RESUMO
The prefrontal cortex is believed to play a major role in depression and suicidal behavior through regulation of cognition, memory, recognition of emotion, and anxiety-like states, with numerous post-mortem studies documenting a prefrontal serotonergic dysregulation considered to be characteristic of depressive psychopathology. This study was carried out to detect changes in gene expression associated with both suicide and major depression using oligonucleotide microarrays (Affymetrix HG-U133 chip set) summarizing expression patterns in primarily ventral regions of the prefrontal cortex (BA44, 45, 46 and 47). A total of 37 male subjects were included in this study, of which 24 were suicides (depressed suicides=16, nondepressed suicides=8) and 13 were matched controls. All subjects were clinically characterized by means of psychological autopsies using structured interviews. Unique patterns of differential expression were validated in each of the cortical regions evaluated, with group-specific changes highlighting the involvement of several key neurobiological pathways that have been implicated in both suicide and depression. An overrepresentation of factors involved in cell cycle control and cell division (BA44), transcription (BA44 and 47) and myelination (BA46) was seen in gene ontology analysis of differentially expressed genes, which also highlights changes in the expression of genes involved in ATP biosynthesis and utilization across all areas. Gene misexpression in BA46 was most pronounced between the two suicide groups, with many significant genes involved in GABAergic neurotransmission. The pronounced misexpression of genes central to GABAergic signaling and astrocyte/oligodendrocyte function provides further support for a central glial pathology in depression and suicidal behavior.
Assuntos
Trifosfato de Adenosina/biossíntese , Transtorno Depressivo Maior , Regulação da Expressão Gênica/fisiologia , Córtex Pré-Frontal/metabolismo , Suicídio , Transmissão Sináptica/fisiologia , Ácido gama-Aminobutírico , Adolescente , Adulto , Análise de Variância , Estudos de Casos e Controles , Transtorno Depressivo Maior/patologia , Transtorno Depressivo Maior/fisiopatologia , Transtorno Depressivo Maior/psicologia , Perfilação da Expressão Gênica/métodos , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Reprodutibilidade dos Testes , Transtornos Relacionados ao Uso de Substâncias/genética , Adulto JovemRESUMO
Random amplification of polymorphic DNA (RAPD) is widely used to detect polymorphisms in many organisms. Individual (or strain) specific amplified bands are generated with single or pairs of primers in PCR reactions and can serve as genetic markers. We have used this method to generate a large number of reproducible bands with single primers, random and retroviral related, on 92 human DNA samples. Theoretically, RAPD PCR presents a logical approach for assessing variability among individuals. We used ten retroviral related primers (12, 20 and 22 bp) and eight random primers (10 bp) to assess individual differences in the context of testing the retroviral hypothesis for schizophrenia. Three pairs of discordant monozygotic twins, four pairs of discordant full sibs and 53 schizophrenic individuals with 25 of their unrelated matched controls were analyzed. Ten of these primers resulted in a total of approx. 850 amplified bands (65-110 bands per primer). Almost all of these bands were identical among each individual analyzed. However, the results are inconclusive with respect to the retroviral hypothesis for schizophrenia. The general lack of RAPD polymorphism in this study may argue for mechanisms other than rearrangements such as inversions, associated with the evolution of the human genome.
Assuntos
DNA Viral/genética , Reação em Cadeia da Polimerase/métodos , Esquizofrenia/genética , Primers do DNA , DNA Viral/análise , Evolução Molecular , Genoma Humano , Humanos , Polimorfismo Genético , Esquizofrenia/virologia , Moldes Genéticos , GêmeosRESUMO
Recently, unstable trinucleotide repeats have been shown to be the etiologic factor in seven neuropsychiatric diseases, and they may play a similar role in other genetic disorders which exhibit genetic anticipation. We have tested one polymerase chain reaction (PCR)-based and two hybridization-based methods for direct detection of unstable DNA expansion in genomic DNA. This technique employs a single primer (asymmetric) PCR using total genomic DNA as a template to efficiently screen for the presence of large trinucleotide repeat expansions. High-stringency Southern blot hybridization with a PCR-generated trinucleotide repeat probe allowed detection of the DNA fragment containing the expansion. Analysis of myotonic dystrophy patients containing different degrees of (CTG)n expansion demonstrated the identification of the site of trinucleotide instability in some affected individuals without any prior information regarding genetic map location. The same probe was used for fluorescent in situ hybridization and several regions of (CTG)n/(CAG)n repeats in the human genome were detected, including the myotonic dystrophy locus on chromosome 19q. Although limited at present to large trinucleotide repeat expansions, these strategies can be applied to directly clone genes involved in disorders caused by large expansions of unstable DNA.
