Characterization of human mosaic Rett syndrome brain tissue by single-nucleus RNA sequencing (Total 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. Total RNA sequencing was performed on human occipital cortex from Rett syndrome brain donors