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.
The non-native oxidation
of horse heart myoglobin with hydrogen
peroxide produces compound II which autoreduces by utilizing an internal
oxidation site. Here, we utilize full UV–visible time-dependent
kinetics with global kinetic singular value decomposition analysis
to explore the mechanism and uncover more detail about the high-valent
heme spectral features. By varying the hydrogen peroxide and myoglobin
concentration, we were able to uncover more detailed spectra of myoglobin
compound II and the autoreduction rate under several different pH
conditions. The compound II spectra demonstrate pH-dependent features
with an inflection point around pH 5.7 ± 0.1. The rate of autoreduction
of compound II,
k
2
, increases with lower
pH with a half-power proton dependence and no indication of a p
K
a
> 3.9 ± 0.2, indicating that the autoreduction
is still dependent on the protonation of the ferryl oxo species. The
k
2
also demonstrates both hydrogen peroxide and
myoglobin dependency. At myoglobin concentrations greater than 6.6
μM, the
k
2
is myoglobin-independent,
but for lower concentrations, a pH-sensitive concentration dependence
is seen.
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