RESUMO
PURPOSE: Posterior polymorphous corneal dystrophy (PPCD) is an autosomal dominant disorder, affecting both the corneal endothelium and Descemet's membrane. In the Czech Republic, PPCD is one of the most prevalent corneal dystrophies. The purpose of this study was to determine the chromosomal locus of PPCD in two large Czech families, by using linkage analysis. METHODS: Linkage analysis was performed on 52 members of two Czech families with PPCD and polymorphic microsatellite markers and lod scores were calculated. The candidate gene VSX1 was also screened for mutations. RESULTS: Significant lod scores were obtained with microsatellite markers on chromosome 20. Linkage analysis delineated the Czech PPCD locus to a 2.7-cM locus on chromosome 20, region p11.2, between flanking markers D20S48 and D20S139, which excluded VSX1 as the disease-causing gene in both families. In addition, the exclusion of VSX1 was confirmed by sequence analysis. CONCLUSIONS: This study reports the localization of PPCD in patients of Czech origin to chromosome 20 at p11.2. Linkage data and sequence analysis exclude VSX1 as causative of PPCD in two Czech families. This refined locus for PPCD overlaps the congenital hereditary endothelial dystrophy (CHED1) disease interval, and it is possible that these corneal dystrophies are allelic.
Assuntos
Mapeamento Cromossômico , Cromossomos Humanos Par 20/genética , Distrofias Hereditárias da Córnea/genética , Lâmina Limitante Posterior/patologia , Endotélio Corneano/patologia , Proteínas do Olho/genética , Proteínas de Homeodomínio/genética , Distrofias Hereditárias da Córnea/epidemiologia , Distrofias Hereditárias da Córnea/patologia , República Tcheca/epidemiologia , Feminino , Marcadores Genéticos , Humanos , Escore Lod , Masculino , Linhagem , Locos de Características QuantitativasRESUMO
BACKGROUND: Despite the continuous production of genome sequence for a number of organisms, reliable, comprehensive, and cost effective gene prediction remains problematic. This is particularly true for genomes for which there is not a large collection of known gene sequences, such as the recently published chicken genome. We used the chicken sequence to test comparative and homology-based gene-finding methods followed by experimental validation as an effective genome annotation method. RESULTS: We performed experimental evaluation by RT-PCR of three different computational gene finders, Ensembl, SGP2 and TWINSCAN, applied to the chicken genome. A Venn diagram was computed and each component of it was evaluated. The results showed that de novo comparative methods can identify up to about 700 chicken genes with no previous evidence of expression, and can correctly extend about 40% of homology-based predictions at the 5' end. CONCLUSIONS: De novo comparative gene prediction followed by experimental verification is effective at enhancing the annotation of the newly sequenced genomes provided by standard homology-based methods.
Assuntos
Biologia Computacional/métodos , Genoma , Animais , Galinhas , Mapeamento Cromossômico , Primers do DNA , DNA Complementar/metabolismo , Interpretação Estatística de Dados , Bases de Dados Genéticas , Éxons , Etiquetas de Sequências Expressas , Perfilação da Expressão Gênica , Íntrons , Modelos Estatísticos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Análise de Sequência de DNA , Software , Distribuição TecidualRESUMO
We have developed a systematic approach to generating cDNA clones containing full-length open reading frames (ORFs), exploiting knowledge of gene structure from genomic sequence. Each ORF was amplified by PCR from a pool of primary cDNAs, cloned and confirmed by sequencing. We obtained clones representing 70% of genes on human chromosome 22, whereas searching available cDNA clone collections found at best 48% from a single collection and 60% for all collections combined.
Assuntos
Clonagem Molecular/métodos , Genoma Humano , Genômica/métodos , Fases de Leitura Aberta/genética , Proteoma/genética , Cromossomos Humanos Par 22/genética , Biologia Computacional , DNA Complementar/genética , Bases de Dados Genéticas , Humanos , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único/genética , Projetos de Pesquisa , Análise de Sequência de DNARESUMO
We report a second-generation gene annotation of human chromosome 22. Using expressed sequence databases, comparative sequence analysis, and experimental verification, we have extended genes, fused previously fragmented structures, and identified new genes. The total length in exons of annotation was increased by 74% over our previously published annotation and includes 546 protein-coding genes and 234 pseudogenes. Thirty-two potential protein-coding annotations are partial copies of other genes, and may represent duplications on an evolutionary path to change or loss of function. We also identified 31 non-protein-coding transcripts, including 16 possible antisense RNAs. By extrapolation, we estimate the human genome contains 29,000-36,000 protein-coding genes, 21,300 pseudogenes, and 1500 antisense RNAs. We suggest that our revised annotation criteria provide a paradigm for future annotation of the human genome.
