NCM 2023 Singapore: Measuring skewed X inactivation by adaptive nanopore sequencing

X-linked genetic disorders tend to affect females less severely than males due to the presence of a second X chromosome that does not carry the deleterious variant. However, the phenotypic expression in females is very variable, which may be explained by which allele is silenced through the dosage compensation mechanism of X chromosome inactivation. Skewed X inactivation, where one allele is preferentially silenced over the other, can significantly impact the severity and penetrance of X-linked mutations in carrier females. When preventative treatment is available, accurate measurement of skewed X inactivation is crucial for predicting disease susceptibility in these individuals. We describe a novel approach using nanopore sequencing to accurately quantify skewed X inactivation in females. By phasing sequence variants and methylation patterns, this single assay detects the disease variant, the skew, and its directionality for any patient, contrary to previous methods that require trio information, polymorphism in certain repeats, and additional genetic testing. Our study includes a cohort of X-linked mutation carrier females affected by various diseases impacting the retina. As retinal DNA cannot be readily obtained, we instead determine the skew from three peripheral tissues (blood, saliva, and buccal swab), and correlated it with disease severity and phenotypic outcomes. By using adaptive sampling on the PromethION to enrich for X-chromosome reads, we could multiplex of samples and improve cost-efficiency. Our method of assessing skewed X inactivation is applicable to all nanopore genomic datasets, providing insights into disease risk and severity and aiding in the development of individualised strategies for X-linked mutation carrier females.