Neuron-specific expression of a synaptotagmin gene in the sea urchin Strongylocentrotus purpuratus

Interest in chordate evolution has emphasized a need for a better understanding of the comparative neuroanatomy of invertebrate deuterostomes. However, molecular and genetic approaches to neurobiological studies in these groups are hampered by a lack of neuron-specific molecular markers. A monoclonal antibody, 1E11, is neuron specific and is useful in identification of neural structures in larvae and adults of echinoderms, hemichordates, and urochordates. To identify a neuron-specific gene product, we have characterized the antigen recognized by 1E11. In immunoblots and immunoprecipitations of neural tissue from adult Strongylocentrotus purpuratus, 1E11 recognizes a 57-kDa band. Tandem mass spectrometry of trypsin digests of the 57-kDa band permitted peptide mass mapping and sequencing of five peptides. All of the sequenced peptides, and 12 additional mass-mapped peptides, are found within the open reading frame of a cDNA encoding synaptotagmin B (Sp-SynB). In situ RNA hybridizations with synaptotagmin B probes with S. purpuratus larvae reveal a pattern of expression that is similar to that revealed by the antibody 1E11. Antibodies produced against a bacterially expressed Sp-SynB protein recognize a 57-kDa protein and colocalize with 1E11. When a full-length Sp-SynB cDNA is expressed in chicken embryonic cells, the cells become immunoreactive to 1E11. We conclude that synaptotagmin B is a gene expressed in neurons that has conserved epitopes in other invertebrate deuterostomes.     

Immunoglobulin gene rearrangement analysis in cerebrospinal fluid of patients with lymphoproliferative processes

OBJECTIVE: To determine the sensitivity and specificity of clonal immunoglobulin heavy chain gene rearrangement (IGHR) analysis in the distinction of benign and malignant lymphoproliferative diseases. METHODS: A retrospective analysis was conducted of patients in whom a malignant lymphoproliferative process was suspected. Cells of CSF samples were collected by centrifugation, resuspended in 100 microl of the supernatant and boiled. A 10 microl aliquot of this lysate served as template for semi-nested polymerase chain reaction using variable and joining region consensus primers. PCR products were analyzed by polyacrylamide gel electrophoresis. Cytopathological diagnosis and flow cytometry results were recorded. Sensitivity and specificity of IGHR analysis, cytopathology and flow cytometry were calculated. RESULTS: Eleven patients (12 specimens) had involvement of leptomeninges at the time of lumbar puncture. Another 25 cases (27 specimens) had normal CSF findings or were diagnosed with benign lymphoproliferative conditions. Sensitivity of CSF cytopathology, flow cytometry and IGHR analysis were 0.27 [95% confidence interval 0.06, 0.61], 0.1 [0.003, 0.45] and 0.58 [0.28, 0.85]. Specificity was 1 [0.86, 1], 0.95 [0.77, 1.0] and 0.85 [0.66, 0.96]. INTERPRETATION: IGHR analysis appears to be a useful addition to morphological and flow cytometry analysis of cerebrospinal fluid in the evaluation of CNS lymphoproliferative processes.     

Genetic background of type I protein C deficiency in Finland

In contrast to other populations the usually rare type II form of protein C deficiency is as common in Finland as type I deficiency. We recently reported that a single mutation explained virtually all cases of type II protein C deficiency in Finland, indicating strong founder effect. We now investigated in the same population the genetic background of type I protein C deficiency. Thirty-eight apparently unrelated families were studied. They represent the vast majority of all families with type I deficiency in Finland. A genetic defect was identified in 23 (61%) families who carried 13 different mutations. Only three of the 13 mutations have been reported in other populations. Unlike in type II deficiency, considerable heterogeneity in mutations was found in type I deficiency. Our results indicate interesting differences in mutational histories of these two different forms of protein C deficiency in Finland.     

