Search

The protein kinase Syk is a key mediator of proximal B-cell receptor (BCR) signaling. Following antigen stimulation, Syk is recruited to the BCR and becomes activated by phosphorylation at Y352. Recently, Syk was found to be constitutively phosphorylated in several common B-cell lymphoma subtypes, indicating a role for antigen-independent Syk activation in the pathogenesis of these diseases. We now report that Syk is constitutively phosphorylated on the activating Y352 residue in chronic lymphocytic leukemia (CLL) B cells. To examine the effects of constitutive Syk activity on intracellular signaling and leukemic cell survival, we performed in vitro studies with the Syk inhibitor R406. Treatment with R406 induced leukemic cell apoptosis in the majority of investigated cases and affected the basal activity or expression of several pro-survival molecules regulated by Syk, including the Akt and extracellular signal-regulated (ERK) kinases, and the anti-apoptotic protein Mcl-1. In addition, R406 prevented the increase in leukemic cell viability induced by sustained BCR engagement and inhibited BCR-induced Akt activation and Mcl-1 upregulation. Collectively, these data identify Syk as a potential target for CLL treatment and suggest that inhibition of this kinase could provide a double therapeutic benefit by disrupting both antigen-dependent and antigen-independent signaling pathways that regulate leukemic cell survival.

A fraction of chronic lymphocytic leukaemia (CLL) cases carry highly homologous B-cell receptors (BCR), i.e. characterized by non-random combinations of immunoglobulin heavy-chain variable (IGHV) genes and heavy-chain complementarity determining region-3 (HCDR3), often associated with a restricted selection of IGVK/L light chains. Such 'stereotyped' BCR occur more frequently in CLL with unmutated (UM) than mutated (M) IGHV genes. We analysed 1426 IG rearrangements (from 1398 CLL cases) by a clustering driven by HCDR3 similarities. Molecular findings were correlated to time-to-treatment (TTT) and presence of known prognosticators. Sixty-nine clusters (319 IG-rearrangements, 22.4%) with stereotyped BCR were identified. Among 30 confirmed clusters (>or=3 IG-rearrangements/cluster), we found 14 novel clusters, of which 11 had M IG rearrangements (M clusters) and predominantly (8/11) used IGHV3 subgroup genes. Recurrent cluster-biased amino acid changes were found throughout IGHV sequences of these 'M clusters'. Regarding clinical outcome: (i) UM CLL from the IGHV1-2/1-3/1-18/1-46/7-4-1/IGKV1-39 cluster had poorer prognosis than UM/M cases, or UM cases using the same IGHV genes but not in clusters; (ii) M CLL from the IGHV3-21/IGLV3-21 cluster had TTT similar to UM CLL, and shorter than M CLL expressing IGHV3-21 but not in cluster. Altogether, our analysis identified additional molecular and clinical features for CLL expressing stereotyped BCR.

We tested the efficacy and safety of oral fludarabine and cyclophosphamide as front-line therapy in chronic lymphocytic leukemia (CLL) and assessed the influence of immunoglobulin variable region heavy chain (IgVH) gene mutation status, interphase cytogenetic abnormalities, and expression of ZAP-70 and CD38 on clinical outcome. Thirty-seven patients with previously untreated CLL received oral fludarabine (30 mg m(2)) and oral cyclophosphamide (250 mg m(2)) for three consecutive days every 4 weeks for six cycles. Eighteen patients had unmutated and 15 had mutated IgVH genes. Nine patients had the 'high risk' cytogenetic abnormality del(11q22.3) or del(17p13.1). Fifteen patients were ZAP-70-positive and eight patients were CD38-positive. Among the 35 valuable patients, 14 patients (40%) obtained a complete response and 13 (37%) a partial response. The median progression-free survival (PFS) was 23 months and median time to re-treatment (TTR) was 38 months. A significantly lower overall response rate (43% vs. 85%, p = 0.011), a shorter PFS (22 vs. 27 months, p = 0.015), and a shorter TTR (22 vs. 40 months, p = 0.031) were noticed in the 'high risk' cytogenetic abnormalities group; TTR was also shorter in IgVH-unmutated than in IgVH-mutated patients (26 vs. 41 months, p = 0.035). Hematologic toxicity included grade IV neutropenia (ten patients) and grade III/IV anemia (three patients). Gastrointestinal toxicity was mild and no patient required hospitalization. The oral combination of fludarabine and cyclophosphamide is an effective, safe, and well-tolerated regimen that, if confirmed with larger series, will be appropriate especially in patients with low risk biological parameters.

Sustained engagement of the B-cell receptor (BCR) increases apoptosis resistance in chronic lymphocytic leukemia (CLL) B cells, whereas transient stimulation usually has an opposite effect. The antiapoptotic BCR signal has been associated with prolonged activation of the PI3K/Akt and MEK/ERK pathways, which are key regulators of survival and proliferation in various cell types. To further define the relative contribution of the Akt and ERK kinases in regulating CLL B-cell survival, we introduced constitutively active mutants of Akt and MEK in primary CLL B cells and evaluated changes in the expression of relevant pro- and antiapoptotic proteins. Sustained activation of Akt resulted in increased leukemic cell viability and increased expression of the antiapoptotic proteins Mcl-1, Bcl-xL, and X-linked inhibitor of apoptosis protein (XIAP), thus largely recapitulating the effects of sustained BCR stimulation. Constitutively active MEK2 also up-regulated XIAP, but did not show a significant impact on leukemic cell survival. Down-regulation of Mcl-1 by siRNA treatment induced rapid and potent apoptosis in CLL B cells and blocked the antiapoptotic effect of sustained BCR stimulation, whereas down-regulation of Bcl-xL and XIAP did not affect leukemic cell viability. These data demonstrate that Akt and Mcl-1 are major components of a survival pathway that can be activated in CLL B cells by antigen stimulation.

Several features of the B-cell receptor (BCR) have emerged as major prognostic factors in chronic lymphocytic leukemia (CLL). In particular, the absence of somatic mutations in the immunoglobulin variable region genes and expression of the protein tyrosine kinase ZAP-70 are strongly associated with an aggressive clinical course, and both features have been correlated with a greater capacity of the BCR to transmit antigen-induced signals. Additionally, differences in BCR structure and reactivity indicate that CLL B-cells from the two prognostic subsets may recognize different types of antigens. Antigens that are not rapidly internalized induce sustained BCR signaling that can promote the survival of the leukemic B-cells. The BCR signal is initially transmitted by the Syk kinase and modulated by ZAP-70, which shows inefficient or absent tyrosine kinase activation in B-cells. However, both ZAP-70 expression and sustained BCR engagement have been associated with prolonged activation of the Akt and ERK kinases, which is required for the induction of several antiapoptotic proteins, including Mcl-1, Bcl-xL and XIAP. Therefore, targeting components along the BCR signaling pathway may be a promising strategy for the treatment of CLL.

