A complete duplication of X chromosome resulting in a tricentric isochromosome originated by centromere repositioning.

BACKGROUND: Neocentromeres are rare and considered chromosomal aberrations, because a non-centromeric region evolves in an active centromere by mutation. The literature reported several structural anomalies of X chromosome and they influence the female reproductive capacity or are associated to Turner syndrome in the presence of monosomy X cell line. CASE PRESENTATION: We report a case of chromosome X complex rearrangement found in a prenatal diagnosis. The fetal karyotype showed a mosaicism with a 45,X cell line and a 46 chromosomes second line with a big marker, instead of a sex chromosome. The marker morphology and fluorescence in situ hybridization (FISH) characterization allowed us to identify a tricentric X chromosome constituted by two complete X chromosome fused at the p arms telomere and an active neocentromere in the middle, at the union of the two Xp arms, where usually are the telomeric regions. FISH also showed the presence of a paracentric inversion of both Xp arms. Furthermore, fragility figures were found in 56% of metaphases from peripheral blood lymphocytes culture at birth: a shorter marker chromosome and an apparently acentric fragment frequently lost. CONCLUSIONS: At our knowledge, this is the first isochromosome of an entire non-acrocentric chromosome. The neocentromere is constituted by canonical sequences but localized in an unusual position and the original centromeres are inactivated. We speculated that marker chromosome was the result of a double rearrangement: firstly, a paracentric inversion which involved the Xp arm, shifting a part of the centromere at the p end and subsequently a duplication of the entire X chromosome, which gave rise to an isochromosome. It is possible to suppose that the first event could be a result of a non-allelic homologous recombination mediated by inverted low-copy repeats. As expected, our case shows a Turner phenotype with mild facial features and no major skeletal deformity, normal psychomotor development and a spontaneous development of puberty and menarche, although with irregular menses since the last follow-up.

Identification of a small supernumerary marker chromosome, r(2)(p10q11.2), and the problem of determining prognosis.

The identification of small supernumerary marker chromosomes (SMCs) and the elucidation of their clinical significance remain two of the problems in classical human cytogenetics. We observed a small supernumerary ring in amniotic fluid cell cultures and identified its origin as r(2)(p10q11.2) and its extent by means of fluorescent in situ hybridisation (FISH). Uniparental disomy (UPD) was excluded by microsatellite analysis using polymorphic markers localised in the same region. On the basis of normal ultrasonographic checks, the patient decided to continue the pregnancy. A normal female was delivered at term and subsequent neonatal follow-ups confirmed the normal phenotype and development. In the present case, genetic counselling was not helpful because of the absence of reference cases. Detailed characterisation made it possible to correlate the normal baby phenotype with the trisomic 2p10-2q11.2 genomic region. Further molecular cytogenetic investigations of SMCs classified by DNA content and pregnancy outcome data should improve genetic counselling and risk evaluation.

Decreased platelet glutamate uptake and genetic risk factors in patients with Parkinson's disease.

Genetic risk factors seem to play a role in sporadic Parkinson's disease (PD), maybe triggering oxidative stress and excitotoxicity within substantia nigra. However, genetic factors act at systemic level: reduced activity of mitochondrial enzymes and decreased glutamate uptake have been shown in platelets from PD patients. In this study we investigated glutamate uptake in platelets from 38 sporadic PD patients, 13 patients with parkinsonian syndromes and 28 controls and assessed polymorphisms of alpha-synuclein and ApoE genes. A 48% reduction of glutamate uptake p)<0.0001) was observed in PD patients which, with respect to control groups, correlated with the disease severity (r = -0.44, p < 0.05). Genetic studies of this population did not show differences between PD and controls, nor correlations with platelet glutamate uptake.

Deletion of parental GST genes as a possible susceptibility factor in the etiology of infant leukemia.

Infant leukemia below the age of 12 months is a rare disease that exhibits a high frequency of 11q23 rearrangements. We assessed the presence of polymorphisms in several metabolic genes in 23 families of infants diagnosed with leukemia under 12 months of age in Italy. When polymorphism frequencies were calculated within families, frequencies of GST gene deletions were significantly higher than expected only among the parents of infants without the 11q23 rearrangement. These data suggest that the deletion of GST genes in parents may affect the risk of infant leukemia through a pathway independent of the MLL gene.

