RESUMO
Genotyping of single nucleotide polymorphisms (SNPs) in large populations presents a great challenge, especially if the SNPs are embedded in GC-rich regions, such as the codon 112 SNP in the human apolipoprotein E (apoE). In the present study, we have used immobilized locked nucleic acid (LNA) capture probes combined with LNA-enhancer oligonucleotides to obtain efficient and specific interrogation of SNPs in the apoE codons 112 and 158, respectively. The results demonstrate the usefulness of LNA oligonucleotide capture probes combined with LNA enhancers in mismatch discrimination. The assay was applied to a panel of patient samples with simultaneous genotyping of the patients by DNA sequencing. The apoE genotyping assays for the codons 112 and 158 SNPs resulted in unambiguous results for all patient samples, concurring with those obtained by DNA sequencing.
Assuntos
Apolipoproteínas E/genética , Sondas de Oligonucleotídeos/genética , Polimorfismo de Nucleotídeo Único/genética , Sequência de Bases , DNA Complementar/genética , Genótipo , Humanos , Dados de Sequência Molecular , Desnaturação de Ácido Nucleico , Hibridização de Ácido Nucleico/métodos , Sondas de Oligonucleotídeos/química , Fotoquímica , Sensibilidade e EspecificidadeRESUMO
Colonoscopy is recommended for persons with a familial risk of colorectal cancer (CRC). A familial risk is identified by a family history with CRC and/or predisposing mutation(s). However, such information may not be available. We analysed whether MSI (MicroSatellite Instability) and/or CIN (Chromosome INstability=LOH (loss of heterozygosity) and/or DNA-aneuploidy (abnormal nuclear DNA contents)) could be used as predictors of familial CRC. Formalin-fixed tissue from 97 patients with CRC (29 patients with 2 or more affected first-degree relatives (="cases"), 29 matched CRC controls without a family history, and 39 relatives to cases) were analysed for MSI and CIN. In this small case-control study, no significant differences in the frequencies of MSI and CIN were observed between cases with a family history and their controls without a family history. MSI+;CIN- was observed in 6/29 cases and in 0/29 controls (p=0.02), most frequently in cases with affected siblings, only (3/7). However, for 13 patients from whom several CRC tumours were analysed, concordant results for MSI/LOH/DNA-ploidy were obtained only in 10/9/9. Among cases and relative(s), concordant results for MSI, LOH and DNA-ploidy were obtained in 16/26, 16/26, and 14/25 families, respectively.Although MSI+;CIN- appeared to predict familial CRC with a high specificity, neither MSI, CIN, or MSI+;CIN- are likely to be sufficiently sensitive predictors of familial CRC.