Common and rare myocilin variants: Predicting glaucoma pathogenicity based on genetics, clinical and laboratory misfolding data

Abstract

Rare variants of the olfactomedin domain of myocilin are considered causative for inherited, early-onset open angle glaucoma, with a misfolding toxic gain of function pathogenic mechanism detailed by 20 years of laboratory research. Myocilin variants are documented in the scientific literature and identified through large-scale genetic sequencing projects such as those curated in the Genome Aggregation Database (gnomAD). In the absence of key clinical and laboratory information, however, the pathogenicity of any given variant is not clear, because glaucoma is a heterogeneous and prevalent age-onset disease, and common variants are likely benign. In this review, we reevaluate the likelihood of pathogenicity for the ~100 non-synonymous missense, insertion-deletion, and premature termination myocilin olfactomedin variants documented in the literature. We integrate available clinical, laboratory cellular, biochemical and biophysical data, the olfactomedin domain structure, and population genetics data from gnomAD. Of the variants inspected, ~50% can be binned based on a preponderance of data, leaving many of uncertain pathogenicity that motivate additional studies. Ultimately, the approach of combining metrics from different disciplines will likely resolve outstanding complexities regarding the role of this misfolding-prone protein within the context of a multifactorial and prevalent ocular disease, and pave the way for new precision medicine therapeutics.

Graphical Abstract

Introduction

For numerous disorders, genetics studies of pedigrees have identified rare causal gene variants, and subsequent laboratory studies have contributed key knowledge of biological function, regulation, and pathogenic mechanisms used to develop new therapeutics (Chong et al., 2015). We focus here on the inherited subtype of the ocular disease glaucoma. On the whole, glaucoma is a heterogeneous age-onset neurodegenerative ocular disorder affecting ~60 million individuals, and a leading cause of irreversible blindness world-wide. Regardless of underlying etiology, in the clinic, glaucoma is characterized by optic disc cupping and decreased visual field (Weinreb, Aung, & Medeiros, 2014; Youngblood, Hauser, & Liu, 2019). Vision loss is also usually preceded by an increase in intraocular pressure (IOP), a causal yet painless risk factor. Eye pressure is maintained in anatomical region of the eye called the trabecular meshwork (TM) (Bill, 1975; Gasiorowski & Russell, 2009; Rohen, Lutjen-Drecoll, Flugel, Meyer, & Grierson, 1993). Glaucoma progression is slowed by reducing IOP through pharmacological agents and surgical techniques, but eventual optic nerve damage, retinal ganglion cell death, and vision loss, are inevitable (Kass et al., 2002).

Since 1997 (Stone et al., 1997), ophthalmologists have identified families with early onset glaucoma that segregate with rare heterozygous nonsynonymous coding mutations in the myocilin gene, MYOC (RefSeq transcript NM_000261.2, https://www.ncbi.nlm.nih.gov/nuccore/NM_000261, 17,321 bp). Despite extensive glaucoma genetics studies for over 20 years (Fingert, Stone, Sheffield, & Alward, 2002; Liu & Allingham, 2017; Tamm, 2002), these rare MYOC variants remain the strongest genetic link to glaucoma, accounting for 1-4% of cases (Fingert, 2011; Gong, Kosoko-Lasaki, Haynatzki, & Wilson, 2004; Resch & Fautsch, 2009) of the most common glaucoma subtype primary open-angle glaucoma (POAG) (Tham et al., 2014; Wiggs & Pasquale, 2017; Youngblood et al., 2019), and 4-30% of juvenile-onset open angle glaucoma (JOAG) cases (Resch & Fautsch, 2009). Available data converge on the pathogenic mechanism by which myocilin variants acquire a toxic gain of function (GOF), which hastens the onset of glaucoma (see below). It is not yet known to what extent understanding myocilin-associated glaucoma pathogenesis might inform sporadic cases of glaucoma, but given precedents from other diseases (Blauwendraat, Nalls, & Singleton, 2020; Kathiresan & Srivastava, 2012; Mathis, Goizet, Soulages, Vallat, & Masson, 2019) and the fact that mutant myocilin dysfunction is localized to the TM tissue whose dysfunction is associated with most forms of glaucoma (Rohen et al., 1993), it likely that at least some molecular insights will be transferrable and lead to new therapeutic directions.

The MYOC gene encodes a ~55 kDa polypeptide chain composed of three exons that encode a modular protein with two multimerization domains, namely, a coiled-coil (Gobeil, Letartre, & Raymond, 2006) and a specialized coiled-coil called a leucine-zipper (Kubota et al., 1997; Resch & Fautsch, 2009) plus a 31 kDa olfactomedin (OLF) domain (Fig. 1A). OLF houses over 90% of reported mutations (Fingert et al., 2002; Hewitt, Mackey, & Craig, 2008; Resch & Fautsch, 2009), which are distributed throughout the sequence and structure, and is the focus of the variants reviewed here. Our working model of the supramolecular structure based on biophysical experiments and structural data (Donegan et al., 2015; Hill et al., 2017) involves the coiled-coil forming a tetramer, which diverges into two pairs of leucine-zipper dimers that in turn connect to two pairs of OLF domains by a ~60 residue, yet-uncharacterized linker (Fig. 1B). Myocilin is expressed in the TM, other eye tissues, and other neuromuscular and skeletal tissues (Kulkarni, Karavanich, Atchley, & Anholt, 2000), and is known to be upregulated upon steroid treatment (Polansky et al., 1997). No bona fide interacting partners or particular function has been established for wild-type (WT) myocilin.

Figure 1. Myocilin gene structure and supramolecular arrangement.

(A) Modular gene structure showing signal peptide (SP), coiled coil (CC), leucine zipper (LZ), linker regions, and OLF domain. Five Cys residues participate in intermolecular disulfide bonds. Cys47 and Cys61 within CC stabilize the tetrameric by forming disulfide bonds with corresponding residues on other protomers, as observed biochemically; Cys185 in LZ stabilizes a dimer with another protomer, observed crystallographically; and Cys245 is disulfide bonded to Cys433 within the OLF domain, also observed crystallographically. (B) Supramolecular arrangement based on biochemical and biophysical studies. The N-terminal CC domain is a tetramer, which separates into two LZ dimers, which connect to the OLF propellers via a predicted unstructured linker.

Large-scale genotyping datasets have identified new variants of Mendelian disease-associated genes, including MYOC. The lack of clinical data on individuals and their relatives to establish a pattern of heritability for all known gene variants of myocilin confounds the interpretation of myocilin variations and their relationship to glaucoma, however. Most of the disease-causing MYOC alleles are very rare and are therefore not necessarily represented in databases such as the Genome Aggregation Database (gnomAD; https://gnomAD.broadinstitute.org/) (Karczewski et al., 2020). For myocilin variants described in both gnomAD and in clinical literature, significant ambiguity arises regarding the contribution of the mutation to glaucoma.