Assuntos
Southern Blotting/métodos , Hibridização in Situ Fluorescente/métodos , Reação em Cadeia da Polimerase/métodos , Repetições de Trinucleotídeos , DNA/análise , DNA/genética , Humanos , Mapeamento por RestriçãoRESUMO
Almost 10% of the human genome consists of DNA sequences that share homology with retroviruses. These sequences, which represent a stable component of the human genome (although some may retain the ability to transpose), remain poorly understood. We used degenerate primers specific to the two conserved regions (boxes 4 and 5) of the retroviral pol gene, common to all retroviruses, and PCR-amplified related sequences from individuals representing two distinct populations: Caucasians and Dogrib Indians. The large number of sequences that are reproducibly amplified represent numerous sites of retroviral integration in the human genome. In both populations studied, one of the two primers yielded a polymorphic band, present in approximately 30% of the samples, that has probably been present in the human genome since before the divergence of the two populations approximately 10,000 years ago. It was established that this polymorphism was due to priming-site differences and not to deletions. Further, this priming site is duplicated at two genomic sites (representing 341- and 343-bp fragments) with at least two alleles each. Such novel polymorphisms should provide useful markers and permit assessment of evolutionary mechanisms associated with retroviral-related genomic evolution.
Assuntos
DNA/genética , Retrovirus Endógenos/genética , Polimorfismo Genético , Alelos , Sequência de Bases , Clonagem Molecular , Primers do DNA/genética , Evolução Molecular , Genes pol , Genética Populacional , Humanos , Indígenas Norte-Americanos/genética , Dados de Sequência Molecular , Territórios do Noroeste , Ontário , Reação em Cadeia da Polimerase , Homologia de Sequência do Ácido Nucleico , População Branca/genéticaRESUMO
Retroviral-related amplicons were used in modified RDA to identify four sequences from affected members of three pairs of monozygotic twins discordant for schizophrenia. One sequence (schizophrenia associated retrovirus, SZRV-1, GenBank Accession No. AF135487) is characterized here. It is similar to two known sequences of retroviral origin: multiple sclerosis-associated retrovirus, MSRV (GenBank Accession No. AF009668), and ERV-9 (GenBank Accession No. S77575). It is present in multiple copies in the human genome and has been localized to six different chromosomal sites. A zooblot shows that this multicopy sequence is predominant in the primate lineage and present in rhesus monkeys and humans. SZRV-1 is expressed as a 9-kb RNA band in the placenta. This could offer support to the hypothesis that retroviral sequences transposing during fetal growth may alter neurodevelopmental genes and cause diseases, although its direct involvement in the causation of schizophrenia remains to be established.
Assuntos
Esquizofrenia/genética , Gêmeos Monozigóticos/genética , Southern Blotting , Retrovirus Endógenos/genética , Humanos , Hibridização in Situ Fluorescente , Cariotipagem , Dados de Sequência Molecular , Reação em Cadeia da PolimeraseRESUMO
Evidence for genetic anticipation has recently become an important subject of research in clinical psychiatric genetics. Renewed interest in anticipation was evoked by molecular genetic findings of a novel type of mutation termed "unstable DNA." The unstable DNA model can be construed as the "best fit" for schizophrenia twin and family epidemiological data. We have performed a large-scale Southern blot hybridization, asymmetrical PCR-based, and repeat expansion-detection screening for (CAG)n/(CTG)n and (CCG)n/(CGG)n expansions in eastern Canadian schizophrenia multiplex families demonstrating genetic anticipation. There were no differences in (CAG)n/(CTG)n and (CCG)n/(CGG)n pattern distribution either between affected and unaffected individuals or across generations. Our findings do not support the hypothesis that large (CAG)n/(CTG)n or (CCG)n/(CGG)n expansions are the major etiologic factor in schizophrenia. A separate set of experiments directed to the analysis of small (30-130 trinucleotides), Huntington disease-type expansions in individual genes is required in order to fully exclude the presence of (CAG)n/(CTG)n- or (CCG)n/(CGG)n-type unstable mutation.