Instability of a 550-base pair DNA segment and abnormal methylation in fragile X syndrome.

The fragile X syndrome, a common cause of inherited mental retardation, is characterized by an unusual mode of inheritance. Phenotypic expression has been linked to abnormal cytosine methylation of a single CpG island, at or very near the fragile site. Probes adjacent to this island detected very localized DNA rearrangements that constituted the fragile X mutations, and whose target was a 550-base pair GC-rich fragment. Normal transmitting males had a 150- to 400-base pair insertion that was inherited by their daughters either unchanged, or with small differences in size. Fragile X-positive individuals in the next generation had much larger fragments that differed among siblings and showed a generally heterogeneous pattern indicating somatic mutation. The mutated allele appeared unmethylated in normal transmitting males, methylated only on the inactive X chromosome in their daughters, and totally methylated in most fragile X males. However, some males had a mosaic pattern. Expression of the fragile X syndrome thus appears to result from a two-step mutation as well as a highly localized methylation. Carriers of the fragile X mutation can easily be detected regardless of sex or phenotypic expression, and rare apparent false negatives may result from genetic heterogeneity or misdiagnosis.

Expression of BCL-2 proto-oncogene in adult acute lymphoblastic leukemia.

The products of the BCL-2 gene prolong survival of lymphohematopoietic cells by inhibition of programmed cell death. We studied bcl-2 protein expression in a series of 43 adult acute lymphoblastic leukemia (ALL) at diagnosis, using a specific monoclonal antibody and flow cytometry. All samples expressed bcl-2 with a mean percentage of positive cells of 77.9. The level of bcl-2 in positive cells expressed as mean equivalent of soluble fluorescence (MESF) was highly variable ranging from 5 x 10(3) to 552 x 10(3) (mean +/- s.d.: 96.5 +/- 109 x 10(3)). Neither the percentage of positive cells nor bcl-2 MESF levels were correlated with initial characteristics including blood counts, immunological phenotype, or cytogenetics. The survival of leukemic cells maintained in cytokine-free liquid culture was not correlated with bcl-2 expression. However, cells from ALL with higher white blood cell (WBC) counts, with t(9;22) translocation, or expressing myeloid surface antigens exhibited significantly longer survival in this culture system. The outcome after intensive chemotherapy did not differ according to bcl-2 expression. Factors associated with poor outcome included WBC counts, presence of t(9;22) translocation, presence of myeloid antigens and prolonged survival of cultured cells. These results indicate that high levels of bcl-2 are not associated with distinct clinical or biological characteristics in ALL.

Expression and activity of caspases 1 and 3 in myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) are characterized by abnormal growth of committed progenitors in clonogenic assay, with reduced number of colonies and decreased colony/cluster ratio. It has been suggested that excessive apoptosis is the cause of marrow failure in MDS. We studied the expression of caspase-1 (interleukin-1beta-converting enzyme, ICE) and caspase-3 (CPP32/apopain) in marrow mononuclear cells, and the growth pattern of committed progenitors in a series of 83 MDS cases. The percentage of apoptotic cells as detected by TUNEL technique, and the percentage of caspase-3-positive cells were significantly higher in refractory anemia (RA) and RA with ringed sideroblasts (RAS) than in chronic myelomonocytic leukemia (CMML), refractory anemia with excess of blasts (RAEB) and RAEB in transformation (RAEB-T). Spontaneous growth of CFU-GM was associated with a higher percentage of blasts, and with a lower expression of caspase-3 and caspase-1. The yield of CFU-E, BFU-E, and CFU-GM (in the presence of growth factors) was decreased by comparison to normal marrow, but large individual differences were observed in all cytological categories. Inhibition of caspase-1 and caspase-3 activities by specific inhibitors resulted in a significant increase of the production of all types of colonies (up to 50-fold of control). In the presence of caspase-3 inhibitor, the number of BFU-E and CFU-E was in the range of normal values in most cases of RA and RAS. In addition, caspase-1 and -3 protease activities were detectable by fluorogenic assay in all cases studied. Western blot analysis confirmed the expression of caspase-3, including the cleaved (activated)-p17 form in most cases of RA/RAS analyzed. It is concluded that caspase-3 is implicated in the increased apoptosis observed in MDS and that inhibition of its activity can restore at least partially the growth of committed progenitors.

