunosuppressive drug OKT3. This monoclonal antibody has come to be widely used in recent years both to prevent and to treat rejection after cardiac transplantation. METHODS: In order to identify variables that predict the development of post-transplantation lymphoproliferative disorder, we analyzed retrospectively a series of 154 consecutive cardiac-transplant recipients at a single institution. Univariate analyses and multivariate analysis by logistic regression were performed. RESULTS: Among 75 patients who did not receive OKT3, post-transplantation lymphoproliferative disorder developed in 1 (1.3 percent), as compared with 9 of 79 patients who received the drug (11.4 percent); the incidence among the OKT3-treated patients was ninefold higher (odds ratio, 9.5; 95 percent confidence interval, 1.6 to 54.7). According to multivariate analysis, the only factor significantly associated with the development of post-transplantation lymphoproliferative disorder was the use of OKT3 (P = 0.001). A significant increase in risk with increasing doses was also apparent: 4 of 65 patients who received a cumulative dose of 75 mg of OKT3 or less (6.2 percent) had post-transplantation lymphoproliferative disorder, whereas 5 of 14 patients who received more than 75 mg had the disorder (35.7 percent; P less than 0.001). CONCLUSION: The addition of OKT3 to the immunosuppressive regimen increases the incidence of post-transplantation lymphoproliferative disorder after cardiac transplantation, and the risk increases sharply after cumulative doses greater than 75 mg. We suggest that the risks and benefits of prophylactic OKT3 administration be reassessed in the light of these findings, particularly since the value of prophylactic immunotherapy in cardiac-transplant recipients remains to be clearly established.

Pseudoleukemia in Down's syndrome. Analysis of immunophenotype and gene rearrangement.

This report demonstrates a case of transient abnormal myelopoiesis (TAM) evolving in a patient with Down's syndrome. A diagnosis was established after the patient's blast cell count decreased considerably three weeks after the initial leukemic phase. The blast population in the authors' case expressed Leu-9 (CD7), 6D1, and TdT+. Cytochemistries showed some of the blast population to be peroxidase positive and Sudan black positive. Platelet peroxidase by electron microscopic examination showed some positive blasts. Therefore, surface markers and cytochemical studies in this case suggested an abnormal proliferation involving a pluripotential stem cell capable of expressing myeloid and lymphoid characteristics. Cytogenetics was performed at birth and showed 47,XY,+21/48,XY,+21,+mar, confirming the diagnosis of Down's syndrome. The origin of the chromosomal fragment was uncertain. It was of interest that during the remission phase of his pseudoleukemia there was a concomitant decrease in the extra chromosomal fragment. Immunoglobulin and T-cell antigen receptor gene rearrangement studies showed only germline patterns, indicating that the lymphoid cells in the blast population were not clonally expanded. Therefore, immunoglobulin and T-cell antigen receptor rearrangement analysis and immunophenotyping are extremely valuable techniques in distinguishing between TAM and acute lymphoblastic leukemia in patients with Down's syndrome.

Acute lymphoblastic leukemia-hand mirror variant. Analysis for endogenous retroviral antibodies in bone marrow plasma.

Two unusual cases of acute lymphoblastic leukemia-hand mirror variant (ALL-HMV) are presented. One patient demonstrated a mixed immunophenotype with HLA-DR, My7, transferrin receptor surface markers, and terminal deoxynucleotidyl transferase positivity. To the authors' knowledge, this is the first ALL-HMV reported with myeloid antigen. The patient died during induction and did not demonstrate the indolent course noted in the female subgroup with this disorder. The second case initially was not an ALL-HMV but presented as a non-T non-B ALL, and the patient had a relapse six years later with numerous hand mirror cells (HMCs). In the authors' experience, this is the first case of ALL that presented as a non-HMC and relapsed as an ALL-HMV. The patient's immunophenotype revealed he was HLA-DR, transferrin receptor, and TdT positive. Both patients' leukemic cells showed a diffuse granular periodic acid-Schiff on a clear background and acid phosphatase-positive pattern. Immunogenetics revealed a clonal rearrangement of one of the two Ig heavy chain loci in the one patient evaluated. Western blot analysis of the bone marrow plasma of both patients with ALL-HMV showed an increase of cross-reactive IgG to the envelope gp70 and IgM against the core p30 proteins of the baboon endogenous virus (BaEV) and simian sarcoma-associated virus (SSAV). Furthermore, their bone marrow plasma demonstrated IgM antibodies to the gp70 that were not present in any of the other non-hand mirror leukemic patients or the normal controls. These findings strengthen the concept that HMCs in ALL are formed in relation to an immunologic response to increased proteins related to BaEV and/or SSAV.