A nomenclature system for metabolic gene polymorphisms.

A nomenclature system for all human metabolic gene polymorphisms is suggested. This system should replace the various nomenclatures used in the literature to describe polymorphisms in many of the cytochrome P450 (CYP) genes, as well as the glutathione S-transferase (GST) and N-acetyltransferase (NAT) genes. The system is based on two published papers proposing nomenclatures for the various alleles of CYP2D6 and NAT. The gene name is followed by an asterisk, followed by an arabic number designating the specific polymorphism in chronological order of first publication. The final number may be followed by letters A,B, etc. when allelic subtypes exist. A table is presented showing nomenclature for 72 polymorphisms in 12 genes, including detailed descriptions and commonly used previous designations.

Different risks of thrombosis in four coagulation defects associated with inherited thrombophilia: a study of 150 families.

Deficiency of the naturally occurring anticoagulant proteins, such as antithrombin, protein C and protein S, and activated protein C resistance due to the factor V Leiden gene mutation is associated with inherited thrombophilia. So far, no direct comparison of the thrombotic risk associated with these genetic defects is available. In this study, we wish to compare the lifetime probability of developing thrombosis, the type of thrombotic symptoms, and the role of circumstantial triggering factors in 723 first- and second-degree relatives of 150 index patients with different thrombophilic defects. We found higher risks for thrombosis for subjects with antithrombin (risk ratio 8.1, 95% confidence interval [CI], 3.4 to 19.6), protein C (7.3, 95% CI, 2.9 to 18.4) or protein S deficiency (8.5, 95% CI, 3. 5 to 20.8), and factor V Leiden (2.2, 95% CI, 1.1 to 4.7) than for individuals with normal coagulation. The risk of thrombosis for subjects with factor V Leiden was lower than that for those with all three other coagulation defects (0.3, 95% CI, 0.1 to 1.6), even when arterial and superficial vein thromboses were excluded and the analysis was restricted to deep vein thrombosis (0.3, 95% CI, 0.2 to 0.5). No association between coagulation defects and arterial thrombosis was found. The most frequent venous thrombotic manifestation was deep vein thrombosis with or without pulmonary embolism (90% in antithrombin, 88% in protein C, 100% in protein S deficiency, and 57% in factor V Leiden), but a relatively mild manifestation such as superficial vein thrombosis was common in factor V Leiden (43%). There was a predisposing factor at the time of venous thromboembolism in approximately 50% of cases for each of the four defects. In conclusion, factor V Leiden is associated with a relatively small risk of thrombosis, lower than that for antithrombin, protein C, or protein S deficiency. In addition, individuals with factor V Leiden develop less severe thrombotic manifestations, such as superficial vein thrombosis.

Lack of mRNA and dystrophin expression in DMD patients three months after myoblast transfer.

We report our experience on myoblast transplantation in three Duchenne muscular dystrophy patients. Pure myoblasts (55 x 10(6) per patient) from HLA-matched donors, were injected into a tibialis anterior and the controlateral muscle was sham injected. Three months after transplantation, biopsies from the injected muscles were negative for dystrophin expression by immunocytochemistry. Reverse transcriptase-PCR (RT-PCR) failed to amplify any fragments of the deleted regions. This result confirms that myoblast transplantation is feasible, although the efficacy of this therapeutic approach is poor.

Changes in peripheral blood lymphocyte subset frequencies in myasthenia gravis patients are related to immunosuppression.

Surface antigens on peripheral blood lymphocytes from myasthenia gravis patients were investigated. The expression of DR+ and CD8+/DR+ T lymphocytes was increased and the expression of CD4+ T cells reduced. Neither thymectomy, clinical condition nor anti-acetylcholine receptor antibody titre correlated with any of the changes in peripheral blood lymphocyte subsets observed. However, immunosuppressive therapy correlated with the significant reduction in CD4+ and CD2+/CD4+ T cells in these patients.

Signal transduction in human tumor infiltrating lymphocytes.

