Measuring X inactivation skew for retinal diseases with adaptive nanopore sequencing

Abstract

X-linked genetic disorders typically affect females less severely than males due to the presence of a second X chromosome not carrying the deleterious variant. However, the phenotypic expression in females is highly variable, which may be explained by an allelic skew in X chromosome inactivation. Accurate measurement of X inactivation skew is crucial to understand and predict disease phenotype in carrier females, with prediction especially relevant for degenerative conditions. We propose a novel approach using nanopore sequencing to quantify skewed X inactivation accurately. By phasing sequence variants and methylation patterns, this single assay reveals the disease variant, X inactivation skew, its directionality, and is applicable to all patients and X-linked variants. Enrichment of X-chromosome reads through adaptive sampling enhances cost-efficiency. Our study includes a cohort of 16 X-linked variant carrier females affected by two X-linked inherited retinal diseases: choroideremia and RPGR-associated retinitis pigmentosa. As retinal DNA cannot be readily obtained, we instead determine the skew from peripheral samples (blood, saliva and buccal mucosa), and correlate it to phenotypic outcomes. This revealed a strong correlation between X inactivation skew and disease presentation, confirming the value in performing this assay and its potential as a way to prioritise patients for early intervention, such as gene therapy currently in clinical trials for these conditions. Our method of assessing skewed X inactivation is applicable to all long-read genomic datasets, providing insights into disease risk and severity and aiding in the development of individualised strategies for X-linked variant carrier females.

Retinal vascular reactivity in carriers of X-linked inherited retinal disease – a study using optical coherence tomography angiography

“…Female carriers of X-linked IRDs may present with a spectrum of retinal changes ranging from near normal retinae to severe retinal degeneration, the latter known as “male-pattern” degeneration ( Figure 1 ) ( 1 – 3 ). This variability in retinal phenotype has been found to be attributed to X-chromosome inactivation ( 4 ), involving the random inactivation of one of two X-chromosomes in females (XX individuals) during early embryonic development ( 5 ).…”