Differential expression of the two human arginase genes in hyperargininemia. Enzymatic, pathologic, and molecular analysis.

Previous studies in our laboratory and others have demonstrated in humans and other mammals two isozymes of arginase (AI and AII) that differ both electrophoretically and antigenically. AI, a cytosolic protein found predominantly in liver and red blood cells, is believed to be chiefly responsible for ureagenesis and is the one missing in hyperargininemic patients. Much less is known about AII because it is present in far smaller amounts and localized in less accessible deep tissues, primarily kidney. We now report the application of enzymatic and immunologic methods to assess the independent expression and regulation of these two gene products in normal tissue extracts, two cultured cell lines, and multiple organ samples from a hyperargininemic patient who came to autopsy after an unusually severe clinical course characterized by rapidly progressive hepatic cirrhosis. AI was totally absent (less than 0.1%) in the patient's tissues, whereas marked enhancement of AII activity (four times normal) was seen in the kidney by immunoprecipitation and biochemical inhibition studies. Immunoprecipitation-competition and Western blot analysis failed to reveal presence of even an enzymatically inactive cross-reacting AI protein, whereas Southern blot analysis showed no evidence of a substantial deletion in the AI gene. Induction studies in cell lines that similarly express only the AII isozyme indicated that its activity could be enhanced severalfold by exposure to elevated arginine levels. Our findings suggest that the same induction mechanism may well be operative in hyperargininemic patients, and that the heightened AII activity may be responsible for the persistent ureagenesis seen in this disorder. These data lend further support to the existence of two separate arginase gene loci in humans, and raise possibilities for novel therapeutic approaches based on their independent manipulation.

Effects of deletions in mouse chromosome 7 on expression of genes encoding the urea-cycle enzymes and phosphoenolpyruvate carboxykinase (GTP) in liver, kidney, and intestine.

Chromosomal deletions at and around the albino locus on chromosome 7 of the mouse affect the enzyme activities and steady-state levels of mRNAs for five urea-cycle enzymes in liver. In newborn c3H homozygotes, activities of these enzymes were 43-62% of normal, while corresponding mRNA levels were 14-29% of normal. c14CoS deletion homozygotes expressed mRNA levels for these enzymes which were 32-48% of normal. However, transcription rates of these genes in hepatic nuclei of c3H/c3H mice were reduced only to 57-84% of normal. Since effects of the deletions had previously been noted in the kidney, mRNA levels for three enzymes expressed also in the kidney were examined. Mice homozygous for the c3H deletion, shown previously to have drastically reduced mRNA levels for phosphoenolpyruvate carboxykinase in the liver, expressed the same deficiency in the kidney, while mRNA levels for argininosuccinate synthetase and argininosuccinate lyase were reduced in the liver but remained unaffected in the kidney. However, mRNA levels for phosphoenolpyruvate carboxykinase, carbamyl phosphate synthetase I, and ornithine transcarbamylase were unaffected in the intestine of c3H homozygotes. The results suggest that a regulatory factor(s) encoded in the DNA encompassed by the deletion is involved in the normal developmental maturation of hepatocytes and certain cells in the kidney.

Regulation of mRNA levels for five urea cycle enzymes in rat liver by diet, cyclic AMP, and glucocorticoids.

Adaptive changes in levels of urea cycle enzymes are largely coordinate in both direction and magnitude. In order to determine the extent to which these adaptive responses reflect coordinate regulatory events at the pretranslational level, measurements of hybridizable mRNA levels for all five urea cycle enzymes were carried out for rats subjected to various dietary regimens and hormone treatments. Changes in relative abundance of the mRNAs in rats with varying dietary protein intakes are comparable to reported changes in enzyme activities, indicating that the major response to diet occurs at the pretranslational level for all five enzymes and that this response is largely coordinate. In contrast to the dietary changes, variable responses of mRNA levels were observed following intraperitoneal injections of dibutyryl cAMP and dexamethasone. mRNAs for only three urea cycle enzymes increased in response to dexamethasone. Levels of all five mRNAs increased severalfold in response to dibutyryl cAMP at both 1 and 5 h after injection, except for ornithine transcarbamylase mRNA which showed a response at 1 h but no response at 5 h. Combined effects of dexamethasone and dibutyryl cAMP were additive for only two urea cycle enzyme mRNAs, suggesting independent regulatory pathways for these two hormones. Transcription run-on assays revealed that transcription of at least two of the urea cycle enzyme genes--carbamylphosphate synthetase I and argininosuccinate synthetase--is stimulated approximately four- to fivefold by dibutyryl cAMP within 30 min. The varied hormonal responses indicate that regulatory mechanisms for modulating enzyme concentration are not identical for each of the enzymes in the pathway.