Here we review approximately 100 missense, insertion-deletion (indel), and premature termination variants of myocilin with literature precedent and sort them into for likelihood of pathogenicity (Table 1, Table 2, Supp. Table S1, Supp. Table S2). At the time of the writing of this review, gnomAD lists 155 variants in the myocilin olfactomedin (OLF) domain including 114 (Supp. Table S3) of unknown pathogenicity that are not described in the clinical literature. Compared to prior review articles (Cheng et al., 2012; Fingert et al., 2002; Gong et al., 2004; H. Wang et al., 2019) and databases (Hewitt et al., 2008; Rangachari et al., 2019) that focus on clinical or biochemical aspects myocilin variants of myocilin, we have taken a holistic approach: we consider available clinical metrics, related statistical analyses, as well as biological, cellular and biochemical behavior, and atomic-detail inferences from the OLF structure. We present the challenges in differentiating glaucoma variants from non-disease variants in this multifactorial disease and suggest paths forward to resolve ambiguities.

Table 1.

Categories of and Criteria for Myocilin (Refseq NM_000261.2) Variants

Category	Supporting information			List of variants
	Clinical data	Laboratory data	Structure
Benign	High prevalence in gnomAD (allele count >50, allele frequency >2e-4). Ample clinical information, laboratory data, and insights from structure.			p.Thr293Lys, p.Val329Met, p.Glu352Lys, p.Thr353Ile, p.Lys398Arg, p.Ala445Val, p.Lys500Arg
Pathogenic	Early onset, familial POAG or JOAG. Literature reports often include IOP values and cup-disc ratios.	Support for a misfolding phenotype includes extent of secretion, solubility of intracellular aggregates in detergent, and/or thermal destabilization of isolated OLF domain variant	Intuition based on effects of mutation on local structure suggests perturbation leading to destabilization and aggregation	p.Cys245Tyr, p.Gly246Arg, p.Gly252Arg, p.Arg272Gly, p.Glu323Lys, p.Gly364Val, p.Gly367Arg, p.Gly367_Gln368delinsVal, p.Pro370Leu, p.Thr377Met, p.Thr377Lys, p.Asp380Ala, p.Lys423Glu, p.Val426Phe, p.Cys433Arg, p.Tyr437His, p.Ile477Asn, p.Ile477Ser, p.Tyr479His, p.Asn480Lys, p.Pro481Leu, p.Pro481Thr, p.Ile499Phe, p.Ile499Ser, p.Ser502Pro
Likely Pathogenic	Supports early onset, familial POAG or JOAG, not necessarily including explicit age of diagnosis, IOP values, and/or cup-disc ratios.	Incomplete or missing support for a misfolding phenotype	Intuition based on effects of mutation on local structure suggests perturbation leading to destabilization and aggregation	p.Val251Ala, p.Pro254Arg, p.Leu255Pro, p.Pro274Arg, p.Gln337Arg, p.Ser341Pro, p.Ala363Thr, p.Phe369Leu, p.Tyr371Asp, p.Thr377Arg, p.Asp380His, p.Asp384Gly, p.Glu385Lys, p.Thr438Ile, p.Asn450Tyr, p.Leu486Phe
Uncertain Significance– Lean Pathogenic	POAG association, but not necessarily Mendelian inheritance patterns and/or early onset diagnosis	May be absent	Intuition based on effects of mutation on local structure suggests perturbation leading to destabilization and aggregation	p.Gly244Val, p.Thr285Met, p.Trp286Arg, p.Glu300Lys, p.Gly326Ser, p.Leu334Pro, p.Ile360Asn, p.Asp378Gly, p.Asp384Asn, p.Gly387Asp, p.Ser393Arg, p.Asp395_Glu396insAspPro, p.Glu396DUP, Gly399Asp, p.Gly399Val, p.Ser425Pro, p.Phe430Leu, p.Thr448Pro, p.Arg470Cys
Uncertain Significance—Lean Benign	Identified in a control group or glaucoma onset significantly later than age 40	Where present, do not indicate a strong misfolding phenotype	Intuition based on effects of mutation on local structure suggests these are conservative substitutions unlikely to appreciably affect OLF structure and stability	p.Thr256Met, p.Glu261Lys, p.Ile288Met, p.Thr290Ala, p.Arg296Cys, p.Gln297His, p.Leu303Ile, p.Ser313Phe, p.Ser331Thr, p.Ser333Cys, p.Arg342Lys, p.Ile345Met, p.Pro361Ser, p.Gly374Val, p.Val402Ile, p.Glu414Lys, p.Thr419Ala, p.Arg422Cys, p.Ala427Thr, p.Asn428Ser, p.Gly434Ser, p.Asp446Tyr, p.Ala447Thr, p.Ile465Met, p.Tyr471Cys, p.Tyr473Cys, p.Met476Arg, p.Val495Ile
Uncertain Significance—Premature termination	Identified in control group or glaucoma onset significantly later than age 40	Where present, misfolding phenotype	OLF domain is not fully translated so it cannot adopt a native structure	p.Gln337Rfs*9, p.Gly362Efs*45, p.Gln368*, p.Trp373*, p.Tyr453Mfs*11, p.Glu483*, p.Trp489Cfs*10

Table 2.

Summary of data for mOLF-resident myocilin (Refseq NM_000261.2) variants predicted to be benign, pathogenic, or likely pathogenic.