Automated counting of in situ hybridization dots in interphase cells of leukemia samples.

Twenty-seven samples (cell cultures prepared for routine cytogenetics) of leukemia patients with known cytogenetic abnormalities were stained by in situ hybridization for interphase cytogenetics with centromere specific probes for chromosome Nos 4, 6, 7, 8, 9, 12, 17, 18, X and Y. The number of hybridization domains per nucleus was quantified using a semi-automated system developed in our laboratory. Results of this automated counting procedure (with and without verification of the counting results by the operator) were compared with conventional cytogenetic data and with visual scoring of the number of hybridization dots. The findings show that the system is capable of analysing 1000 cell nuclei in less than 30 min, including the necessary verification of the results by the operator. Automated counting and visual scoring were in good agreement. Conventional cytogenetics and interphase cytogenetics agreed in only 50% of the cases, confirming other studies showing that conventional cytogenetic results are not always representative for the majority of the cell population.

Cytogenetic heterogeneity in t(11;19) acute leukemia: clinical, hematological and cytogenetic analyses of 48 patients--updated published cases and 16 new observations.

We report on 16 cases of t(11;19) acute leukemia and review data of published observations: altogether updated data of 48 patients are analyzed. Four hematological groups could be distinguished: (i) 13 cases of acute lymphoblastic leukemia (ALL) of B lineage, mostly CD19+; (ii) eight cases of biphenotypic leukemia: CD19+ (most often) ALL but with simultaneous or inducible expression of differentiation marker of monocytic lineage. The B lineage and biphenotypic leukemias were predominantly found in female infants; (iii) four cases of T-ALL in children; and (iv) 23 acute non-lymphocytic leukemia (ANLL) cases generally of M4 or M5 subtype, predominantly in males. Cytogenetically, at least two subtypes were observed with possibly an identical breakpoint on 11q23 but discrete breakpoints on 19p: lymphoid, biphenotypic, and most congenital myeloid cases showed a distal breakpoint on 19p13 producing 11q- and 19p+ derivatives, while most older myeloid cases showed 11q+ and 19p- derivatives as a result of a more proximal breakpoint on 19p12 or p13.1. The latter type was clearly detected using R bands but barely visible using Q or G bands while the other translocation was easy to detect with G bands but could be missed with R bands. The white blood cell count is usually high in these t(11;19) acute leukemias and prognosis is poor, except for T-ALL cases.

Good correlation between RT-PCR analysis and relapse in Philadelphia (Ph1)-positive acute lymphoblastic leukemia (ALL).

We sequentially performed cytogenetic analysis and RT-PCR analysis of BCR-ABL transcripts in 17 cases of Ph1-positive ALL who had achieved hematological complete remission (CR) with intensive chemotherapy (CT). Sixteen cases were studied prospectively. All but one of the patients had reached cytogenetic CR, but cytogenetic has low sensitivity in predicting relapse. Twelve patients relapsed, three died in first CR and two were alive in first CR. Two of five, two of four, and five of nine patients who were allografted (in first or second CR), autografted and received consolidation CT, respectively, achieved negative two-round PCR in the bone marrow (BM): three died in CR, three remained in CR with negative two-step PCR in the BM and three relapsed after 22 to 28 months. In all cases, relapse was preceded by switch to PCR positivity in the BM by 4 to 6 months. The remaining nine patients remained PCR-positive in the BM and relapsed after 2 to 16 months. In the four autografted cases, PCR was positive at the time of bone marrow harvest. The two patients who received a purged transplant achieved negative PCR and prolonged CR, whereas the two patients who received an unpurged transplant remained PCR positive and relapsed. In 34% of the samples where analysis was concomitant, sensitivity of PCR proved lower in the blood than in the BM. These findings show that RT-PCR is a useful tool in the monitoring of MRD in Ph1 positive ALL.