IGHV3-21-using chronic lymphocytic leukemia (CLL) is a distinct entity with restricted immunoglobulin gene features and poor prognosis and is more frequently encountered in Northern than Southern Europe. To further investigate this subset and its geographic distribution in the context of a country (Italy) with both continental and Mediterranean areas, 37 IGHV3-21 CLLs were collected out of 1076 cases enrolled by different institutions from Northern or Central Southern Italy. Of the 37 cases, 18 were identified as homologous (hom)HCDR3-IGHV3-21 CLLs and were found almost exclusively (16 of 18) in Northern Italy; in contrast, 19 nonhomHCDR3-IGHV3-21 cases were evenly distributed throughout Italy. Clinically, poor survivals were documented for IGHV3-21 CLLs as well as for subgroups of mutated and homHCDR3-IGHV3-21 CLLs. Negative prognosticators CD38, ZAP-70, CD49d, and CD79b were expressed at higher levels in homHCDR3 than nonhomHCDR3-IGHV3-21 cases. Differential gene expression profiling (GEP) of 13 IGHV3-21 versus 52 non-IGHV3-21 CLLs identified, among 122 best-correlated genes, TGFB2 and VIPR1 as down- and up-regulated in IGHV3-21 CLL cases, respectively. Moreover, GEP of 7 homHCDR3 versus 6 nonhomHCDR3-IGHV3-21 CLLs yielded 20 differentially expressed genes, with WNT-16 being that expressed at the highest levels in homHCDR3-IGHV3-21 CLLs. Altogether, IGHV3-21 CLLs, including those with homHCDR3, had a peculiar global phenotype in part explaining their worse clinical outcome.

Expression of ZAP-70 is an important negative prognostic factor in chronic lymphocytic leukemia (CLL). This protein tyrosine kinase is a key mediator of T-cell receptor (TCR) signaling and is structurally homologous to Syk, which plays an analogous role in B-cell receptor (BCR) signaling. Recent studies indicate that ZAP-70 may participate in BCR signaling as well, but the mechanism of action is not completely understood. We have now compared antigen receptor-induced activation of ZAP-70 in B cells and T cells by analyzing phosphorylation of critical regulatory tyrosine residues. We show that BCR-mediated activation of ZAP-70 is very inefficient in CLL and lymphoma B cells and is negligible when compared to activation of Syk. Despite the inefficient catalytic activation, the ability of ZAP-70 to recruit downstream signaling molecules in response to antigen receptor stimulation appeared relatively preserved. Moreover, ectopic expression of ZAP-70 enhanced and prolonged activation of several key mediators of BCR signaling, such as the Syk, ERK, and Akt kinases, and decreased the rate of ligand-mediated BCR internalization. We conclude that the role of ZAP-70 in BCR signaling is quite distinct from its role in TCR signaling and is likely mediated by inhibition of events that terminate the signaling response.

Elevated levels of TNF-alpha have been associated with progressive disease in patients with chronic lymphocytic leukemia (CLL). Thalidomide has been shown to inhibit production of TNF-alpha. We investigated the effects of thalidomide on clinical outcome and TNF-alpha serum levels in five pre-treated CLL patients. The schedule consisted on daily thalidomide (Thal), oral fludarabine (Flu) and oral cyclophosphamide (CTX). Median duration of treatment was 60 days; four patients stopped treatment for disease progression and one patient for neurological toxicity. Serum TNF-alpha levels did not show any decrease during treatment. Low-dose thalidomide is not effective in CLL patients with refractory disease.

Recent studies indicate that V(H) gene usage in B-CLL may have prognostic impact independently of V(H) gene mutation status. The V1-69 gene is the most frequently rearranged V(H) gene in B-CLL and is almost always unmutated. We therefore investigated whether patients with a V1-69 gene rearrangement differ in clinical course and outcome with respect to patients expressing other unmutated V(H) genes. We show that V1-69 B-CLLs constitute a uniform group of patients that more often present at advanced clinical stages and require early treatment, but their survival does not differ significantly from patients with other unmutated V(H) genes.

Chronic lymphocytic leukemia (CLL) B-cells are hyporesponsive to many proliferative signals that induce activation of normal B-lymphocytes. However, a heterogeneous response has recently been observed with immunostimulatory CpG-oligodeoxynucleotides (CpG ODN). We now show that CpG ODN induce proliferation mainly in CLL B-cells from patients with progressive disease and unmutated immunoglobulin V(H) genes, whereas G(1)/S cell cycle arrest and apoptosis are induced in leukemic B-cells from stable/V(H) mutated CLL. Examination of early signaling events demonstrated that all CLL B-cells respond to CpG ODN stimulation by degradation of the NF-kappaB inhibitor IkappaB and activation of the Akt, ERK, JNK and p38 MAPK kinases, but the magnitude and duration of the signaling response was greater in the proliferating cases. Pharmacological inhibition of these pathways showed that simultaneous activation of Akt, ERK and JNK is required for cell cycle progression and proliferation. Conversely, introduction of constitutively active Akt in nonproliferating CLL B-cells resulted in induction of cyclin A following CpG ODN stimulation, indicating that increased Akt activation is sufficient to overcome the hyporesponsiveness of these cells to proliferative signals. Thus, the magnitude of Akt signaling may determine the distinct responses observed in leukemic B-cells belonging to the different prognostic subgroups.

The frequent expression of the otherwise rare IgVH3-72 gene was demonstrated in highly stable B-cell chronic lymphocytic leukaemias (B-CLLs). Here we describe the immunophenotypic profiles of eleven IgVH3-72 B-CLLs, by investigating expression of ZAP-70 and of a set of surface antigens previously demonstrated to represent the signature of distinct B-CLL prognostic groups. All IgVH3-72 B-CLLs revealed a homogeneous immunophenotypic profile, expressing all the markers associated with good prognosis, but not those representing the signature of bad prognosis B-CLLs, including ZAP-70. Our results corroborate the notion that IgVH3-72 B-CLLs represent a molecularly and immunophenotypically homogeneous disease group with a good prognosis.

