Pyrosequencing for detection of mutations in the connexin 26 (GJB2) and mitochondrial 12S RNA (MTRNR1) genes associated with hereditary hearing loss

Hereditary hearing loss (HHL) is one of the most common congenital disorders and is highly heterogeneous. Mutations in the connexin 26 (CX26) gene (GJB2) account for about 20% of all cases of childhood deafness, and approach 50% in documented recessive cases of non-syndromic hearing loss. In addition, a single mitochondrial DNA mutation, mt1555A>G, in the 12S rRNA gene (MTRNR1), is associated with familial cases of progressive deafness. Effective screening of populations for HHL necessitates rapid assessment of several of these potential mutation sites. Pyrosequencing links a DNA synthesis protocol for determining sequence to an enzyme cascade that generates light whenever pyrophosphate is released during primer strand elongation. We assessed the ability of Pyrosequencing to detect common mutations causing HHL. Detection of the most common CX26 mutations in individuals of Caucasian (35delG), Ashkenazi (167delT), and Asian (235delC, V37I) descent was confirmed by Pyrosequencing. A total of 41 different mutations in the CX26 gene and the mitochondrial mt1555A>G mutation were confirmed. Genotyping of up to six different adjacent mutations was achieved, including simultaneous detection of 35delG and 167delT. Accurate and reproducible results were achieved taking advantage of assay flexibility and experimental conditions easily optimized for a high degree of standardization and cost-effectiveness. The standardized sample preparation steps, including target amplification by PCR and preparation of single-stranded template combined with automated sequence reaction and automated genotype scoring, positions this approach as a potentially high throughput platform for SNP/mutation genotyping in a clinical laboratory setting. .     

Neuroprotective effects of IGF-I against TNFalpha-induced neuronal damage in HIV-associated dementia

Human immunodeficiency virus type 1 (HIV-1) infection often results in disorders of the central nervous system, including HIV-associated dementia (HAD). It is suspected that tumor necrosis factor-alpha (TNFalpha) released by activated and/or infected macrophages/microglia plays a role in the process of neuronal damage seen in AIDS patients. In light of earlier studies showing that the activation of the insulin-like growth factor I receptor (IGF-IR) exerts a strong neuroprotective effect, we investigated the ability of IGF-I to protect neuronal cells from HIV-infected macrophages. Our results demonstrate that the conditioned medium from HIV-1-infected macrophages, HIV/CM, causes loss of neuronal processes in differentiated PC12 and P19 neurons and that these neurodegenerative effects are associated with the presence of TNFalpha. Furthermore, we demonstrate that IGF-I rescues differentiated neurons from both HIV/CM and TNFalpha-induced damage and that IGF-I-mediated neuroprotection is strongly enhanced by overexpression of the wt IGF-IR cDNA and attenuated by the antisense IGF-IR cDNA. Finally, IGF-I-mediated antiapoptotic pathways are continuously functional in differentiated neurons exposed to HIV/CM and are likely supported by TNFalpha-mediated phosphorylation of I(kappa)B. All together these results suggest that the balance between TNFalpha and IGF-IR signaling pathways may control the extent of neuronal injury in this HIV-related experimental setting.     

Inhibition of HIV-1 expression by HIV-2

HIV-1 and HIV-2 are co-endemic in certain geographic areas. HIV-2 is more weakly pathogenic than HIV-1, and progression to AIDS occurs less frequently and over a longer period of time. Recent epidemiologic studies suggest that individuals infected with HIV-2 have a lower risk of HIV-1 infection. Both immune mechanisms and various modes of viral interference have been proposed to account for these results. Our findings, described in this paper, suggest that HIV-2 inhibits HIV-1 replication. To study the molecular interactions between HIV-1 and HIV-2, proviral clones were transfected alone or in combination into the human T cell line CEM. LTR-CAT indicator constructs were included for the purpose of monitoring viral promoter activity. Viral replication in transfected cells was monitored by p24 antigen capture assay of cell culture supernatants and Western blot analysis of cell extracts. HIV-2 inhibited HIV-1 replication as determined by intracellular and extracellular p24 antigen levels. Similar results were obtained with simultaneous virus infection using HIV-1 and HIV-2, rather than transfections of proviral DNA. Using cotransfection of HIV-1 and HIV-2 LTR indicator gene constructs, the mechanism of inhibition was found to be suppression of the HIV-1 LTR by HIV-2. The inhibitory effect of HIV-2 is not due to Tat-2, but appears to discriminate between the HIV-1 and HIV-2 LTRs based on differences in the Tat activation response element, TAR. These results suggest both a molecular mechanism for HIV-2 interference with HIV-1 replication and a potential molecular approach to therapy.     

