Abstract：
The majority of genes in the human genome is present in two copies but the expression levels of both alleles is not equal. Allelic imbalance is an aspect of gene expression relevant not only in the context of genetic variation, but also to understand the pathophysiology of genes implicated in genetic disorders, in particular, dominant genetic diseases where patients possess one normal and one mutant allele. Polyglutamine (polyQ) diseases are caused by the expansion of CAG trinucleotide tracts within specific genes. Spinocerebellar ataxia type 3 (SCA3) and Huntington\'s disease (HD) patients harbor one normal and one mutant allele that differ in the length of CAG tracts. However, assessing the expression level of individual alleles is challenging due to the presence of abundant CAG repeats in the human transcriptome, which make difficult the design of allele-specific methods, as well as of therapeutic strategies to selectively engage CAG sequences in mutant transcripts.
To precisely quantify expression in an allele-specific manner, we used SNP variants that are linked to either normal or CAG expanded alleles of the ataxin-3 (ATXN3) and huntingtin (HTT) genes in selected patient-derived cell lines. We applied a SNP-based quantitative droplet digital PCR (ddPCR) protocol for precise determination of the levels of transcripts in cellular and mouse models. For HD, we showed that the process of cell differentiation can affect the ratio between endogenous alleles of HTT mRNA. Additionally, we reported changes in the absolute number of the ATXN3 and HTT transcripts per cell during neuronal differentiation. We also implemented our assay to reliably monitor, in an allele-specific manner, the silencing efficiency of mRNA-targeting therapeutic approaches for HD. Finally, using the humanized Hu128/21 HD mouse model, we showed that the ratio of normal and mutant HTT transgene expression in brain slightly changes with the age of mice.
Using allele-specific ddPCR assays, we observed differences in allele expression levels in the context of SCA3 and HD. Our allele-selective approach is a reliable and quantitative method to analyze low abundant transcripts and is performed with high accuracy and reproducibility. Therefore, the use of this approach can significantly improve understanding of allele-related mechanisms, e.g., related with mRNA processing that may be affected in polyQ diseases.
To precisely quantify expression in an allele-specific manner, we used SNP variants that are linked to either normal or CAG expanded alleles of the ataxin-3 (ATXN3) and huntingtin (HTT) genes in selected patient-derived cell lines. We applied a SNP-based quantitative droplet digital PCR (ddPCR) protocol for precise determination of the levels of transcripts in cellular and mouse models. For HD, we showed that the process of cell differentiation can affect the ratio between endogenous alleles of HTT mRNA. Additionally, we reported changes in the absolute number of the ATXN3 and HTT transcripts per cell during neuronal differentiation. We also implemented our assay to reliably monitor, in an allele-specific manner, the silencing efficiency of mRNA-targeting therapeutic approaches for HD. Finally, using the humanized Hu128/21 HD mouse model, we showed that the ratio of normal and mutant HTT transgene expression in brain slightly changes with the age of mice.
Using allele-specific ddPCR assays, we observed differences in allele expression levels in the context of SCA3 and HD. Our allele-selective approach is a reliable and quantitative method to analyze low abundant transcripts and is performed with high accuracy and reproducibility. Therefore, the use of this approach can significantly improve understanding of allele-related mechanisms, e.g., related with mRNA processing that may be affected in polyQ diseases.
摘要：
背景：人类基因组中的大多数基因以两个拷贝存在，但两个等位基因的表达水平不相等。等位基因失衡是基因表达的一个方面，不仅在遗传变异的背景下，还要了解与遗传疾病有关的基因的病理生理学，特别是,显性遗传疾病，患者具有一个正常和一个突变等位基因。聚谷氨酰胺（polyQ）疾病是由特定基因中CAG三核苷酸片段的扩展引起的。脊髓小脑性共济失调3型（SCA3）和亨廷顿氏病（HD）患者具有一个正常和一个突变等位基因，其CAG束的长度不同。然而,由于人类转录组中存在丰富的CAG重复序列，因此评估单个等位基因的表达水平具有挑战性，这使得等位基因特异性方法的设计变得困难，以及将CAG序列选择性地结合在突变转录物中的治疗策略。
结果：为了以等位基因特异性方式精确量化表达，我们在选定的患者来源细胞系中使用了与ataxin-3(ATXN3)和亨廷顿蛋白(HTT)基因的正常或CAG扩增等位基因相关的SNP变异体.我们应用了基于SNP的定量液滴数字PCR（ddPCR）方案，用于精确确定细胞和小鼠模型中转录本的水平。对于HD，我们表明，细胞分化的过程可以影响HTTmRNA的内源性等位基因之间的比例。此外,我们报道了神经元分化过程中每个细胞ATXN3和HTT转录本的绝对数量的变化.我们还实施了我们的检测来可靠地监测，以等位基因特异性的方式，mRNA靶向治疗HD的沉默效率。最后,使用人性化的Hu128/21HD小鼠模型，我们发现，正常和突变的HTT转基因在大脑中的表达比例随小鼠的年龄略有变化。
结论：使用等位基因特异性ddPCR检测，我们观察到SCA3和HD的等位基因表达水平存在差异.我们的等位基因选择性方法是分析低丰度转录本的可靠和定量方法，并且具有高准确性和可重复性。因此,使用这种方法可以显着提高对等位基因相关机制的理解，例如，与mRNA加工有关，可能在polyQ疾病中受到影响。
结果：为了以等位基因特异性方式精确量化表达，我们在选定的患者来源细胞系中使用了与ataxin-3(ATXN3)和亨廷顿蛋白(HTT)基因的正常或CAG扩增等位基因相关的SNP变异体.我们应用了基于SNP的定量液滴数字PCR（ddPCR）方案，用于精确确定细胞和小鼠模型中转录本的水平。对于HD，我们表明，细胞分化的过程可以影响HTTmRNA的内源性等位基因之间的比例。此外,我们报道了神经元分化过程中每个细胞ATXN3和HTT转录本的绝对数量的变化.我们还实施了我们的检测来可靠地监测，以等位基因特异性的方式，mRNA靶向治疗HD的沉默效率。最后,使用人性化的Hu128/21HD小鼠模型，我们发现，正常和突变的HTT转基因在大脑中的表达比例随小鼠的年龄略有变化。
结论：使用等位基因特异性ddPCR检测，我们观察到SCA3和HD的等位基因表达水平存在差异.我们的等位基因选择性方法是分析低丰度转录本的可靠和定量方法，并且具有高准确性和可重复性。因此,使用这种方法可以显着提高对等位基因相关机制的理解，例如，与mRNA加工有关，可能在polyQ疾病中受到影响。
