Demonstration of phosphoglucomutase 1 in a subclone of the K-562 cell line.

Phosphoglucomutase 1, an enzyme mapping on the short arms of chromosome 1, is constantly missing in the leukemic cell line K-562 in spite of the presence of three No. 1 chromosomes. In the present work, a subclone of the cell line, K-562 (S)P, is described, where the enzyme can be demonstrated, thus excluding a small deletion as the cause for the lack of expression of phosphoglucomutase 1. The relationship between the presence of the enzyme and the karyotype changes in this subclone is analyzed. Addition of several inducers to the standard K-562 line failed to elicit expression of the enzyme.

Regulation of acetylcholinesterase expression in the K-562 cell line.

Acetylcholinesterase, an erythroid marker constitutively expressed in K-562 cells, can be further induced by sodium butyrate. The highest level of acetylcholinesterase induction is reached in approximately equal to 3 days, in parallel with increased hemoglobin expression. Acetylcholinesterase induction is reversible, and repeated addition of butyrate is necessary to maintain a high level of the enzyme. Actinomycin D inhibits the induction.

Terminal erythroid differentiation in the K-562 cell line by 1-beta-D-arabinofuranosylcytosine: accompaniment by c-myc messenger RNA decrease.

Two erythroid markers, acetylcholinesterase and hemoglobin, can be reversibly induced in the K-562 cell line after sodium butyrate treatment. In the present paper we show that 1-beta-D-arabinofuranosylcytosine (ara-C), induces the coordinate, irreversible expression of these two erythroid markers. This induction occurs at an ara-C concentration (0.05 mM) that results in K-562 cytostasis and is accompanied by deep morphological changes of cells. The differentiated phenotype is independent of the K-562 cell clone used [K-562, K-562 (S), K-562 (S)P] and is associated with the loss of cell renewal capacity. Continuous presence of the inducer is not necessary to achieve terminal differentiation. In contrast to what is seen for other inducers (sodium butyrate and hemin), one of the early effects of ara-C treatment is the marked decrease of c-myc mRNA expression after the first 4 hours of induction, whereas N-ras and histone 4 expression remain constant during the first 48 h. Our results suggest that ara-C treatment can irreversibly activate the erythroid differentiative program of K-562 cells.

Expression of erythroid acetylcholinesterase in the K-562 leukemia cell line.

Differentiation-dependent expression of enzyme loci was evaluated in two human leukemic cell lines, the pluripotent leukemia cell line K-562 and the promyelocytic-like cell line HL-60. Acetylcholinesterase, a marker of erythroid differentiation, was present in K-562 cells and absent in HL-60 cells. This difference between the two lines was apparently unrelated to dosage effect; other enzymes carried on trisomic chromosomes in K-562 cells did not show dosage effect. Acetylcholinesterase activity was higher in subclone K-562 (S), which shows higher expression of hemoglobin. Electrophoretic mobility of acetylcholinesterase from K-562 (S) was of fetal type.

Early and long term follow-up with minisatellite probes in bone marrow transplanted patients.

Twenty-five patients who received bone marrow transplantation (BMT) for chronic granulocytic leukemia (CGL), acute leukemia and severe aplastic anemia were studied before and after BMT in order to document and characterize the events following transplantation. DNA analysis was performed using minisatellite probes, which give rise to extremely polymorphic Southern blot band patterns specific to each individual and are regarded as "genetic fingerprint." Sensitivity studies using a mixture of donor and recipient cells could distinguish the presence of 1% of cells from one individual. Blood specimens were obtained from donor recipient before BMT and at days 10, 30, 90, and 270 after BMT. Karyotype analysis was also performed in CGL patients at the same time of DNA analysis. Engraftment was identified by DNA analysis as early as 10 days posttransplant and correlated with cytogenetic findings. This confirmed that a single hybridization filter is informative in 100% of patients and is easily applicable for early and long term studies of chimerism in BM transplanted patients.

Effects of fibroblasts on the growth of erythroid progenitor cells in vitro.

The effect of embryo fibroblasts on the growth of erythroid colony-forming cells in vitro (CFU-e) from mouse bone marrow was investigated. First, the maintenance of CFU-e number in suspension culture was assayed. CFU-e recovered from suspension culture fell rapidly to values below 30% of the initial number. When erythropoietin (EP) was added, the initial decline during the first day was followed by a rise to 80%. In cultures supplemented with irradiated fibroblasts, the number of CFU-e did not show an abortive fall, but there was a slight increase during 3 days of culturing. The influence of fibroblasts on the colony-forming ability of CFU-e was studied in a semisolid culture system composed of an agar underlayer and a methylcellulose overlayer. The number of erythroid colonies scored after 5 days of culture in the presence of different levels of EP was proportional to the number of added fibroblasts and the colony size (depending on the number of fibroblasts) increased to macroscopic dimensions. Fibroblasts alone, without EP, induced colony formation by CFU-e if added in concentrations of 1 X 10(5) or higher. EP was not detectable in medium conditioned by the fibroblasts. These data indicate that fibroblasts may stimulate erythroid colony formation (in the absence of EP) and enhance the colony-forming ability of CFU-e in the presence of EP. From these results, it is suggested that fibroblasts exert proliferation activating effects on CFU-e target cells.

