Genetic abnormalities associated with the t(12;21) and their impact in the outcome of 56 patients with B-precursor acute lymphoblastic leukemia.

The ETV6/RUNX1 rearrangement is found in 20-30% of children with B-cell precursor acute lymphoblastic leukemia and is associated with a good outcome. To determine the cytogenetic and molecular abnormalities associated with the ETV6/RUNX1 rearrangement and the influence of this rearrangement in patients' evolution, we analyzed the molecular cytogenetic profiles of 56 children with this rearrangement and B-cell precursor acute lymphoblastic leukemia. Secondary changes detected with conventional cytogenetics and with fluorescence in situ hybridization were found in 71.4% of cases, the most frequent being the loss of the normal ETV6 allele, 12p aberrations, duplication of the fusion gene, and trisomy 21, as in replicating the results of previous studies. In this preliminary series, with a mean follow-up of 69.3 months, secondary abnormalities did not influence patients' outcome. It seems therefore that the prognostic value of the t(12;21) does not vary and that ETV6/RUNX1 rearrangement is an independent indicator of good prognosis.

Changes in the attitudes towards psychiatry among Spanish medical students during training in psychiatry

Background: To gain an understanding of the process of recruitment, studyingchanges in attitudes and views towards psychiatry among Spanish medical studentsduring their fourth academic year.Methods: A 33-item questionnaire was administered to 48 medical students before andafter having completed training in psychiatry. Comparative data analysis was carried outusing the Wilcoxon test.Results: The comparison showed that there was a reduction in the number of studentsreporting that “psychiatrists abuse their legal power”, that “for most specialists in thisarea, psychiatry was not their preferred choice” and that “those students interested in psychiatryare regarded as odd or peculiar”. However, the view that “psychiatry is an expandingfrontier of medicine” decreased among the students. The percentage of students consideringpsychiatry as a future career rose from 4.2% to 10.4% after training.Conclusions: The students’ opinions change with the experience of training in psychiatryand become more realistic. Alongside these changes in attitudes, there is an increasein the proportion of students willing to consider psychiatry as a future career, whichsuggests that there is no reduction in vocations for psychiatry among Spanish students (AU)

ETV6/RUNX1 rearrangement in childhood B-precursor acute lymphoblastic leukemia with normal karyotypes or without cytogenetic results.

The ETV6/RUNX1 rearrangement (also known as TEL/AML1) was evaluated in 39 children with B-precursor acute lymphoblastic leukemia (ALL) who had a normal karyotype or lack of mitoses. Forty-one point six percent of patients with normal karyotypes and 66.6% of patients without mitoses presented with the ETV6/RUNX1 rearrangement. In addition to this rearrangement, eight patients showed loss of the normal ETV6 allele; of three patients without mitoses, two showed an extra signal of the RUNX1 gene and the third showed the fusion gene duplicated and loss of the normal ETV6 allele. One patient without the ETV6/RUNX1 rearrangement and without mitoses showed two extra signals of the RUNX1 gene.

AML1 amplification in a child with acute lymphoblastic leukemia.

We report a case of de novo acute lymphoblastic leukemia with tandem amplification of the AML1 gene located in a chromosome marker that originated from chromosome 21 and a long event-free survival.

Chromatid segregation analysis in native human lymphocyte anaphases using sequential fluorescence in situ hybridization.

A sequential multiprobe fluorescence in situ hybridization technique was developed to study the 13, 18, 21, X and Y chromatid segregation in human lymphocytes anaphases cultures without antimitotic treatment. This method was used to know if exist any different chromosomes segregation in lymphocytes from Down syndrome patients and compared it with controls. The results show that the prevalent sequence of centromere separation was X, 13, 21, Y and 18 in Down syndrome patients and Y, 13, X, 21 and 18 in controls. Chromatid segregation in early anaphase was asynchronic for all chromosome pairs studied. Late anaphase showed a frequency of non-disjunction of 4.5% in the controls, affecting only chromosomes 18 and Y; in the Down syndrome patients, the frequency was higher (20.3%) and affected all chromosomes studied. This technique could be applicated to know the incidence of non disjunction in couples with repetitive abortions or in cases with different aneuploidies in the offspring.

Follow-up by cytogenetic and fluorescence in situ hybridization analysis of allogeneic bone marrow transplantation in two children with Fanconi's anaemia in transformation.

Results of bone marrow transplantation (BMT) in patients with Fanconi's anaemia (FA) in transformation are very poor and only a few cases with favourable outcome have been reported. We present the follow-up of two FA-myelodysplastic syndrome (MDS) patients with monosomy 7 and complex karyotype implicating chromosome 1. Both relapsed with acute myeloid leukaemia (AML) following an allogeneic BMT from an HLA-identical brother. The patients showed clonal cytogenetic evolution coinciding with the leukaemic transformation. In one patient, fluorescence in situ hybridization using X and Y chromosome probes detected an increase of host cells before clinical relapse. Both patients received a successful second allogeneic BMT from the same donor using a more intensive treatment regimen and remain in clinical and cytogenetic remission more than 3 years later.

SRY gene transferred to the long arm of the X chromosome in a Y-positive XX true hermaphrodite.

Yp-specific sequences, including the testicular determinant gene SRY, have been detected and located in a 46,XX true hermaphrodite individual, using PCR amplification and fluorescent in situ hybridization (FISH). Among different Y chromosome loci tested, it was only possible to detect Yp sequences. The Y-centromere and Yq sequences were absent. Unexpectedly, the Y fragment was translocated to the long arm of one of the X chromosomes, at the Xq28 level, and the derivative (X) chromosome of the patient lacked q-telomeric sequences. To our knowledge, this is the first Yp/Xq translocation reported. The coexistence of testicular and ovarian tissue in the patient may have arisen by differential inactivation of the Y-bearing X chromosome, in which Xq telomeric sequences are missing. The possible origin of the Yp/Xq translocation, during paternal meiosis or in somatic paternal cells, is discussed.

