Análisis molecular de la longitud telomérica en linfomas foliculares: Su participación en la progresón tumoral / Molecular analysis of telomere length in follicular lymphomas: Its participation in tumor progression

Los telómeros son estructuras esenciales para el mantenimiento de la integridad cromosómica y la capacidad replicativa de la célula. La reducción de la longitud telomérica (LT) aumenta la probabilidad de producir errores capaces de generar cambios genómicos importantes para el desarrollo neoplásico, determinando desbalances de material genético. En este trabajo se evaluó la LT mediante el análisis de fragmentos de restricción terminal (TRF) en médula ósea y/o biopsia ganglionar de 36 pacientes (edad media: 54.2 años; rango 29-77 años; 21 varones): 29 con linfoma folicular (LF) al diagnóstico y 7 con linfoma B difuso a células grandes secundario a LF (LBDCG-S). Se efectuó el análisis del rearreglo molecular del gen BCL-2 por PCR anidada y de larga distancia. Las medias de TRF en LF (4.18±0.18 Kb) y LBDCG-S (3.31±0.25 Kb) resultaron significativamente menores que en controles (8.50±0.50 Kb) (p<0.001), encontrándose diferencias entre ambos subtipos histológicos (p=0.036). Las muestras negativas para el rearreglo BCL-2 mostraron LT menores (3.39±0.30 Kb) que las positivas (4.25±0.19 Kb) (p=0.023), observándose una tendencia a valores menores en pacientes negativos para el rearreglo BCL-2, intermedios en positivos para mcr, minor cluster region, (3.84±0.45 Kb) y mayores en los positivos para MBR, Major Breakpoint Region, (4.35±0.21 Kb). Nuestros resultados muestran una reducción de la LT en LF y LBDCG-S, con TRFs significativamente más cortos en estos últimos, sugiriendo la participación del acortamiento telomérico em la progresión tumoral. Asimismo, las diferencias detectadas entre los casos BCL-2 positivos y negativos sustentarían la presencia de diferentes mecanismos patogénicos propuestos para estos distintos LF.

Análisis molecular de la longitud telomérica en linfomas foliculares: Su participación en la progresón tumoral / Molecular analysis of telomere length in follicular lymphomas: Its participation in tumor progression

Los telómeros son estructuras esenciales para el mantenimiento de la integridad cromosómica y la capacidad replicativa de la célula. La reducción de la longitud telomérica (LT) aumenta la probabilidad de producir errores capaces de generar cambios genómicos importantes para el desarrollo neoplásico, determinando desbalances de material genético. En este trabajo se evaluó la LT mediante el análisis de fragmentos de restricción terminal (TRF) en médula ósea y/o biopsia ganglionar de 36 pacientes (edad media: 54.2 años; rango 29-77 años; 21 varones): 29 con linfoma folicular (LF) al diagnóstico y 7 con linfoma B difuso a células grandes secundario a LF (LBDCG-S). Se efectuó el análisis del rearreglo molecular del gen BCL-2 por PCR anidada y de larga distancia. Las medias de TRF en LF (4.18±0.18 Kb) y LBDCG-S (3.31±0.25 Kb) resultaron significativamente menores que en controles (8.50±0.50 Kb) (p<0.001), encontrándose diferencias entre ambos subtipos histológicos (p=0.036). Las muestras negativas para el rearreglo BCL-2 mostraron LT menores (3.39±0.30 Kb) que las positivas (4.25±0.19 Kb) (p=0.023), observándose una tendencia a valores menores en pacientes negativos para el rearreglo BCL-2, intermedios en positivos para mcr, minor cluster region, (3.84±0.45 Kb) y mayores en los positivos para MBR, Major Breakpoint Region, (4.35±0.21 Kb). Nuestros resultados muestran una reducción de la LT en LF y LBDCG-S, con TRFs significativamente más cortos en estos últimos, sugiriendo la participación del acortamiento telomérico em la progresión tumoral. Asimismo, las diferencias detectadas entre los casos BCL-2 positivos y negativos sustentarían la presencia de diferentes mecanismos patogénicos propuestos para estos distintos LF. (AU)

[Telomeres and telomerase activity: their role in aging and in neoplastic development]. / Telómeros y actividad de telomerasa: su participación en el envejecimiento y el desarrollo neoplásico.

