E. M. E. Smit

Replication pattern of the X chromosomes in three X/autosomal translocations

Three X/autosomal translocations, two familial and one de novo, were analyzed. Late-replicating chromosomes and chromosome regions were studied with R-banding techniques after BrdU incorporation. The first translocation, t(X;4)(q21;q13), was a de novo translocation, found in a woman with amenorrhea. The structurally normal X was late replicating in all cells. The second translocation, t(X;6)(p21;q26), was found in an unbalanced form, 46,XX,der(6), in a phenotypically abnormal girl; her mother carried the balanced translocation. In the mothers blood culture inactivation of the Xs followed two modes: In 85% of the cells the normal X was late replicating, and in the remaining 15% the der(X) was inactivated, including the attached fragment of chromosome 6. The third translocation, t(X;17)(p11;q24), was found in three generations. In the phenotypically normal mother, who carried the balanced translocation, the late-replicating X was always the normal X. In her daughters, who had an unbalanced karyotype, 46,X,der(X), and multiple congenital abnormalities, the X part of the translocation chromosome was always late replicating. No spreading of inactivation over the attached autosomal region was observed, resulting for these patients in a partial trisomy of 17q. Their peculiar phenotype is described.

Rapid detection of chromosome 16 inversion in acute nonlymphocytic leukemia, subtype M4: regional localization of the breakpoint in 16p

The pericentric inversion of chromosome 16 characteristic for acute nonlymphocytic leukemia, subtype M4, was detected in five patients by means of nonradioactive in situ hybridization of complete cosmids. First, five cosmids situated along the short arm of chromosome 16 were used to map the breakpoint of the inversion distal to the rare folate-sensitive fragile site FRA16A. Then, the use of two cosmids on either side of the breakpoint, combined with a probe specific for the centromeric region of chromosome 16, readily detected the inversion, even in poor metaphase spreads.

Bone Marrow Karyotypes of Children with Nonlymphocytic Leukemia

Summary: Bone marrow (BM) karyotypes from 16 consecutive children presenting with nonlymphocytic leukemia were established with the use of banding techniques, before therapy. The two patients with chronic myeloid leukemia (CML) showed the Philadelphia (PhI) translocation (9q+;22q−). Five of the 14 patients with an acute nonlymphocytic leukemia (ANLL) presented no acquired cytogenetic abnormalities, but one of these five showed a high level of hypodiploidy. One patient with AML evidenced a variant of the Ph1 chromosome originated as a translocation (12p+;22q−). Nonrandom abnormalities (−7; 7q−; +8; t(8;21); −21) were found in six patients, isolated or in association with other aberrations. Among the random abnormalities, apparently balanced translocations and chromosomal deletions were observed.In ANLL, no correlation could be found between morphologic diagnosis and cytogenetic findings. On the other hand, the presence of BM cells with a normal karyotype at diagnosis was associated with an improved remission rate and survival time. Followup studies were performed in four ANLL patients with an abnormal cell clone at diagnosis. Three of them achieved hema-tologic remission; their BM karyotype was found to be normal at that stage. In the 4th patient, generalization of the abnormal karyotype in BM cells was seen in the terminal phase of the disease.Speculation: Chromosome identification techniques have greatly improved our knowledge of acquired BM aneuploidy found in leukemia. Nonrandom patterns of abnormalities have been found. Analyses of larger series of patients using advanced banding techniques and systematic followup studies will ascertain the significance of these cytogenetic abnormalities for diagnosis, prognosis, and neoplastic transformation.

Cytogenetic clonality analysis: typical patterns in myelodysplastic syndrome and acute myeloid leukaemia

