RESUMO
Several linkage studies provided evidence for the presence of the hereditary prostate cancer locus, HPCX1, at Xq27-q28. The strongest linkage peak of prostate cancer overlies a variable region of ~750 kb at Xq27 enriched by segmental duplications (SDs), suggesting that the predisposition to prostate cancer may be a genomic disorder caused by recombinational interaction between SDs. The large size of SDs and their sequence similarity make it difficult to examine this region for possible rearrangements using standard methods. To overcome this problem, direct isolation of a set of genomic segments by in vivo recombination in yeast (a TAR cloning technique) was used to perform a mutational analysis of the 750 kb region in X-linked families. We did not detect disease-specific rearrangements within this region. In addition, transcriptome and computational analyses were performed to search for nonannotated genes within the Xq27 region, which may be associated with genetic predisposition to prostate cancer. Two candidate genes were identified, one of which is a novel gene termed SPANXL that represents a highly diverged member of the SPANX gene family, and the previously described CDR1 gene that is expressed at a high level in both normal and malignant prostate cells, and mapped 210 kb of upstream the SPANX gene cluster. No disease-specific alterations were identified in these genes. Our results exclude the 750-kb genetically unstable region at Xq27 as a candidate locus for prostate malignancy. Adjacent regions appear to be the most likely candidates to identify the elusive HPCX1 locus.
Assuntos
Cromossomos Humanos X/genética , DNA de Neoplasias/genética , Loci Gênicos , Neoplasias da Próstata/genética , Autoantígenos/genética , Mapeamento Cromossômico , Cromossomos Humanos X/química , Análise Mutacional de DNA , Família , Feminino , Ligação Genética , Predisposição Genética para Doença , Humanos , Masculino , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Próstata/metabolismo , Próstata/patologia , Neoplasias da Próstata/diagnóstico , Recombinação Genética , Saccharomyces cerevisiae/genética , Duplicações Segmentares GenômicasRESUMO
BACKGROUND: Previous genetic linkage studies identified a locus for susceptibility to prostate cancer called HPCX at Xq27. The candidate region contains two clusters of SPANX genes. The first cluster called SPANX-A/D includes SPANX-A1, SPANX-A2, SPANX-B, SPANX-C, and SPANX-D; the second cluster called SPANX-N includes SPANX-N1, SPANX-N2, SPANX-N3, and SPANX-N4. The SPANX genes encode cancer-testis (CT) specific antigens. Previous studies identified SPANX-B and SPANX-D variants produced by gene conversion events, none of which are associated with X-linked prostate cancer. METHODS: In this study we applied transformation-associated recombination cloning (TAR) in yeast to analyze sequence variations in SPANX-A1, SPANX-A2, and SPANX-C genes that are resided within large chromosomal duplications. A SPANX-N1/N4 cluster was analyzed by a routine PCR analysis. RESULTS: None of the sequence variations in the coding regions of these genes is associated with susceptibility to prostate cancer. CONCLUSIONS: Therefore, genetic variation in the SPANX genes is not the actual target variants explaining HPCX. However, it is possible that they play a modifying role in susceptibility to prostate cancer through complex recombinational interaction.
Assuntos
Cromossomos Humanos X/genética , Proteínas Nucleares/genética , Neoplasias da Próstata/genética , Análise Mutacional de DNA , DNA de Neoplasias/genética , Feminino , Predisposição Genética para Doença , Variação Genética , Humanos , Masculino , Mutação Puntual , Reação em Cadeia da Polimerase , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
Genetic linkage studies indicate that germline variations in a gene or genes on chromosome Xq27-28 are implicated in prostate carcinogenesis. The linkage peak of prostate cancer overlies a region of approximately 750 kb containing five SPANX genes (SPANX-A1, -A2, -B, -C, and -D) encoding sperm proteins associated with the nucleus; their expression was also detected in a variety of cancers. SPANX genes are >95% identical and reside within large segmental duplications (SDs) with a high level of similarity, which confounds mutational analysis of this gene family by routine PCR methods. In this work, we applied transformation-associated recombination cloning (TAR) in yeast to characterize individual SPANX genes from prostate cancer patients showing linkage to Xq27-28 and unaffected controls. Analysis of genomic TAR clones revealed a dynamic nature of the replicated region of linkage. Both frequent gene deletion/duplication and homology-based sequence transfer events were identified within the region and were presumably caused by recombinational interactions between SDs harboring the SPANX genes. These interactions contribute to diversity of the SPANX coding regions in humans. We speculate that the predisposition to prostate cancer in X-linked families is an example of a genomic disease caused by a specific architecture of the SPANX gene cluster.