Telomeres are specialized structures at the ends of eukaryotic chromosomes, composed of tandem repeats of a repetitive DNA sequence (TTAGGG)n and associated proteins. They have a number of important functions including the protection of chromosomes from end-to-end fusion and degradation. When telomeres become critically short, telomere separation in mitosis cannot be performed properly leading to metaphase telomeric associations (tas) and chromosome instability. This instability can be relevant for neoplastic transformation because it increases the probability of errors that can generate genetic changes critical in the multistep process of transformation, like gene amplification and loss of heterozygosity. The mechanisms involved in tas are unknown, but it could be because of failure in the enzymatic activity of telomerase, a ribonucleoprotein enzyme with an RNA template that directs synthesis of telomeric repeats at chromosome extremities, producing telomeric length stabilization. A progressive telomere shortening with ageing has been shown to occur both in vitro and in vivo. Recent studies have shown an association between the presence of tas and telomeric shortening, and also a correlation between telomere reduction and increased telomerase activity in both solid tumors and hematologic malignancies. The evidence that most human malignancies have telomerase activity would indicate that telomerase could be a prevalent and specific tumor marker, and thus may be a novel and excellent target for anti-cancer therapy.

Telómeros y actividad de telomerasa: su participación en el envejecimiento y el desarrollo neoplásico. / [Telomeres and telomerase activity: their role in aging and in neoplastic development]

Telomeres are specialized structures at the ends of eukaryotic chromosomes, composed of tandem repeats of a repetitive DNA sequence (TTAGGG)n and associated proteins. They have a number of important functions including the protection of chromosomes from end-to-end fusion and degradation. When telomeres become critically short, telomere separation in mitosis cannot be performed properly leading to metaphase telomeric associations (tas) and chromosome instability. This instability can be relevant for neoplastic transformation because it increases the probability of errors that can generate genetic changes critical in the multistep process of transformation, like gene amplification and loss of heterozygosity. The mechanisms involved in tas are unknown, but it could be because of failure in the enzymatic activity of telomerase, a ribonucleoprotein enzyme with an RNA template that directs synthesis of telomeric repeats at chromosome extremities, producing telomeric length stabilization. A progressive telomere shortening with ageing has been shown to occur both in vitro and in vivo. Recent studies have shown an association between the presence of tas and telomeric shortening, and also a correlation between telomere reduction and increased telomerase activity in both solid tumors and hematologic malignancies. The evidence that most human malignancies have telomerase activity would indicate that telomerase could be a prevalent and specific tumor marker, and thus may be a novel and excellent target for anti-cancer therapy.

Evidence of chromosome instability in chronic pancreatitis.

In the current study we analyzed chromosome instability on peripheral blood lymphocytes cultured from 7 untreated patients with chronic pancreatitis (CP) by assessing telomeric associations (TAS), chromosome aberrations (CA) and sister chromatid exchanges (SCE). Seven healthy individuals were also analyzed. Mean frequencies of TAS were significantly higher in CP patients (X +/- SE: 11.00 +/- 2.37) compared to controls (1.00 +/- 0.30) (p<0.001). Chromosomes preferentially involved in TAS were: 9, 20, 16 and 21, being the most affected arms: 9p, 20q, 16p, 9q and 21q. All these terminal bands were coincident with cancer breakpoints (p<0.03), two of them (40%) were specifically associated to pancreatic carcinoma rearrangements. Three bands (60%) were coincident with oncogene location. The mean frequency of CA was significantly higher in patients (3.88 +/- 0.80) compared to controls (0.63 +/- 0.49) (p<0.001). Chromosomes 1, 2 and 13 were the most damaged. No specifically affected breakpoints were found. SCE analysis showed higher levels in patients (8.33 +/- 0.70) than in controls (6.62 +/- 0.34) (p<0.025), but no differences were observed in cell cycle kinetics. Our results clearly indicate that CP patients exhibit chromosome instability, showing the presence of an unstable genome that could be related to the cancer development observed in this disease.

Translocation t(1;10) (q21;q26) in a case of acute nonlymphocytic leukemia.