In the present study we demonstrate that the CD28 activation pathway was functional in human tumor infiltrating lymphocytes (TIL), as it could induce a strong proliferation. On the other hand, a mitogenic combination of anti-CD2 monoclonal antibodies (MoAb) induced a slight proliferation of TIL isolated from lung but not from renal cell carcinoma (RCC). It is to note that CD28 triggering led to calcium mobilization, whereas stimulation via CD2 did not. Moreover, in most cases no synergistic effect between CD2 and CD28 activation pathways could be observed. Phenotypic analysis showed that freshly isolated TIL were mostly CD3+, LAM1+ and CD45RO+. Upon stimulation with anti-CD28 MoAb, the majority of cells lost LAM1 antigen and coexpressed both high (CD45RA) and low (CD45RO) molecular weight isoforms of CD45 molecule. By contrast, among CD2-activated TIL, a small fraction was LAM1+ and less than 10% coexpressed CD45RA and CD45RO antigens. Noteworthy, the CD45 molecule could regulate the CD28-induced calcium mobilization, as demonstrated by cross-linking of CD45RO, but not CD45RA, isoforms before challenging TIL with anti-CD28 MoAb.

Signalling in human tumour infiltrating lymphocytes: the CD28 molecule is functional and is physically associated with the CD45R0 molecule.

The CD28 T cell activation pathway was functional in human tumour infiltrating lymphocytes (TIL) and can induce strong proliferation, lymphokine release and calcium mobilisation. Conversely, TIL responded poorly to stimulation via CD2, and CD28 did not synergise with CD2, which is at variance with that observed using peripheral lymphocytes from the same patients. On stimulation with anti-CD28 the monoclonal antibody, most TILs, which were CD3+, CD28+ and CD45R0+ at the beginning of culture, co-expressed both high (CD45RA) and low (CD45R0) molecular weight isoforms of CD45. CD28 was associated with the CD45R0 isoform at the cell surface of activated TIL, as demonstrated by immunoprecipitation and immunoenzymatic assay. Thus CD28 can substitute for CD3 in TIL leading to the expansion of functional lymphocytes and to the amplification of antitumour immune response.

Impairment of lymphocyte suppressive system in recent onset insulin-dependent diabetes mellitus. Correlation with blood glucose and serum insulin levels.

In a previous study, we observed an impairment of the theophylline-induced suppressive system in recent onset IDDM patients, and demonstrated also a correlation with metabolic derangement. The aim of this study was to better investigate the relationship between theophylline sensitivity (ThS) and blood glucose/plasma insulin levels in recent onset IDDM patients subjected to preprogrammed variations by an insulin/glucose clamp with artificial pancreas. Eight patients were studied within 8 weeks from the onset of IDDM. ThS was evaluated as the ability of theophylline to inhibit blastogenic response of peripheral blood lymphocytes (PBL) to Concanavalin A (ConA), after 120 min preincubation of the cells. All patients were connected to an artificial pancreas. Through i.v. continuous insulin infusion (0.02 U/kg/h) and/or i.v. continuous glucose and saline infusion, the following experimental conditions, lasting at least 1h, were obtained: T1: relative euglycemia and normal insulinemia; T2: relative euglycemia and hyperinsulinemia; T3: hyperglycemia and normal insulinemia; T4: hyperglycemia and hyperinsulinemia. ThS was maintained in 6/8 patients at T1 and in 8/8 patients at T4. ThS was lost in 4/8 patients at T2 and T3. These data suggest that the loss of ThS induced by hyperglycemia can be corrected by hyperinsulinemia, and that it is maintained when euglycemia is accompanied by hypoinsulinemia. It is lost when these two parameters lose their interrelationship.

Thymopentine treatment improves clinical and immunological functions in aged subjects with chronic bronchitis.