Additional evidence that "plasmacytoid T-cell lymphoma" associated with chronic myeloproliferative disorders is of macrophage/monocyte origin

Plasmacytoid T-cell lymphoma (PTL) is a rare lymphoma with unique morphologic, immunologic, and clinical features. Thus far, only three cases have been reported, each terminating in myeloid leukemia. The macrophage/monocyte rather than T-cell origin of "plasmacytoid T-cells" in reactive lymph nodes has been suggested in the past, but there has been no extensive investigation to demonstrate whether the PTLs are also of this lineage. The authors now report on a patient with PTL who had a long history of clinically stable idiopathic myelofibrosis. Immunocytochemical staining of the neoplastic plasmacytoid cells, with a large panel of monoclonal antibodies used on fresh-frozen and paraffin-embedded tissue sections, showed that the neoplastic cells expressed several macrophage/monocyte-associated markers, i.e., CD31, CD36 (thrombospondin receptor), and CD68 (KP1). Other markers of the macrophage/monocyte lineage (e.g., CD11b, CD11c, CD16) were absent. The neoplastic cells lacked B-cell-associated antigens and lacked most T-cell-associated markers, with the exception of CD2 and CD4. These findings are in close agreement with those of previous studies on normal plasmacytoid T-cells and support the macrophage/monocytic origin of PTL. Molecular hybridization studies provided additional support for the nonlymphoid origin of the plasmacytoid cells by demonstrating the absence of T-cell-receptor beta-chain and immunoglobulin heavy-chain gene rearrangements in the neoplastic cells. The results of the authors' studies indicate that "plasmacytoid T-cell lymphoma" associated with a chronic myeloproliferative disorder is of macrophage/monocyte lineage.     

Schizophrenia and Evoked Potentials: Maximum Amplitude, Frequency of Peaks, Variability, and Phenothiazine Effects

The purpose of this study was to compare averaged visual evoked potentials AVEPs) in normal subjects and in schizophrenics off and on phenothiazine medication. Flashes of 4 intensities were used. Ss were tested 3 times within a 1 month period. Measures of maximum amplitude (Am), frequency of peaks I FOP), and variability (V) were obtained- With increases in stimulus intensity all Ss showed increases in Ams and decreases in FOPs and V. Schizophrenics had smaller Ams. greater FOPs, and grear V than normals. Schizoprenies on phenothiazines generally had less FOPs initially and after 1 wk mi medication but not after about I month on medication. There was no consistently significant effect of phenothiazines on maximum amplitude. NO drug effect on variability was observed. Schizophrenics showed a decrease in FOPs over time while normals showed an increase. A relationship was found between variability and overall thought disturbance. Changes in clinical condition over a month were not associated with discernible AVEP changes. Relationships between Am, FOP, unit V are presented.     

Peak Identification in Visual Evoked Potentials

Waveform patterns evoked by 4 intensities of flash in normal subjects were studied in relation to intersubject variability. Time-frequency distribution curves of all peaks occurring between 11 and 280 msec after flash onset and meeting minimal criteria were obtained from 46 males. These distributions closely corresponded to similar data reported by others for single intensity stimulation. An algorithm was developed which identified in 67 to 100% of instances a single “peak event’ within the time ranges of each of 6 peak distributions. Many peak events appeared and disappeared within the 4 intensity sets of individuals. Latencies were obtained for these peak events. Application of the algorithm to a replicate sample of 29 Ss, which included 8 females, indicated generalizability. Test-retest data on 15 Ss showed its reliability. The data suggest that methodology significantly contributes to the variability of peak identification among subjects. This may be reduced by employing multiple intensities of stimulation.