A. Benign
Variant	Clinical Data	Cell Assaysa	Thermal Stability	Structure Analysis	Additional Variants	Other
p.Thr293Lys c.878C>A	Diagnosis ● POAG (Alward et al., 1998; Alward et al., 2002; Ennis et al., 2010; Faucher et al., 2002; Fingert et al., 1999; Millá et al., 2013; Williams-Lyn et al., 2000) ● Exfoliation Syndrome (Alward et al., 2002) ● NTG (Weisschuh, Neumann, Wolf, Wissinger, & Gramer, 2005) ● OHT (Faucher et al., 2002) ● PACG (X. Huang et al., 2014) ● Control Group (Faucher et al., 2002) Country/Ethnicity ● German ● Spanish ● Caucasian ● African American ● Chinese ● English ● Canadian Family History of POAG/JOAG? ● Yes (Millá et al., 2013) ● No (Ennis et al., 2010; Faucher et al., 2002; X. Huang et al., 2014; Williams-Lyn et al., 2000) gnomAD? ● Allele count 157, allele frequency 5.6e-4  ● European, Ashkenazi, Latino	Secretion Assay ● 37 °C  ○ +++ (Gobeil, Letartre, & Raymond, 2006) ● 30 °C  ○ n/a Triton X-100 Solubility Assay ● n/a	● WT-like  ○ Tm=52.9 °C (Donegan et al., 2015)	Location ● Surface exposed ● Internal loop of Blade C Interactions ● None Predicted consequence of substitution ● Tolerated	● p.Thr293Met (c.878C>T)  ○ gnomAD: Allele count 3, allele frequency 1.21e-5  ○ Tolerated ● p.Thr293Ala (c.877A>G)  ○ gnomAD: Allele count 1, allele frequency 4.02e-6  ○ Tolerated	n/a
p.Val329Met c.985G>A	Diagnosis ● JOAG (Svidnicki et al., 2018) ● POAG (Fingert et al., 1999; Liu et al., 2012; Shimizu et al., 2000) ● Control Group (Fingert et al., 1999) Country/Ethnicity ● African American ● Brazilian Family History of POAG/JOAG? ● Yes (Shimizu et al., 2000) ● gnomAD? ● Allele count 72, allele frequency 2.55e-4  ○ African/African American	Secretion Assay ● 37 °C  ○ n/a ● 30 °C  ○ n/a Triton X-100 Solubility Assay ● Soluble(Shimizu et al., 2000)	● Slightly destabilized, mouse WT-like (Patterson-Orazem et al., 2019)  ○ Tm=49.6 °C (Donegan et al., 2015)	Location ● Buried ● Innermost strand of Blade B Interactions ● Hydrophobic pocket with Blade A Predicted consequence of substitution ● Tolerated, rearrangement likely ● Met is residue in D. rerio myocilin	n/a	n/a
p.Glu352Lys c.1054G>A	Diagnosis ● POAG (Alward et al., 1998; Faucher et al., 2002; Fingert et al., 1999; Liu et al., 2012; Whigham et al., 2011; Williams-Lyn et al., 2000) ● Control Group (Alward et al., 1998; Fingert et al., 1999; Liu et al., 2012; Svidnicki et al., 2018; Whigham et al., 2011) ● PCG (Millá et al., 2013) Country/Ethnicity ● Caucasian ● African American ● African Caribbean ● Canadian ● Black South African ● Spanish ● Brazilian Family History of POAG/JOAG? ● Yes (Williams-Lyn et al., 2000) ● No (Faucher et al., 2002; Williams-Lyn et al., 2000) gnomAD? ● Allele count 337, allele frequency 1.19e-3  ○ African/African American	Secretion Assay ● 37 °C  ○ +++ (Gobeil et al., 2006) ● 30 °C  ○ n/a Triton X-100 Solubility Assay ● Soluble (Z. Zhou & Vollrath, 1999) ● Insoluble (Shimizu et al., 2000)	● WT-like based on Glu352Gln (Burns, Turnage, Walker, & Lieberman, 2011)	Location ● Surface exposed ● Exterior loop of Blade B Interactions ● None Predicted consequence of substitution ● Tolerated	n/a	n/a
p.Thr353Ile c.1058C>T	Diagnosis ● JOAG (Bhattacharjee et al., 2007; Lam et al., 2000; Park et al., 2016; Rose et al., 2011) ● POAG (Banerjee, Bhattacharjee, Ponda, Sen, & Ray, 2012; B. Fan et al., 2002; B. J. Fan et al., 2005; Fingert et al., 1999; Jia, Tam, et al., 2009; Lam et al., 2000; Pang et al., 2002; Yoon, Kim, Moon, Lim, & Joo, 1999; X. M. Zhou et al., 2013) ● NTG (B. J. Fan et al., 2005) ● PACG (Aung et al., 2005) ● Control Group(Aung et al., 2005; B. Fan et al., 2002; B. J. Fan et al., 2005; Lam et al., 2000; Liu et al., 2012; Pang et al., 2002) Country/Ethnicity ● Korean ● Japanese ● Chinese ● Indian Family History of POAG/JOAG? ● Yes (B. Fan et al., 2002; Park et al., 2016; Rose et al., 2011; Yoon et al., 1999; X. M. Zhou et al., 2013) ● No (Bhattacharjee et al., 2007) gnomAD? ● Allele count 191, allele frequency 6.75e-4  ○ Asian	Secretion Assay ● 37 °C  ○ +++ (Gobeil et al., 2006) ● 30 °C  ○ n/a Triton X-100 Solubility Assay  ○ n/a	WT-like  ○ Tm=53.1 °C (Donegan et al., 2015)	Location ● Surface exposed, outer strand of Blade B Interactions ● Thr351 side chain, solvent Predicted consequence of substitution ● Tolerated	n/a	● Homozygous carrier had more severe symptoms than heterozygous family members (Yoon et al., 1999) ● Individual heterozygous for Thr353Ile and Asp384Asn had more severe symptoms than father with only Thr353Ile (X. M. Zhou et al., 2013) ● One individual heterozygous for Thr353Ile and Asn480Lys (Rose et al., 2011)
p.Lys398Arg c.1193G>A	Diagnosis ● POAG (Alward et al., 1998; Alward et al., 2002; Fingert et al., 1999; Hulsman et al., 2002; Jansson, Marknell, Tomic, Larsson, & Wadelius, 2003; Liu et al., 2012; McDonald et al., 2010; O’Gorman et al., 2019; Rakhmanov et al., 2005; Vázquez, Herrero, Bastús, & Pérez, 2009; Vincent et al., 2002; Williams-Lyn et al., 2000) ● Control Group (Alward et al., 1998; Fingert et al., 1999; Hulsman et al., 2002; Liu et al., 2012; Vázquez et al., 2009) ● Exfoliative glaucoma (Jansson et al., 2003) ● Pigmentary glaucoma (Alward et al., 1998; Alward et al., 2002; Fingert et al., 1999) Country/Ethnicity ● Russian ● Mexican ● Dutch ● Swedish ● English/Scottish ● Canadian ● Spanish ● UK  ○ African American ● Australian ● Caucasian Family History of POAG/JOAG?  ○ n/a gnomAD? ● Allele count 992, allele frequency 3.51e-3 ● European	Secretion Assay ● 37 °C  ○ +++ (Gobeil et al., 2006; Jacobson et al., 2001) ● 30 °C  ○ n/a Triton X-100 Solubility Assay ● Soluble (Shimizu et al., 2000; Z. Zhou & Vollrath, 1999)	WT-like  ○ Tm=53.8 °C (Burns et al., 2011)	Location ● Surface exposed, loop connecting inner strands of Blade C Interactions ● None Predicted consequence of substitution ● Tolerated	n/a	n/a