B cell chronic lymphocytic leukemia (B-CLL) is a clonal overgrowth of CD5(+) B lymphocytes. In this disease, the B cell antigen receptor (BCR) is intimately linked to disease severity, because patients with BCRs, comprised of unmutated V(H) genes, follow a much more aggressive course. This and related observations suggest that B-CLL derives from a B cell subset comprised of restricted BCR structural diversity and that antigen-selection and drive are major factors promoting the disease. Nevertheless, the initiating event(s) that lead to the development of B-CLL are still unclear, in part because of the lack of an animal model that spontaneously evolves the molecular abnormalities that occur in the human disease. Because overexpression of the TCL1 gene in murine B cells leads to a CD5(+) B cell lymphoproliferative disorder with many of the features of human B-CLL, we studied leukemias emerging in these mice to examine the extent to which their BCRs resemble those in B-CLL. Our data indicate that the immunoglobulin heavy and light chain rearrangements in TCL1 mice display minimal levels of somatic mutations and exhibit several molecular features found in the human disease. Like human B-CLL, TCL1 leukemic rearrangements from different mice can be very similar structurally and closely resemble autoantibodies and antibodies reactive with microbial antigens. Antigen-binding analyses confirm that selected TCL1 clones react with glycerophospholipid, lipoprotein, and polysaccharides that can be autoantigens and be expressed by microbes. This (auto)antigen-driven mouse model reliably captures the BCR characteristics of aggressive, treatment-resistant human B-CLL.

Background and Objectives: The protein tyrosine kinase ZAP-70 has recently emerged as a major prognostic indicator in chronic lymphocytic leukemia (CLL). ZAP-70 is structurally and functionally homologous to Syk, a key mediator of B-cell receptor signaling. We therefore evaluated ZAP-70 expression in CLL B cells using Syk as an intracellular standard., Design and Methods: The relative amounts of ZAP-70 and Syk were determined in purified B cells from 92 CLL patients using a novel reverse transcriptase/polymerase chain reaction (RT-PCR) procedure that co-amplifies both transcripts with equal efficiency. The ZAP-70/Syk mRNA ratio was correlated with VH gene mutation status, median treatment-free survival and FACS analysis of ZAP-70 expression., Results: ZAP-70 was expressed in the majority of cases with unmutated VH genes (88%), but also at lower levels in a substantial fraction of cases with mutated VH genes (44%). High levels of ZAP-70, defined as ZAP-70/Syk mRNA ratios above 0.25, were observed mainly in cases with unmutated VH genes and correlated with short treatment-free survival. In contrast, no difference was observed in the median treatment-free survival between patients with low ZAP-70/Syk ratios (0.05-0.25) and patients with no or negligible ZAP-70 expression (ZAP-70/Syk<0.05). In 73 cases ZAP-70 expression was investigated by RT/PCR and FACS analysis; concordance with VH gene mutation status was 86% and 71%, respectively., Interpretation and Conclusions: ZAP-70 is frequently expressed in CLL B cells, but only high levels correlate with unmutated VH gene status and progressive disease. Expression of ZAP-70 can be accurately assessed by analysis of the ZAP-70/Syk mRNA ratio, thus providing an alternative to FACS analysis.

The clinical course of chronic lymphocytic leukemia (CLL) differs significantly between patients with mutated (M-CLL) and unmutated (U-CLL) immunoglobulin (Ig) variable heavy-chain (V(H)) genes, implying a role for B-cell receptor (BCR) signaling in the pathogenesis of this disease. We have now investigated activation of downstream BCR signaling pathways in U-CLL and M-CLL B cells using soluble anti-IgM (sol-IgM) and immobilized anti-IgM (imm-IgM) antibodies as models for antigenic stimulation. Ligation of the BCR with sol-IgM induced incomplete responses in both CLL subsets, resembling the pattern described for tolerant B cells. This response was characterized by transient phosphorylation of extracellular signal-related kinase (ERK) and Akt (protein kinase B [PKB]), lack of activation of c-JUN NH2-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), and variable activation of phospholipase Cgamma2 (PLCgamma2) and nuclear factor-kappaB (NF-kappaB). Stimulation with imm-IgM elicited a more complete BCR signal and significantly prolonged phosphorylation of ERK and Akt, indicating persistent or repetitive BCR signaling. Moreover, this type of stimulation increased the levels of the antiapoptotic protein myeloid cell leukemia-1 (Mcl-1) and protected from chemotherapy-induced apoptosis, whereas induction of apoptosis and down-regulation of Mcl-1 was observed following stimulation with sol-IgM. These data demonstrate that only sustained BCR signaling can promote survival of CLL B cells and indicate that the main difference between CLL with mutated and unmutated V(H) genes may reside in the availability of such stimulation.

Previous studies suggest that the diversity of the expressed variable (V) region repertoire of the immunoglobulin (Ig)H chain of B-CLL cells is restricted. Although limited examples of marked constraint in the primary structure of the H and L chain V regions exist, the possibility that this level of restriction is a general principle in this disease has not been accepted. This report describes five sets of patients, mostly with unmutated or minimally mutated IgV genes, with strikingly similar B cell antigen receptors (BCRs) arising from the use of common H and L chain V region gene segments that share CDR3 structural features such as length, amino acid composition, and unique amino acid residues at recombination junctions. Thus, a much more striking degree of structural restriction of the entire BCR and a much higher frequency of receptor sharing exists among patients than appreciated previously. The data imply that either a significant fraction of B-CLL cells was selected by a limited set of antigenic epitopes at some point in their development and/or that they derive from a distinct B cell subpopulation with limited Ig V region diversity. These shared, stereotyped Ig molecules may be valuable probes for antigen identification and important targets for cross-reactive idiotypic therapy.

CTLA-4 is a CD28 homologue that plays an important role in negative regulation of T-cell responses. Its transient expression on the surface of activated T cells antagonizes the activating signals and terminates the T-cell response. An A to G polymorphism at position 49 of the CTLA-4 first exon has recently been associated with several autoimmune disorders. In the present study we have examined the prevalence of the A and G alleles of the CTLA-4 gene in 50 patients with autoimmune hemolytic anemia (AIHA), of which 20 had idiopathic AIHA and 30 had AIHA and chronic lymphocytic leukemia (CLL), and in 60 patients with immune thrombocytopenic purpura (ITP). Control subjects were 100 healthy individuals and 100 CLL patients without clinical evidence for an autoimmune disease. The G allele was present at a significantly higher frequency among the patients with AIHA (P = 0.003), whereas no difference was observed between patients with ITP and controls. The G allele frequency was highest among CLL patients who had developed AIHA. The obtained data indicate that the G allele of CTLA-4 predisposes to the development of AIHA, particularly among patients with CLL., (Copyright 2003 Wiley-Liss, Inc.)

