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Sézary syndrome (SS) is a rare form of cutaneous T-cell lymphomas (CTCL) manifested by generalized exfoliative erythroderma, intense pruritus, peripheral lymphadenopathy, and abnormal hyperchromatic mononuclear cells in the skin, lymph nodes, and peripheral blood. Previous studies have revealed complex genetic aberrations affecting almost all chromosomes with up- and down-regulation of several genes in this subtype of CTCL. It is still unclear, however, which of these genetic alterations are the primary changes and which are the secondary ones. We have long thought that a key molecular defect should be consistently present at DNA, RNA, and protein levels. We therefore assume that some chromosome copy number changes (CNC) seen in cancer cells may have a direct impact on gene expression pattern and that these CNC are presumably functional. To test this hypothesis, we designed a simple but novel boinformatic method that included analysis of SS gene expression microarray raw data, generation of gene lists from the chromosomal regions showing significant CNC in SS, and data remaining to establish if the CNC gene lists affected gene clustering in terms of separation of SS cases from the normal controls or not. The bioinformatic analysis of 17 selected gene lists with GeneSpring software showed that four (24%) from copy number losses at 1p36p22, 6q24, and 15q11.2, as well as gains at 22q11.2q13.3, were capable of separating all six SS cases from two controls (P<0.05), and some of the genes, such as LCK at 1p34.3, may have implications in CTCL. The remaining 13 gene lists produced a mixed gene clustering patterns between SS cases and normal control samples (P<<0.01). These findings suggest that CNC from these chromosomal regions may be functional and relevant to SS.

Addiction is a complex psychiatric disorder considered to be a disease of the brain's natural reward reinforcement system. Repeated stimulation of the 'reward' pathway leads to adaptive changes in gene expression and synaptic organization that reinforce drug taking and underlie long-term changes in behaviour. The primitive nature of reward reinforcement pathways and the near universal ability of abused drugs to target the same system allow drug-associated reward and reinforcement to be studied in non-mammalian species. Zebrafish have proved to be a valuable model system for the study of vertebrate development and disease. Here we demonstrate that adult zebrafish show a dose-dependent acute conditioned place preference (CPP) reinforcement response to ethanol or nicotine. Repeated exposure of adult zebrafish to either nicotine or ethanol leads to a robust CPP response that persists following 3 weeks of abstinence and in the face of adverse stimuli, a behavioural indicator of the establishment of dependence. Microarray analysis using whole brain samples from drug-treated and control zebrafish identified 1362 genes that show a significant change in expression between control and treated individuals. Of these genes, 153 are common to both ethanol- and nicotine-treated animals. These genes include members of pathways and processes implicated in drug dependence in mammalian models, revealing conservation of neuro-adaptation pathways between zebrafish and mammals.

Acquired mutations activating Janus kinase 3 (jak3) have been reported in Down syndrome (DS) and non-DS patients with acute megakaryoblastic leukaemia (AMKL). This highlighted jak3-activation as an important event in the pathogenesis of AMKL, and predicted inhibitors of jak3 as conceptual therapeutics for AMKL. Of 16 DS-transient myeloproliferative disorder (TMD)/AMKL patients tested, seven showed JAK3 mutations. Three mutations deleted the kinase (JH1) domain, abolishing the main function of jak3. Another patient displayed a mutation identical to a previously reported inherited loss-of-function causing severe combined immunodeficiency. Our data suggest that both gain-, and loss-of function mutations of jak3 can be acquired in DS-TMD/AMKL.