p.Ala445Val c.1334C>T	Diagnosis ● POAG (Alward et al., 1998; Faucher et al., 2002; Fingert et al., 1999; Lopez-Martinez et al., 2007) ● NTG (Weisschuh et al., 2005) ● Pigmentary glaucoma (Faucher et al., 2002) ● OHT (Vincent et al., 2002) Country/Ethnicity ● German ● Canadian ● French-Canadian ● Spanish ● Australian Family History of POAG/JOAG? ● Yes (Vincent et al., 2002) ● No (Faucher et al., 2002) gnomAD? ● Allele count 57, allele frequency 2.01e-4	Secretion Assay ● 37 °C  ○ +++ (Gobeil et al., 2006) ● 30 °C  ○ n/a Triton X-100 Solubility Assay  ○ n/a	● WT-like  ○ Tm=54.2 °C (Burns et al., 2011)	Location ● Surface exposed, on loop connecting inner strands of Blade D Interactions ● Thr443 Predicted consequence of substitution ● Tolerated	n/a	n/a
p.Lys500Arg c.1499A>G	Diagnosis ● POAG (Alward et al., 1998; Fingert et al., 1999; Liu et al., 2012; Whigham et al., 2011; Williams, Carmichael, Wainstein, Hobbs, & Ramsay, 2015; Williams-Lyn et al., 2000) ● Control (Liu et al., 2012) Country/Ethnicity  ○ African American ● Black South African Family History of POAG/JOAG? ● Yes (Williams et al., 2015) gnomAD? ● Allele count 161, allele frequency 5.69e-4	Secretion Assay ● 37 °C  ○ n/a ● 30 °C  ○ n/a Triton X-100 Solubility Assay ● n/a	n/a	Location ● C-terminal strand within molecular clasp of Blade E, surface exposed Interactions ● None Predicted consequence of substitution ● Tolerated	p.Lys500Thr (c.1499A>C) ● gnomAD: Allele count 1, allele frequency 3.98e-6 ● Tolerated in structure	One control individual was compound heterozygous for p.Lys500Arg and p.Asp446Tyr (Liu et al., 2012)
B. Pathogenic
Variant	Clinical Data	Cell Assays	Thermal Stability	Structure Analysis	Additional Variants	Other
p.Cys245Tyr c.734G>A	Diagnosis ● JOAG (B. J. Fan et al., 2006) ● OHT (B. J. Fan et al., 2006) Country/Ethnicity ● Chinese Family History of POAG/JOAG? ● Yes (B. J. Fan et al., 2006) gnomAD?  ○ n/a	Secretion Assay ● 37 °C  ○ - (Hogewind et al., 2007)  ○ 0.28% of WT (Zadoo, Nguyen, Zode, & Hulleman, 2016) ● 30 °C  ○ −/+ (Zadoo et al., 2016) Triton X-100 Solubility Assay ● Insoluble (Hogewind et al., 2007; Yam, Gaplovska-Kysela, Zuber, & Roth, 2007) ● Partially insoluble (Jia, Gong, et al., 2009)	Likely destabilized  ○ n/a (but see Cys433Arg)	Location ● Blade E, molecular clasp, surface exposed Interactions ● Disulfide bonded to Cys433 Predicted consequence of substitution ● Deleterious	n/a	n/a
p.Gly246Arg c.736G>A	Diagnosis ● JOAG (Adam et al., 1997; Brézin et al., 1997) Country/Ethnicity ● French Family History of POAG/JOAG? ● Yes (Adam et al., 1997; Brézin et al., 1997) gnomAD?  ○ n/a	Secretion Assay ● 37 °C  ○ - (Gobeil et al., 2006; Vollrath & Liu, 2006)  ○ 0.47% of WT (Zadoo et al., 2016) ● 30 °C  ○ ++ (Gobeil et al., 2006; Vollrath & Liu, 2006; Zadoo et al., 2016) Triton X-100 Solubility Assay  ○ n/a	Destabilized  ○ Tm=~42.6 °C (Burns et al., 2011; Donegan, Hill, Turnage, Orwig, & Lieberman, 2012)	Location ● Blade E, molecular clasp, surface exposed Interactions ● None Predicted consequence of substitution ● Deleterious, charged side chain like Arg would interfere with proper positioning of Cys245 for disulfide bond formation	n/a	n/a
p.Gly252Arg c.754G>A	Diagnosis ● POAG (Hewitt et al., 2007) ● JOAG (Booth et al., 2000; Rozsa et al., 1998; Shimizu et al., 2000; Vincent et al., 2002; Willoughby et al., 2004) ● OHT (Hewitt et al., 2007) Country/Ethnicity ● Australian ● Chinese ● Scottish ● US/Caucasian Family History of POAG/JOAG? ● Yes (Booth et al., 2000; Hewitt et al., 2007; Rozsa et al., 1998; Shimizu et al., 2000; Vincent et al., 2002) gnomAD?  ○ n/a	Secretion Assay ● 37 °C ● – (Gobeil et al., 2006) ● 30 °C  ○ +++ (Gobeil et al., 2006) Triton X-100 Solubility Assay ● Insoluble(Shimizu et al., 2000)	Destabilized  ○ Tm=44.8 °C (Burns et al., 2011) or 43.0 °C (Donegan et al., 2012), buffer dependent	Location ● Blade E, molecular clasp, surface exposed Interactions ● Main chain NH H-bonded to carbonyl of Asp498 Predicted consequence of substitution ● Deleterious, Arg would clash with neighboring Glu253 and Trp250, causing destabilization of the molecular clasp	● p.Gly252Glu (c.755G>A)  ○ gnomAD: allele count 2, allele frequency 8.25e-6  ○ Likely deleterious to structure ● p.Gly252Ala (c.755G>C)  ○ gnomAD: allele count 3, allele frequency 1.15e-5  ○ Likely deleterious to structure	● One individual compound heterozygous for Gly252Arg and Gly244Val (Hewitt et al., 2007) ● One individual also heterozygous for OPTN mutation (Willoughby et al., 2004)
p.Arg272Gly c.814C>G	Diagnosis ● JOAG (Shimizu et al., 2000) ● POAG (Shimizu et al., 2000) Country/Ethnicity ● US/Caucasian Family History of POAG/JOAG? ● Yes (Shimizu et al., 2000) gnomAD?  ○ n/a	Secretion Assay ● 37 °C  ○ - (Gobeil et al., 2006) ● 30 °C  ○ ++ (Vollrath & Liu, 2006) Triton X-100 Solubility Assay ● Insoluble (Shimizu et al., 2000)	Destabilized  ○ Tm=44.7 °C (Burns et al., 2011) or 41 °C (Donegan et al., 2012), buffer dependent	Location ● Blade A, innermost β-strand, near bottom face, not solvent exposed Interactions ● Sidechain guanidinium group forms multiple stabilizing interactions with Glu483 on Blade E, and Thr285 on the adjacent β-strand of Blade A Predicted consequence of substitution ● Deleterious, removal of side chain interactions	● p.Arg272Gln (c.815G>A)  ○ gnomAD: allele count 2, allele frequency 8.01e-6  ○ Likely deleterious to structure ● p.Arg272* (c.814C>T)  ○ gnomAD: allele count 2, allele frequency 8.02e-6  ○ Uncertain	n/a