Vaccination with immunogenic formulations of lymphoma-derived immunoglobulin can elicit strong anti-idiotypic immune responses which have proved effective in murine B cell tumor challenge experiments and suggested possible benefits in recent human clinical trials. Naked plasmid DNA vaccines encoding the Id determinants as scFv fragments provide the most promising alternative to protein immunization. With this approach the addition of an immunogenic domain linked to the scFv has proved essential for the induction of a protective immune response. In this study we have produced a scFv gene construct linked to the CH3 exon of the human IgG1 constant region and tested its efficacy in inducing protective immunity against the mouse BCL1 lymphoma. We have also generated a second construct in which the BCL1 VL gene was deleted to investigate whether the VH region domain contains sufficient antigenic determinants for a protective immune response. Both constructs induced anti-idiotypic antibodies that specifically reacted with the BCL1 IgM protein in ELISA and with BCL1 tumor cells in flow cytometry assays. Protection against tumor challenge was fully achieved with the complete scFv construct whereas immunization with the construct lacking the VL gene resulted in only a slight prolongation of the survival. We therefore conclude that a plasmid DNA vaccine containing the VH and VL genes of the lymphoma Ig linked to the human IgG1 CH3 exon is highly effective in inducing a protective immune response in the BCL1 model. We also demonstrated that VH gene immunization can induce strong anti-idiotypic antibody responses.

Background/aim: In previous studies we demonstrated that all patients affected by HCV-positive type II mixed cryoglobulinaemia have a monoclonal B-cell population in peripheral blood mononuclear cells, and that a large fraction of HCV-infected patients develop a monoclonal B-cell expansion, even in the absence of dosable serum cryoglobulins. However, the prevalence of Type II mixed cryoglobulinaemia in HCV-infected individuals seems to be high in Italy, whereas it is very low in Japan. This study was performed to investigate whether there are ethnic differences in the prevalence of asymptomatic HCV-associated monoclonal B-cell expansions., Methods: Forty-four Japanese patients affected by HCV-positive chronic liver disease (two healthy carriers, 31 chronic hepatitis and 11 cirrhosis) were compared with a group of 60 Italian patients (one healthy carrier, 49 chronic hepatitis, and 10 cirrhosis) without dosable levels of cryoglobulins. The monoclonality of peripheral blood mononuclear cells was investigated by RT/PCR analysis of Immunoglobulin gene rearrangements. Liver function tests, rheumatoid factor, cryocrit level, anti-HCV antibodies, HCV-RNA, and HCV genotype were performed according to standard methodology., Results: A B-cell monoclonal population was found in 26% of Italian patients, whereas all Japanese patients were negative. No correlation was found between B-cell monoclonality and severity of liver disease, length or source of the infection, HCV genotype, sex, clinical and biochemical parameters., Conclusions: This study indicates that a monoclonal B-cell proliferation in peripheral blood mononuclear cells is common in HCV infection, but only in Italy, whereas it is absent in Japan. This explains the very low prevalence of Type II mixed cryoglobulinaemia in HCV-positive Japanese subjects, and suggests that HCV is able to determine a B-cell expansion only in the presence of, presently undetermined, host factors.

The aetiology of non-Hodgkin's lymphomas remains a controversial matter, but, recently, evidence has emerged showing that these neoplastic aberrations of the immune system may be due to viruses, at least in some cases. In fact, patients affected by an inherited immune deficiency, and those presenting disease characterized by autoimmune dysfunctions, show an increased risk for the development of non-Hodgkin's lymphomas. Several viruses have been identified as potential aetiologic agents for of non-Hodgkin's lymphomas: one of these is the Epstein-Barr virus, which has been detected in cultures of tumour cells from patients with Burkitt's lymphoma: this virus seems to be involved also in the pathogenesis of some histological variants of Hodgkin's disease. In addition, the human T-cell lymphotrophic virus family members have also been recognized as possible aetiologic agents for several lymphomas, such as cutaneous T-cell lymphomas, T-cell leukaemia and T-cell hairy cell leukaemia. Recently, hepatitis C virus has been recognized as the aetiologic agent of mixed cryoglobulinaemia, which can be considered as a benign lymphoproliferative disorder. Since mixed cryoglobulinaemia can frequently evolve into more aggressive haematological disorders, an increased prevalence of hepatitis C virus infection in non-Hodgkin's lymphomas has been found, especially in low-grade non-Hodgkin's lymphomas. The possible aetiopathogenetic role of hepatitis C virus in non-Hodgkin's lymphomas is discussed on the basis of molecular, clinical and epidemiological considerations.

A high prevalence of chronic hepatitis C virus (HCV) infection has recently been shown in a subset of B-cell non-Hodgkin's lymphomas, most of which belong to the lymphoplasmacytoid lymphoma/immunocytoma subtype and are characterized by the production of a monoclonal IgM cryoglobulin with rheumatoid factor activity. To better define the stage of differentiation of the malignant B cell and to investigate the role of chronic antigen stimulation in the pathogenesis of the HCV-associated immunocytomas, we analyzed the variable (V) region gene repertoire in 16 cases with this type of tumor. The lymphoma-derived V gene sequences were successfully determined in 8 cases; 5 of them expressed the 51p1 VH gene in combination with the kv325 VL gene. Moreover, a monoclonal 51p1-expressing B-cell population was detected in 4 of the remaining immunocytomas by an allele-specific Ig gene fingerprinting assay, indicating that HCV-associated immunocytomas represent clonal proliferations of a highly selected B-cell population. Somatic mutations and intraclonal diversity were observed in all of the lymphoma V genes, and clonally related IgM and IgG VH transcripts indicative of isotype switching were present in one case. These findings are consistent with an antigen-driven process and support a role for chronic antigen stimulation in the growth and clonal evolution of HCV-associated immunocytomas.

The increased number of CD5+ B-cells in some human autoimmune diseases, the frequent commitment of CD5+ B-cells to the production of natural autoantibodies, and the apparent involvement of these cells in the pathogenesis of the autoimmune hemolytic anemia (AIHA) in certain mouse models suggests a causal relationship between the CD5+ chronic lymphocytic leukemia (CLL) B-cell and the AIHA which frequently develops in this malignant disorder. In support of this conclusion is our recent finding that the VH region gene repertoire of the leukemic B-cells from CLL patients with AIHA is rather biased and characterised by the over-representation of the 51p1 VH gene. On the other hand, it appears relatively certain that the pathogenic anti-erythrocyte antibodies in CLL patients with AIHA are produced by remnant normal B-cells, and that the antibodies expressed by the leukemic CD5+ B-cells do not directly bind red blood cells (RBC). Of interest, the antibodies produced by the leukemic B-cells from CLL patients with AIHA might have in common rheumatoid factor (RF) activity. These data indicate that the antibodies produced by the leukemic B-cells from CLL patients with AIHA are not directly involved in red blood cell destruction, but may be involved in the induction or amplification of a polyclonal anti-RBC response. Finally, we discuss the possible clinical implications of our finding that CLL patients with leukemic cells expressing the 51p1 VH gene may be at a higher risk to develop autoimmune hemolytic anemia.

