Quantitative magnetic resonance imaging in a naturally occurring canine model of spinal cord injury.

STUDY DESIGN: Retrospective cohort study. OBJECTIVES: To analyze magnetic resonance imaging (MRI) evaluator agreement in dogs with spinal cord injury (SCI) caused by intervertebral disk herniation (IVDH) using semiautomated and manual lesion segmentation and to analyze the associations between MRI and functional outcome. SETTING: United States of America. METHODS: T2-weighted MRIs from dogs with SCI resulting from thoracolumbar IVDH were identified from a database. Evaluators categorized MRIs on the basis of the presence or absence of a T2-hyperintense spinal cord lesion in axial and sagittal images. A semiautomated segmentation algorithm was developed and used to estimate the lesion volume. Agreement between evaluators and between semiautomated and manual segmentation was analyzed. The relationships of qualitative and quantitative MRIs with behavioral functional outcome were analyzed. RESULTS: Axial images more commonly depicted lesions compared with sagittal images. Lesions in axial images had more consistent associations with functional outcome compared with sagittal images. There was imperfect qualitative agreement, and lesion volume estimation was imprecise. However, there was improved precision using semiautomated segmentation compared with manual segmentation. CONCLUSION: Lesion volume estimation in dogs with naturally occurring SCI caused by IVDH is challenging, and axial images have important advantages compared with sagittal images. The semiautomated segmentation algorithm described herein shows promise but may require further refinement.

A trithorax-like gene is interrupted by chromosome 11q23 translocations in acute leukaemias.

Some acute lymphocytic leukaemias, particularly those in young children, are associated with a t(4;11)(q21;q23) reciprocal translocation. We have cloned the translocation breakpoint on chromosome 11q23 and isolated corresponding RNA transcripts from this region. The translocation occurs within a cluster of Alu repetitive elements located within an intron of a gene that gives rise to 11.5 (kb) transcript spanning the translocation breakpoint. The 11.5 kb transcript encodes a protein that is highly homologous to the Drosophila trithorax gene, a developmental regulator. An analysis of a series of leukaemic patients carrying t(4;11) and t(9;11) translocations indicate that the majority of breakpoints in infant leukaemias lie within a 5 kb region.

High-resolution genomic profiling of human papillomavirus-associated vulval neoplasia.

BACKGROUND: The incidence of human papillomavirus-associated vulval neoplasia is increasing worldwide; yet the associated genetic changes remain poorly understood. METHODS: We have used single-nucleotide polymorphism microarray analysis to perform the first high-resolution investigation of genome-wide allelic imbalance in vulval neoplasia. Our sample series comprised 21 high-grade vulval intraepithelial neoplasia and 6 vulval squamous cell carcinomas, with paired non-lesional samples used to adjust for normal copy number variation. RESULTS: Overall the most common recurrent aberrations were gains at 1p and 20, with the most frequent deletions observed at 2q, 3p and 10. Copy-neutral loss of heterozygosity at 6p was a recurrent event in vulval intraepithelial neoplasia. The pattern of genetic alterations differed from the characteristic changes we previously identified in cutaneous squamous cell carcinomas. Vulval neoplasia samples did not exhibit gain at 5p, a frequent recurrent aberration in a series of cervical tumours analysed elsewhere using an identical protocol. CONCLUSION: This series of 27 vulval samples comprises the largest systematic genome-wide analysis of vulval neoplasia performed to date. Despite shared papillomavirus status and regional proximity, our data suggest that the frequency of certain genetic alterations may differ in vulval and cervical tumours.

Magnetic resonance imaging in dogs with neurologic impairment due to acute thoracic and lumbar intervertebral disk herniation.

