We studied the subcellular distribution of mitochondria and superoxide dismutase-1 (SOD1) in whole mounts of microdissected motor axons of rats expressing the ALS-linked SOD1-G93A mutation. The rationale was to determine whether physical interactions between the enzyme and mitochondria were linked to the axonopathy of motor fibers occurring in amyotrophic lateral sclerosis (ALS). Mitochondria and SOD1 displayed a homogeneous distribution along motor axons both in nontransgenic rats and in those overexpressing wild-type SOD1. In contrast, axons from SOD1-G93A rats (older than 35 days) showed accumulation of mitochondria in discrete clusters located at regular intervals. Most of SOD1 immunoreactivity was enriched in these clusters and colocalized with mitochondria, suggesting a recruitment of SOD1-G93A to the organelle. The SOD1/mitochondrial clusters were abundant in motor axons but scarcely seen in sensory axons. Clusters also were stained for neuronal nitric oxide synthase, nitrotyrosine, and cytochrome c. The later also was detected surrounding clusters. Ubiquitin colocalized with clusters only at late stages of the disease. The cytoskeleton was not overtly altered in clusters. These results suggest that mutant SOD1 and defective mitochondria create localized dysfunctional domains in motor axons, which may lead to progressive axonopathy in ALS.

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The conclusive demonstration of RNA in vertebrate axons by in situ hybridization (ISH) has been elusive. We review the most important reasons for difficulties, including low concentration of axonal RNAs, localization in specific cortical domains, and the need to isolate axons. We demonstrate the importance of axon micro-dissection to obtain a whole mount perspective of mRNA distribution in the axonal territory. We describe a protocol to perform fluorescent ISH in isolated axons and guidelines for the preservation of structural and molecular integrity of cortical RNA-containing domains (e.g., Periaxoplasmic Ribosomal Plaques, or PARPs) in isolated axoplasm.

4832 Background Myeloproliferatives neoplasms (MPN) are clonal stem cell disorders characterized by proliferation of one or more of the myeloid lineages, associated with genetic abnormalities that include translocations or point mutations of genes that encode cytoplasmic or tyrosin quinase (TK) receptor proteins. This produces an abnormal constitutively activation of signal transduction pathways, leading to an unregulated proliferation. According to the WHO criteria, MPN are classified into BCR-ABL+/Philadelfia Ph+: chronic myeloid leukemia (CML) and MPN BCR-ABL fusion-/Ph-. A single acquired point mutation, JAK2V617F, has been described in 95% of Polycytemia vera (PV), in 50 % of essencial thrombocythemia (ET) and idiopathic myelofivrosis (IMF), and generally absent in MPN Ph+. In the last years, it has been described the co-ocurrence of both BCR-ABL and V617F mutation in few cases of CML patients. We report here the rare and concomitant ocurrence of JAK2V617F mutation with BCR-ABL translocation at presentation in atypical CML. Methods Blood samples from six patients with clinical suspicion of MPN diagnosis, were referred to our laboratory to cytogenetic studies and molecular analysis of BCR-ABL fusion gene expression by conventional RT-PCR and JAK2V617F status mutation by ASO-PCR in three of them. All patients showed a slightly elevated white blood cells level (7200-25900), trombocythosis (700 -1036 platelets) and small splenomegaly. Three patients, after CML diagnosis, recieved Imatinib therapy and were monitored by quantitative BCR-ABL real time PCR. Due to persistent thrombocytosis, slightly elevated white blood cells level and small splenomegaly; JAK2 status was analized in these blood specimens, and later retrospectively in the stored initial diagnosis samples. Results BCR-ABL rearrangement (b3a2 isoform) and JAK2V617F mutation were identified in all 6 patients at diagnosis. Three cases showed lack of Ph chromosome, 1 patient showed 15 % Ph+ metaphases and in the remained two patients no data was available. Quantitative PCR for BCR-ABL expression performed in 3 patients during follow-up (8-12 months) showed BCR-ABL/BCR ratio < = 0.0018 % (scored according to the International Scale) and the presence of JAK2V617F mutation. Retrospective assessment of stored bood samples showed that JaK mutation was already present at the time of the diagnosis of CML. Conclusions The coexistence of both genetic defects, BCR-ABL fusion gene and JAK2V617F mutation in NMP patients is a rare and uncommon feature. We found 6 MPN patients hourboring both genetics features in blood dignosis samples. In three cases JAK2V617F mutation was detected in MPN patients BCR-ABL positive after the remission induction with Imatinib. The rapid remission of BCR-ABL transcript, after a short period of Imatinib treatment, led us to think that BCR-ABL fusion gene expression was present in a low burden at diagnosis. The complete reduction of BCR-ABL rearrangement, after the imatinib therapy, and the persistence of JAK 2 mutation suggests two possible mechanisms for this double genetic alteration: 1) a haematopoyetic cell subclone with a pre-existing JAK2V617F acquires the BCR-ABL fusion gene, which confers a selective advantage to double mutant progenitor; 2) two clons, one of them having BCR-ABL rearrangement, and the other one the JAK2V617F mutation (biclonal origin). These cases intend to contribute to the discussion about the onset of the molecular alterations, and their correlation with the differente phenotypes and clinical management. Disclosures: No relevant conflicts of interest to declare.