The human C epsilon gene produces a number of alternatively spliced heavy chain transcripts of which some encode functional IgE isoforms. We now show that differentially processed epsilon mRNA variants also exist in the mouse and are generated by differential polyadenylation and alternative splicing of primary epsilon chain transcripts. The two poly(A) sites of the mouse membrane transcripts were identified in the present study by RACE-PCR analysis. The first poly(A) site is located 743 nt downstream from the beginning of the second membrane exon (M2) and contains the same non-consensus AGTAAA signal sequence as the single poly(A) site of the human membrane transcripts. The second poly(A) site is located almost 500nt further downstream and is characterized by an AAGAAA hexamer. This poly(A) site contains a (G+T) rich element downstream to the site of cleavage and polyadenylation and is preferentially utilized by the membrane epsilon transcripts. Additional diversity of epsilon transcripts is generated by alternative splicing between the last constant region exon (CH4) and the two membrane exons (M1 and M2). The alternatively spliced transcripts include two variants that skip the first membrane exon and encode epsilon heavy chains that lack the transmembrane domain. The third variant is generated by splicing to an internal site in M2 and codes for a membrane isoform that is 10 amino acids shorter in the cytoplasmic domain than the classical membrane IgE. Although little amino-acid sequence homology exists between the murine epsilon chain isoforms and their human counterparts, the pattern of splicing is rather conserved between the two species.

The human C epsilon gene expresses two membrane IgE heavy chain mRNAs which differ in the sequence that encodes their extracellular membrane-proximal domain. In the long IgE isoform (mLIgE), this domain contains a stretch of 52 amino acids which are absent in the short variant (mSIgE). We have now generated B cell transfectoma cell lines that express these two isoforms and show that both types of mIgE form functional B cell antigen receptors (BCR). Both receptors associate with the Ig-alpha/Ig-beta heterodimer, as well as with protein kinases that are capable of phosphorylating this complex. Upon their cross-linking, both receptors can activate protein tyrosine kinases that phosphorylate the same substrate proteins. Both IgE receptors also associate with two novel proteins that do not bind to mIgM. Apart from these similarities, the two IgE-BCRs show several differences of which some are analogous to the differences between the IgM- and IgD-BCRs. First, the mSIgE is transported to the cell surface at a higher rate than the mLIgE. Second, the two IgE-BCRs associate with differently glycosylated Ig-alpha proteins, the mLIgE associates with the completely glycosylated form, whereas the mSIgE associates with an Ig-alpha glycoform that is partially sensitive to endoglycosidase H. Third, the kinetics of protein tyrosine phosphorylation induced by receptor cross-linking is significantly different for the two IgE-BCRs. Finally, cross-linking of the mSIgE-BCR leads to growth inhibition of the B cell transfectoma, whereas signaling through the mLIgE-BCR does not affect the cellular proliferation. These data show that the two human membrane IgE isoforms assemble into functionally distinct antigen receptors which can induce different cellular responses.

The partial molecular characterization of a large deletion present in two members of an Indonesian-Malay family with beta-thalassemia trait is described. Polymerase chain reaction and sequencing analyses of the breakpoint identified a sequence which has previously been described in patients with the 45 kb Filipino beta 0-thalassemia deletion, i.e. a 5' breakpoint at position -4279 nucleotides 5' from the Cap site of the beta-globin gene. The 3' breakpoint is located in an L1 family of repetitive sequences at an unknown distance from the beta-globin gene. The hematological and hemoglobin data of the patients with this beta 0-thalassemia deletion further supports the concept that the unusually high Hb A2 levels are unique to deletions removing the 5' beta-globin gene region, and points to the importance of the 3' junction sequences for the regulation of Hb F levels in patients with deletional defects of the beta-globin gene cluster.

The malignant B cells in chronic lymphocytic leukemia (CLL) typically express low-density membrane IgM or IgM/IgD. In vitro experiments have shown that the CLL cells can be induced to differentiate into cells that secrete immunoglobulin (Ig) and can occasionally undergo heavy (H) chain class switching. We now show that the CLL cells also undergo isotype-switching in vivo, since gamma and/or alpha H chain transcripts with identical FW3/CDR3/FW4 regions as the mu CLL transcripts were detected in all of the 13 investigated patients with IgM+ CLL. In most cases switching had occurred to alpha1 and gamma3, but CLL transcripts corresponding to the other gamma chain isotypes were also detected. In one case both the productively and nonproductively rearranged allele were found to undergo H chain class switching. CLL gamma transcripts were also present in surface IgG+ sorted B cells, demonstrating that a small subset of the CLL cells express membrane IgG. In addition, transcripts encoding secretary gamma2 and gamma3 H chains were detected in two cases, which suggests that some serum IgG could be produced by the leukemic clone. Analysis of sorted PBL showed that isotype-switching occurs in CLL cells that express the CD5 antigen. Finally, nucleotide sequence analysis showed that the mu, alpha, and gamma CLL transcripts are identical, demonstrating that the CLL cells do not accumulate somatic mutations in their variable region genes after the H chain class switching. These data provide in vivo evidence that isotype-switching is a frequent phenomenon in CLL, and indicate that a subset of the CLL lymphocytes progress to later stages of B cell differentiation.

Background: Several authors have reported on the effectiveness of alpha-interferon (IFN-alpha) in the treatment of patients with mixed cryoglobulinemia. This prompted the authors to investigate the long term effects of this drug on clinical, hematologic, and virologic parameters in a group of 20 patients (13 women and 7 men) affected by mixed cryoglobulinemia., Methods: In all patients, bone marrow biopsy, phenotyping of marrow cells, and polymerase chain reaction (PCR) immunoglobulin gene rearrangement in peripheral blood lymphocytes were performed before therapy and at the end of the follow-up. A liver biopsy was obtained in patients with biochemical signs of chronic liver disease. The presence of hepatitis C virus (HCV) RNA in serum was assessed by detection of anti-HCV antibodies, and by PCR amplification of the 5' untranslated region of HCV. The HCV genotype was also determined by PCR amplification of the core region of the virus with type-specific primers. The treatment schedule followed by all patients was 3 million units of recombinant IFN-alpha 2b 3 times weekly for 1 year., Results: In 6 patients, the marrow histology before therapy showed a massive (more than 50%) monomorphous infiltration by plasmacytoid lymphocytes, indicating the presence of low grade non-Hodgkin's lymphoma. Anti-HCV antibodies were present in 19 (95%) subjects, and HCV-RNA was detectable in all patients. In addition, all patients affected by Type II mixed cryoglobulinemia showed a monoclonal B-cell expansion in peripheral blood mononuclear cells (PBMC). With therapy, 5 patients (25%) achieved a complete response and 11 patients (55%) a partial response, whereas minor responses were observed in the remaining 4 patients (20%). One of the complete responders and all patients showing partial responses relapsed a few months after therapy withdrawal. At the end of the follow-up, four patients had obtained a complete remission. Bone marrow examination showed that B-lymphocytic monoclonal infiltrate disappeared in three patients. Moreover, these three patients had become negative for B-cell expansion in PBMC. Lack of response, or relapse, was associated with the presence of Type II HCV., Conclusions: HCV may be the cause of mixed cryoglobulinemia. The disease is associated with a high prevalence of bone marrow B-cell lymphomas. IFN-alpha appears to be an effective agent for the treatment of mixed cryoglobulinemia. It also seems able to determine regression of the lymphoproliferative disorder. The HCV genotype appears to be the most important predictive factor for the response to antiviral therapy.

