RESUMO
OBJECTIVE: To develop a novel prenatal assay based on selective analysis of cell-free DNA in maternal blood for evaluation of fetal Trisomy 21 (T21) and Trisomy 18 (T18). METHODS: Two hundred ninety-eight pregnancies, including 39 T21 and seven T18 confirmed fetal aneuploidies, were analyzed using a novel, highly multiplexed assay, termed digital analysis of selected regions (DANSR™). Cell-free DNA from maternal blood samples was analyzed using DANSR assays for loci on chromosomes 21 and 18. Products from 96 separate patients were pooled and sequenced together. A standard Z-test of chromosomal proportions was used to distinguish aneuploid samples from average-risk pregnancy samples. DANSR aneuploidy discrimination was evaluated at various sequence depths. RESULTS: At the lowest sequencing depth, corresponding to 204,000 sequencing counts per sample, average-risk cases where distinguished from T21 and T18 cases, with Z statistics for all cases exceeding 3.6. Increasing the sequencing depth to 410,000 counts per sample substantially improved separation of aneuploid and average-risk cases. A further increase to 620,000 counts per sample resulted in only marginal improvement. This depth of sequencing represents less than 5% of that required by massively parallel shotgun sequencing approaches. CONCLUSION: Digital analysis of selected regions enables highly accurate, cost efficient, and scalable noninvasive fetal aneuploidy assessment.
Assuntos
DNA/sangue , Síndrome de Down/diagnóstico , Complicações na Gravidez/diagnóstico , Gravidez/sangue , Diagnóstico Pré-Natal/métodos , Trissomia/diagnóstico , Adulto , Cromossomos Humanos Par 18/genética , Cromossomos Humanos Par 21/genética , Análise Custo-Benefício , Síndrome de Down/sangue , Síndrome de Down/genética , Feminino , Feto , Testes Genéticos/métodos , Humanos , Processamento de Imagem Assistida por Computador , Complicações na Gravidez/genética , Diagnóstico Pré-Natal/economia , Estudos Prospectivos , Reprodutibilidade dos Testes , Trissomia/genéticaRESUMO
Tight control of the number and distribution of crossovers is of great importance for meiosis. Crossovers establish chiasmata, which are physical connections between homologous chromosomes that provide the tension necessary to align chromosomes on the meiotic spindle. Understanding the mechanisms underlying crossover control has been hampered by the difficulty in determining crossover distributions. Here, we present a microarray-based method to analyze multiple aspects of crossover control simultaneously and rapidly, at high resolution, genome-wide, and on a cell-by-cell basis. Using this approach, we show that loss of interference in zip2 and zip4/spo22 mutants is accompanied by a reduction in crossover homeostasis, thus connecting these two levels of crossover control. We also provide evidence to suggest that repression of crossing over at telomeres and centromeres arises from different mechanisms. Lastly, we uncover a surprising role for the synaptonemal complex component Zip1 in repressing crossing over at the centromere.