The detection of large deletions or duplications in genomic DNA

While methods for the detection of point mutations and small insertions or deletions in genomic DNA are well established, the detection of larger (>100 bp) genomic duplications or deletions can be more difficult. Most mutation scanning methods use PCR as a first step, but the subsequent analyses are usually qualitative rather than quantitative. Gene dosage methods based on PCR need to be quantitative (i.e., they should report molar quantities of starting material) or semi-quantitative (i.e., they should report gene dosage relative to an internal standard). Without some sort of quantitation, heterozygous deletions and duplications may be overlooked and therefore be under-ascertained. Gene dosage methods provide the additional benefit of reporting allele drop-out in the PCR. This could impact on SNP surveys, where large-scale genotyping may miss null alleles. Here we review recent developments in techniques for the detection of this type of mutation and compare their relative strengths and weaknesses. We emphasize that comprehensive mutation analysis should include scanning for large insertions and deletions and duplications.     

Prevalent SLC26A4 mutations in patients with enlarged vestibular aqueduct and/or Mondini dysplasia: a unique spectrum of mutations in Taiwan, including a frequent founder mutation

OBJECTIVES/HYPOTHESIS: The purpose of the study is to elucidate the mutation spectrum of SLC26A4 among patients with enlarged vestibular aqueduct and/or Mondini dysplasia in Taiwan and to explore the origin of the most common mutation, IVS7-2A>G. The correlation between the genotypes and the phenotypes is also investigated, with special emphasis placed on comparison between the genotypes and hearing levels. STUDY DESIGN: A 3-year prospective clinical genetic study at a tertiary care university hospital. METHOD: Mutations on SLC26A4 were screened in 38 families that fulfilled the criteria of enrollment, and single nucleotide polymorphisms (SNPs) in the vicinity of IVS7-2A>G were typed. The presence of goiter, radiologic findings, and audiologic results of the probands were then compared according to the genotypes. RESULTS: A total of eight mutations were detected in 33 families, and IVS7-2A>G accounted for 84% (48/57) of the mutated alleles. SNP analysis confirmed the founder effect of IVS7-2A>G. Meanwhile, no obvious correlation was observed between SLC26A4 genotypes and phenotypes. CONCLUSION: The present study disclosed the unique SLC26A4 mutation spectrum in Taiwan, confirmed that IVS7-2A>G arose from a common ancestor, and demonstrated the lack of correlation between genotypes and phenotypes. High prevalence of certain SLC26A4 mutations in East Asians, as revealed here and previously, might largely facilitate mutation screening and genetic counseling in these areas.     

The gene expression profile of extraskeletal myxoid chondrosarcoma

Extraskeletal myxoid chondrosarcoma (EMC) is a soft tissue tumour that occurs primarily in the extremities and is characterized by a balanced translocation most commonly involving t(9;22) (q22;q12). The morphological spectrum of EMC is broad and thus a diagnosis based on histology alone can be difficult. Currently, no systemic therapy exists that improves survival in patients with EMC. In the present study, gene expression profiling has been performed to discover new diagnostic markers and potential therapeutic targets for this tumour type. Global gene expression profiling of ten EMCs and 26 other sarcomas using 42,000 spot cDNA microarrays revealed that the cases of EMC were closely related to each other and distinct from the other tumours profiled. Significance analysis of microarrays (SAM) identified 86 genes that distinguished EMC from the other sarcomas with 0.25% likelihood of false significance. NMB, DKK1, DNER, CLCN3, and DEF6 were the top five genes in this analysis. In situ hybridization for NMB gene expression on tissue microarrays (TMAs) containing a total of 1164 specimens representing 62 different sarcoma types and 15 different carcinoma types showed that NMB was highly expressed in 17 of 22 EMC cases and very rarely expressed in other tumours and thus could function as a novel diagnostic marker. High levels of expression of PPARG and the gene encoding its interacting protein, PPARGC1A, in most EMCs suggest activation of lipid metabolism pathways in this tumour. Small molecule inhibitors for PPARG exist and PPARG could be a potential therapeutic target for EMC.