Balanced complex chromosomal rearrangements with more than four breakpoints: report of a new case.

Congenital complex chromosomal rearrangement (CCR) compatible with life are rare in man. We report on a new case of apparently balanced CCR in a 30-month-old boy with mental retardation and minor anomalies. This CCR consists in a 3-way reciprocal translocation (2;3;16) and an insertion (6;7), as it was analyzed by different banding and high resolution techniques. It involves 6 breakpoints: 2q11, 13q12, 16p11, 6p21.3, 7q21.3 and 7q35.

Male pseudohermaphroditism, partial androgen receptors defect, 11p13 deletion: indication of gene localization.

A partial androgen receptor defect was found in a boy with male pseudohermaphroditism and an 11p13 deletion. We hypothesize that a gene responsible for the function or structure of androgen receptors might be localized in the 11p13 band or in close proximity to it.

A case of myelodysplastic syndrome with abnormal megakaryocytes and ins(8;3)(q24;q21q26).

We report a 56-year-old male patient with refractory anemia with excess of blasts in transformation (RAEB-T) who had an ins(8;3)(q24;q21q26) as the sole chromosome abnormality in bone marrow (BM) cells. The findings of disturbed thrombocytopoiesis with numerous micromegakaryocytes suggest that it could be a variant of the classic ins(3;3)(q26;q21q26) described in hematologic malignancies with abnormal thrombopoiesis.

Persistence of t(4;11) in cytologically normal bone marrow treated ex vivo with Asta-Z.

The chromosomal anomaly t(4;11) is closely related to a specific type of acute leukemia: occurrence in young children, hyperleukocytosis with a particular immunologic phenotype, and poor response to therapy. Allogeneic bone marrow (BM) transplantation has been done in a few cases. We report a case in which a complete remission was obtained after intensive therapy. Because no donor was available, an autologous BM transplantation was performed after purge ex vivo of the BM collection by Asta-Z. Relapse occurred at day 45.

A complex t(3;8;17) involving breakpoint 8p11 in a case of M5 acute nonlymphocytic leukemia with erythrophagocytosis.

The t(8;16)(p11;p13) is a recently described new chromosome rearrangement of acute nonlymphocytic leukemia (ANLL). It appears to be specifically associated with acute monoblastic (AML-M5) or unusual myelomonocytic leukemia with prominent erythrophagocytosis in the leukemic cells. A complex t(3;8;17)(q27;p11;q12) is reported in a case of acute monoblastic leukemia with erythrophagocytosis. Sixteen cases of this t(8;16) and two other variant translocations are reviewed. The pathogenetic mechanism of the variant translocations is discussed, suggesting that the der(8) is a consistent recombinant.

t(2;18) and t(18;22) variant chromosomal translocations in B cell malignancies.

Variant translocations (2;18 and 18;22) are described in this review. The chromosomal and molecular findings of these translocation of BCL2 and their effect on possible BCL2 gene activation is discussed. Unanswered questions still remain and these include why this is so rare compared to the 25% incidence recorded for translocations in Burkitt's lymphoma. Further studies are obviously still needed in order to determine the true frequency of these findings and their distribution in the various B-cell disorders.

[Constitutional translocation t(1;6) and Burkitt's lymphoma]. / Translocation constitutionnelle t(1;6) et lymphome de Burkitt.

BACKGROUND: The relationship between constitutional chromosome aberrations and a predisposition to malignancy has already been established. CASE REPORT: A 4 year-old boy was admitted suffering from abdominal mass. Biopsies of the tumor and bone marrow showed a stage IV Burkitt's lymphoma. Karyotype showed a constitutional t(1;6)(p36;q22) in initial bone marrow samples and peripheral lymphocytes. No classic Burkitt's type translocation was demonstrated in the malignant bone marrow cells. CONCLUSION: Our case is similar to four other reported cases. The break-points 1p36 and 6q22 seem to be involved in several malignant tumors, especially lymphomas.