BACKGROUND: Magnetic resonance imaging (MRI) is a correlate to physical examination in various myelopathies and a predictor of functional outcome. OBJECTIVES: To describe associations among MRI features, neurological dysfunction before MRI, and functional outcome in dogs with disk herniation. ANIMALS: One hundred and fifty-nine dogs with acute thoracolumbar disk herniation. METHODS: Retrospective case series. Signalment, initial neurological function as assessed by a modified Frankel score (MFS), and ambulatory outcome at hospital discharge and >3 months (long-term) follow-up were recorded from medical records and telephone interview of owners. Associations were estimated between these parameters and MRI signal and morphometric data. RESULTS: Dogs with intramedullary T2W hyperintensity had more severe pre-MRI MFS (median 2, range 0-4) and lower ambulatory proportion at long-term follow-up (0.76) than those dogs lacking hyperintensity (median MFS 3, range 0-5; ambulatory proportion, 0.93) (P=.001 and .013, respectively). Each unit of T2W length ratio was associated with a 1.9 times lower odds of long-term ambulation when adjusted for pre-MRI MFS (95% confidence interval 1.0-3.52, P=.05). Dogs with a compressive length ratio >1.31 (which was the median ratio within this population) had more severe pre-MRI MFS (median 3, range 0-5) compared with those with ratios < or =1.31 (median MFS 3, range 0-4; P=.006). CONCLUSIONS AND CLINICAL IMPORTANCE: MRI features were associated with initial injury severity in dogs with thoracolumbar disk herniation. Based on results of this study, the T2W length ratio and presence of T2W intramedullary hyperintensity appear to be predictive of long-term ambulatory status.

Magnetic resonance imaging characteristics of necrotizing meningoencephalitis in Pug dogs.

BACKGROUND: The magnetic resonance imaging (MRI) characteristics of necrotizing meningoencephalitis (NME) are not well documented. OBJECTIVES: To describe common MRI features of NME, to compare the MRI features to histopathologic findings, and to determine whether or not MRI lesions are predictive of survival time. ANIMALS: Eighteen Pugs with NME. METHODS: Retrospective MRI case study of Pugs identified by a search of medical records at 6 veterinary institutions. Eighteen dogs met inclusion criteria of histopathologically confirmed NME and antemortem MRI exam. MRI lesions were characterized and compared with histopathology with the kappa statistic. Survival times were compared with MRI findings by use of Mann-Whitney U-tests and Spearman's rho. RESULTS: Twelve of 18 lesions were indistinctly marginated with mild parenchymal contrast enhancement. Prosencephalic (17/18) lesion distribution included the parietal (16/18), temporal (16/18), and occipital (16/18) lobes. There were cerebellar (4/18) and brainstem (3/18) lesions. Asymmetric lesions were present in both gray and white matter in all dogs. Falx cerebri shift was common (11/18), and 6 dogs had brain herniation. Leptomeningeal enhancement was present in 9/18 dogs. A moderate positive association was found between parenchymal contrast enhancement and both necrosis (kappa= 0.45; P= .045) and monocytic inflammation (kappa= 0.48; P= .025). Higher MRI lesion burden was correlated with longer time from disease onset to MRI (P= .045). MRI lesion burden did not correlate to survival time. CONCLUSIONS AND CLINICAL IMPORTANCE: Asymmetric prosencephalic grey and white matter lesions with variable contrast enhancement were consistent MRI changes in Pugs with confirmed NME. While not pathognomonic for NME, these MRI characteristics should increase confidence in a presumptive diagnosis of NME in young Pugs with acute signs of neurologic disease.

MicroRNA miR-181a correlates with morphological sub-class of acute myeloid leukaemia and the expression of its target genes in global genome-wide analysis.

MicroRNAs (miRNAs) are short single-stranded RNAs that have a potentially important role in gene regulation. Using a quantitative real-time polymerase chain reaction assay specific to the mature miRNA, the expression level of a selected group of haematopoietic tissue-specific miRNAs was measured across a set of 30 primary adult acute myeloid leukaemia (AML) with a normal karyotype. The expression levels of each miRNA were correlated with the genome-wide mRNA expression profiles in the same leukaemias. This revealed that miR-181a correlated strongly with the AML morphological sub-type and with the expression of genes previously identified through sequence analysis as potential interaction targets. Three other miRNAs, miR-10a, miR-10b and miR-196a-1, showed a clear correlation with HOX gene expression.

Genome-wide detection of recurring sites of uniparental disomy in follicular and transformed follicular lymphoma.

