The X chromosome harbors an unusually high density of immune genes and is a major biological contributor to sex differences in immune function and autoimmune disease susceptibility. In females, X-chromosome inactivation (XCI) produces two principal functional consequences: modulation of X-linked gene dosage through XCI escape, and determination of cellular exposure to heterozygous X-linked variants through XCI skew. Because XCI generates a mosaic of cells expressing different parental X chromosomes, these properties have been difficult to quantify in individual women and are typically measurable only when XCI is non-random or after aggregation across large cohorts. This study addresses that gap by introducing a single-cell phasing approach that resolves chromosome-scale X haplotypes from transcriptome data, enabling patient-resolved measurement of escape, skew and variant exposure.
The authors present scDaisyChain, a graph-based computational framework that reconstructs chromosome-scale X haplotypes directly from heterozygous single-nucleotide polymorphisms (SNPs) observed in single-cell long-read transcriptomes. scDaisyChain links heterozygous SNPs across long reads to phase alleles at chromosome scale and thereby infers which parental X chromosome is expressed in individual cells. This approach permits direct measurement of lineage- and cell-resolved XCI escape and skew without relying on non-random XCI or large population aggregation.
According to the source, scDaisyChain achieves near-ground-truth accuracy in highly polymorphic mouse hybrids. In human samples, the framework shows strong concordance with orthogonal long-read whole-genome phasing. These validation results support scDaisyChain as a reliable method for reconstructing X haplotypes and inferring allele-specific expression at single-cell resolution as reported by the authors.
Applied to peripheral blood immune cells from healthy women, scDaisyChain revealed a lineage-specific escape program. Lymphoid cells were observed to escape XCI more broadly than monocytes. This broader escape in lymphoid lineages was associated with increased chromatin accessibility on the inactive X and with female-biased expression of escaped genes. In contrast, monocytes showed the lowest degree of XCI escape in health, indicating a cell-type-specific regulation of inactive X transcriptional permissiveness.
Beyond escape, the authors document lineage-specific XCI skew, meaning that within a lineage the proportion of cells expressing one parental X relative to the other can vary. They formalize the downstream consequence of skew as variant exposure: the proportion of cells in which a given heterozygous X-linked variant is expressed. Variant exposure therefore depends on both which genes escape XCI and the distribution of parental X expression across cells in a given lineage.
The study reports that predicted deleterious X-linked variants are preferentially found in low-exposure states. As an illustrative case, the authors identify a splice-altering variant in TLR8 that is expressed in relatively few cytotoxic T cells, suggesting limited cellular exposure to this deleterious allele in that lineage. This observation links single-cell-resolved allele expression patterns to potential modulation of variant penetrance across cell types.
In rheumatoid arthritis (RA) patients, the monocyte compartment—highlighted because it shows the lowest escape in healthy donors—exhibits reproducible inactive X dysregulation. This dysregulation converges on a trained-immunity program associated with disease flare and synovial macrophage activation. Within this context, the authors report elevated escape of specific X-linked genes, including IL13RA1 and HDAC8, in RA monocytes. These disease-associated changes underscore how lineage-specific XCI dynamics can be altered in autoimmune states and may intersect with disease-relevant immune programs.
The findings position lineage-specific escape, skew and variant exposure as quantifiable, patient-resolved determinants of sex-biased immune gene dosage and X-linked variant penetrance in both health and autoimmune disease. By enabling measurement of XCI properties at single-cell resolution within individual donors, scDaisyChain resolves a dimension of female biology that was previously inaccessible, with implications for understanding inter-individual variation in immune responses and the clinical impact of X-linked variation.
The source reports use of single-cell long-read transcriptomes and heterozygous SNP phasing to reconstruct X haplotypes with scDaisyChain. Validation included highly polymorphic mouse hybrids (near-ground-truth accuracy) and concordance with orthogonal long-read whole-genome phasing in human samples. Peripheral blood immune cells from healthy women and samples from rheumatoid arthritis patients were profiled to identify lineage-specific escape and disease-associated inactive X dysregulation, as described by the authors. The source lists funding and author affiliations but does not provide additional experimental protocol details, sequencing depths, cohort sizes, or statistical metrics in the provided text.
The source text does not report specific cohort sizes, sequencing depths, quantitative accuracy metrics, statistical significance values, or detailed computational parameters for scDaisyChain within the excerpt provided. Where such details are required for interpretation or replication, they were not reported in the provided source content and should be consulted in the full preprint.