p.Glu323Lys c.967G>A	Diagnosis ● JOAG (Rozsa et al., 1998; Shimizu et al., 2000) ● POAG (Shimizu et al., 2000) Country/Ethnicity ● Panamanian Family History of POAG/JOAG? ● Yes (Rozsa et al., 1998; Shimizu et al., 2000) gnomAD?  ○ n/a	Secretion Assay ● 37 °C  ○ - (Gobeil et al., 2006)/+(Vollrath & Liu, 2006) ● 30 °C  ○ +++ (Gobeil et al., 2006; Vollrath & Liu, 2006) Triton X-100 Solubility Assay ● Insoluble (Shimizu et al., 2000; Z. Zhou & Vollrath, 1999)	Destabilized  ○ Tm=44 °C (Donegan et al., 2012) or 45.6 °C (Burns et al., 2011), buffer dependent	Location ● Loop connecting Blade A to Blade B, top face, but not surface exposed Interactions ● Main chain NH forms H-bonding interaction with carbonyl of Gln337 ● Side chain forms polar contact with NH Glu340 Predicted consequence of substitution ● Deleterious, charge inversion	● p.Glu323_Val329dup (c.967_987dup)  ○ gnomAD: allele count 1, allele frequency 3.98e-6  ○ Likely deleterious to structure	n/a
p.Gly364Val c.1091G>T	Diagnosis ● POAG (Alward et al., 1998; Fingert et al., 1999; Stone et al., 1997) Country/Ethnicity ● US ● US/Caucasian Family History of POAG/JOAG? ● Yes(Stone et al., 1997) gnomAD?  ○ n/a	Secretion Assay ● 37 °C  ○ - (Gobeil et al., 2006)/++ (Jacobson et al., 2001; Vollrath & Liu, 2006) ● 30 °C  ○ +++ (Gobeil et al., 2006; Jacobson et al., 2001; Vollrath & Liu, 2006) Triton X-100 Solubility Assay ● Partially insoluble (Z. Zhou & Vollrath, 1999)	Destabilized  ○ Tm=~45 °C (Burns et al., 2011; Donegan et al., 2012)	Location ● Long loop connecting Blade B to Blade C, surface exposed Interactions ● Main chain NH forms H-bonding interaction with carbonyl of Ser393 Predicted consequence of substitution ● Deleterious, steric clash with Gln368 and Phe369	● p.Gly364Ser (c.1090G>A)  ○ gnomAD: allele count 2, allele frequency 7.95e-6 ● Likely deleterious to structure	● Adenovirus-injected myocilinG364V into mouse eyes led to significant IOP elevation (Shepard et al., 2007)
p.Gly367Arg c.1099G>A	Diagnosis ● JOAG (Cobb et al., 2002; Gupta et al., 2020; Iliev et al., 2008; Kanagavalli, Krishnadas, Pandaranayaka, Krishnaswamy, & Sundaresan, 2003; Souzeau et al., 2015; Vincent et al., 2002; Yao et al., 2018) ● POAG (J. Chen et al., 2011; Faucher et al., 2002; Mansergh et al., 1998; Melki, Belmouden, Brezin, & Garchon, 2003; K. G. Michels-Rautenstrauss et al., 1998; Suzuki et al., 1997; Fumiko Taniguchi, Suzuki, Shirato, & Araie, 2000) Country/Ethnicity ● Japanese ● German ● Irish ● Indian ● Swiss ● Australian ● Scottish ● Chinese ● French ● Canadian ● Italian/French Family History of POAG/JOAG? ● Yes (J. Chen et al., 2011; Cobb et al., 2002; Faucher et al., 2002; Gupta et al., 2020; Iliev et al., 2008; Mansergh et al., 1998; K. G. Michels-Rautenstrauss et al., 1998; Souzeau et al., 2015; Fumiko Taniguchi et al., 2000; Vincent et al., 2002; Yao et al., 2018) ● No (Gupta et al., 2020; Kanagavalli et al., 2003) gnomAD?  ○ n/a	Secretion Assay ● 37 °C  ○ - (Gobeil et al., 2006; Kanagavalli, Pandaranayaka, Krishnadas, Krishnaswamy, & Sundaresan, 2007) ● 30 °C  ○ +++ (Gobeil et al., 2006) Triton X-100 Solubility Assay  ○ n/a	Destabilized  ○ Tm= 42.7 °C (Donegan et al., 2012) or 45.7 °C (Burns et al., 2011), buffer dependent	Location ● Long loop connecting Blade B to Blade C, surface exposed Interactions ● Main chain NH forms polar contact with side chain of Asp378 Predicted consequence of substitution ● Deleterious, large size of arginine would be sterically incompatible with neighboring Tyr376, which forms a solvent-exposed π-π stacking interaction with His366	See p.Gly367_Gln368delinsVal	n/a
p.Gly367_Gln368delinsVal c.1101_1103delGACAinsT	Diagnosis ● JOAG (Angius, De Gioia, et al., 1998; Angius, Pisano, et al., 1998) ● POAG (Angius, De Gioia, et al., 1998; Angius, Pisano, et al., 1998) Country/Ethnicity ● Italian Family History of POAG/JOAG? ● Yes (Angius, De Gioia, et al., 1998; Angius, Pisano, et al., 1998) gnomAD?  ○ n/a	Secretion Assay ● 37 °C  ○ - (Vollrath & Liu, 2006) ● 30 °C ● + (Vollrath & Liu, 2006) Triton X-100 Solubility Assay ● Insoluble (Z. Zhou & Vollrath, 1999)	n/a	Location ● Long loop connecting Blade B to Blade C, surface exposed Interactions ● Main chain NH forms polar contact with side chain of Asp378 Predicted consequence of substitution ● Deleterious, changes to this loop are generally problematic	See p.Gly367Arg	n/a
