RÉSUMÉ
Recentes relatos vêm demonstrando a ocorrência de um número crescente de transcritos antisenso naturais (NATs) no genoma humano e a ocorrência de expressão alélica diferencial (ADE) em genes autossômicos não submetidos a imprinting. Devido aos diversos mecanismos pelos quais podem afetar a expressão gênica, alterações na transcrição dos NATs podem estar envolvidas no desenvolvimento de patologias, como o câncer... Neste trabalho apresentamos duas estratégias inéditas para identificar em larga escala novos transcritos antisenso e genes que apresentam expresssão alélica diferencial... A metodologia de GLGI-MPSS foi aplicada em 96 dessas 4.308 tags, permitindo a sua extensão em um fragmento maior de cDNA correspondente a extremidade 3 do transcrito... Dessa maneira, foi possível inferir a expressão de cada um dos alelos de um gene a partir da freqüência das tags alelo específicas representadas nas diferentes bibliotecas de SAGE. Assim, de um total de 20.034 genes, 1.372 (6,85%) apresentaram tags alelo específicas e, de acordo com o padrão de expressão dessas tags, esses genes foram classificados em 3 categorias principais: a) 554 genes (40,4%) foram classificados com expressão alélica diferencial; b) 440 genes (20,8%) foram classificados com expressão monoalélica, dos quais 285 estavam representados em mais de 10 bibliotecas de SAGE; e por fim, b) 378 genes (32,0%) foram classificados com expressão bialélica. Nossos dados sugerem que pelo menos 60,0% (554+285/1.372) dos genes humanos apresentam expressão alélica diferencial... Em conjunto, nossos resultados demonstraram que a estratégia computacional utilizando os dados de MPSS foi eficaz para a identificação de novos transcritos antisenso no genoma humano e, ainda, que tags alelo específicas de SAGE podem ser eficientemente utilizadas na identificação de genes humanos que apresentam expressão alélica diferencial.
Recent reports have demonstrated the occurrence of an increasing number of natural antisense transcripts (NATs) in the human genome and the occurrence of allelic differential expression (ADE) in non-imprinted autosomal genes. Due to the diverse mechanisms by which NATs can affect gene expression, their abnormal expression may be involved in the development of pathological states, such as cancer. Similarly, genes displaying ADE have been associated with phenotypic variability and may also contribute to the development of complex genetic diseases. In this work, we present two unpublished strategies to high-throughput identification of new NATs and genes displaying ADE. The first strategy was based on the use of computational tools for the identification of MPSS tags that mapped on the opposite strand of known human genes represented by mRNA sequences, and for which the MPSS tag represents the only evidence of the existence of the NAT. Thus, from a total of 340,829 unique and distinct MPSS tags present in 41 MPSS libraries, 4,308 tags indicated the existence of a new NAT. The GLGI-MPSS methodology was applied for 96 out of these 4,308 tags, allowing their extension into a longer cDNA fragment corresponding to the 3' end of the transcript. The alignment of these fragments against the human genome sequence using BLAT, confirmed that 46/96 GLGI-MPSS fragments corresponded to 3' especific extensions with antisense orientation. Interestingly, we observed that 41.3% (19/46) of these GLGI-MPSS presented at their 3' end a poly(A) tail aligned to the human genome sequence. We demonstrated that a fraction of these transcripts are artifacts generated by internal priming in contaminating DNA and that another fraction of these transcripts are real and could be attributed to retroposition events in the human genome. The expression of the remaining 25/27 GLGI-MPSS fragments was evaluated by strand-specific RT-PCR, and the existence of 17/25 was confirmed by this methodology. The second strategy was based on the use of computational tools that allowed the integration of data from expressed sequences (mRNA and SAGE) and polymorphisms (SNPs) to the human genome sequence for the creation of a database containing allele-specific SAGE tags. In this way, it was possible to infer the expression of each allele of a gene from the frequency of each allelespecific tag represented in different SAGE libraries. So, from a total of 20,034 genes, 1,295 (6.46%) genes presented allele-specific SAGE tags and according to their expression pattern genes were classified into 3 major categories: a) 481 (37.2%) genes were classified with allelic differential expression; b) 442 (34.1%) genes were classified with monoallelic expression, of which 242 were represented in more than 10 SAGE libraries; and c) 372 (28.7%) genes were classified with bialleic expression. Twenty genes were chosen for experimental validation by gDNA and cDNA direct sequencing, of which 13 presented more than 5 individual heterozygotes. From these 13 genes, 10 (77%) demonstrated ADE in at least 20% of the heterozygotes evaluated. Interestingly, for PHC1 we observed monoallelic expression in all heterozygotes. Taking into account our experimental validation efficiency (77%), our analysis suggests that at least 43% of all human genes (481+242 x 0.77/1,295) display ADE. Taken together, our results demonstrated that the computational strategy using MPSS data was effective in the identification of new NATs in the human genome and that allele-specific SAGE tags can be efficiently used to expedite the identification of human genes displaying ADE.