A number of alternatively spliced epsilon transcripts have been detected in IgE-producing B cells, in addition to the mRNAs encoding the classical membrane and secreted IgE heavy (H) chains. In a recent study, we examined the protein products of three of these alternatively spliced isoforms and found that they are intracellularly retained and degraded because of their inability to assemble into complete IgE molecules. We have now similarly examined a more recently described epsilon mRNA species that is generated by splicing between a donor splice site immediately upstream of the stop codon in the H-chain constant region exon 4 (CH4) and an acceptor site located in the 3' part of the second membrane exon. We show that this isoform is efficiently secreted by both plasma cells and B lymphocytes and therefore represents a second secreted IgE isoform (epsilon S2). The epsilon S2 H chain is only six amino acids longer than the classical secreted Ig H chain (epsilon S1) and contains a C-terminal cysteine, which is a characteristic sequence feature of mu and alpha H chains. However, unlike IgM and IgA, the epsilon S2 C-terminal cysteine (Cys-554) does not induce polymerization of H2L2 molecules (where L is light chain), but rather creates a disulfide bond between the two H chains that increases the rate of association into covalently bound H2L2 monomers. This C-terminal cysteine also does not function as an intracellular retention element because the epsilon S2 isoform was secreted in amounts equal to that of the epsilon S1, both in B lymphocytes and in plasma cells. The epsilon S2 H chains secreted by B lymphocytes differed from the epsilon S1 H chains in the extent of glycosylation. Interestingly, a difference in glycosylation between B-lymphocytes and plasma cells was also noted for both isoforms. The presence of the Cys-554 also allowed the identification of a distinctive asymmetric pathway of IgE assembly, common to both types of epsilon H chains.

Several IgE heavy (H) chain transcripts are produced by alternative splicing between constant region (CH3 and CH4) and membrane (M1 and M2) exons and by differential cleavage-polyadenylation at poly(A) sites downstream of the CH4 and M2 exons. We have now characterized the poly(A) signal of the epsilon transcripts that contain membrane exon sequences (epsilon CH4-M1'-M2, epsilon CH4-M1-M2, epsilon CH4-M2' and epsilon CH4-M2") and have determined the complete sequence of the M2 exon and 1.4 kb of downstream genomic DNA. The membrane locus poly(A) site was identified by RACE-PCR analysis of epsilon transcripts obtained from IgE-producing myeloma cells and normal peripheral blood lymphocytes (PBL). All membrane exon transcripts were found to be polyadenylated following a CA dinucleotide located 1046 nt from the beginning of the M2 exon. An AGTAAA hexamer, located 13 nt upstream from the site of cleavage and polyadenylation, was the only poly(A) signal sequence present in the 1.4 kb of genomic DNA downstream of the M2 exon. A (G+T)-rich region, which is also conserved in most poly(A) signals, was present 50 nt downstream of the AGTAAA hexamer. Northern blot analysis confirmed that this poly(A) site is used by the membrane exon epsilon mRNAs expressed by the U266 myeloma. The four membrane exon transcripts were detected in different relative amounts in PBL and IgE-producing myeloma cells, which could reflect different epsilon mRNA splicing patterns during B-cell differentiation.

Clonal expansions of IgM-producing B cells were investigated in 38 patients with a chronic hepatitis C virus infection. Eight patients were affected with type II mixed cryoglobulinaemia (two of whom also had non-Hodgkin's lymphoma and one had Waldenström's disease), one with type III mixed cryoglobulinaemia, one with Waldenström's disease, and 28 with chronic liver disease. To detect the clonal B-cell expansions we used a RT/PCR procedure in which the CDR3/FW4 regions of the IgM heavy chain mRNAs were amplified and resolved in sequencing polyacrylamide gels. Clonal Ig gene rearrangements were detected in all patients with type II mixed cryoglobulinaemia and also at a high frequency (24%) in the HCV-infected patients without cryoglobulinaemia. A polyclonal pattern was present in the patient with type III mixed cryoglobulinaemia and in the 15 normal individuals and 16 age-related patients with HCV-negative alcoholic liver disease which were investigated as controls. No association was found between the presence of a clonal B-cell expansion and age, sex, liver histology, or levels of serum aminotransferase. The serum levels of rheumatoid factor were increased in all patients with a clonal expansion, suggesting that the expanded B-cell clones belong to the rheumatoid factor producing B-cell subset.

We have investigated the IgE heavy chain isoforms produced in vivo by analyzing the epsilon mRNA species present in unstimulated PBL and expressing them individually in a myeloma cell line. Seven epsilon mRNA species were identified by using reverse transcription-PCR, cloning, and sequencing analysis. These species included the classical secreted (epsilon CH4-S) and membrane-bound (epsilon CH4-M1'-M2) IgE and five alternatively spliced epsilon transcripts. At the protein level, the five alternatively spliced epsilon transcripts (epsilon CH4*, epsilon CH4-M2', epsilon CH4'-1, epsilon CH4'-1-M2, and epsilon CH3-13-CH4) corresponded to four epsilon heavy chain isoforms, in which various parts of the CH4 domain were replaced by new stretches of amino acids at the carboxyl termini. The same epsilon mRNA species also were present in the IgE producing myeloma cell line U266. However, except for the classical membrane and secreted IgE, the corresponding proteins could not be identified. To further characterize the epsilon CH4-S, epsilon CH4*, epsilon CH4-M2', epsilon CH4'-1, and epsilon CH4-M1'-M2 species, we expressed them as chimeric mouse/human anti-4-hydroxy-5-iodo-3-nitrophenacetyl Abs in a mouse myeloma cell line. Only the classical secreted and membrane isoforms were found to be secreted or expressed on the cell surface, respectively, and the other forms were retained within the cells and degraded. These data suggest that some of the epsilon mRNA isoforms produced by PBL are aberrantly spliced mRNAs, the protein products of which are eliminated by post-translational events.