Assuntos
Mapeamento Cromossômico , Cromossomos Humanos X/genética , Duplicação Gênica , Ligação Genética/genética , Predisposição Genética para Doença , Proteínas Nucleares/genética , Neoplasias da Próstata/genética , Sequência de Bases , Clonagem Molecular , Componentes do Gene , Genômica/métodos , Humanos , Masculino , Dados de Sequência Molecular , Recombinação Genética/genética , Análise de Sequência de DNA , LevedurasRESUMO
Human sperm protein associated with the nucleus on the X chromosome (SPANX) genes comprise a gene family with five known members (SPANX-A1, -A2, -B, -C, and -D), encoding cancer/testis-specific antigens that are potential targets for cancer immunotherapy. These highly similar paralogous genes cluster on the X chromosome at Xq27. We isolated and sequenced primate genomic clones homologous to human SPANX. Analysis of these clones and search of the human genome sequence revealed an uncharacterized group of genes, SPANX-N, which are present in all primates as well as in mouse and rat. In humans, four SPANX-N genes comprise a series of tandem duplicates at Xq27; a fifth member of this subfamily is located at Xp11. Similarly to SPANX-A/D, human SPANX-N genes are expressed in normal testis and some melanoma cell lines; testis-specific expression of SPANX is also conserved in mouse. Analysis of the taxonomic distribution of the long and short forms of the intron indicates that SPANX-N is the ancestral form, from which the SPANX-A/D subfamily evolved in the common ancestor of the hominoid lineage. Strikingly, the coding sequences of the SPANX genes evolved much faster than the intron and the 5' untranslated region. There is a strong correlation between the rates of evolution of synonymous and nonsynonymous codon positions, both of which are accelerated 2-fold or more compared to the noncoding sequences. Thus, evolution of the SPANX family appears to have involved positive selection that affected not only the protein sequence but also the synonymous sites in the coding sequence.
Assuntos
Amplificação de Genes , Hominidae/genética , Proteínas de Neoplasias/genética , Neoplasias Testiculares/genética , Cromossomo X/genética , Sequência de Aminoácidos , Animais , Antígenos de Neoplasias/genética , Mapeamento Cromossômico , Sequência Conservada , Primers do DNA , Evolução Molecular , Éxons , Gorilla gorilla/genética , Humanos , Macaca mulatta/genética , Masculino , Dados de Sequência Molecular , Pongo pygmaeus/genética , Isoformas de Proteínas/genética , Roedores , Saguinus/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Testículo/fisiologiaRESUMO
The reported draft human genome sequence includes many contigs that are separated by gaps of unknown sequence. These gaps may be due to chromosomal regions that are not present in the Escherichia coli libraries used for DNA sequencing because they cannot be cloned efficiently, if at all, in bacteria. Using a yeast artificial chromosome (YAC)/ bacterial artificial chromosome (BAC) library generated in yeast, we found that approximately 6% of human DNA sequences tested transformed E. coli cells less efficiently than yeast cells, and were less stable in E. coli than in yeast. When the ends of several YAC/BAC isolates cloned in yeast were sequenced and compared with the reported draft sequence, major inconsistencies were found with the sequences of those YAC/BAC isolates that transformed E. coli cells inefficiently. Two human genomic fragments were re-isolated from human DNA by transformation-associated recombination (TAR) cloning. Re-sequencing of these regions showed that the errors in the draft are the results of both missassembly and loss of specific DNA sequences during cloning in E. coli. These results show that TAR cloning might be a valuable method that could be widely used during the final stages of the Human Genome Project.