p.Pro370Leu c.1109C>T	Diagnosis ● JOAG (Adam et al., 1997; Bayat et al., 2008; Campos-Mollo et al., 2007; X. Chen et al., 2009; Y. Chen, Jiang, Wan, Yu, & Sun, 2006; Damji et al., 1999; Hamanaka et al., 2017; C. Huang et al., 2018; X. Huang et al., 2014; Li et al., 2014; Mukhopadhyay et al., 2002; Rose et al., 2011; Rozsa et al., 1998; Shimizu et al., 2000; Stoilova et al., 1998; Suzuki et al., 1997; Svidnicki et al., 2018; F. Taniguchi, 1999; Vasconcellos et al., 2000; Vincent et al., 2002; Wei et al., 2011; Wiggs et al., 1998) ● POAG (Wei et al., 2005) Country/Ethnicity ● Colombian ● Chinese ● Iranian ● Japanese ● French-Canadian ● Canadian ● Indian ● Brazilian ● Greek ● French ● US ● US/Caucasian Family History of POAG/JOAG? ● Yes (Adam et al., 1997; Campos-Mollo et al., 2007; X. Chen et al., 2009; Y. Chen et al., 2006; Damji et al., 1999; Hamanaka et al., 2017; X. Huang et al., 2014; Li et al., 2014; Mukhopadhyay et al., 2002; Rozsa et al., 1998; Shimizu et al., 2000; Stoilova et al., 1998; Suzuki et al., 1997; F. Taniguchi, 1999; Vincent et al., 2002; Wei et al., 2005; Wei et al., 2011; Wiggs et al., 1998) ● No (Bayat et al., 2008) gnomAD?  ○ n/a	Secretion Assay ● 37 °C  ○ - (Gobeil et al., 2006; Vollrath & Liu, 2006; Yam et al., 2007) ● 30 °C  ○ - (Gobeil et al., 2006; Vollrath & Liu, 2006) Triton X-100 Solubility Assay ● Insoluble (Shimizu et al., 2000; Yam et al., 2007; Z. Zhou & Vollrath, 1999)	Destabilized ● Could not be isolated in folded state for measurement (Burns et al., 2011) ● When combined with stabilizing variant D478S to yield OLFP370L/D478S, Tm = 47°C compared to OLFD478S alone, which is 58 °C (Hill et al., 2019)	Location ● Long loop connecting Blade B to Blade C, surface exposed Interactions ● None Predicted consequence of substitution ● Deleterious, adjacent to Tyr371, likely Pro likely properly organizes this region to promote the interaction between the backbone carbonyl of Phe369 and Nε of Lys423, and orient Tyr371 for cation-π	● p.Pro370Ser (c.1108C>T)  ○ gnomAD: allele count 1, allele frequency 3.19e-5  ○ Likely deleterious to structure	● Often serves as a “positive control” for pathogenic variants ● MyocilinP370L accumulates in the ER(Lopez-Martinez et al., 2007) ● MyocilinP370L downregulates homeostatic ER-stress response (L. Wang et al., 2007) ● MyocilinP370L impairs mitochondrial function (decreased transmembrane potential, increased ROS production, increased intracellular cytoplasm) (He, Leung, Zhuo, & Ge, 2009) ● Detailed histochemical analysis (Hamanaka et al., 2017)
p.Thr377Met c.1130C>T	Diagnosis ● JOAG (Puska, Lemmela, Kristo, Sankila, & Jarvela, 2005; Shimizu et al., 2000; Young et al., 2012; Zgaga et al., 2008) ● POAG (Allingham et al., 1998; Alward et al., 1998; Fingert et al., 1999; Hamanaka et al., 2017; Kanagavalli et al., 2003; Kitsos et al., 2010; Liu et al., 2012; Mackey et al., 2003; Melki, Idhajji, et al., 2003; Petersen et al., 2006; Puska et al., 2005; Shimizu et al., 2000; Wiggs et al., 1998; Wilkinson et al., 2003; Wirtz et al., 2008) ● NTG (Mackey et al., 2003; Puska et al., 2005; Wilkinson et al., 2003) ● OHT (Hamanaka et al., 2017; Kitsos et al., 2010; Mackey et al., 2003; Petersen et al., 2006; Puska et al., 2005; Wilkinson et al., 2003; Wirtz et al., 2008) Country/Ethnicity ● Croatian ● US ● US/Caucasian ● Indian ● Australian  ○ African American ● Finnish ● Greek ● Dutch/Croatian ● English ● Greek/Macedonian ● Japanese ● Moroccan Family History of POAG/JOAG? ● Yes(Allingham et al., 1998; Alward et al., 1998; Hamanaka et al., 2017; Kanagavalli et al., 2003; Kitsos et al., 2010; Mackey et al., 2003; Petersen et al., 2006; Puska et al., 2005; Shimizu et al., 2000; Wiggs et al., 1998; Wilkinson et al., 2003; Wirtz et al., 2008; Young et al., 2012; Zgaga et al., 2008) gnomAD? ● Allele count 5, allele frequency 1.99e-5	Secretion Assay ● 37 °C ● + (Gobeil et al., 2006)/++ (Vollrath & Liu, 2006) ● 30 °C  ○ +++ (Gobeil et al., 2006; Vollrath & Liu, 2006) Triton X-100 Solubility Assay ● Partially insoluble (Z. Zhou & Vollrath, 1999) ● Insoluble (Shimizu et al., 2000)	Destabilized ● Tm = 44.3 °C (Donegan et al., 2012) or 47.7°C (Burns et al., 2011), buffer dependent	Location ● Long loop connecting Blade B to Blade C Interactions ● Main chain carbonyl makes polar contact with Nε of Lys423 ● Side chain makes polar contacts with Gly374 carbonyl and main chain NH of Tyr371 Predicted consequence of substitution ● Deleterious, loss of numerous polar interactions would propagate to weaken cation-π and metal ion site	● p.Thr377Lys (c.1130C>A) (Vincent et al., 2002)  ○ Likely deleterious to structure ● p.Thr377Arg (c.1130C>G) (Waryah, Narsani, Sheikh, Shaikh, & Shahani, 2013)  ○ Likely deleterious to structure ● p.Thr377Ala (c.1129A>G)  ○ gnomAD: allele count 2, allele frequency 7.96e-6  ○ Uncertain	● Detailed histochemical analysis (Hamanaka et al., 2017) ● Four family members compound heterozygous for p.Thr377Met and p.Gln368* have higher IOP than family members with only p.Gln368* (Young et al., 2012) ● Two family members compound heterozygotes for p.Thr377Met and p.Val495Ile (Alward et al., 1998)
p.Asp380Ala c.1139A>C	Diagnosis ● JOAG (Campos-Mollo et al., 2007; Kennan et al., 1998; Stoilova et al., 1998) Country/Ethnicity ● Spanish ● English Family History of POAG/JOAG? ● Yes (Campos-Mollo et al., 2007; Kennan et al., 1998; Stoilova et al., 1998) gnomAD?  ○ n/a	Secretion Assay ● 37 °C  ○ - (Gobeil et al., 2006)/+(Vollrath & Liu, 2006) ● 30 °C  ○ ++ (Gobeil et al., 2006)/+++ (Vollrath & Liu, 2006) Triton X-100 Solubility Assay ● Partially insoluble (Z. Zhou & Vollrath, 1999)	Destabilized ● Tm = ~46.6 °C (Burns et al., 2011; Donegan et al., 2012)	Location ● Innermost strand, Blade C Interactions ● Ca2+, Na+, Tyr371 Predicted consequence of substitution ● Deleterious, loss of ionic interactions	● p.Asp380Gly (c.1139A>G) (Alward et al., 1998)  ○ Likely deleterious to structure ● p.Asp380His (c.1138G>C) (Wirtz, Samples, Choi, & Gaudette, 2007)  ○ Likely deleterious to structure ● p.Asp380Asn (c.1138G>A) (Challa et al., 2002)  ○ Likely deleterious to structure	● Aggregation of OLFD380A is facile at 37 °C, pH 7.2 (Hill, Donegan, & Lieberman, 2014)
