The spectrum of CFTR mutations in south-west German cystic fibrosis patients.

The cystic fibrosis transmembrane conductance regulator (CFTR) gene of 110 cystic fibrosis (CF) patients from the south-west of Germany was screened for 12 different mutations. This analysis resulted in an identification of 79% of all CF mutations and a complete genotype in 66% of the families. The most common mutation found was delta F508 (67%). Another 5 mutations accounted for a further 12.5% (4% G542X; 3% R553X; 3% N1303K; 2% 1717-1 G-->A; 0.5% G551D) whereas 6 mutations (R117H, A455E, delta I507, S549I, S549N, and R1162X) were not found. Fifty-four (49%) patients were delta F508 homozygotes and 18 (16.5%) were compound heterozygotes for delta F508 and one of the rarer mutations. These frequencies differ slightly from those found in the north of Germany and considerably from those reported from the south of Europe, which seems to be consistent with a north to south decline of the relative abundance of delta F508. Two patients, age 6 and 25 years, were compound heterozygotes for G542X and N1303K. The clinical features of the 6 year old were characterised by severe gastrointestinal and as yet only mild pulmonary complications whereas the 25 year old manifested severe pulmonary and gastrointestinal symptoms indicating that the N1303K mutation of the C-terminal CFTR nucleotide binding fold significantly impairs protein function in both the pancreas and the lungs.

Use of polymerase chain reactions to monitor minimal residual disease in acute lymphoblastic leukemia patients.

T-cell receptor (TCR) delta gene rearrangements are observed in more than 80% of acute lymphoblastic leukemia (ALL) patients. Moreover, a preferential usage of specific genetic elements has been shown in different ALL subtypes: V delta 1 DJ delta 1 rearrangements predominate in T-ALL, while most B-precursor ALLs show a recombination of V delta 2 to D delta 3. Recently we have proposed a strategy for the detection of minimal residual disease (MRD) based on the isolation of clonospecific probes following the in vitro amplification of V delta 1 DJ delta 1 junctions by polymerase chain reaction (PCR) and now have adapted this method to the preparation of specific V delta 2 D delta 3 fragments. In the present study, clonospecific probes were generated from 11 T-ALL and 16 cALL patients (21 children, 6 adults). The sensitivity of these 27 probes in detecting residual leukemia cells varied between 10(-4) to 10(-6) as determined by semiquantitative evaluation of dilution experiments. PCR analysis of 55 bone marrow (BM) and peripheral blood (PB) samples obtained from the 27 patients during complete clinical remission showed the following results: (1) Evidence for MRD was obtained in the BM of all patients (eight of eight) investigated 2 to 6 months after remission induction and also in 6 of 11 cases on maintenance therapy 7 to 19 months after diagnosis. (2) In contrast, all patients but one (10 of 11) analyzed 6 to 41 months after the termination of treatment lacked apparent evidence for leukemia DNA; the exception was a girl exhibiting 10(-4) to 10(-5) residual cells in her PB 5.5 years after diagnosis. (3) Longitudinal analysis in nine patients disclosed marked individual differences in the intervals between achievement of clinical remission and complete eradication of the leukemia cell clone. (4) Differences in the duration of MRD were not associated with distinct clinical-hematologic features. (5) Detection of residual disease by PCR proceeded clinical relapse in two cases.

HLA-haploidentical bone marrow transplantation in three infants with adenosine deaminase deficiency: stable immunological reconstitution and reversal of skeletal abnormalities.

Three infants with severe combined immunodeficiency and adenosine deaminase (ADA) deficiency were treated by T-cell depleted bone marrow transplantation (BMT), using human leukocyte antigen (HLA)-haploidentical parents as donors. In the first patient, two initial transplants failed to engraft and no change of the immunodeficiency was observed. In order to overcome this graft resistance, cytoreductive conditioning was used prior to a third transplant. In the other two patients, similar conditioning was used prior to initial transplants. In all three patients, complete and permanent immunological reconstitution was observed and they survive from 3.5 to 5 years after transplantation. In biopsies obtained from iliac bones prior to BMT, osteochondral abnormalities characteristic of ADA-deficiency were noted in all three patients. After successful transplantation, these abnormalities had completely resolved. Our results demonstrate that cytoreductive conditioning prior to HLA-haploidentical BMT is useful in order to obtain stable engraftment and reversal of abnormalities associated with ADA deficiency.

Clonal analysis of myelodysplastic syndromes: evidence of multipotent stem cell origin.