The cell morphology and karyotype of bone marrow samples from 24 patients with myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML) were studied simultaneously with a combined technique of May‐Grünwald‐Giemsa (MGG) staining and fluorescence in situ hybridization (FISH) with chromosome‐specific DNA probes. This enabled us to investigate cell lineage involvement in three malignant conditions: MDS (n = 12), leukaemia‐transformed MDS (LT‐MDS) (n = 5) and de novo AML (n=7). In MDS we found blasts and often significant proportions of mature granulocytic and erythroid cells to be cytogenetically abnormal. Percentages of granulocytic and erythroid cells with cytogenetic aberrations were generally less than those of blasts. These data support the involvement of a transformed pluripotent stem cell that has retained maturation abilities. In two patients with chronic myelomonocytic leukaemia (CMMoL) the clonal involvement of monocytes was predominant. Results in the five patients with LT‐MDS were similar to those in MDS. In the bone marrow of five of the seven de novo AML patients the cytogenetic abnormalities were restricted to the blasts and did not include the more mature granulocytic or erythroid populations. In the other two patients with AML, both with a t(8;21) and a loss of the Y chromosome, high percentages of mature neutrophils were cytogenetically abnormal. These patterns of clonal lineage involvement in MDS, LT‐MDS, t(8;21) AML and AML appear typical and may be of clinical use, for example, for distinguishing LT‐MDS from de novo AML in newly presenting patients.

C-Group Chromosome Abnormalities in Bone Marrow Cells of Three Children with Dyshaematopoiesis of Unknown Origin

Clinical and cytogenetic findings in three children with dyshaematopoiesis and bone marrow aneuploidy are described. Monosomy 7 was found in immature cells of one 10‐year‐old boy with myelofibrosis following a 3 years evolution of severe thrombocytopenia and anaemia. Trisomy 8 was found in 80% of the bone marrow metaphases of a 5 1/2year‐old girl with aplastic anaemia and Australia antigen positivity. During a 3 year observation period the number of cells with trisomy 8 regressed and eventually disappeared. Improvement of her clinical condition is present but still limited. Trisomy 8 was also found in all bone marrow cells of an 8‐year‐old girl with an undefined myeloproliferative disorder. Her disease was apparently related to collagen‐vascular disorders like periarteritis or other necrotizing angiitis and presented bvith periods of exacerbation and periods of chronic evolution. Periods of exacerbation were accompanied by excessive myeloid proliferation. Repeated bone marrow cytogenetic analysis during the acute and chronic phases showed trisomy 8 in all the metnphases analysed. During the last episode of acute illness, further clonal evolution was observed, characterized by a translocation (8; 17).

Two-colour FISH detection of the inv(16) in interphase nuclei of patients with acute myeloid leukaemia

The inv(16)(p13q22) and t(16;16)(p13;q22) in acute myeloid leukaemia are associated with a relatively good prognosis but are difficult to detect using classic cytogenetics. We have designed a two‐colour fluorescence in situ hybridization approach that uses two DNA probes that map close to and on either side of the inv(16) p‐arm breakpoint region. This new strategy clearly detected the inv(16)(p13q22)/t(16;16)(p13;q22) on both metaphase chromosomes and in interphase nuclei, even when they are of poor quality. This procedure also detected the inv(16) in cases with an additional deletion of sequences proximal to the 16p‐arm breakpoint which is present in 20% of all cases.

Isolation, properties and chromosomal localization of four closely linked hamster interferon-alpha-encoding genes.

Three recombinant phages containing hamster interferon-alpha-encoding genes (Ha Ifa) were isolated from a Ha genomic library, using a murine (Mu) Ifa probe. The phage inserts contained overlapping genomic fragments which span a total length of approx. 30 kb, on which four Ha Ifa genes are localized. The Ifa gene cluster could be assigned to hamster chromosome 2q. The nt sequences of the four Ifa genes were determined. Two of the genes are functional (Ha Ifa-1 and Ifa-3) and two are pseudogenes (Ifa-ps2 and Ifa-ps4). Ha Ifa-1 and -3 were transiently expressed in COS cells and they gave rise to protein products (A1 and A3, respectively) with antiviral properties on hamster CHO cells. In addition, Ha A1 revealed high antiviral activity on murine L929 cells.

Translocation (5p; 17q) in blast crisis of chronic myeloid leukemia.

A new abnormality of chromosome 17, distinct from i(17q), was observed in bone marrow cells of two patients with CML during a blast crisis. The marker was identified as a translocation of the short arm of chromosome 5 onto the long arm of chromosome 17, i.e., t(5p; 17q). In one case, the marker was clearly dicentric. The clinical history of these two patients showed some similarities. A slightly different marker has previously been observed in a cell line derived from a solid tumor.