Claus R. Bartram

RAG Mutations in Human B Cell-Negative SCID

Patients with human severe combined immunodeficiency (SCID) can be divided into those with B lymphocytes (B+ SCID) and those without (B− SCID). Although several genetic causes are known for B+ SCID, the etiology of B− SCID has not been defined. Six of 14 B− SCID patients tested were found to carry a mutation of the recombinase activating gene 1 (RAG-1), RAG-2, or both. This mutation resulted in a functional inability to form antigen receptors through genetic recombination and links a defect in one of the site-specific recombination systems to a human disease.

The acute lymphoblastic leukaemia cell line SEM with t(4;11) chromosomal rearrangement is biphenotypic and responsive to interleukin-7

Summary A cell line, designated SEM, was established from the peripheral blood of a 5‐year‐old girl in relapse with acute lymphoblastic leukaemia (ALL). Both the lymphoblasts of the patient and the cells of the cell line SEM showed the t(4:11) chromosomal rearrangement. The analysis of the immunophenotype of the SEM cell line revealed the B‐cell differentiation antigens CD19, CD22 and CDw75 in the absence of CD20. CD24 and immunoglobulin expression. Besides B‐lineage antigens. SEM cells were positive for the myeloid antigens CD13, CD15, CD33 and CDw65. Immunogenotypic analysis of SEM cells showed a monoclonal rearrangement of immunoglobulin heavy‐chain (IgH), T‐cell receptor (TCR) γ and δ genes. Addition of interleukin (IL)‐7 promoted the growth of the patients lymphoblasts in culture and enhanced the proliferation of SEM cells. The SEM cells also express messenger RNA (mRNA) for the IL‐7 receptor (IL‐7R), but no evidence for autocrine production of IL‐7 by the cell line was found. Addition of IL‐4, tumour necrosis factor (TNF)‐α, interferon (IFN)‐α, or IFN‐γ resulted in a profound inhibition of SEM growth. Thus, these cytokines may have important growth regulatory activities for biphenotypic leukaemic ALL cells.

Rapid and reliable detection of N- ras mutations in acute lymphoblastic leukemia by melting curve analysis using LightCycler technology

We applied a new strategy for the detection of N-ras gene mutations based on LightCycler technology. We designed two sets of amplimers and internal hybridization probes representing N-ras codons 12/13 and codon 61, respectively. Genomic DNAs from 134 childhood acute lymphoblastic leukemia (ALL) patients (83 common ALL, nine pre-pre-B ALL, 19 pre-B ALL, 23 T-ALL) were amplified, followed by the analysis of the melting temperatures of the PCR products on the LightCycler. PCR products exhibiting an abnormal melting characteristic were directly sequenced. Sequence analyses unravelled nucleotide substitutions at codon 12 in 10 patients, at codon 13 in three, and at codon 61 in one case. The incidence of N-rasmutations (10%) is compatible with previous reports. The LightCycler technology facilitates the rapid analysis of other genes exhibiting hot spot mutations in human malignancies.

Rapid and non-radioactive prenatal diagnosis of β thalassaemia and sickle cell disease: application of the polymerase chain reaction (PCR)

The standard method for the prenatal diagnosis of the haemoglobinopathies is by restriction enzyme mapping of chorionic villus DNA using Southern blotting and radioactively labelled gene probes. An improvement of the procedure which involves the selective amplification of DNA fragments by the polymerase chain reaction allows one to visualize restriction fragments directly without the use of radioactivity and within 2 d after obtaining the sample.

High rate of chromosome abnormalities detected by fluorescence in situ hybridization using BCR and ABL probes in adult acute lymphoblastic leukemia

The value of dual-color fluorescence in situ hybridization (FISH) with BCR and ABL probes for the detection of the Philadelphia (Ph) translocation and of other alterations involving ABL and/or BCR was evaluated in adult acute lymphoblastic leukemia (ALL). One hundred and four patients were studied prospectively using interphase nuclei FISH, chromosome analysis (CA), and PCR assays for the chimeric BRC/ABL transcript. FISH detected a Ph translocation in 24 cases (23.1%), as was confirmed by CA and/or PCR. FISH revealed a false positive diagnosis of a Ph translocation in four cases (5% false positive rate). Among 54 cases with combined FISH, CA and PCR assays, FISH failed to establish a correct diagnosis in 3.7%, PCR in 5.6%, and CA in 7.4%. The combination of two screening methods led to discrepant results in 9.3% (FISH + PCR), 11.1% (FISH + CA), or 13% (CA + PCR) of the cases. In seven of 80 (8.8%) Ph-negative patients, gain of BCR and/or ABL was identified. Overall, FISH detected alterations of the BCR and/or ABL genes with an incidence of 29.8% of the current study. Due to the possibility of false positive diagnosis of a Ph translocation using dual-color FISH the combination with chromosome and/or RT-PCR analyses is recommended in adult ALL patients.

Immune thrombocytopenia more than a year after allogeneic marrow transplantation due to antibodies against donor platelets with anti-PlA1 specificity: evidence for a host-derived immune reaction.