Single-nucleotide polymorphism (SNP) array analysis was performed using the 10K GeneChip array on a series of 26 paired follicular lymphoma (FL) and transformed-FL (t-FL) biopsies and the lymphoma cell lines SCI-1, DoHH2 and RL2261. Regions of acquired homozygosity were detected in 43/52 (83%) primary specimens with a mean of 1.7 and 3.0 aberrations in the FL and t-FL, respectively. A notable feature was the occurrence of recurring sites of acquired uniparental disomy (aUDP) on 6p, 9p, 12q and 17p in cell lines and primary samples. Homozygosity of 9p and 17p arose predominantly in t-FL and in three cases rendered the cell homozygous for a pre-existing mutation of either CDKN2A or TP53. These data suggest that mutation precedes mitotic recombination, which leads to the removal of the remaining wild-type allele. In all, 18 cases exhibited abnormalities in both FL and t-FL samples. In 10 cases blocks of homozygosity were detected in FL that were absent in the subsequent t-FL sample. These differences support the notion that FL and t-FL may arise in a proportion of patients by divergence from a common malignant ancestor cell rather than by clonal evolution from an antecedent FL.

Fingerprinting genomic instability in oral submucous fibrosis.

BACKGROUND: Oral submucous fibrosis (OSF) is a high-risk pre-cancerous condition where 7-13% of these patients develop head and neck squamous cell carcinoma (HNSCC). To date there is no cancer predictive markers for OSF patients. Genomic instability hallmarks early genetic events during malignant transformation causing loss of heterozygosity (LOH) and chromosomal copy number abnormality. However, to date there is no study on genomic instability in OSF. Although this condition is known as a high-risk pre-cancerous condition, there is no data regarding the genomic status of this disease in terms of genetic susceptibility to malignant transformation. METHODS: In this study, we investigated the existence of genetic signatures for carcinogenesis in OSF. We employed the high-resolution genome-wide Affymetrix Mapping single nucleotide polymorphism microarray technique to 'fingerprint' global genomic instability in the form of LOH in 15 patient-matched OSF-blood genomic DNA samples. RESULTS: This rapid high-resolution mapping technique has revealed for the first time that a small number of discrete hot-spot LOH loci appeared in 47-53% of the OSF tissues studied. Many of these LOH loci were previously identified regions of genomic instability associated with carcinogenesis of the HNSCC. CONCLUSION: To our knowledge, this is the first evidence that genomic instability in the form of LOH is present in OSF. We hypothesize that the genomic instability detected in OSF may play an important role in malignant transformation. Further functional association studies on these putative genes may reveal potential predictive oral cancer markers for OSF patients.

siRNA-mediated AML1/MTG8 depletion affects differentiation and proliferation-associated gene expression in t(8;21)-positive cell lines and primary AML blasts.

The chromosomal translocation t(8;21) is associated with 10-15% of all cases of acute myeloid leukaemia (AML). The resultant fusion protein AML1/MTG8 interferes with haematopoietic gene expression and is an important regulator of leukaemogenesis. We studied the effects of small interfering RNA (siRNA)-mediated AML1/MTG8 depletion on global gene expression in t(8;21)-positive leukaemic cell lines and in primary AML blasts using cDNA arrays, oligonucleotide arrays and real-time reverse transcription-polymerase chain reaction (RT-PCR). Suppression of AML1/MTG8 results in the increased expression of genes associated with myeloid differentiation, such as AZU1, BPI, CTSG, LYZ and RNASE2 as well as of antiproliferative genes such as IGFBP7, MS4A3 and SLA both in blasts and in cell lines. Furthermore, expression levels of several genes affiliated with drug resistance or indicative of poor prognosis AML (BAALC, CD34, PRG2, TSPAN7) are affected by AML1/MTG8 depletion. In conclusion, siRNA-mediated suppression of AML1/MTG8 cause very similar changes in gene expression pattern in t(8;21)-positive cell lines and in primary AML blasts. Furthermore, the results suggest that the specific targeting of AML1/MTG8 function may be a promising approach for complementing existing treatment strategies.

