Expression signature as a biomarker for prenatal diagnosis of trisomy 21. Homo sapiens

To date, a universal biomarker panel with a potential to predict high-risk pregnancies or adverse pregnancy outcome does not exist. Transcriptomic approach, a measure of gene expression levels across the genome, is a powerful tool to capture differentially expressed genes (DEG) in various conditions, including human trisomy of chromosome 21 (Ts21). DEG can be used to design a biomarker set as a diagnostic-predictive tool for various conditions of heterogeneous aetiology in a prenatal setting. In the search of novel biomarker set to predict high-risk pregnancies, we performed global expression profiling to find DEG in Ts21 used as a model. Subsequently we performed targeted validation and diagnostic performance evaluation on a larger group of case and control samples. Initially, transcriptomic profiles of 10 cultivated amniocyte samples with Ts21 and 9 with normal euploid constitution were determined using Agilent 4x44K expression microarrays. DEG were discovered using linear regression modelling implemented in limma package. Datasets from Ts21 transcriptomic studies available at GEO repository were incorporated to select our preliminary top DEG. Subsequently, selected top DEG were validated using RT-PCR quantification on independent sample of 16 cases with Ts21 and 32 controls, as well as new datasets from previously performed expression studies in Ts21. The classification was performed using support vector machine classification kernel and evaluated using leave-one-out cross validation approach. Overall design: Transcriptomic profiles of 10 cultivated amniocyte samples with Ts21 and 9 with normal euploid constitution were determined using Agilent 4x44K expression microarrays.

迄今为止，尚未存在可用于预测高危妊娠或不良妊娠结局的通用生物标志物组（biomarker panel）。转录组学方法（transcriptomic approach）是一种检测全基因组基因表达水平的手段，可有效捕获多种条件下的差异表达基因（differentially expressed genes, DEG），包括人类21号染色体三体（trisomy of chromosome 21, Ts21）。差异表达基因可用于构建生物标志物集，作为产前场景下针对多种异质性病因疾病的诊断预测工具。

为筛选可预测高危妊娠的新型生物标志物集，本研究以21号染色体三体为模型，开展全基因组表达谱分析以筛选差异表达基因。随后，针对更大规模的病例与对照样本开展靶向验证及诊断性能评估。

初始阶段，本研究使用安捷伦4x44K表达芯片（Agilent 4x44K expression microarrays）对10例培养的21号染色体三体羊膜细胞样本及9例染色体核型正常的羊膜细胞样本进行转录组谱分析。差异表达基因通过limma软件包（limma package）中的线性回归模型进行筛选。我们纳入了GEO数据库（GEO repository）内已公开的21号染色体三体转录组学研究数据集，以筛选初步的核心差异表达基因。随后，采用实时定量聚合酶链反应（RT-PCR）对独立样本集（16例21号染色体三体病例及32例对照样本）以及此前已发表的21号染色体三体表达谱研究新数据集，对筛选得到的核心差异表达基因进行验证。本研究使用支持向量机（support vector machine）分类核函数开展分类建模，并采用留一法交叉验证（leave-one-out cross validation）进行模型性能评估。

实验整体设计：使用安捷伦4x44K表达芯片对10例培养的21号染色体三体羊膜细胞样本及9例染色体核型正常的羊膜细胞样本进行转录组谱分析。