Summary We report on a male patient transplanted from his HLA‐matched sister for Ph1‐chromosome positive chronic myelogenous leukaemia who developed immune thrombocytopenia more than 1 year after transplantation. The platelet antibody reacted with the platelet specific antigen PIA1 on donor platelets, and also on recipient platelets after engraftment. A presumed host‐versus‐donor induced thrombocytopenia was supported by Southern blot analysis using a Y‐chromosome specific probe demonstrating residual hostorigin cells in the patients excised spleen.

A phase I/II study of recombinant interferon alpha 2a and hydroxyurea for chronic myelocytic leukemia

SummaryNine previously untreated patients with Philadelphia chromosome-positive chronic myelocytic leukemia (CML) were treated with recombinant interferon alpha 2a (rIFN-alpha 2a) and hydroxyurea. Patients received 6×106 U rIFN-alpha 2a daily for the first week and 3×106 U rIFN-alpha 2a daily for the second week. As maintenance treatment starting on day 15, patients received 3×106 U rIFN-alpha 2 a 3 times a week. Simultaneously, hydroxyurea was given, starting at a dose of 40 mg/kg on day one. The maintenance dosage was adjusted to the white blood cell count. Two patients responded with complete hematological remissions but without cytogenetic and molecular-genetic improvements. Seven patients responded with partial hematological remissions. Response to therapy was rapid; normal white blood cell counts were reached after a median of 12 days. The doses of rIFN-alpha 2a and hydroxyurea needed to keep the leucocyte count in the normal range were low (3×106 U rIFN-alpha 2a 3 times per week, 0.5–1.5 g hydroxyurea/day). Acute toxicity of the combination therapy consisted of fever (9 of 9 patients), flulike symptoms (7 of 9 patients), pruritus and/or rash (3 of 9 patients) and evidence of a tumor cell lysis syndrome (1 of 9 patients). The side effects were not dose-limiting. Combination therapy with rIFN-alpha 2a and hydroxyurea for CML is well tolerated and allows quick and effective hematological control of the disease.

Prospective BCR-ABL analysis by polymerase chain reaction (RT-PCR) in adult acute B-lineage lymphoblastic leukemia: reliability of RT-nested-PCR and comparison to cytogenetic data.

The reliability of routine BCR-ABL RT-nested-PCR was evaluated in 1453 B-lineage ALL or hybrid leukemia at initial diagnosis by RT-nested-PCR. All BCR-ABL-positive (nu2009=u2009642) and 176 BCR-ABL-negative samples underwent a second RT-PCR. In 518 patients, karyotyping and/or FISH was compared to the BCR-ABL status. The second RT-PCR revealed in 155/642 initially positive samples a divergent result (153 BCR-ABL-negative, two other transcripts) that in most cases turned out to be caused by contaminations in the first RT-nested-PCR. Confirmatory RT-PCR detected 2/176 false negative first RT-nested-PCR results. Thirty-nine specimens remained ambiguous despite different RT-PCR approaches. As far as cytogenetic evaluation and FISH is available (nu2009=u200923), the majority but not all patients with an ambiguous RT-PCR result were Ph-negative (nu2009=u200918). RT-nested-PCR and cytogenetics yielded in 346 of 383 evaluable samples a concordant result. Differing results are given and account in part to the lower sensitivity of karyotyping. Taken together, confirmed RT-PCR detected BCR-ABL fusion transcripts consistently in 487 out of 1453 ALL samples (c-ALL: 43%, pre-B ALL: 34%, pro-B ALL: 5%, B-ALL: 0%, hybrid leukemia: 5/11). Since false positive initial RT-nested-PCR data were frequent, either confirmatory second RT-PCR or FISH analysis is warranted to guarantee sensitive and reliable results of utmost clinical relevance.

Philadelphia-positive chronic myelogenous leukemia with breakpoint 5′ of the breakpoint cluster region but within the bcr gene

SummaryWe report on the first Philadelphia chromosome (Ph) positive chronic myelogenous leukemia (CML) characterized by a rearrangement within the 5′ part of the bcr gene on chromosome 22, but outside the restricted breakpoint cluster region. In situ hybridization studies revealed a translocation of the c-abl oncogene to the Ph chromosome and Northern blot analysis identified a chimeric 8 kb bcr/abl RNA transcript in leukemic cells. These data suggest that 1. less bcr coding sequences than previously assumed may be essential for the putative transforming activity of the rearranged bcr/abl gene and 2. the bcr probes currently used for diagnostic purposes could miss Ph-positive CML cases.

Two novel missense and frameshift mutations in exons 5 and 6 of the purine nucleoside phosphorylase (PNP) gene in a severe combined immunodeficiency (SCID) patient.

Abstract Four percent of human severe combined immunodeficiency cases are caused by a deficiency of the enzyme purine nucleoside phosphorylase (PNP). In this study we investigated the molecular basis for this rare autosomal recessive disease. Sequence analyses led to the identification of two new mutations in the PNP gene: an A to G transition in exon 5, which leads to the substitution of tyrosine 192 by a cysteine residue, and a 1-bp deletion in exon 6, which causes premature translation termination of the PNP protein. Both PNP mutations affect predicted major structural motifs of the protein and result in posttranslational instability of the enzyme.