Follow-up of chimerism in children with hematological diseases after allogeneic hematopoietic progenitor cell transplants.

Using in situ hybridization with an X and Y chromosome probe mixture, 106 bone marrow samples from 38 patients with malignant and non-malignant hematological diseases who received sex-mismatched allogeneic hematopoietic progenitor cell transplants (PCT) in a single institution within short-term intervals (1, 3, 6, 12, 24 and >24 months) have been sequentially studied. The patients received either HLA-identical (n = 31) or non-identical (n = 7) PCT. Twenty-six children showed donor chimerism, 10 children showed mixed chimerism (MC) and two children presented autologous reconstitution. Chimerism status with different parameters has been related (age, sex, donor, disease status before PCT, conditioning regimen, GVHD prophylaxis, relapse, GVHD and survival). Our results indicate that female patients (P = 0.011) and a less intensive conditioning regimen (P = 0.039) are significantly associated with the MC status. Mixed chimerism is not, per se, significantly associated with leukemia relapse but an increase of the MC is indicative of clinical relapse.

Cytogenetic analysis using simultaneous and sequential fluorescence in situ hybridization.

A sequential fluorescence in situ hybridization (FISH) technique is described. This method allows the detection of up to eighteen chromosome pairs in consecutive hybridizations (8 steps) on the same metaphase using centromeric, whole chromosome painting, and single copy DNA probes with different fluorochromes. The technique may be used with diagnostic purposes in cases with poor cytogenetic material.

Characterization by FISH of a t(5;13) in a patient with bilateral retinoblastoma.

We have used fluorescence in situ hybridization (FISH) with a biotinylated cosmid probe (13q14) to screen 25 unrelated cases with bilateral retinoblastoma and one case with unilateral retinoblastoma. In 25 cases no deletion or chromosome rearrangements were, found. One constitutional mutation resulting from a de novo balanced chromosome translocation (5;13) in a patient with bilateral retinoblastoma was detected.

Analysis of human sperm-derived pronuclei by three-colour fluorescent in-situ hybridization.

We describe a modification of the human sperm-zona-free hamster egg fusion method that permits the study of aneuploidy in sperm-derived pronuclei by multicolour fluorescent in-situ hybridization (FISH). Zona-free hamster eggs and human spermatozoa were fused and cultured for 15 h in the presence of colcemid (1 microg/ml of medium) to obtain hamster oocytes arrested at metaphase II and human spermatozoa at the pronuclear stage. By applying a whole human genomic DNA probe we confirmed that 100% of pronuclei tested (372/372) were of human origin. One-colour fluorescent in-situ hybridization using a centromeric 18 probe was applied to 919 pronuclei with different dithiothreitol (DTT) pretreatments: 50 mM (10 min) or 25 mM (20 and 25 min). The highest hybridization efficiency was obtained with treatment with 25 mM DTT for 20 min (90.3%). Sex chromosome aneuploidy was analysed by three-colour FISH in a total of 2596 pronuclei from a normal donor. Hybridization efficiency was 98.6%. The disomy rates for X, Y and XY chromosomes (0.11, 0.04 and 0.08% respectively) were similar to data reported for sperm nuclei by three-colour FISH and to those obtained in sperm chromosomes. These results suggest that selection of potentially fertile spermatozoa (spermatozoa able to fertilize zona-free hamster eggs and produce a pronucleus) does not imply chromosomal selection.

A case with 47,XXY,del(15)(q11;q13) karyotype associated with Prader-Willi phenotype.

Herein we present the case of a 12-year-old boy who attended our clinic for obesity and hyperphagia. As a newborn he was noted to have diffuse muscular hypotonia and poor sucking response. At the age of 11 years, he was admitted to hospital for respiratory insufficiency. He had personality disorders characterized by temper tantrums and violent outbursts including self-mutilation. Physical evaluation revealed marked central obesity, he had small hands and feet, and also genital hypoplasia. Of the biochemical parameters, hyperglycemia and a low serum testosterone level must be emphasized. The patient fulfills the clinical criteria of typical Prader-Willi syndrome. Cytogenetic and fluorescence in situ hybridization analysis showed a karyotype 47,XXY, del(15)(q11;q13). To our knowledge this is the first report of the aforementioned genotype expressed as Prader-Willi phenotype in childhood.

Isochromosome 14q in childhood acute lymphoblastic leukemia.

We report a case of childhood acute lymphoblastic leukemia (ALL) with an isochromosome 14q as the sole abnormality. A review of the literature revealed that isochromosome 14 has not been previously reported in ALL. The prognostic significance of this abnormality is compared with that of other hematologic disorders with trisomy 14.

Appraisal of fluorescence in situ hybridization (FISH) techniques in prenatal diagnosis.

The reliability of FISH was appraised using probes for the X and Y chromosome and for chromosomes 12 and 18 in prenatal and adult interphase nuclei. Detection of a single hybridization spot proved to be quite reliable (80-92% positive nuclei). Detection of two hybridization spots was more difficult; percentages of nuclei showing two signals varied between 62-72%. The percentages of nuclei with the correct number of spots was higher in the metaphases occasionally found. Thus, FISH may complement but not replace cytogenetic analysis. For sex determination and for the detection of mosaicism, we suggest the use of two different probes in separate regions of the same preparation.