Human arginase isozymes.

Studies in experimental animals and humans demonstrate the existence of two arginase isozymes. One, designated AI (or A1), has a high pI, is located in the cytosol, is most abundant in liver, and is thought to be primarily responsible for ammonia detoxification as urea. The gene coding for this isozyme is mutated in human hyperargininemia. A second isozyme, designated AII (or A4), has a neutral pI, is located in the mitochondrial matrix, and is thought to be involved primarily in the production of ornithine as a precursor of proline and glutamate. It appears to be expressed in most but not all tissues and in more nearly equal amounts. The two isozymes are immunologically distinct and are coded for by two separate genes. The great similarity in all measured kinetic and some physicochemical properties implies a high degree of structural similarity at the active site, but the lack of immunological cross-reactivity and DNA cross-hybridization implies substantial compositional differences in other parts of the enzyme molecules.

Isolation of human liver arginase cDNA and demonstration of nonhomology between the two human arginase genes.

A human liver cDNA library was screened by colony hybridization with a rat liver arginase cDNA. The number of positive clones detected was in agreement with the estimated abundance of arginase message in liver, and the identities of several of these clones were verified by hybrid-select translation, immunoprecipitation, and competition by purified arginase. The largest of these human liver arginase cDNAs was then used to detect arginase message on northern blots at levels consistent with the activities of liver arginase in the tissues and cells studied. The absence of a hybridization signal with mRNA from a cell line expressing only human kidney arginase demonstrated the lack of homology between the two human arginase genes and indicated considerable evolutionary divergence between these two loci.

The gene for human liver arginase (ARG1) is assigned to chromosome band 6q23.

The human liver arginase gene, whose deficiency is responsible for argininemia (McKusick no. 20780), has been assigned to 6q23 through a combination of somatic cell hybrid analysis and in situ hybridization using a 1,550-base pair (bp) human DNA probe for this gene.

Cloning of rat liver arginase cDNA and elucidation of regulation of arginase gene expression in H4 rat hepatoma cells.

In order to study the regulation of expression of the two arginase genes in mammalian tissues, we undertook to clone cDNA specific for rat liver arginase. mRNA was isolated from rat liver polysomes enriched for the arginase message by immunopurification and was used to produce an 800-member cDNA library carried in pBR322. Four arginase clones were identified by hybrid selection, and one was used to find two others following colony hybridization. Clonal identity was verified by its enrichment in the cDNA made from immunopurified mRNA; by hybrid selection, immunoprecipitation, and competition by purified arginase; hybridization on Northern analysis with liver-derived RNA (high in arginase) and its absence with mRNA from tissues low in arginase; and independent identification by hybrid selection and colony hybridization. Northern analysis of mRNA from H4-II-E-C3 (H4) rat hepatoma cells in which arginase activity was induced by hydrocortisone demonstrated equal, eightfold augmentation of both arginase activity and arginase mRNA levels. Southern blot analysis of DNA from these cells indicated that no change in arrangement or copy number accompanied induction. Southern analysis also suggested that the gene for rat liver arginase is present in a single copy, without pseudogenes, and that a high degree of homology exists between it and its mouse counterpart.

Cloning and regulation of messenger RNA for mouse apolipoprotein E.

A cDNA clone for mouse apolipoprotein E has been identified from a mouse liver cDNA library by a combination of differential colony hybridization and hybrid selection-translation. The identity of the clone was unambiguously established by partial sequencing and comparison with human apolipoprotein E nucleotide and amino acid sequences. In conjunction with an in vitro translation assay for apolipoprotein E, the clone has been used to examine the relative levels of apolipoprotein E mRNA in various tissues of the mouse and the regulation of apolipoprotein E synthesis in response to a diet rich in saturated fat and cholesterol. In the tissues examined, the clone was found to hybridize to a polyadenylated RNA species of approximately 1400 nucleotides. Of the tissues involved in lipoprotein synthesis, liver is very rich (about 1% of total) in apolipoprotein E mRNA while intestine contains only trace amounts. Appreciable levels of active apolipoprotein E mRNA (up to 10% of that in liver) are also detected in peripheral tissues not associated with lipoprotein synthesis, including lung, kidney, spleen, and heart. Thus, extrahepatic apolipoprotein E synthesis may contribute significantly to the levels present in plasma, and a possible function in "reverse cholesterol transport" is considered. When mice were placed on a high lipid diet there was no discernible change in the level of apolipoprotein E mRNA in liver or intestine, although the level of the circulating protein increased about 3-fold. We conclude that in mice the effect of diet on apolipoprotein E levels in blood does not result from induction of mRNA in these tissues.