[Follow-up of allogenic bone marrow graft with DNA polymorphisms. Use of two minisatellite MS31 and MS43 probes. Comparison with data of chromosomal polymorphisms]. / Suivi de greffe allogénique de moelle osseuse par les polymorphismes de l'ADN. Utilisation de deux sondes minisatellites MS 31 et MS 43. Comparaison avec les données des polymorphismes chromosomiques.

OBJECTIVES: Allogenic bone marrow transplantation is widely used to treat many diseases of the haemopoietic system as well as metabolic disorders. Follow-up is essential to assess acceptance, rejection or post-graft relapse. This study was undertaken to evaluate the usefulness of the minisatellite probes MS31 and MS43 used as a routine follow-up test after bone marrow transplantation. METHODS: Twenty receivers of allogenic bone marrow transplants were followed-up. Two monoclonal minisatellite probes, MS31 and MS43, were used for comparison with the classical polymorphism methods. RESULTS: Fourteen cases of total chimeras, 3 cases of rejections and 3 cases of mixed chimeras were observed with the molecular probe techniques. In 19 of the 20 cases, this technique gave results compatible with classical polymorphism results. CONCLUSIONS: The minisatellite probes MS31 and MS43 were found to be sensitive, effective tests for bone marrow transplants which can be used in routine follow-up.

[Acquired myelodysplastic syndromes after treatment of melanoma with fotemustine and dacarbazine]. / Syndromes myélodysplasiques acquis après traitement par fotémustine et dacarbazine pour mélanome.

INTRODUCTION: Secondary myelodysplasia after antimitotic therapy is a rare complication usually observed with alkylating agents. The condition usually progresses to acute leukaemia with very poor short term prognosis. CASE REPORT: We report the cases of 2 women who developed myelodysplasia 2 and 9 months after treatment associating dacarbazine and fotemustine for visceral metastases of a malignant melanoma. DISCUSSION: The frequency of these rare complications is probably underestimated because of the rapid unfavourable outcome of metastatic malignant melanoma. We were unable to determine whether dacarbazine, fotemustine or their combination was incriminated in this complication. Risk could be reduced by carefully determining the cumulative doses of these antimitotics.

Collagen biosynthesis in cell culture from chorionic villi.

After chorionic villus biopsy of human placenta, cell cultures were propagated with Ham's F10 medium or Eagle's minimum essential medium (MEM). It was possible to study the morphology of the cells by transmission electron microscopy (TEM) after a special culture of the cells in a collagen gel. The cells embedded in a collagen gel were able to contract the gel and to organize collagen fibres, as fibroblast cultures do. TEM showed vacuolization and well-developed cisternae of the endoplasmic reticulum, especially in the case of MEM culture. The aim was to determine whether cells cultivated from early placenta were able to synthesize enough collagen for a metabolic study. A high level of collagen biosynthesis could be quantified. Types I and III collagen can be determined which is useful for studying the abnormalities of collagen synthesis in suspected cases of osteogenesis imperfecta or Ehlers-Danlos type IV syndrome. The hydroxylation of lysine can also be studied with respect to Ehlers-Danlos type VI syndrome. Moreover, these cells, in contrast to fibroblast cultures, made it possible to study the biosynthesis of type IV collagen.

Six cases of partial duplication-deficiency 21 syndrome: 21(dupq22delp23) due to maternal pericentric inversion: inv(21)(p12;q22). A family study.

Six probands, apparently not related, with a minimal phenotype of Down's syndrome were investigated between 1970 and 1984 in our laboratory. We found in all of them an identical chromosomal abnormality 46,XX or XY,-21,+ der21(dupq22delp23). The der 21 was due to aneusomie de recombinaison, each mother having an abnormal chromosome 21: inv(21)(p12;q22). The fathers' caryotypes were normal. All parents were young and healthy. Pedigrees were established in order to find a relationship between these families. Four of our probands could be related. Familial investigations are still in progress for the last two cases; the ancestors being born in the same small geographical area (within 50 km2) we think that we shall be able to establish a relationship with the others families.