Restriction fragment length polymorphisms (RFLPs) of the X-chromosome genes hypoxanthine phosphoribosyl transferase (HPRT) and phosphoglycerate kinase (PGK) were studied in 34 female patients with primary myelodysplastic syndromes (MDS). Twelve patients (35%) were heterozygous at the HPRT or PGK loci for BamHI or BglI RFLPs, respectively. In eight patients showing PGK polymorphisms, clonality was determined by X-chromosome inactivation analysis. These included patients from different morphologic subtypes: four with refractory anemia (RA), two with RA and ring sideroblasts (RARS), one patient with RA with excess of blasts (RAEB), and one with chronic myelomonocytic leukemia (CMML). A monoclonal pattern of X-chromosome inactivation was observed in seven cases. In a further case characterized by bone marrow hypoplasia, peripheral blood (PB) leukocytes were polyclonal in origin. Following low-dose cytarabine therapy, reversion to polyclonal hematopoiesis was observed in a case of RAEB indicating the presence of residual normal hematopoietic stem cells with the capacity for marrow reconstitution. The clonal relation of lymphoid and granulocyte/monocyte lineages was studied directly in two cases of CMML exhibiting somatic mutations of N-ras or Ki-ras oncogenes. By selective oligonucleotide hybridization to ras gene sequences amplified in vitro by the polymerase chain reaction, a mutated ras allele was demonstrated in PB granulocytes, monocytes, and B and T lymphocytes of both patients. We conclude that MDS arise from a multipotent hematopoietic stem cell with the potential for myeloid and lymphoid differentiation.

[Diagnosis and treatment of aplastic anemia]. / Diagnostik und Behandlung der aplastischen Anämie.

Severe aplastic anemia is a rare disorder in childhood. Among various therapeutical strategies bone marrow transplantation (BMT) and immunosuppressive treatment with antithymocyte globulin (ATG) have proven to be most successful. Priority should be given to BMT over ATG treatment for patients with HLA-identical donors. Patients with Fanconi's anemia require a reduced conditioning program with cyclophosphamide and irradiation for BMT. Own experiences indicate that cooperative studies are highly needed to improve medical care for patients with severe aplastic anemia.

[Diagnostic and therapeutic applications of gene technology methods in thalassemia]. / Diagnostik und Therapieansätze mit gentechnologischen Methoden bei Thalassämien.

By applying gene technology, great progress has been made in defining the molecular basis of thalassemias. While the prevention of thalassemias through prenatal diagnosis has improved significantly, advances for molecular therapy have been rather limited until now.

[Treatment of Fanconi anemia by bone marrow transplantation]. / Die Behandlung der Fanconi-Anämie durch Knochenmarktransplantation.

We report on our experience with allogenic bone marrow transplantation in the treatment of Fanconi anemia. Eight patients were treated, ranging in age from 5 to 17 years. Beside severe hemopoietic insufficiency, all patients exhibited typical cytogenetic abnormalities with an increased rate of chromosomal breaks, while constitutional signs of the disorder were rather variable. Marrow donors were HLA-identical siblings. For conditioning, we used cyclophosphamide at 5 mg/kg on 4 consecutive days followed by thoraco-abdominal irradiation at 5 Gy with full lung shielding. For prophylaxis of graft versus host disease, cyclosporin A was given except in 3 cases who received T-cell depleted marrow. In 2 of the latter cases, graft failure was observed, successfully reversed in one by retransplantation. All others showed prompt and stable engraftment of donor cells. Complications of graft versus host disease developed in 2, requiring prolonged immunosuppressive treatment. Of 8 transplanted patients, 7 survive. With the exception of a recently treated girl, they have normal stable marrow functions. Our results confirm that successful treatment of Fanconi anemia is possible in a majority of patients with HLA-identical donors.

bcr rearrangement and translocation of the c-abl oncogene in Philadelphia positive acute lymphoblastic leukemia.

The Philadelphia (Ph1) chromosome, the cytogenetic hallmark of chronic myeloid leukemia (CML), has also been detected in a significant number of acute lymphoblastic leukemias (ALL). Using in situ hybridization, we demonstrate that in accordance with observations in CML the Ph1 chromosome in ALL patients is the result of a consistent translocation of the c-abl oncogene to the Ph1 chromosome. Southern blot analysis using bcr probes, however, suggests that Ph1-positive ALL includes heterogeneous leukemic subtypes: six ALL patients showed bcr rearrangements as observed in CML; in three other patients recombination involving 5' bcr sequences could be demonstrated, but the corresponding translocated 3' bcr sequences were not detectable. A third group of five patients did not show any bcr rearrangements at all. Northern blot analysis using RNA from three Ph1-positive ALL patients revealed that in the leukemic cells of two patients larger c-abl mRNA transcripts were present, as in CML. In the RNA of one patient without a detectable bcr rearrangement, only the normal c-abl mRNA transcripts are present. The observed heterogeneity in bcr rearrangements of this group of Ph1-positive ALL patients is in contrast with the consistent results obtained in more than 50 Ph1-positive CML patients investigated in chronic and acute states.