Molecular characterisation of the t(1;15)(p22;q22) translocation in the prostate cancer cell line LNCaP.

Although chromosome translocations are well-documented recurrent events in hematological malignancies and soft tissue sarcomas, their significance in carcinomas is less clear. We report here the molecular characterization of the reciprocal translocation t(1;15)(p22;q22) in the prostate carcinoma cell line, LNCaP. The chromosome 1 breakpoint was localized to a single BAC clone, RP11-290M5, by sequential FISH analysis of clones selected from the NCBI chromosome 1 map. This was further refined to a 580-bp region by Southern blot analysis. A 2.85-kb fragment spanning the der(1) breakpoint was amplified by long-range inverse PCR. The breakpoint on chromosome 1 was shown to lie between the CYR61 and the DDAH1 genes with the der(1) junctional sequence linking the CYR61 gene to the TSPAN3 (TM4SF8) gene on chromosome 15. Confirmatory PCR and FISH mapping of the der(15) showed loss of chromosome material proximal to the breakpoint on chromosome 15, containing the PSTPIP1 and RCN2 genes. On the available evidence we conclude that this translocation does not result in an in-frame gene fusion. Comparative expressed sequence hybridization (CESH) and comparative genomic hybridization (CGH) analysis, showed relative down-regulation of gene expression surrounding the breakpoint, but no gross change in genomic copy number. Real-time quantitative RT-PCR for genes around the breakpoint supported the CESH data. Therefore, here we may have revealed a gene down-regulation mechanism associated with a chromosome translocation, either through small deletion at the breakpoint or through another means of chromosome domain related gene regulation.

Breakpoint heterogeneity in t(10;11) translocation in AML-M4/M5 resulting in fusion of AF10 and MLL is resolved by fluorescent in situ hybridization analysis.

Ten AML-M4/M5 patients' samples containing a t(10;11) translocation, but with different cytogenetic breakpoints on chromosome 11q (11q13-23), were studied by G- and R-banding and fluorescent in situ hybridization. Southern blotting analysis, studied in five patients, revealed a rearranged MLL gene. Reverse transcription-PCR analysis carried out in six patients showed a 5' MLL-3' AF-10 fusion transcript. Fluorescent in situ hybridization studies suggested that in 8 of 10 patients, the rearrangement/fusion transcript resulted from an inversion of a part of 11q (q13q23) translocated to 10p12. In the other two patients, it is assumed that an inversion/translocation has occurred of a part of 10p to the der(11). The results suggest that the orientation of the AF-10 gene on 10p is 5' telomeric and 3' centromeric. This is the first example of opposite-oriented genes being involved in translocation to yield fusion transcripts.

A new member of the proprotein convertase gene family (LPC) is located at a chromosome translocation breakpoint in lymphomas.

A new member of the proprotein convertase gene family (LPC) has been identified at a chromosome translocation breakpoint occurring in a high grade lymphoma. The translocation t(11;14)(q23;q32) has been molecularly cloned and shown to be the result of a fusion between an intron in the 3' -untranslated region of LPC with a sequence close to the switch region S gamma 4 of the IGH locus. The LPC gene encodes a protein of 785 amino acids with substantial homology to furin and the other members of the proprotein convertase family and represents a novel target for chromosome translocation and subsequent deregulation.

Normal c-abl gene protein--a nuclear component.

The subcellular distribution of the c-abl and bcr-abl gene products from KG1A and K562 cells has been studied by two different techniques. Firstly, physical disruption followed by subcellular fractionation was used to demonstrate that normal c-abl (p145) was recovered from the cytosol and the nuclear fractions of KG1A cells. In contrast, bcr-abl products were recovered exclusively from the cytosol fraction of K562 cells. Secondly, indirect immunofluorescence was used to localize c-abl protein to the cytoplasm, nuclear membrane and infrequently to the nucleus of KG1A cells and bcr-abl protein to only the cytoplasm of K562 cells. Thus both the approaches indicate that there is a component of normal c-abl products which appears to be nuclear and this is not reflected in the distribution of the bcr-abl 210 kDa protein, which remains cytosolic.