Channels formed by connexins (Cx), the major protein subunits of gap junctions, allow passage of ions and molecular messengers between cells to provide a mechanism of synchronized cellular response. Twenty human Cx isoforms have been identified and mutations in the gene GJB3 encoding the 31 kDa isoform, Cx31, can cause dominant or recessive skin disease, dominant or recessive deafness or dominant neuropathy with deafness. Cx31 is expressed in differentiating keratinocytes in skin. Here, we also demonstrate endogenous Cx31 expression in human neuronal cell lines, particularly in differentiated neurones. Exogenous Cx31 expression induced neurite outgrowth in human neuronal cell lines, but not differentiation in primary human keratinocytes. Though neither the neuropathy and hearing loss mutation (66delD)Cx31 nor the skin disease associated mutation (R42P)Cx31 is able to traffic to the plasma membrane, the R42P mutant induced neurite outgrowth to a level equal to wild-type Cx31. In contrast, there was significantly reduced neurite outgrowth after (66delD)Cx31 expression. In addition to indicating a potential disease mechanism for the neuropathy/deafness mutation, this work demonstrates a tissue-specific function for Cx31.

Chronic lymphocytic leukaemia (CLL) has a strong hereditary component, but an understanding of predisposition genes is poor. Anticipation with familial CLL has been reported, although the molecular mechanism is unknown. Expansion of trinucleotide repeat sequences underlies anticipation observed in neurodegenerative disease. A polymerase chain reaction-based assay was used to analyse the stability of ten CCG- and CAG-trinucleotide repeat tracts in 18 CLL families and 140 patients with the sporadic form of the disease. The study suggests that anticipation, if it occurs in CLL, is not linked to CCG- and CAG-repeat expansion, however, variation in repeat length at certain loci (FRA16A) may permit identification of susceptible family members. In addition, polymorphisms with prognostic significance were identified. These were high length (but not expanded) repeats at FRA11B (P = 0.01), ATXN1 (P = 0.032) and ATXN3 (P = 0.022), all associated with poor risk disease.

Objective: Placental insufficiency is a primary cause of intrauterine growth restriction (IUGR). In our study, microarray technology was used to identify genes, which may impair placentation resulting in IUGR., Study Design: The RNA was isolated from both IUGR term placentas and normal term placentas. Microarray experiments were used to identify differentially expressed genes between the 2 cohorts. Real-time quantitative reverse transcriptase polymerase chain reaction and immunohistochemistry were used in follow-up experiments., Results: Microarray experiments identified increased expression of certain genes including leptin, soluble vascular endothelial growth factor receptor, human chorionic gonadotropin, follistatin-like 3, and hypoxia-inducible factor 2alpha in the IUGR. Real-time quantitative polymerase chain reaction confirmed these results., Conclusion: The upregulation of soluble vascular endothelial growth factor receptor and hypoxia-inducible factor 2alpha at this period in pregnancy indicate that placental angiogenesis is altered in IUGR and that hypoxia is a major contributor to maldevelopment of the placental vasculature.

Deletions of 11q in chronic lymphocytic leukemia (CLL) are usually associated with progressive disease and poor prognosis. A novel translocation within the previously identified 11q minimal region has been defined in a patient with CLL. The breakpoint is between genes POU2AF1 and BTG4. POU2AF1 is a B-cell-specific transcriptional coactivator, and BTG4 is a member of the BTG family of negative regulators of the cell cycle, making both of them good candidate genes for the pathogenesis of 11q- CLL. POU2AF1 was observed to be differentially expressed in the cells of patients with CLL compared to its expression in normal B cells in the absence of mutations. This may reflect ongoing stimulation and active accessory signaling in CLL cells. BTG4 could contribute to CLL pathogenesis following inactivation by haploinsufficiency., (Copyright 2005 Wiley-Liss, Inc.)