Acute undifferentiated leukemia: implications for cellular origin and clonality suggested by analysis of surface markers and immunoglobulin gene rearrangement.

Although B cell leukemias and, recently, T cell leukemias can be identified both by surface marker and molecular analysis, there remains a population of acute undifferentiated leukemias (AUL) that cannot be allocated definitively to a single cell lineage. AUL was diagnosed in nine patients according to stringent criteria. We combined both immunologic and molecular approaches to analyze further the ambiguous origin of AUL cells. Southern blot analysis revealed rearranged Ig heavy-chain genes in seven patients and indicated a biclonal or oligoclonal leukemic cell population in three of them, including one case of AUL with translocation (4;11). Analysis of cell surface markers showed expression of at least one early B cell-associated antigen (BA-1, BA-2, B4, UL-38) in six of these seven patients, with coexpression of a myeloid antigen (VIM-2) in three patients. Leukemic cells of two other patients neither exhibited Ig chain gene rearrangements nor expressed B cell-associated antigens. T cell receptor beta-chain genes showed germline configuration in all nine cases. Our results demonstrate heterogeneity among AUL patients based on molecular and surface marker analyses and suggest that most AUL blast cells are derived from a precursor cell that shares phenotypic and genotypic characteristics of early B cells with certain surface antigens of myeloid cells, in some cases of AUL more than one abnormal cell clone or subclone may exist, and the cellular origin, at least of some AULs exhibiting t(4;11), may be truly B cell lineage committed.

Conversion of acute undifferentiated leukemia phenotypes: analysis of clonal development.

The cellular origin of acute undifferentiated leukemia (AUL) is still a matter of controversy. We report on two cases in which the diagnosis of AUL was established according to restricted criteria. Blast cells of both patients showed phenotypic conversion during the course of disease. In one case, within 24 days from starting treatment, the leukemic phenotype changed from AUL to acute myelomonocytic leukemia (FAB L1, TdT+ to FAB M4, TdT-). The initial phenotype of this acute leukemia was characterized by the co-expression of both B-lymphoid and myeloid markers on the same cell. Moreover, analysis of esterase isoenzyme pattern showed the whole spectrum of isoenzymes typically seen in myelomonocytic leukemias already at diagnosis, yet blast cells additionally contained all three isoenzymes of beta-hexosaminidase typically seen in AUL. However, examination of immunoglobulin (Ig) heavy chain gene rearrangement initially and after conversion revealed an identical monoclonal configuration of Ig heavy chain sequences in both samples. The second AUL patient relapsed after allogeneic bone marrow transplantation with common ALL-antigen (CALLA) positive acute leukemia. Subsequent Southern blot analysis showed a novel rearranged Ig fragment compared to the analysis before transplantation indicating that the leukemic clones prior to and after transplantation were not identical. No chromosomal abnormalities were observed in both cases. These data support the view that AUL cells originate from a pluripotent stem cell that is capable to differentiate in the myelomonocytic lineage (patient 1), and confirm the value of Ig gene analysis as marker for cellular clonality.

Additional c-abl/bcr rearrangements in a CML patient exhibiting two ph1 chromosomes during blast crisis.

Recent data suggest that two human genes, c-abl on chromosome 9 and bcr on chromosome 22, are involved in the generation of Ph1-positive CML. To examine a possible role of these sequences in transition from chronic towards blastic phase, rearrangements within bcr were analysed in 4 patients with Ph1-positive CML during chronic and acute phase. In 3 patients bcr rearrangements were identical in both phases, while in a fourth patient with duplicated Ph1 an amplified additional bcr fragment was detected in acute phase. Northern blot analysis of blast cells of the latter patient showed a novel 10.3 kb RNA species that replaced the altered 8 kb RNA transcript usually found in Ph1-positive CML.

[Cytomegalovirus hyperimmune globulin prophylaxis in bone marrow transplants in children with various diseases]. / Zytomegaliehyperimmunglobulinprophylaxe nach Knochenmarktransplantation bei Kindern mit verschiedenen Grunderkrankungen.