Molecular analysis of an unstable genomic region at chromosome band 11q23 reveals a disruption of the gene encoding the alpha2 subunit of platelet-activating factor acetylhydrolase (Pafah1a2) in human lymphoma.

A region of 150 kb has been analysed around a previously isolated, lymphoma associated, translocation breakpoint located at chromosome band 11q23. This balanced and reciprocal translocation, t(11;14)(q32;q23), has been shown to result in the fusion between chromosome 11 specific sequence and the switch gamma4 region of the IGH locus. The LPC gene, encoding a novel proprotein convertase belonging to the furin family, has been identified in this region. In order to characterize further the region surrounding the translocation, we have determined the detailed structure of LPC. Here we show that LPC consists of at least 16 exons covering 25 kb, and that there is a partial duplication, involving mobile genetic elements and containing LPC exons 13-17 in a tail-tail configuration at 65 kb downstream. Since the chromosomal breakpoint lay between these two structures, the intervening region was further analysed and shown to contain at least two unrelated genes. The previously known SM22 gene was localized close to the 3' tail of LPC. Furthermore, we identified the gene encoding the alpha2 subunit of platelet-activating factor acetylhydrolase (Pafah1a2) at the chromosomal breakpoint. The position of another previously identified breakpoint was also located to within the first intron of this gene. Altogether, our results give evidence of a genomic instability of this area of 11q23 and show that Pafah1a2 and not LPC is the gene disrupted by the translocation, suggesting that deregulated Pafah1a2 may have a role in lymphomagenesis.

Identification of two normal bcr gene products in the cytoplasm.

A monoclonal antibody (7C6) has been derived against a synthetic bcr peptide and used to study normal bcr gene products. The expression of a bcr phosphoprotein of 130 kd was demonstrated, in addition to the previously identified bcr phosphoprotein of 160 kd. Sequential immunoprecipitation demonstrated that both p160 and p130 had determinants from two separate regions of the putative bcr translated sequence. The synthesis of bcr products in Philadelphia positive and negative cells was examined by metabolic labelling and it was shown that the rate of synthesis of the p210 bcr-abl product was comparable with that of the normal bcr products. The in vivo phosphorylation of the p160 exceeded that of the p130 and both normal products were unaffected by the increased phosphorylation of the p210 bcr-abl. There was no evidence with the 7C6 antibody of any normal bcr products larger than 160 kilodaltons. Immunofluorescence analysis by conventional and confocal microscopy identified normal bcr products as cytoplasmic proteins with relatively high expression in the myeloid cell line KGl.

Bcl-2/JH rearrangements in benign lymphoid tissues with follicular hyperplasia.

The t(14; 18)(q32;q21) chromosomal translocation, characteristic of follicular lymphoma, couples the bcl-2 protooncogene on chromosome 18 to the immunoglobulin heavy-chain joining region (JH). This results in a deregulated transcription rate of bcl-2, suggesting a major role of the t(14;18) translocation in lymphomagenesis. By using a sensitive polymerase chain reaction technique specific for the major breakpoint region t(14;18), we now demonstrate the presence of bcl-2/JH rearrangements in lymph nodes and tonsils with follicular hyperplasia in 13 of 24 cases (54%). The approximate frequency was one translocation-positive cell in 10(5) cells. No bcl-2/JH rearrangements were detected in reactive lymph nodes without follicular hyperplasia or in bone marrow cells. Sequence analysis showed the amplified bcl-2/JH fragments to be unique to each individual sample and distinct from 24 sequenced follicular lymphoma-derived t(14;18) junctions, thus excluding contamination artifacts. The presence of random nucleotide insertions at the breakpoint junctions suggests a pre-B-cell origin of the t(14;18) translocation, in analogy with follicular lymphomas. We conclude that the t(14;18) translocation can occur in non-malignant tissue and will not, on its own, lead to malignancy.

Molecular cloning of a novel 11q23 breakpoint associated with non-Hodgkin's lymphoma.