Chromosome banding studies carried out on bone marrow cells from a 16 year-old boy with an M1 acute nonlymphocytic leukemia (ANLL) revealed an unbalanced translocation involving chromosomes 1 and 10: der(10) t(1;10) (q21;q26) that results in a partial trisomy 1q between bands 1q21-1qter. Marker del(6)(q21) and trisomies of chromosomes 18, 21 and 22 were also observed. To our knowledge, this der(10) is the first to be reported in a patient with ANLL.

Proximal trisomy 1q in a girl with developmental delay and minor anomalies.

We report on a girl with developmental delay, macrocephaly, facial asymmetry, small downturned palpebral fissures, high and narrow palate, micrognathia, short neck, a heart defect, and unilateral renal agenesis. Cytogenetic analysis showed a proximal tandem duplication of the long arm of chromosome one (1q12-->q21.3). This abnormality was suggested by G- and C-banding but it was specifically characterized by fluorescent in situ hybridization (FISH). Clinical findings in our patient are compared with those of the literature in an attempt to delineate the phenotype in patients with proximal 1q duplication.

[Molecular genetics of hemophilia A]. / Genética molecular de la hemofilia A.

Hemophilia A (HemA), an X linked genetic disease, is the most common coagulation disorder with an incidence of about 1-2 in 10,000 males and is caused by mutations in the factor VIII (FVIII) coagulation gene. Firstly, some clinical aspects of the HemA are presented: the current methods to assess both the amount and activity of FVIII, the severity range observed and the presence of inhibitor antibodies against the therapeutic FVIII. Follows a discussion of the relationship of the structural domains of the FVIII protein (Figure 1), the aminoacid sequence and their functions. An activation-inactivation model of the successive peptide bonds cleavages of the FVIII is also presented (Figure 2). After the cloning of the FVIII gene in 1984, almost all types of HemA causing mutations have been characterized. However, the size and complexity of this gene prevented a screening of the full range of mutations for an accurate molecular diagnosis. Moreover, most of the patients with moderate and mild disease have missense mutations whereas approximately half of severe patients have nonsense, frameshift, and some missense mutations. There are also less frequently mutations such as deletions and insertions leading to severe phenotype and mutations affecting mRNA splicing and duplications causing both severe and mild HemA. In order to give genetic counselling in HemA families, studies at the DNA level using intragenic and/ or extragenic polymorphism analysis have been used. But this approach is not entirely satisfactory because it fails in several situations. Most of the causing mutations described above are private, and they have been found in only a few unrelated families. Recently, a common molecular inversion of the FVIII gene was identified in 50% of unrelated patients with severe HemA. The copies of a particular DNA sequence (termed F8A gene). One copy is located within intron 22 of the FVIII gene and the other two, 500 kb upstream. An homologous recombination mechanism was proposed for the inversion between an intragenic copy of the F8A gene and either the distal (80% of the inversion) or the proximal copy (20%). Both of these inversions lead to severe HemA because no intact FVIII is produced and can be easily diagnosed by Southern blot analysis. This inversion originates almost exclusively in male germ cells, because pairing Xq with its homologous in female meiosis would probably inhibit the proposed intrachromosome recombination. The molecular analysis of the inversion of intron 22 is now considered as the first line for families with severe HemA patients. In recent years the treatment of patients with hemophilia A and B has been intravenous injection of FVIII or FIX concentrates, respectively. This regimen of regular injection of plasmatic proteins bears a high risk of infection by contaminating viruses (HIV, HBV, etc). Future treatment for patients with hemophilia may include the use of either gene therapy or recombinant coagulation factors. Both strategies would completely avoid the infection risk offering a safe and effective treatment for the disease. Recombinant factors, obtained by genetic engineering methods, provide a renewable and unlimited source of FVIII or FIX. The clinical trials of recombinant factors have already started in mid-1995 giving positive results. On the other hand, gene therapy for hemophilia is now in the pre-clinical stage but offers the prospect of a cure for the disease, thus potentially freeing patients from regular injections of the lacking protein. However, experiments in animal models suggest that it may be difficult to obtain adequate therapeutic levels of factors for long periods of time. Recently, a retroviral-mediated gene delivery of human FVIII in mice has been reported using the ex vivo strategy of gene therapy. Therapeutic levels of FVIII in the circulation were obtained for > 1 week and it was also observed that the capacity of primary cells to deliver FVIII in blood was strongly dependent on

Ag-NOR staining and satellite association in bone marrow cells from patients with mycosis fungoides.

