Evaluation of X-chromosome inactivation in a large cohort of females with X-linked retinal diseases

Abstract

Purpose : X-chromosome inactivation (XCI) is the stable, epigenetic transcriptional usually random silencing of an X-chromosome in female diploid cells. It has been proposed that the degree to which female carriers are affected may be dependent on skewed XCI in some X-linked diseases. Here, we used an XCI assay to evaluate a large cohort of female carriers with variants that typically cause X-linked inherited retinal diseases (IRDs) to investigate the role that XCI plays in the expression of these diseases.

Methods : The XCI assay was performed on 73 female carriers with pathogenic variants in CHM, CACNA1F, NYX, OPN1LW/OPN1MW, RS1, RP2, and RPGR. Review of clinical phenotype and family history was performed to determine affected status and disease severity. Blood-derived DNA samples were undigested or digested with HpaII and amplified in a multiplex reaction for the RP2 GAAA repeats (RP2), human AR-CAG repeats, and ZFX control. PCR products were analyzed by capillary electrophoresis and the peak size and area under the curve were measured. Human AR-CAG alleles that had ratios of 80% or more were considered to be skewed, and ratios higher than 90% were considered to be extremely skewed. RP2 alleles were evaluated in the case of uninformative human AR-CAG alleles.

Results : Skewing and extreme XCI skewing was observed in 26% (n=19/73) of this cohort, including 9.6% (n=7/73) with extreme skewing, and 2.7% (n=2/73) with an apparently silenced allele. Of those with skewed XCI ratios, 60% (n=6/10) were considered affected, and 77.8% (n=7/9) of those with extreme skewing were considered affected. Further investigation of the two individuals that displayed 100% XCI revealed that both patients had structural X chromosome abnormalities. One individual with a severe, early onset choroideremia presentation harbored an Xq22;10q21 balanced translocation with a breakpoint in intron 8 of CHM. Another individual with classical retinoschisis harbored a pathogenic RS1 variant and an Xp21.3 terminal deletion.

Conclusions : Skewed XCI was noted in a significant proportion of affected female individuals with pathogenic variants in X-linked IRD genes. Complete inactivation was associated with cytogenetic abnormalities. While the majority with skewed XCI exhibit retinal phenotypes, the predictive value of XCI is limited. Additional studies of gene-specific phenotype expression of X-linked IRDs in female carriers are needed.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.