Marrow colony stimulating activity in chronic myelogenous leukemia.

Colony stimulating activity (CSA) from bone marrow adherent cells was tested in various myeloproliferative disorders in comparison with chronic myelogenous leukemia (CML). A higher level of medullary CSA was found in acute leukemias with monoblastic component, as contrasted with low values in other acute non lymphocytic leukemias (ANLL). In CML, while the majority of patients had CSA values within normal interval, few patients had consistently higher level of this activity. Comparative analysis of growth pattern in agar or in liquid culture suggests that significant increase in medullary CSA production can be related to an abnormal regulation of granulopoiesis.

A novel mutation (D305V) in the early growth response 2 gene is associated with severe Charcot-Marie-Tooth type 1 disease.

Hereditary motor and sensory neuropathies (HMSN) comprises a wide clinical spectrum of related disorders with defects in peripheral nerve myelination. Charcot-Marie-Tooth type 1 (CMT1) is the most common form and is usually a mild disease with onset in the first or second decade; however there is a interfamilial and intrafamilial clinical variation, ranging from asymptomatic expression to severe muscular weakness and atrophy. Recently point mutations in the early growth response 2 gene (EGR2/Krox-20) have been associated with hereditary myelinopathies. We investigated for mutations at the EGR2 gene a patient with severe CMT1 phenotype. Direct sequencing of EGR2 gene showed a heterozygous A T transversion at nucleotide 1064 that predicts an Asp305Val substitution within the first zinc-finger domain. The finding of a novel EGR2 mutation associated with a different phenotype confirms that peripheral neuropathies represent a continuum spectrum of related disorders due to an underlying defect in myelination.

A SOD1 gene mutation in a patient with slowly progressing familial ALS.

We report a new missense mutation (Gly12Arg) [corrected] in exon 1 of the Cu/Zn superoxide dismutase (SOD1) gene in a 67-year-old patient with familial ALS (FALS). The clinical course showed an unusually slow progression. The enzymatic activity of the mutated SOD1 was 80% of normal. At the molecular level, the Gly12Arg [corrected] mutation occurs in a region outside the active site and may lead to local distortion strain in the protein structure.

Involvement of chromosomal region 9q34 in a case of variant Ph1 translocation t(22;22).

In a patient with chronic myelocytic leukemia chromosome analysis showed a translocation (22;22) (q13;q11). Chromosomes 9 were apparently not involved. Using somatic cell hybrids and a v-abl probe, we demonstrated the translocation of c-abl sequences from chromosome 9 to chromosome 22q-. This confirms the hypothesis that the translocation of c-abl oncogene is essential for the development of Ph1 positive CML.

Genetic analysis of Huntington disease in Italy.

Twelve Italian families with Huntington disease were tested with 10 probes known to be linked to the disease locus and able to detect polymorphisms at the following loci on chromosome 4: D4S10, D4S127, D4S95, D4S43, D4S115, D4S111, D4S90. The results confirmed the applicability of the linkage approach for presymptomatic diagnosis in Italian families. Positive lod scores were found between D4S10, D4S95, D4S43 and the disease, whereas D4S90 did not indicate significant linkage values. With the limitations due to the small size of the tested sample, no genetic heterogeneity was detected in the families examined for loci D4S10, D4S95/S127, D4S43.

Non-random association between DNA markers and Huntington disease locus in the Italian population.

A group of Huntington disease (HD) families of Italian ancestry was analyzed for 11 RFLPs from genetic loci mapped in 4p16 and genetically linked to the HD gene. We found a statistically significant difference of allele distributions in HD vs normal chromosomes for loci D4S10, D4S127, and D4S43. This observation increases the number of loci in linkage disequilibrium with HD. However, the amount of disequilibrium does not allow either a finer localization of the HD gene or a substantial improvement in risk calculations.

Parental origin of chromosome 4p deletion in Wolf-Hirschhorn syndrome.

We report on molecular studies in 7 patients with Wolf-Hirschhorn syndrome (WHC) not showing an obvious chromosome 4p deletion. Analysis of a set of polymorphic probes mapping in the 4p16.3 region showed the absence of paternal haplotypes in 5 cases, and maternal haplotypes in 2. These observations corroborate evidence for preferential paternal origin of the de novo 4p chromosome deletion. The overall results of molecular studies suggest that the preponderance of paternally derived WHC could be due, rather than to imprinting of this region, to an excess of structural rearrangements in the male meiosis, related to differences between the mechanisms of sperm and egg production.