The effect of hyperimmune globulin for the prevention of cytomegalovirus (CMV) infections following bone marrow transplantation (BMT) was evaluated in 21 children with various diseases and compared to a historical group of 23 children without prophylaxis. A higher incidence of interstitial pneumonia (19%) as well as CMV-associated infections with or without pneumonitis (29%) could be demonstrated in patients with CMV-prophylaxis as against the rate of interstitial pneumonia (4%) and CMV-infections (8%) in children without prophylaxis. This surprising observation was very likely due to selection of patients with high risk features and higher incidence of GVHD in the prophylaxis group. The analysis of patients with prophylaxis failure shows a low CMV-infection rate in initially seronegative marrow recipients and a high risk for CMV-infections in seropositive patients. Therefore, even though CMV-infections could not be completely abrogated, hyperimmune globulin administration might have reduced CMV-complications in seronegative patients. In CMV-seropositive marrow recipients, however, this type of prophylaxis remains unsatisfactory in preventing severe CMV-infections caused by virus reactivation.

Ultrasound characteristics of the pancreas in children with cystic fibrosis.

Eighteen patients (3 months to 16 years) with cystic fibrosis (CF) were examined with a real-time mechanical sector-scanner (5 MH transducer). Compared with age-matched controls, each CF patient showed morphologic changes in the pancreas on abdominal ultrasound examination. A very common finding was a decrease in organ anterior-posterior diameters (CF versus controls: head, 1.25 +/- 0.38 cm versus 2.44 +/- 0.39 cm; body, 0.56 +/- 0.35 cm versus 0.98 +/- 0.3 cm; tail, 0.83 +/- 0.28 cm versus 1.81 +/- 0.38 cm) and a pronounced, age-independent increase in tissue echogenicity. In younger patients, small cystic degenerations could be observed in the pancreatic tail. No correlation could be found between ultrasound morphology, especially pancreatic duct imaging, and exocrine pancreatic function.

C-abl and bcr are rearranged in a Ph1-negative CML patient.

Chromosomal analysis of a patient with chronic myelocytic leukemia (CML) revealed a translocation (9;12) (q34;q21) without a detectable Philadelphia chromosome (Ph1). Using molecular approaches we demonstrate (i) a rearrangement within the CML breakpoint cluster region (bcr) on chromosome 22, and (ii) a joint translocation of bcr and c-abl oncogene sequences to the derivative chromosome 12. These observations support the view that sequences residing on both chromosome 9 (c-abl) and 22 (bcr) are involved in the generation of CML and suggest that a subset of Ph1-negative patients may in fact belong to the clinical entity of Ph1-positive CML.

Involvement of chromosome 9 in variant Ph1 translocation.

Cytogenetic analysis of a patient with chronic myelocytic leukemia revealed a translocation (21; 22) (q 22; q 11) without a detectable involvement of chromosome 9. By in-situ hybridization studies, however, we demonstrate a reciprocal translocation of sequences from chromosome 9 (c-abl) to Ph1 and chromosome 22 (bcr) to 9, respectively. These observations suggest a consistent participation of chromosome 9 in the Ph1 translocation, regardless of the cytogenetic subtype.

[Molecular mechanisms of antibody synthesis]. / Molekulare Mechanismen der Antikörpersynthese.

Antibodies or immunoglobulins play a central part in the immune system. The basic unit of an antibody is composed of two identical light and two identical heavy chains; each chain contains two functionally and structurally distinct regions: an amino-terminal variable or antigen-binding site, and a carboxy-terminal constant region responsible for immunological effector functions. Thanks to recombinant DNA technology the paradox of a limited number of genes and a virtually unlimited capacity to generate specific antibodies has now been resolved at least in outline. Immunoglobulin chains are encoded in multiple gene segments of three unlinked gene families scattered along chromosomes 2 (kappa light chain), 14 (heavy chain) and 22 (lambda light chain). During B-cell differentiation these genes are assembled by somatic recombination mechanisms to form active genes. The enormous diversity generated by means of DNA rearrangements is supplemented by mutations somatically introduced into variable region sequences. The medical impact of these discoveries will be substantial. Possible applications include identification of B-cell precursors lacking conventional markes, a molecular classification of lymphomas and a precise distinction between monoclonal and polyclonal lymphoproliferative disorders.

Immunoreconstitution in severe combined immunodeficiency after transplantation of HLA-haploidentical, T-cell-depleted bone marrow.

Immunological reconstitution by transplantation of HLA-haploidentical, paternal bone marrow was attempted in four infants with severe combined immunodeficiency who lacked HLA-identical donors. To prevent graft-versus-host disease, T lymphocytes were removed from the grafts by agglutination with soybean agglutinin and rosette formation with sheep red blood cells. None of the patients received conditioning treatment before transplantation. Normal, donor-derived cellular immune functions developed in all four patients within several months of transplantation. Threatening complications of graft-versus-host reactions were not seen. All four patients remain in excellent health 12-15 months after discharge home, with persisting normal T-cell functions.