The Hb F levels in beta-thalassaemia can be affected by factors both linked and unlinked to the beta-globin gene cluster. We have recently analysed a group of patients with a homozygosity for the IVS-I-6 (T-->C) mutation, showing a wide variation in Hb F levels (2-47%) which could not be accounted for by any sequence variation within regulatory elements of the beta-globin gene cluster. In order to further investigate factors underlying this phenotypic difference we have developed a competitive reverse transcription/polymerase chain reaction procedure and used this method to determine the relative amounts of gamma- and beta-mRNAs in 10 patients with the IVS-I-6 homozygosity and 15 heterozygous parents, two IVS-I-6/delta beta-thalassaemia compound heterozygotes, five homozygotes for the beta(+) IVS-I-110 (G-->A) mutation, and in two with a homozygosity for the beta(0) codon 39 (C-->T) mutation. Three heterozygotes were also included. The percentages of gamma/(gamma(+) beta) mRNA were 10-73% in the IVS-I-6 homozygotes and < 2% to 10% in their heterozygous parents. A direct relationship existed between the level of mRNA and the % Hb F. However, the relative gamma-mRNA levels in the IVS-I-6 homozygotes were higher than their Hb F levels, indicating a possible competition between the gamma and beta transcripts for translational factors with a less efficient initiation of protein synthesis on the gamma-mRNA or a preferential survival of cells with mainly beta-globin gene expression at the post-reticulocyte stage. The gamma-mRNA levels in the two IVS-I-6/delta beta-thalassaemia compound heterozygotes were 71% and 62%, similar to their Hb F levels (63% and 59%), and averaged 82% (range 65-91%) in the five IVS-I-110 homozygotes, and 97.5% in the two codon 39 homozygotes. The correlation between these values and the % Hb F could not be evaluated because of the transfusion regimens; however, the levels of gamma-mRNA were as expected for patients with these beta-thalassaemia alleles.

We have used a quantitative reverse transcription/polymerase chain reaction (RT/PCR) procedure to evaluate the relative amounts of beta A and beta S mRNA transcripts in eight subjects with a simple Hb S heterozygosity, in six with Hb S-beta(+)-thalassaemia (thal), and in three individuals with Hb S-beta(+)-HPFH [hereditary persistence of fetal haemoglobin (Hb)] [two with the Atlanta type and one with the G gamma-202 (C-->G) substitution]. A balanced synthesis of beta A and beta S mRNAs was observed in all Hb S heterozygotes, whereas the beta A mRNA levels were reduced to approximately 16% of that of the beta S mRNA in the six Hb S-beta(+)-thal compound heterozygotes, to approximately 43% in the two subjects with Hb S-beta(+)-HPFH (Atlanta type), and to 23.8% in the one individual with Hb S-beta(+)-HPFH [G gamma-202 (C-->G) substitution]. The higher Hb A versus Hb S levels observed in all groups of the patients studied, further confirm a post-translational control mechanism in determining the levels of Hb A and Hb S in the peripheral blood of these individuals. The procedure described here provides an accurate and easy method for studying the relative expression of particular globin genes at the transcriptional level in patients with various haemoglobinopathies.

We have collected haematological, haemoglobin (Hb) and DNA sequence data for 29 patients with a homozygosity for the IVS-I-6 (T-->C) mutation with the intention of identifying factors contributing to the observed variability in the severity of the disease. None of the patients had received blood transfusion therapy for at least 6 months prior to the study. Hb levels varied from 5.0 to 9.9 g/dl. Patients with high Hb F (more than 1.5 g/dl or > 20%) had high total Hb levels (7.5-9.7 g/dl) but some with low Hb F also had high total Hb levels; two had a concomitant alpha-thalassaemia-2 (alpha-thal-2) heterozygosity. An inverse correlation between the Hb F and Hb A2 levels was observed. The majority of the patients were homozygous for haplotype VI (49/58 chromosomes) but haplotypes IV (2/58) and VII (7/58) were also present. The only haplotype IV homozygote had high Hb F levels with high G gamma values and the C-->T mutation at position -158 in the G gamma promoter, while both high and low Hb F levels were observed among patients with haplotypes VI and VII. Analysis of sequence variations in regulatory regions included the 5' hypersensitive sites (HS) 4. 3 and 2 of the locus control region (LCR), the G gamma and A gamma 5' flanking regions, the second intervening sequence (IVS-II), and the 5' beta-globin gene region in two patients with high Hb F (one homozygote each for haplotypes VI and IV), and in two patients with low Hb F levels (one homozygote each for haplotypes VI and VII). Haplotype specific differences were observed in the LCR 5' HS-2 and in the G gamma and A gamma flanking and IVS-II regions; however, no differences were present between the low and high Hb F-producing haplotype VI chromosomes, suggesting a major role for factors which are not linked to the beta-globin gene cluster in mediating gamma-globin gene expression in patients with this type of beta-thal.

We studied a heterozygous beta zero-thalassaemia patient from Croatia with an unusually high Hb A2 level of 7.6% and an elevated Hb F level of 5.8%. The same condition was found in his father (Hb A2 8.2%; Hb F 8.5%). Gene mapping and direct sequencing analyses revealed a new deletion of 1605 bp in the 5' beta-globin gene region between positions -984/5 and +620/1. This deletion has not been observed among more than 500 beta-thalassaemia chromosomes from the Balkan countries studied in our laboratory.

The low levels of IgE produced by PBMC from normal individuals has so far prevented an analysis of their IgE H chain repertoire. Using a nested polymerase chain reaction approach, we were able to detect epsilon transcripts in all normal and allergic individuals we investigated. We further cloned epsilon CDR3/FW4 regions from two normal and two atopic individuals with low serum IgE levels. Sequence analysis of 104 clones identified 26 different epsilon CDR3/FW4 regions and an additional number of clonally related transcripts in the two atopic individuals. Preferential usage of DH genes from the DXP family (33%) and of the JH4b gene (35%) were observed, similar to reported findings for the IgM-producing peripheral blood B cell subset. Using CDR3 specific oligonucleotides, we detected the CDR3/FW4 regions of a particular set of clonally related epsilon transcripts in mu and gamma 4 transcripts of the same individual. This finding demonstrates the in vivo production of IgE together with the two other Ig isotypes (IgM and IgG4) by the progeny of a common B cell precursor, and suggests a possible mechanism for regulating the allergic response. The clonally related epsilon transcripts were found to be only of the secreted form. We give also evidence that the IgE-producing B cells undergo somatic mutation because a number of identical mutations were observed in the FW4 regions of epsilon and mu clonally related transcripts. Some of these mutations were shared with other transcripts from the same and other individuals, supporting the existence of sequence-specific hot spots for the somatic hyper-mutation machinery in the JH gene segments.