p.Lys423Glu c.1267A>G	Diagnosis ● JOAG (Bruttini et al., 2003; Faucher et al., 2002; Morissette et al., 1998; Svidnicki et al., 2018) ● POAG (Bruttini et al., 2003; Morissette et al., 1998) ● OHT (Morissette et al., 1998) Country/Ethnicity ● Brazilian ● Italian ● French-Canadian ● Canadian Family History of POAG/JOAG? ● Yes (Bruttini et al., 2003; Faucher et al., 2002; Morissette et al., 1998) ● No (Faucher et al., 2002) gnomAD?  ○ n/a	Secretion Assay ● 37 °C  ○ - (Gobeil et al., 2006; Jacobson et al., 2001; Vollrath & Liu, 2006) ● 30 °C  ○ - (Gobeil et al., 2006; Vollrath & Liu, 2006) Triton X-100 Solubility Assay ● Insoluble (Z. Zhou & Vollrath, 1999)	Destabilized ● 34.2 °C (Donegan et al., 2012) or 40.5 °C (Burns et al., 2011)	Location ● On loop connecting Blade C to Blade D Interactions ● Internal cation-π with Tyr371 Predicted consequence of substitution ● Deleterious, charge inversion internal to protein	n/a	● In family, 4 homozygotes had no symptoms while heterozygotes did have JOAG/POAG (Morissette et al., 1998)
p.Val426Phe c.1276G>T	Diagnosis ● JOAG (Lim, Lichter, Higashi, Downs, & Richards, 2003; Mansergh et al., 1998; Millá et al., 2013; Rozsa et al., 1998; Shimizu et al., 2000) ● POAG (Shimizu et al., 2000) Country/Ethnicity ● Spanish ● US/Caucasian Family History of POAG/JOAG? ● Yes (Lim et al., 2003; Mansergh et al., 1998; Millá et al., 2013; Rozsa et al., 1998; Shimizu et al., 2000) gnomAD?  ○ n/a	Secretion Assay ● 37 °C  ○ - (Gobeil et al., 2006; Vollrath & Liu, 2006) ● 30 °C  ○ ++ (Vollrath & Liu, 2006)/+++ (Gobeil et al., 2006) Triton X-100 Solubility Assay ● Insoluble (Shimizu et al., 2000; Z. Zhou & Vollrath, 1999)	Destabilized  ○ Tm= 41.5 °C (Donegan et al., 2012) or 45.1 °C (Burns et al., 2011), buffer dependent	Location ● On loop connecting Blade C to Blade D Interactions ● Hydrophobic between Blades C and D Predicted consequence of substitution ● Deleterious, would weaken or disrupt the neighboring cation-π interaction between Lys423 and Tyr371 (see Lys423)	n/a	n/a
p.Cys433Arg c.1297T>C	Diagnosis ● JOAG (Povoa, Malta, Rezende Mde, de Melo, & Giannella-Neto, 2006; Svidnicki et al., 2018; Vasconcellos et al., 2000) ● POAG (Povoa et al., 2006) ● OHT (Povoa et al., 2006) Country/Ethnicity ● Brazilian Family History of POAG/JOAG? ● Yes (Povoa et al., 2006; Vasconcellos et al., 2000) gnomAD?  ○ n/a	Secretion Assay ● 37 °C  ○ - (Gobeil et al., 2006) ● 30 °C  ○ - (Gobeil et al., 2006) Triton X-100 Solubility Assay  ○ n/a	Destabilized  ○ Tm=40.4 °C (Burns et al., 2011) or 42.6 °C (Donegan et al., 2012), buffer dependent	Location ● Blade D, β-turn connecting two inner strands on bottom face Interactions ● Disulfide with Cys245, and NH fmors interaction with Glu385; Glu385Ala is misfolded (Donegan et al., 2012) Predicted consequence of substitution ● Deleterious, loss of covalent disulfide bond and interacting with Glu385	n/a	n/a
p.Tyr437His c.1309T>C	Diagnosis ● JOAG (Alward et al., 2002; Lei, Li, Liu, & Zhang, 2019; Wiggs et al., 1998; Yang et al., 2020) ● POAG (Alward et al., 1998; Alward et al., 2002; Fingert et al., 1999; Lei et al., 2019; Stone et al., 1997) ● OHT (Lei et al., 2019) Country/Ethnicity ● Chinese ● US ● US/Caucasian Family History of POAG/JOAG? ● Yes (Alward et al., 1998; Lei et al., 2019; Stone et al., 1997; Wiggs et al., 1998; Yang et al., 2020) gnomAD?  ○ n/a	Secretion Assay ● 37 °C  ○ - (Gobeil et al., 2006; Jacobson et al., 2001; Vollrath & Liu, 2006)  ○ 0.2% of WT (Zadoo et al., 2016) ● 30 °C  ○ −/+ (Vollrath & Liu, 2006; Zadoo et al., 2016) Triton X-100 Solubility Assay ● Insoluble (Hogewind et al., 2007; Z. Zhou & Vollrath, 1999)	Destabilized  ○ Tm=40.3 °C (Donegan et al., 2012) or 41.4 °C (Burns et al., 2011), buffer dependent	Location ● Blade D, near disulfide bond Interactions ● Side chain forms H-bonding interaction with NH of Leu248 in Blade E Predicted consequence of substitution ● Deleterious, His is too small to form inter-blade interaction	n/a	● Detailed histochemical comparison between human donor eyes expressing myocilinY437H or myocilinWT (van der Heide et al., 2018) ● Mouse models with different genetic backgrounds have differing phenotypes (Chou, Tomarev, & Porciatti, 2014; Gould et al., 2006; Joe, Nakaya, Abu-Asab, & Tomarev, 2015; Kasetti, Phan, Millar, & Zode, 2016; Lynch et al., 2018; McDowell et al., 2012; Senatorov et al., 2006; Shepard et al., 2007; Zhuo et al., 2008; Zillig, Wurm, Grehn, Russell, & Tamm, 2005; Zode et al., 2011) ● Aggregated OLFY437H has amyloid character (Orwig et al., 2012)
p.Ile477Asn c.1430T>A	Diagnosis ● JOAG (Richards, 1998; Rozsa et al., 1998; Shimizu et al., 2000) ● POAG (Alward et al., 1998; Fingert et al., 1999; Shimizu et al., 2000) ● Country/Ethnicity ● US ● US/Caucasian Family History of POAG/JOAG? ● Yes (Alward et al., 1998; Richards, 1998; Rozsa et al., 1998; Shimizu et al., 2000; Stokes, 1940) gnomAD?  ○ n/a	Secretion Assay ● 37 °C  ○ - (Gobeil et al., 2006; Vollrath & Liu, 2006) ● 30 °C  ○ - (Gobeil et al., 2006; Vollrath & Liu, 2006) Triton X-100 Solubility Assay ● Insoluble (Shimizu et al., 2000; Z. Zhou & Vollrath, 1999)	Destabilized  ○ Tm=37.7 °C (Burns et al., 2011) or 39.7 °C (Donegan et al., 2012), buffer dependent	Location ● Blade E, innermost strand, part of hydrophobic belt Interactions ● Side chain hydrophobic packing, main chain carbonyl is ligand to Ca2+ Predicted consequence of substitution ● Deleterious, new polarity in hydrophobic environment	See p.Ile477Ser	n/a
