Suzanne McElwaine

Acquired mutations in GATA1 in neonates with Down's syndrome with transient myeloid disorder

Transient myeloid disorder is a unique self-regressing neoplasia specific to Downs 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 Downs syndrome. We aimed to investigate changes in GATA1 in patients with Downs 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.

Role of DNA methylation in the suppression of Apaf-1 protein in human leukaemia.

Apaf-1 protein deficiency occurs in human leukaemic blasts and confers resistance to cytochrome-c-dependent apoptosis. Demethylation treatment with 5-aza-2′-deoxycytidine (5aza2dc) increased the sensitivity of the K562 leukaemic cell line to UV light-induced apoptosis in association with increased Apaf-1 protein levels. There was no correlation between Apaf-1 protein expression and Apaf-1 mRNA levels after the demethylation treatment. Methylation-specific polymerase chain reaction was used to show that the methylation can occur within the Apaf-1 promoter region in leukaemic blasts. Apaf-1 DNA methylation was demonstrated in acute myeloid leukaemia, chronic myeloid leukaemia and acute lymphoid leukaemia, suggesting that it is not specific to a particular leukaemia subtype. Apaf-1 protein expression did not correlate with Apaf-1 mRNA levels in human leukaemic blasts. Some leukaemic cells expressed high levels of Apaf-1 mRNA but low levels of Apaf-1 protein. This study suggests that Apaf-1 DNA promoter methylation might contribute to the inactivation of Apaf-1 expression. However, Apaf-1 protein levels might also be controlled at post-transcription level.

Microarray transcript profiling distinguishes the transient from the acute type of megakaryoblastic leukaemia (M7) in Down's syndrome, revealing PRAME as a specific discriminating marker

Transient myeloproliferative disorder (TMD) is a unique, spontaneously regressing neoplasia specific to Downs 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 Students 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.

Loss‐of‐function JAK3 mutations in TMD and AMKL of Down syndrome

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.

Identification of a potential role for POU2AF1 and BTG4 in the deletion of 11q23 in chronic lymphocytic leukemia

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.

Trinucleotide repeat dynamic mutation identifying susceptibility in familial and sporadic chronic lymphocytic leukaemia

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.

Functional copy number changes in Sézary syndrome: toward an integrated molecular cytogenetic map III

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.

A Novel Phenotype of Glycogen Storage Disease Presenting as Gastrointestinal Neuromuscular Disease

Introduction Gastric smooth muscle layers are electrically excited by slow waves. In the normal stomach, slow waves propagate longitudinally in ring wavefronts from the upper corpus to the distal antrum. Circumferential propagation does not appear to occur, likely because there is no excitable tissue available circumferentially in these rings. However, rapid circumferential propagation has been observed in isolated gastric tissues and during gastric pacing. In this study, the propagation profiles of slow wave behaviors in diabetic gastroparesis were defined at high resolution (HR). The hypothesis was that circumferential propagation occurs during dysrhythmia, presenting a novel marker of gastric electrical dysfunction. Methods HR (multi-electrode) mapping was performed in 7 patients with diabetic gastroparesis undergoing laparotomy for stimulator implantation. Anterior serosal recordings were taken using flexible PCB arrays (256 electrodes; 4 mm spacing; 36cm2), and activation mapping was performed. Velocity fields were calculated using a finite difference approach incorporating a Gaussian filter smoothing function. Amplitudes were calculated using a peak-trough detection algorithm. Longitudinal and circumferential propagation data from corpus recordings were compared with Students t-test. Means±SEM are reported. Results Atypical or dysrhythmic propagation was observed in 6/7 patients, including incomplete conduction block, complete block with escape, ectopic pacemaking in the corpus and antral tachygastria (freq range: 2.7-4.2 cpm). Circumferential propagation was associated with all of these events (circumferential velocity 6.6 ± 0.9 mm/s vs longitudinal velocity 2.9 ± 0.2 mm/s; p<0.01). Extracellular slow wave amplitudes were also ~2.5x higher during circumferential propagation (411±66 uV vs 170±27 uV; p<0.01). Isochronalmapping demonstrated that circumferential propagation led to the rapid restoration of a normal longitudinal wavefront distal to the source of the dysrhythmia. However, circumferential propagation also promoted organized retrograde propagation in the case of antral tachygastria. Conclusions Propagation abnormalities in diabetic gastroparesis include conduction blocks, escape, and ectopic events, potentially as a consequence of known ICC network degradation. Circumferential propagation emerges in many of these abnormal patterns because excitable tissue becomes available in the circumferential direction. Circumferential conduction is found to be associated with high velocities and high amplitudes, which could therefore serve as useful clinical indicators for abnormal slow wave propagation. Functionally, rapid circumferential propagation serves to restore normal slow wave propagation distal to conduction defects, however it can also promote organized retrograde tachygastria.