Silver staining of nucleolus organizing regions (Ag-NORs) of acrocentric chromosomes and the frequency of satellite association (SA) in bone marrow (BM) cells from 7 patients with mycosis fungoides (MF), were studied. BM samples of 7 normal healthy individuals were taken as controls. The mean number of Ag-NORs per metaphase was increased in patients (7.20 +/- 0.25) compared with controls (5.40 +/- 0.16) (p < 0.002), related with the increase of the D group. Moreover, a significant higher percentage of Ag-NOR positive cells in patients (71.7 +/- 3.9) than controls (48.0 +/- 7.8) (p < 0.02), was seen. The analysis of SA revealed a significant increase in the percentage of cells with 1-2 association pairs (ASPs) in patients with respect to their controls (p < 0.05), and a trend to a decrease in the percentage of cells without ASPs. Furthermore, a correlation between the number of Ag-NORs and the mean of ASPs per cell was also found for patients (rk = 0.65; p < 0.05). These results may be associated with a certain degree of immaturity, a high proliferative activity and modifications of the growth rate of BM cells in MF patients.

Chromosome instability in lymphocytes from patients with celiac disease.

Cytogenetic studies were performed in celiac disease (CD) patients to determine if the presence of chromosome instability is related to the predisposition to cancer. Chromosome aberrations (CA) and sister chromatid exchange (SCE) frequencies in peripheral blood lymphocyte cultures from untreated CD patients and healthy controls were analyzed. Patients showed aberrations in 23% of cells, while only 3% were detected in the control group (p < 0.0001). The mean frequencies of gaps, breaks and total CA were found to be higher in CD patients compared to controls (p < 0.0001). Breakpoint distribution was nonrandom among chromosomes from celiac patients (p = 0.01), but not among controls (p = 0.04). The frequency of SCE/cell showed a mean value of 6.9 +/- 0.6 in CD patients and 7.3 +/- 0.2 in controls. No statistical differences were found. Breakpoints involved in CD patients presented a strong coincidence with the location of fragile sites (78.6%) and sites of cancer chromosome rearrangements (57.1%), most of them (75%) associated with malignant non-Hodgkin lymphomas. These results suggest that CD is a condition with increased chromosome instability characterized by a high level of CA and normal SCE frequencies, probably related to the increased incidence of cancer.

[Expression of new fragile sites detected in lymphoproliferative processes]. / Expresión de sitios frágiles nuevos detectados en procesos linfoproliferativos.

Fragile sites are specific chromosomal sites prone to breakage and rearrangements and they are probably related with cancer development. Fragile sites expression induced by 5-fluorodeoxyuridine (FudR) or 5-bromodeoxyuridine (BrdU) was analyzed in 4 healthy individuals and 4 patients affected with lymphoproliferative disorders: one Hodgkin's disease, one mycosis fungoides and two chronic lymphoproliferative disorders. Three standard peripheral lymphocyte cultures with F-10 medium and 5% of fetal calf serum were set up for each individual. For fragile sites induction, 10 micrograms/mL FudR or 50 micrograms/mL BrdU were added 24 hr or 6 hr before harvest. Standard and sequential G banded metaphases were studied for each individual and treatment. Quantitative analysis showed a low incidence of acentric fragments, dicentric, tri- or quadriradials, while gaps and breaks were more frequently observed. Chromosome or chromatid type aberrations were compared, showing similar values in all non-treated cultures. Chromosome type aberrations were increased in patients and controls treated cultures. Patient cultures treated by FudR presented a threefold increase of chromosome type alterations respect to chromatid ones. Moreover, chromosome breaks showed a twofold increase in patient treated cultures respect to control ones. The high number of chromosome breaks detected in these cases could be associated with an increased chromosome instability in cancer patients. Twenty common fragile sites (c-fra) were identified with sequential G banding. Patients and controls individuals have expressed 14 c-fra. Eleven of them were induced, in different proportions, by both chemical agents, showing that fragile sites share a structural homology in DNA.(ABSTRACT TRUNCATED AT 250 WORDS)