Assuntos
Doenças do Desenvolvimento Ósseo/diagnóstico , Calcinose/diagnóstico , Cromossomos Humanos Par 9 , Epífises/anormalidades , Trissomia/diagnóstico , Aborto Eugênico , Adulto , Doenças do Desenvolvimento Ósseo/complicações , Doenças do Desenvolvimento Ósseo/congênito , Doenças do Desenvolvimento Ósseo/genética , Calcinose/complicações , Calcinose/congênito , Calcinose/genética , Epífises/diagnóstico por imagem , Feminino , Humanos , Gravidez , Ultrassonografia Pré-NatalRESUMO
Prenatal diagnosis of fetal trisomy 21 is usually performed by cytogenetic analysis. This requires lengthy laboratory procedures, high costs and is unsuitable for large-scale screening of pregnant women. Today, trisomy 21 can be rapidly diagnosed within 24 h by molecular analysis of uncultured fetal cells using the semi-quantification of fluorescent PCR products from short tandem repeat (STR) polymorphic markers. The aim of our study was to test a chromosome quantification method on the basis of the analysis of fluorescent PCR products derived from non-polymorphic target genes. Co-amplification of a portion of DSCR1 (Down syndrome Critical Region 1) and the reference gene, CFTR (cystic fibrosis transmembrane regulator) enabled molecular detection of trisomy 21. Our method was successfully tested on a total of 154 amniotic fluids in a blind prospective study. Calculation of the DSCR1/CFTR ratio allowed us to distinguish between 152 normal amniotic fluids (mean ratio 0.99) and 2 amniotic fluids presenting a trisomy 21 status (DSCR1/CFTR ratio of 1.53 and 1.61, respectively). The results obtained by conventional cytogenetic analysis and our quantitative PCR method were concordant in every case. Our gene-based fluorescent PCR approach represents an alternative molecular method for rapid and reliable detection of trisomy 21, which can be helpful in the prenatal diagnosis of women at high risk of fetal trisomy 21.
Assuntos
Amniocentese , Líquido Amniótico/química , Síndrome de Down/diagnóstico , Programas de Rastreamento/métodos , Reação em Cadeia da Polimerase/métodos , Espectrometria de Fluorescência/métodos , Adulto , Líquido Amniótico/citologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Proteínas de Ligação a DNA , Síndrome de Down/genética , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Idade Materna , Repetições Minissatélites/genética , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Gravidez , Gravidez de Alto Risco , Estudos Prospectivos , Sequências Repetitivas de Ácido Nucleico , Reprodutibilidade dos Testes , Método Simples-CegoRESUMO
Prenatal diagnosis of chromosomal abnormalities by cytogenetic analysis is time consuming, expensive, and requires highly qualified technicians. Rapid diagnosis of aneuploidies followed by reassurance for women with normal results can be performed by molecular analysis of uncultured foetal cells in less than 24 h. Today, all molecular techniques developed for a fast diagnosis of aneuploidies rely on the semi-quantification of fluorescent PCR products from short tandem repeat (STR) polymorphic markers. Our objective was to test a chromosome quantification method based on the analysis of fluorescent PCR products derived from non-polymorphic target genes. An easy to set up co-amplification of portions of DSCR1 (Down Syndrome Critical Region 1), DCC (Deleted in Colorectal Carcinoma), and RB1 (Retinoblastoma 1) allowed the molecular detection of aneuploidies for chromosomes 21, 18 and 13 respectively. Quantitative analysis was performed in a blind prospective study of 400 amniotic fluids. Four samples (1%) could not be analysed by PCR probably because of a low concentration of foetal DNA. Follow up karyotype analysis was done on all samples and molecular results were in agreement with the cytogenetic data with no false-positive or false-negative results. Our gene based fluorescent PCR approach is an alternative molecular method for a rapid and reliable detection of aneuploidies which can be helpful for the clinical management of high-risk pregnancies.