A simple procedure for nonradioactive labeling of oligonucleotides has recently been developed (1). It consists of 3' end labeling of oligonucleotides with terminal transferase by incorporation of a single digoxigenin labeled dideoxy uridine triphosphate. We used these oligonucleotides for allele specific oligomer hybridization of polymerase chain reaction amplified DNA, followed by an enzyme-linked immunoassay and subsequent enzyme-catalyzed color reaction. We compared this procedure with the standard radioactive oligonucleotide hybridization technique through the detection of the most common Mediterranean beta-thalassemia mutations. This procedure was also used for the confirmation of a new mutation at position -87 (C----A) (2) of the beta-globin gene and for the subsequent family analysis.

We have developed a new nonradioactive method for the identification of mutations causing beta-thalassemia and cystic fibrosis. The method is based on the priming of polymerase chain reaction (PCR) by an oligonucleotide complementary to the DNA sequence containing the mutation of interest, which anneals only to the perfectly matched sequence under high stringency conditions. A more upstream oligonucleotide primer is also included in the reaction to discriminate between homozygosity and heterozygosity for a particular mutation and serves as a control for the efficiency of the amplification.

Analyses of DNA from 64 patients with thalassemia major using the hybridization technique of amplified DNA with radiolabeled synthetic oligonucleotide probes identified 13 different beta-thalassemia mutations. The codon 39 (C----T) and IVS-I-110 (G----A) mutations occurred most frequently but seven additional mutations were observed which were present at frequencies of 3.9 to 10.2%. This broad spectrum of beta-thalassemia alleles complicates the analyses for institutions involved in prenatal diagnosis. Promoter mutations were rare and the frequencies of two other mild mutations [IVS-I-6 (T----C) and the poly A mutation] were relatively low indicating that beta-thalassemia is a severe disease among Bulgarians. The high frequencies of 4.7-5.5% for the four frameshifts at codons 5, 6, 8, and 8/9 may be specific for this population.

This study concerned the evaluation of beta-thalassemia alleles in nearly 50 patients with beta-thalassemia major and in 130 -thalassemia heterozygotes using gene amplification and dot-blot hybridization with synthetic probes. Fourteen different mutations were observed; of these, three (IVS-I-110; IVS-I-6; IVS-I-1) account for some 75% of all beta-thalassemia alleles. Newly discovered variants, i.e. T----C in the initiation codon and AATAAA----AATGAA in the poly A site were observed in a few patients. The poly A mutation with classical beta-thalassemia alleles result in thalassemia intermedia. Hb Lepore is a rather common abnormality and combinations of this variant with beta-thalassemia often result in severe disease; a search for beta-thalassemia mutations among patients affected with this disease should include an analysis to detect this hemoglobin abnormality.

Sequence analyses of amplified DNA from a Yugoslavian patient with Hb Lepore-beta-thalassemia and from his father with a simple beta-thalassemia trait have revealed a T----A mutation within the ATA box at a position 30 base pairs upstream from the Cap site. The nucleotide substitution was confirmed through dot-blot analysis of amplified DNA with specific 32P-labeled synthetic oligonucleotide probes. The patient had a clinically severe condition; his Hb Lepore-beta-thalassemia was of the beta + type, as about 8-10% of the non-alpha chain was normal beta A. The same T----A mutation at nucleotide -30 was present on both chromosomes of a young Turkish patient who suffered from a thalassemia intermedia with a low level of Hb F (13.1%) and a relatively high beta A chain synthesis. These data are similar to those obtained for other types of beta +-thalassemia caused by comparable substitutions at positions 31, 29, and 28 base pairs upstream from the Cap site of the beta-globin gene.

DNA amplification combined with the use of synthetic oligonucleotide probes has become an important tool in the identification of base substitutions. We report the use of this DNA amplification technique for the detection of mutations in beta-thalassemia. A series of oligonucleotide primers are synthesized which span the beta-globin gene; one primer is complementary to the coding strand and the other to the non-coding strand. The primers are chosen so that there is little homology with other DNA segments, especially the delta gene. Each set of primers spans an area of the gene between 100 and 300 bp, while the suspected mutation point is located between these two primers. With the use of such a primer set, the beta-globin gene region is amplified by denaturation, annealing and DNA synthesis. The amplification cycle is repeated 25-30 times, using the Klenow fragment of DNA polymerase I. The resulting amplified DNA is hybridized with normal and synthetic deoxynucleotide probes using a standard dot-blot method. We have designed a set of primers and experimental conditions which should prove useful to diagnostic centers for detection of numerous beta-thalassemia mutations.

One of the easiest and most sensitive methods of detecting mutations in the beta-globin gene leading to beta-thalassemia is by the use of oligonucleotide probes. The current method involves digestion of 5-10 micrograms of genomic DNA followed by gel electrophoresis, and blotting onto nitrocellulose. The membrane is then hybridized with a 32P-radiolabeled oligonucleotide probe containing the specific point mutation of interest. Finally, the membrane is subjected to X-ray film for 3-10 days. We wish to report a method for detecting these mutations which involves 1 microgram of genome DNA or less. The method involves the use of a gene amplification technique. A series of primers are synthesized which span the beta-globin gene. In each primer set, one primer is complementary to the beta-gene and the other primer is complementary to the non-coding strand. The suspected mutation point is located between these two primers. With the use of this primer set, the beta-globin gene region is amplified by denaturing, annealing, and DNA synthesis. The amplification cycle is repeated 25 to 30 times. The amplification is conducted using the Klenow fragment of DNA polymerase I or Taq polymerase in the presence of all four deoxynucleotide triphosphates. The resulting amplified DNA is applied to a nylon membrane with the aid of a dot-blot apparatus and directly hybridized with normal and mutant deoxynucleotide probes. The entire process requires one to two days. More than 300 beta-thalassemia homozygotes have been identified in our laboratories; over 20 different mutations have been observed.

Clinical and haematological observations, made for 10 Yugoslavian patients with the Hb Lepore-beta-thalassaemia condition, suggested a considerable variation from severe disease and complete blood transfusion dependency to a moderate, compensated, anaemia without major complications and without a need for regular blood transfusions. As the type of Hb Lepore was the same in all patients (Lepore-Boston-Washington) and an alpha-globin gene deficiency was absent, it was concluded that the type of beta-thalassaemia determined the severity of the disease. Six patients with severe disease had one of the following three beta-thalassaemia determinants: IVS-1 position 110 G----A, exon 2 codon 39 C----T, and IVS-1 position 1 G----A, while the three patients with mild disease had the Portuguese type of thalassaemia which is caused by the T----C substitution at position 6 of the IVS-1. In one patient with severe disease the beta-thalassaemia determinant remained unknown. Our observations are consistent with those made for thalassaemia patients with a homozygosity for these determinants.