Classifying molecular phenotypes of G6PC variants for pathogenic properties and to guide therapeutic development.

Due to advances in sequencing technologies, identification of genetic variants is rapid. However, the functional consequences of most genomic variants remain unknown. Consequently, variants of uncertain significance (VUS) that appear in clinical DNA diagnostic reports lack sufficient data for interpretation. Algorithms exist to aid prediction of a variant's likelihood of pathogenicity, but these predictions usually lack empiric evidence. To examine the feasibility of generating functional evidence in vitro for a given variant's role in disease, a panel of 29 coding sequence variants in the G6PC gene was assessed. G6PC encodes glucose-6 phosphatase enzyme, and reduction in its function causes the rare metabolic disease glycogen storage disease type 1a (GSD1a). Variants were heterologously expressed as fusion proteins in a hepatocyte-derived cell line and examined for effects on steady-state protein levels, biosynthetic processing, and intracellular distribution. The screen revealed variant effects on protein levels, N-linked glycosylation status, and cellular distribution. Of the eight VUS tested, seven behaved similar to wild-type protein while one VUS, p.Cys109Tyr, exhibited features consistent with pathogenicity for all molecular phenotypes assayed, including significantly reduced protein levels, alteration in protein glycosylation status, and abnormally diffuse protein localization pattern, and has recently been reported in a patient with GSD1a. Our results show that such a screen can add empiric evidence to existing databases to aid in diagnostics, and also provides further classification for molecular phenotypes that could be used in future therapeutic screening approaches for small molecule or gene editing strategies directed at specific variants.

CLIC5 stabilizes membrane-actin filament linkages at the base of hair cell stereocilia in a molecular complex with radixin, taperin, and myosin VI.

Chloride intracellular channel 5 protein (CLIC5) was originally isolated from microvilli in complex with actin binding proteins including ezrin, a member of the Ezrin-Radixin-Moesin (ERM) family of membrane-cytoskeletal linkers. CLIC5 concentrates at the base of hair cell stereocilia and is required for normal hearing and balance in mice, but its functional significance is poorly understood. This study investigated the role of CLIC5 in postnatal development and maintenance of hair bundles. Confocal and scanning electron microscopy of CLIC5-deficient jitterbug (jbg) mice revealed progressive fusion of stereocilia as early as postnatal day 10. Radixin (RDX), protein tyrosine phosphatase receptor Q (PTPRQ), and taperin (TPRN), deafness-associated proteins that also concentrate at the base of stereocilia, were mislocalized in fused stereocilia of jbg mice. TPRQ and RDX were dispersed even prior to stereocilia fusion. Biochemical assays showed interaction of CLIC5 with ERM proteins, TPRN, and possibly myosin VI (MYO6). In addition, CLIC5 and RDX failed to localize normally in fused stereocilia of MYO6 mutant mice. Based on these findings, we propose a model in which these proteins work together as a complex to stabilize linkages between the plasma membrane and subjacent actin cytoskeleton at the base of stereocilia.