4839 Introduction. Chronic myeloid leukemia (CML) is characterized by the Philadelphia chromosome (Ph) observed in more than 90% of patients with CML as a result of t(9;22)(q34;q11), leading to the formation of chimeric gene BCR/ABL encoding for proteins with abnormal tyrosine kinase activity. Cytogenetic variants of Ph chromosome can be identifed in 5 to 10% of CML patients, involving additional chromosomes other than 9 and 22. To explain the formation of variant translocations one-step, two-step and multi-step mechanisms have been proposed. Rarely, the variant Ph chromosome results from a BCR insertion on the ABL region and form a BCR/ABL fusion gene, generally mapping to 9q34, instead of the usual location at 22q11. In very few variant Ph cases, the insertion of the BCR/ABL product in a third chromosome was demonstrated. Case Report 28 year-old man, with bilateral central scotoma and gingivorragia. Physical examination: Grade 4 splenomegaly. Peripheral blood count showed hemoglobin concentration 11.5 g/dl, platelet count: 300.000/mm3, and white blood cell count 590.000/mm3. Blood smear: myelemia exhibiting 30% of myeloid blasts. Bone marrow biopsy: panmyelosis showing 20% of myeloid blasts. Cytogenetic analysis by G-banding performed in peripheral blood verified the following karyotype: 46, XY, t(9;22;10)(q34;q11;q24)[20] The analysis of the BCR-ABL fusion gene according to standard protocols detected the presence of the b3a2 isoform. Fluorescence in situ hybridization (FISH) studies using dual color dual fusion probes in metaphases showed a signal pattern 1F2G1R. The fusion signal mapped to 10q24, the red signal to 9q34, and the normal green signal to chromosome 22, while a second low intensity green signal mapped to the Ph chromosome. No signal was observed in der(9). Interphase FISH analysis in nuclei (n=200) presented the same signal pattern. Instead of using whole chromosome probes for 9 and 22, we hybridised probes used to detect DiGiorge syndrome. These probes detect gene control ARSA (spectrum green) localized at 22q13 and Tuple1 at 22q11 (spectrum orange). Two signals, green and orange were identified in normal chromosome 22. Ph chromosome showed the orange signal, whereas the green signal mapped to der(10). Discussion. The localization of the hybrid BCR/ABL gene on chromosomes other than 22q is a rare event wich can only be detected by FISH techniques. When these unusual translocation occurs, the hypothesis most often put forward is that several consecutive chromosome rearrangements have taken place. In the present case the interpretation of karyotypes, FISH data and molecular evidence lead to the following hypothesis: Insertion of the BCR sequence from chromosome 22 to chromosome 9 may have ocurred, producing a BCR/ABL fusion in der(9). The Ph chromosome detected by G-banding showed a different green fluorescence intensity in the metaphase FISH signal pattern with BCR/ABL dual color dual fusion probes, as a result of an insertion on chromosome 9. This first event was followed by the translocation between the derivative 9 and chromosome 10, being the final localization of the BCR/ABL gene in 10q24. FISH analysis using a DiGeorge syndrome probe, supports the hypothesis of a multistep mechanism underlying insertion and translocations events in the present case. The relocation of BCR/ABL fusion sequence on sites other than chromosme 22q11 represent a rare type of variant Ph translocation. At least 21 cases described in the literature, showed fusion gene BCR/ABL located at 9q24. Only 12 patients with variant Ph were reported bearing BCR/ABL on a third chromosome. All of them involved a masked Ph chromosome. To our best knowledge this is the first report showing a variant Ph chromosome detected by G-banding in a CML patient due to a BCR insertion on ABL sequences and exhibiting the fusion signal in a third chromosome. Disclosures: No relevant conflicts of interest to declare.