Quantitative differentiation of benign and misfolded glaucoma-causing myocilin variants on the basis of protein thermal stability

Abstract

Accurate predictions of pathogenicity for mutations associated with genetic disease are key to the success of precision medicine. Inherited, missense coding mutations in the myocilin gene (MYOC), within its olfactomedin (OLF) domain, comprise the strongest genetic link to primary open angle glaucoma via a toxic gain of function, and MYOC is an attractive precision medicine target. However, not all mutations in MYOC cause glaucoma, and common variants are expected to be neutral polymorphisms. The gnomAD database lists ∼100 missense variants documented within OLF, all of which are relatively rare (allele frequency <0.001%) and nearly all are of unknown pathogenicity. To disambiguate disease from benign OLF variants, we first characterized the most prevalent population-based variants using a suite of cellular and biophysical assays, and identify two variants with features of aggregation-prone familial disease variants. Next, we consider all available biochemical and clinical data to demonstrate that pathogenic and benign variants can be differentiated statistically based on a single metric, thermal stability of OLF. Our results motivate genotyping MYOC in patients for clinical monitoring of this widespread, painless, and irreversible age-onset disease.

Competition between inside-out unfolding and pathogenic aggregation in an amyloid-forming β-propeller

“…At room temperature, OLF WT incubated in 3 M urea, where tertiary structure is no longer detected by near-UV circular dichroism (CD), can be refolded upon dilution to buffer lacking urea (Fig. 2c ) 20 . By contrast, when OLF WT incubated in 5 M urea is diluted, significant precipitation is visible.…”

“…1 ): P1 (G 326 AVVYSGSLYFQ) located in the two innermost strands of blade B and P3 (V 426 ANAFIICGTLYTVSSY) comprising the two innermost strands of blade D. A third stretch, P2 (G 387 LWVIYSTDEAKGAIVLSK) within blade C, was predicted to form amyloid but remained soluble when tested experimentally 19 . Pathogenic mutations can be defined biophysically as those with thermal stability (T m )≤ 47 °C for their OLF domains 20 , 5 °C lower than WT 21 . This decreased stability facilitates amyloid formation at 37 °C and in neutral pH buffers 3 , 19 .…”

“…This decreased stability facilitates amyloid formation at 37 °C and in neutral pH buffers 3 , 19 . In the context of full-length myocilin in a cellular environment, disease variants form amyloid in the endoplasmic reticulum (ER) 20 . The ER Hsp90 paralog Grp94 coaggregates with mutant myocilin, obstructing ER-associated degradation 22 .…”