The effect of in vivo thymopentine treatment (50 mg/subcutaneously every other day for six weeks) on clinical features and in vitro immunological parameters was evaluated in ten aged patients with chronic bronchitis. In vitro immunological studies were performed before and after treatment both on phenotypic (OK monoclonal defined lymphocyte subpopulations) and functional parameters (blastogenic responses to polyclonal mitogens Concanavalin-A and Phytoemagglutinine). Thymopentine did not significantly affect lymphocyte subpopulations, which were reduced before pharmacological treatment, when compared to those of young healthy controls. Peripheral blood lymphocytes blastogenic response to polyclonal mitogens was restored, even though proliferation did not reach the values of young healthy subjects. Thymopentine treatment significantly improved lymphocyte functions, without being able, however, to overcome completely the age-dependent loss of blastogenic responses. Nevertheless the favourable evolution of clinical symptoms we observed in our study may be, at least in part, related to the improvement in vivo of those immunological functions that we studied in vitro. Eight out of ten patients showed an evident improvement of symptoms and signs of chronic bronchitis after thymopentine treatment and four of them arrived to a complete remission from infectious episodes. No side effects were noted. Although still preliminary, these results seem to be encouraging as thymopentine could be used with success as an immunomodulating agent in aged people.

Evidence for adenosine adenosinedeaminase lymphocyte system impairment in ageing.

Age related immune disfunctions are the result of humoral and cellular changes of the immune system and reflect major alterations of T cell subpopulations which concern cyclic nucleotides and their precursors. There are now many reports showing that adenosine can affect some phenotypic and functional lymphocyte characteristics. We have found that a short preincubation (30') with adenosine can inhibit proliferative responses of peripheral blood lymphocytes to polyclonal mitogen Concanavalin A in young healthy controls (p less than 0.05) but not in aged healthy subjects. These data led us to the hypothesis that an impairment of the adenosine-adenosinedeaminase system could play an important role in the age-associated decline of immune responses. Our results show a highly significant reduction (16.45 +/- 3.56 vs 24.42 +/- 9.5 p less than 0.001) of adenosinedeaminase activity in peripheral lymphocytes of aged humans (mean age 75.5 +/- 6.7 range 23-30). Preliminary studies suggest that this alteration could be responsible to some extent, for the decreased mitogenic response of lymphocytes reported in ageing.

Impairment of lymphocyte-suppressive system in recent-onset insulin-dependent diabetes mellitus. Correlation with metabolic control.

Impairment of suppressor-cell activity may be important in the pathogenesis and maintenance of insulin-dependent diabetes mellitus (IDDM). In 23 recent-onset IDDM patients, lymphocyte sensitivity in vitro to theophylline was tested both in basal conditions and after improvement of metabolic control. This pharmacologic agent is mainly effective on a lymphocytic subpopulation with phenotypic and functional suppressive features. Peripheral blood lymphocytes from IDDM patients showed a loss of theophylline sensitivity, identified as inhibition of both E-rosette formation and blastogenic response to polyclonal mitogens concanavalin A (ConA) and phytohemagglutinin (PHA). An inverse relationship was demonstrated between the theophylline-induced suppression of ConA blastogenic response and blood glucose and glycosylated hemoglobin levels (P less than .01). Metabolic control seemed to be important even in relation to lymphocyte subpopulation distribution. In IDDM patients we found a significant (P less than .05) reduction of OKT4+ lymphocytes that is correlated with blood glucose and glycosylated hemoglobin levels (P less than .01). The improvement of metabolic control led to recovery of theophylline sensitivity. We suggest a deficiency in a suppressive system that could be involved in IDDM onset and the possible role of metabolic control in the impairment of some immunologic functions reported with this pathologic condition.

Spontaneous "in vitro" variations of theophylline sensitivity in human peripheral blood lymphocytes.

Theophylline reversibly inhibits E rosette formation by a portion of human circulating T lymphocytes. We investigated the effect of theophylline on E rosette formation and intracellular content of cyclic nucleotides (cAMP, cGMP). When the amine is added to 15 human healthy donors' lymphocytes either before or after 24 hours of culture at 37 degrees C, in absence of mitogens, a portion of theophylline-sensitive T cells spontaneously becomes theophylline-resistant after 24 hours of culture. While the intracellular content of cAMP does not significantly vary, the ability of theophylline to induce an increase of cAMP appears to be impaired after 24 hours of culture. The possible correlation between theophylline resistance and impaired turnover of cAMP in the cultured lymphocytes is discussed.