Autosomal dominant polycystic kidney disease: a linkage evaluation of heterogeneity in Italy. Italian Collaborative Group on Polycystic Kidney Disease.

A survey of 29 families with Adult Polycystic Kidney Disease (ADPKD) was performed to evaluate the genetic heterogeneity of the disease in Italy. The approach was through the linkage between the disease and 2 polymorphic DNA fragments as detected by the probes 3'HVR and 24.1. Linkage between the polymorphic markers and the disease was confirmed, with the following lod scores: between 3'HVR and ADPKD1 = 12.974 at theta = 0.02; between 24.1 and ADPKD = 1.716 at theta = 0.07; between 3'HVR and 24.1 = 2.738 at theta = 0.09. No evidence of significant genetic heterogeneity in the examined Italian regions was detected.

Exclusion of the ninjurin gene as a candidate for hereditary sensory neuropathies type I and type II.

Ninjurin is a protein that is up-regulated in Schwann cells and neurons after peripheral nerve injury. Its role in promoting nerve regeneration and its expression in sensory neurons of dorsal root ganglia, as well as the chromosomal localization of the ninjurin gene, makes this gene a candidate for hereditary sensory neuropathies (HSN). In the present report, the human ninjurin gene was analyzed in 17 unrelated patients with HSN type I, two patients with HSN type II, and 10 normal controls, by single strand conformation polymorphism and by direct sequencing. All three exons and splice junctions of the gene were investigated and no mutations were found in our sample of patients. Our results rule out a mutation in the translated region of the ninjurin gene as a cause of HSN type I and type II.

Correlation between acquired pseudo-Pelger-Huet anomaly and involvement of chromosome 17 in chronic myeloid leukemia.

Acquired pseudo-Pelger-Huet neutrophils appeared in the peripheral blood of 11 of 83 Philadelphia-positive chronic myeloid leukemia patients during the blastic phase of the disease. Chromosomal analysis performed at the time of pseudo-Pelger appearance showed karyotype evolution with involvement of the short arms of a chromosome #17. In eight cases an i(17q) was present, in two cases an unbalanced translocation, and in one case monosomy. All these rearrangements had in common the loss of the distal end of the short arm. The morphologic and chromosomal study of the remaining 72 chronic myeloid leukemia patients demonstrated neither pseudo-Pelger nor 17p involvement.

Masked Philadelphia chromosome caused by translocation (9;11;22).

In a patient with chronic myelocytic leukemia (CML), chromosome analysis revealed a translocation involving chromosomes No. 9, 11, and 22, with three break points, thus giving origin to a so-called "masked" Philadelphia chromosome (Ph1). A review of similar cases reported in the literature indicates that a masked Ph1 is very rare, that the chromosomes involved vary from case to case, and that in most cases the pattern of the rearrangement is quite different from that of two- and three-chromosome variant Ph1 translocations.

Karyotype evolution in a case of chronic myelogenous leukemia with an unusual Philadelphia chromosome translocation, t(4;22), and an additional translocation, t(3;5).

A patient with chronic myelogenous leukemia was found, at the time of diagnosis, to have an unusual Philadelphia chromosome translocation, t(4;22) (q35;q11) and an additional previously unreported translocation, t(3;5) (q27;q22). The blastic crisis, which occurred after 14 months, was characterized by the appearance of i(17q). Ten months later, two different hyperdiploid cell lines with 50 chromosomes were found in about 20% of the metaphases examined.

Marked karyotype abnormalities in two cases of acute myelogenous leukemia.

Two patients with acute myelogenous leukemia with severe chromosome abnormalities are described. The cytogenetic analysis shows the following karyotype: patient No. 1: 41,XY,-1,-2,-4,-5,-13,-15,-17,-18,-22,+5 markers; patient No. 2: 46,XY,-2,-5,-7,-13,+16,-21,-21,+5 markers. In each patient one set of double minute chromosomes was observed.

Karyotype evolution in CML: high frequency of translocations other than the Ph.

The karyotypes of 33 Philadelphia-positive chronic myelogenous leukemia patients during the blastic phase are reported. Only three patients (9%) had a Philadelphia clone without further chromosomal aberrations, whereas, all the others had karyotype evolution. Aside from some nonrandom abnormalities (+8, i(17q), +Ph, +19) we found a higher frequency of clones with random structural rearrangements (13 cases, 39.4%) than previously reported. From a clinical point of view, however, the additional chromosomal (structural) abnormalities do not significantly influence the patients' survival.