Chromosomal analysis of a non-Hodgkin's lymphoma revealed a t(11;14)(q23;q32) translocation amongst other abnormalities. To investigate the molecular basis of this translocation, a cosmid library was constructed from the tumour DNA and the rearranged IGH locus was isolated in a single cosmid. Fluorescence in situ hybridization confirmed that the cloned region contained sequences from chromosome 11q23 fused to chromosome 14q32. Sequence analysis identified the breakpoint as a fusion between a region from the switch segment of the C gamma 4 gene of the IGH locus and an unknown sequence on chromosome 11. The chromosome 11 sequence maps proximal to the CD3 gene cluster and is therefore distinct from both the HTRX1 gene (rearranged in acute leukaemias) and the RCK gene (rearranged in a cell line derived from a histiocytic B-cell lymphoma). This newly identified region contains a cluster of rare cutting restriction enzyme sites located within 200 bases of the breakpoint, suggestive of a CpG island. Although this t(11;14)(q23;q32) translocation and that in the RC-K8 cell line affect different regions on chromosome 11, the breakpoints on chromosome 14 were found to have occurred at equivalent positions of S gamma 2 and S gamma 4 segments.

The synovial sarcoma associated protein SYT interacts with the acute leukemia associated protein AF10.

As a result of the synovial sarcoma associated t(X;18) translocation, the human SYT gene on chromosome 18 is fused to either the SSX1 or the SSX2 gene on the X chromosome. Although preliminary evidence indicates that the (fusion) proteins encoded by these genes may play a role in transcriptional regulation, little is known about their exact function. We set out to isolate interacting proteins through yeast two hybrid screening of a human cDNA library using SYT as a bait. Of the positive clones isolated, two were found to correspond to the acute leukemia t(10;11) associated AF10 gene, a fusion partner of MLL. Confirmation of these results was obtained via co-immunoprecipitation of endogenous and exogenous, epitope-tagged, SYT and AF10 proteins from cell line extracts and colocalization of epitope-tagged SYT and AF10 proteins in transfected cells. Subsequent sequential mutation analysis revealed a highly specific interaction of N-terminal SYT fragments with C-terminal AF10 fragments. The N-terminal interaction domain of the SYT protein was also found to be present in several SYT orthologs and homologs. The C-terminal interaction domain of AF10 is located outside known functional domains. Based on these results, a model is proposed in which the SYT and AF10 proteins act in concert as bipartite transcription factors. This model has implications for the molecular mechanisms underlying the development of both human synovial sarcomas and acute leukemias.

The cloning, mapping and expression of a novel gene, BRL, related to the AF10 leukaemia gene.

The MLL gene is reciprocally translocated with one of a number of different partner genes in a proportion of human acute leukaemias. The precise mechanism of oncogenic transformation is unclear since most of the partner genes encode unrelated proteins. However, two partner genes, AF10 and AF17 are related through the presence of a cysteine rich region and a leucine zipper. The identification of other proteins with these structures will aid our understanding of their role in normal and leukaemic cells. We report the cloning of a novel human gene (BRL) which encodes a protein containing a cysteine rich region related to that of AF10 and AF17 and is overall most closely related to the previously known protein BR140. BRL maps to chromosome 22q13 and shows high levels of expression in testis and several cell lines. The deduced protein sequence also contains a bromodomain, four potential LXXLL motifs and four predicted nuclear localization signals. A monoclonal antibody raised to a BRL peptide sequence confirmed its widespread expression as a 120 Kd protein and demonstrated localization to the nucleus within spermatocytes.

A new estimate of human ribosomal gene number.

Radioactively labelled DNAs (5 X 10(6) cpm/mug) complementary to human 18 S and 28 S ribosomal RNA were synthesized using RNA-directed DNA polymerase (EC 2.7.7.7). These complementary DNAs were used to measure human ribosomal gene numbers by two independent methods, both of which indicated numbers at least four-fold lower than those previously reported. First, the kinetics of the annealing of the complementary DNAs with total human placental DNA indicated that the number of both 18-S and 28-S ribosomal genes per haploid genome is approximately 50. Second, saturation experiments in which a constant amount of DNA was annealed with increasing amounts of complementary DNA also indicated that the number of 28 S ribosomal RNA genes in human placental and spleen DNA is is about 50 per haploid genome.