RLIM-specific activity reporters define variant pathogenicity in Tonne-Kalscheuer syndrome.

Abstract

Tonne-Kalscheuer syndrome (TOKAS, OMIM # 300978) is an X-linked recessive disorder with devastating consequences for patients such as intellectual disability, developmental delay, and multiple congenital abnormalities. TOKAS is associated with hemizygous variants in the RLIM gene that encodes a RING-type E3 ubiquitin ligase. The current sustained increase in reported RLIM variants of uncertain significance creates an urgent need to develop assays that can screen these variants and experimentally determine their pathogenicity and disease association. Here, we engineered flow cytometry-based RLIM-specific reporters to measure RLIM activity in TOKAS. This paper describes the design and use of RLIM-specific reporters to determine the pathogenicity of a TOKAS RLIM gene variant. Our data demonstrates that RLIM-specific flow cytometry reporters based on either the full length or a degron region of the substrate REX1 measure RLIM activity in cells. Further, we describe the TOKAS variant RLIM p.Asn581Lys and using reporter assays, determine that it disrupts RLIM catalytic activity. This data reveals how the p.Asn581Lys variant impairs RLIM function and suggests pathogenic mechanisms. The use of RLIM-specific reporters will greatly accelerate the resolution of variants of uncertain significance and disease association in TOKAS.