Characterization of human mosaic Rett syndrome brain tissue by single-nucleus RNA sequencing (Single-cell or single-nuclei RNA sequencing)

In females with X-linked genetic disorders, wild-type and mutant cells coexist within brain tissue because of random X-chromosome inactivation. This cellular mosaicism leads to phenotypic variability and poses significant challenges for interpreting the effects of X-linked mutant alleles on gene expression. We present a single-nucleus RNA sequencing approach that resolves mosaicism by using single nucleotide polymorphisms in genes that are expressed in cis with the X-linked mutation to determine whether individual nuclei express the wild-type or mutant allele even when the mutant gene is not directly detected. This approach enables genome-wide comparisons of gene expression between mutant and wild-type cells within the same individual and eliminates the variability introduced by comparisons to controls with different genetic backgrounds. We apply this approach to mosaic female mouse models and humans with Rett syndrome, an X-linked neurodevelopmental disorder caused by mutations in the methyl-DNA-binding protein MECP2, and reveal that cell-type-specific DNA methylation patterns largely predict the degree of gene upregulation by MECP2 in specific neuronal subtypes. The approach described here can be broadly applied to the characterization of gene expression in additional mosaic X-linked conditions. Single-cell or single-nuclei RNA sequencing was performed on male and female MeCP2 mutant mice or human occipital cortex from Rett syndrome brain donors.对于罹患X连锁遗传疾病的女性而言，由于随机X染色体失活（X-chromosome inactivation）现象，脑组织内会同时存在野生型（wild-type）与突变型（mutant）细胞。这种细胞镶嵌现象会引发表型异质性，同时为解析X连锁突变等位基因对基因表达的调控效应带来极大挑战。本研究提出一种单细胞核RNA测序（single-nucleus RNA sequencing）方法，可通过分析与X连锁突变位点处于顺式的基因内的单核苷酸多态性（single nucleotide polymorphisms），判断单个细胞核所表达的是野生型还是突变型等位基因，即便无法直接检测到突变基因。该方法可实现在同一受试者体内，对突变型与野生型细胞的基因表达进行全基因组水平比对，同时消除了因使用不同遗传背景的对照样本所引入的异质性偏差。我们将此方法应用于罹患瑞特综合征（Rett syndrome）的镶嵌型雌性小鼠模型与人类样本——瑞特综合征是一种由甲基DNA结合蛋白MECP2（methyl-DNA-binding protein MECP2）突变引发的X连锁神经发育疾病——并揭示：细胞类型特异性的DNA甲基化模式，可在很大程度上预测特定神经元亚型中MECP2介导的基因上调程度。本文所提出的方法可被广泛应用于其他X连锁镶嵌疾病的基因表达特征解析。研究对携带MeCP2突变的雌雄小鼠，以及瑞特综合征脑供体的人类枕叶皮层样本，开展了单细胞或单细胞核RNA测序实验。