Transient myeloproliferative disorder (TMD) is a unique, spontaneously regressing neoplasia specific to Down's syndrome (DS), affecting up to 10% of DS neonates. In 20-30% of cases, it reoccurs as progressive acute megakaryoblastic leukaemia (AMKL) at 2-4 years of age. The TMD and AMKL blasts are morphologically and immuno-phenotypically identical, and have the same acquired mutations in GATA1. We performed transcript profiling of nine TMD patients comparing them with seven AMKL patients using Affymetrix HG-U133A microarrays. Similar overall transcript profiles were observed between the two conditions, which were only separable by supervised clustering. Taqman analysis on 10 TMD and 10 AMKL RNA samples verified the expression of selected differing genes, with statistical significance (P < 0.05) by Student's t-test. The Taqman differences were also reproduced on TMD and AMKL blasts sorted by a fluorescence-activated cell sorter. Among the significant differences, CDKN2C, the effector of GATA1-mediated cell cycle arrest, was increased in AMKL but not TMD, despite the similar level of GATA1. In contrast, MYCN (neuroblastoma-derived oncogene) was expressed in TMD at a significantly greater level than in AMKL. MYCN has not previously been described in leukaemogenesis. Finally, the tumour antigen PRAME was identified as a specific marker for AMKL blasts, with no expression in TMD. This study provides markers discriminating TMD from AMKL-M7 in DS. These markers have the potential as predictive, diagnostic and therapeutic targets. In addition, the study provides further clues into the pathomechanisms discerning self-regressive from the progressive phenotype.

Transient myeloid disorder is a unique self-regressing neoplasia specific to Down's syndrome. The transcription factor GATA1 is needed for normal growth and maturation of erythroid cells and megakaryocytes. Mutations in GATA1 have been reported in acute megakaryoblastic leukaemia in Down's syndrome. We aimed to investigate changes in GATA1 in patients with Down's syndrome and either transient myeloid disorder (n=10) or acute megakaryoblastic leukaemia (n=6). We recorded mutations eliminating exon 2 from GATA1 in all patients with transient myeloid disorder (age 0-24 days) and in all with acute megakaryoblastic leukaemia (age 14-38 months). The range of mutations did not differ between patients with each disorder. Patients with transient myeloid disorder with mutations in GATA1 can regress spontaneously to complete remission, and mutations do not necessarily predict later acute megakaryoblastic leukaemia.

Standard identification systems usually ensure that biopsy material is correctly associated with a given patient. Sometimes, as when a tumor is unexpectedly found, the provenance (proof of origin) of a tissue sample may be questioned; the tissue may have been mislabelled or contaminated with tissue from another patient. Techniques used to confirm tissue provenance include comparing either tissue markers of gender or ABO blood groups; however, these methods have weak confirmatory power. Recently, the use of DNA-based polymerase chain reaction (PCR) techniques has been reported. Paired, formalin-fixed, paraffin-embedded, 10 microns tissue sections were selected from 17 patients, 8 of whom had carcinoma, either by dividing a biopsy section, using sequential biopsies, or sequential biopsy and autopsy tissue. The resulting 36 samples were coded before analysis. In two additional cases, 1-mm fragments of tumor from one patient were included in the tissue block of benign tissue from another patient, the tumor fragments were identified on hematoxylin-and-eosin-stained sections, separately scraped off the glass slide, and analyzed. Tissue from two clinical cases, one of suspected mislabelling and one with a suspected carry-over of malignant tissue were also investigated. Short tandem repeat sequences (STR) or microsatellites, are 2-5 base pair repeats that vary in their repeat number between individuals. This variation (polymorphism) can be assessed using a PCR. A panel of markers of 3 STRs; ACPP, INT 2, and CYP 19 (on chromosomes 3, 11, and 15, respectively) were used. DNA was isolated from the samples after xylene deparaffinization and proteinase digestion, and was then amplified in a radioactive PCR using primers selected to give a product size ranging from 136-178 bases. Amplified products were electrophoresed on denaturing polyacrylamide gels, dried, and autoradiographed. DNA segments were successfully extracted from all samples but one, which was fixed in Bouin's fluid. By comparing allele sizes from the panel, all tissue pairs (other than the Bouin's pair) were successfully matched, the 1-mm tumor fragments were correctly assigned, and the two clinical problems were solved. STRs are highly informative and robust markers, well suited to PCR of small portions of tissue sections, and are an effective method to confirm the provenance of benign and malignant biopsy and autopsy material.