RESUMO
Homozygosity mapping is a powerful procedure that is capable of detecting recessive disease-causing genes in a few patients from families with a history of inbreeding. We report here a homozygosity mapping algorithm for high-density single nucleotide polymorphism arrays that is able to (i) correct genotyping errors, (ii) search for autozygous segments genome-wide through regions with runs of homozygous SNPs, (iii) check the validity of the inbreeding history, and (iv) calculate the probability of the disease-causing gene being located in the regions identified. The genotyping error correction restored an average of 94.2% of the total length of all regions with run of homozygous SNPs, and 99.9% of the total length of them that were longer than 2 cM. At the end of the analysis, we would know the probability that regions identified contain a disease-causing gene, and we would be able to determine how much effort should be devoted to scrutinizing the regions. We confirmed the power of this algorithm using 6 patients with Siiyama-type α1-antitrypsin deficiency, a rare autosomal recessive disease in Japan. Our procedure will accelerate the identification of disease-causing genes using high-density SNP array data.
Assuntos
Algoritmos , Genoma/genética , Genômica/métodos , Homozigoto , Modelos Genéticos , Polimorfismo de Nucleotídeo Único/genética , Consanguinidade , Genes Recessivos/genética , Genótipo , Humanos , Japão , Reprodutibilidade dos Testes , Deficiência de alfa 1-Antitripsina/genéticaRESUMO
RATIONALE: Pulmonary alveolar microlithiasis is an autosomal recessive disorder in which microliths are formed in the alveolar space. OBJECTIVES: To identify the responsible gene that causes pulmonary alveolar microlithiasis. METHODS: By means of a genomewide single-nucleotide polymorphism analysis using DNA from three patients, we have narrowed the region in which the candidate gene is located. From this region, we have identified a gene that has mutations in all patients with pulmonary alveolar microlithiasis. MEASUREMENTS AND MAIN RESULTS: We identified a candidate gene, SLC34A2, that encodes a type IIb sodium phosphate cotransporter and that is mutated in six of six patients investigated. SLC34A2 is specifically expressed in type II alveolar cells, and the mutations abolished the normal gene function. CONCLUSION: Mutations in the SLC34A2 gene that abolish normal gene function cause pulmonary alveolar microlithiasis.
Assuntos
Litíase/genética , Litíase/patologia , Pneumopatias/genética , Pneumopatias/patologia , Alvéolos Pulmonares/patologia , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIb/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Feminino , Humanos , Imuno-Histoquímica , Hibridização In Situ , Masculino , Dados de Sequência Molecular , Mutação , Alvéolos Pulmonares/química , Alvéolos Pulmonares/diagnóstico por imagem , RNA Mensageiro/análise , Radiografia Torácica , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIb/análise , Xenopus laevisRESUMO
Lung cancer is one of the leading causes of the cancer death worldwide. Gefitinib is an inhibitor of the tyrosine kinase activity of the epidermal growth factor receptor (EGFR) and has been introduced in the treatment of advanced lung cancers. The responsiveness to gefitinib has been linked to the presence of EGFR mutations. Clinical samples contain many normal cells in addition to cancer cells. A method capable of detecting EGFR mutations in a large background of wild-type EGFR genes could provide a superior clinical test. We developed a rapid and sensitive detection system for EGFR mutations named the peptide nucleic acid-locked nucleic acid (PNA-LNA) PCR clamp that can detect EGFR mutations in the presence of 100-to 1,000-fold background of wild-type EGFR. We used this method to screen 30 non-small cell lung cancer cell lines established from Japanese patients. In addition to 11 cell lines that have mutations, we found 12 cell lines in which specific mutations are observed only in the subpopulation(s) of the cells. Genetic heterogeneity of EGFR suggests that the EGFR gene is unstable in established cancers and the heterogeneity may explain variable clinical responses of lung cancers to gefitinib.