p.Ile477Ser c.1430T>G	Diagnosis ● POAG (Adam et al., 1997) Country/Ethnicity ● French Family History of POAG/JOAG? ● Yes (Adam et al., 1997) gnomAD?  ○ n/a	Secretion Assay ● 37 °C  ○ - (Izumi et al., 2003; Jacobson et al., 2001; Vollrath & Liu, 2006) ● 30 °C  ○ +++ (Vollrath & Liu, 2006) Triton X-100 Solubility Assay ● Insoluble (Z. Zhou & Vollrath, 1999)	Destabilized  ○ Tm = 40.1 °C (Donegan et al., 2012) or 41.9 °C (Burns et al., 2011), buffer dependent	Location ● Blade E, innermost strand, part of hydrophobic belt Interactions ● Side chain hydrophobic packing, main chain carbonyl is ligand to Ca2+ Predicted consequence of substitution ● Deleterious, new polarity in hydrophobic environment	See p.Ile477Asn	n/a
p.Tyr479His c.1435T>C	Diagnosis ● POAG (Lopez-Martinez et al., 2007; Millá et al., 2013) Country/Ethnicity ● Spanish Family History of POAG/JOAG? ● Yes (Millá et al., 2013) gnomAD?  ○ n/a	Secretion Assay ● 37 °C  ○ −/+ (Lopez-Martinez et al., 2007) ● 30 °C  ○ n/a Triton X-100 Solubility Assay  ○ n/a (Z. Zhou & Vollrath, 1999)	n/a	Location ● Blade E, innermost strand, near bottom face Interactions ● Tyr479 side chain forms short (2.6 Å) H-bonding interaction with Glu385, located on Blade C, otherwise in hydrophobic environment Predicted consequence of substitution ● Deleterious, His is too small to form interaction with Glu385, and Glu385Ala is misfolded (Donegan et al., 2012), indicating the H-bonding interaction is important.	n/a	● MyocilinY479H accumulates in ER (Lopez-Martinez et al., 2007)
p.Asn480Lys c.1440C>A,G	Diagnosis ● JOAG (Brézin et al., 1998; Mimivati, Nurliza, Marini, & Liza-Sharmini, 2014; Rose et al., 2011) ● POAG (Adam et al., 1997; Brézin et al., 1998; Guevara-Fujita et al., 2008; Hulsman et al., 2002; Melki, Belmouden, et al., 2003) Country/Ethnicity ● Andean ● Malay ● French ● Dutch ● Indian Family History of POAG/JOAG? ● Yes (Adam et al., 1997; Brézin et al., 1998; Guevara-Fujita et al., 2008; Hulsman et al., 2002; Mimivati et al., 2014; Rose et al., 2011) gnomAD?  ○ n/a	Secretion Assay ● 37 °C ● – (Gobeil et al., 2006)/+(Vollrath & Liu, 2006) ● 30 °C ● + (Gobeil et al., 2006)/++(Vollrath & Liu, 2006) Triton X-100 Solubility Assay ● Insoluble (Z. Zhou & Vollrath, 1999)	Destabilized  ○ Tm=42.4 °C (Donegan et al., 2012) or 46.1 °C (Burns et al., 2011), buffer dependent	Location ● Blade E, on β-turn between internal strands, side chain buried Interactions ● Asn480 side chain forms polar interactions with main chain NH of Leu483 and Glu483 Predicted consequence of substitution ● Deleterious, buried point charge unfavorable	n/a	● One individual is compound heterozygous for Asn480Lys and Thr353Ile (Rose et al., 2011)
p.Pro481Thr c.1441C>A	Diagnosis ● POAG (Fingert et al., 1999) ● JOAG (Alward et al., 2002) Country/Ethnicity ● US Family History of POAG/JOAG?  ○ n/a gnomAD?  ○ n/a	Secretion Assay ● 37 °C  ○ n/a ● 30 °C  ○ n/a Triton X-100 Solubility Assay  ○ n/a		Location ● Blade E, on β-turn between internal strands, side chain buried Interactions ● Pro481 forms H-bonding interaction to Lys484 Nε in same loop Predicted consequence of substitution ● Deleterious, removal of constraint within β-turn	● See p.Pro481Leu ● p.Pro481Arg (c.1442C>G) (Jansson et al., 2003) ● Likely deleterious to structure ● p.Pro481Ser (c.1441C>T) (Mabuchi et al., 2001) ● gnomAD: allele count 1, frequency 3.98e-6 ● Uncertain	n/a
p.Pro481Leu c.1442C>T	Diagnosis ● POAG (Faucher et al., 2002; Fingert et al., 1999) ● OHT (Faucher et al., 2002) ● Angle Closure Glaucoma (Faucher et al., 2002) Country/Ethnicity  ○ African American ● Canadian Family History of POAG/JOAG? ● Ye s(Faucher et al., 2002) gnomAD?  ○ n/a	Secretion Assay ● 37 °C  ○ - (Gobeil et al., 2006) ● 30 °C  ○ ++ (Gobeil et al., 2006) Triton X-100 Solubility Assay  ○ n/a	Destabilized  ○ Tm= 45.5 °C (Donegan et al., 2012) or 46.6 °C (Burns et al., 2011), buffer dependent	Location ● Blade E, on β-turn between internal strands, side chain buried Interactions ● Pro481 forms H-bonding interaction to Lys484 N in same loop Predicted consequence of substitution ● Deleterious, removal of constraint within β-turn	See p.Pro481Thr	n/a
p.Ile499Phe c.1495A>T	Diagnosis ● POAG (Adam et al., 1997; Melki, Belmouden, et al., 2003) Country/Ethnicity ● French Family History of POAG/JOAG? ● Yes (Adam et al., 1997) gnomAD?  ○ n/a	Secretion Assay ● 37 °C  ○ - (Gobeil et al., 2006)/+ (Vollrath & Liu, 2006) ● 30 °C  ○ +++ (Gobeil et al., 2006; Vollrath & Liu, 2006) Triton X-100 Solubility Assay ● Partially Insoluble (Z. Zhou & Vollrath, 1999)	Destabilized  ○ Tm =42.8°C (Donegan et al., 2012) or 44.4°C (Burns et al., 2011), buffer dependent	Location ● Blade E, exterior strand, side chain buried in hydrophobic belt Interactions ● Hydrophobic Predicted consequence of substitution ● Deleterious, side chain to large	See p.Ile499Ser	n/a
p.Ile499Ser c.1496T>G	Diagnosis ● JOAG (Shimizu et al., 2000) Country/Ethnicity ● US/Caucasian Family History of POAG/JOAG? ● Yes (Shimizu et al., 2000) gnomAD?  ○ n/a	Secretion Assay ● 37 °C  ○ n/a ● 30 °C  ○ n/a Triton X-100 Solubility Assay ● Insoluble (Shimizu et al., 2000)	Likely destabilized  ○ n/a (but see Ile499Phe)	Location ● Blade E, exterior strand, side chain buried in hydrophobic belt Interactions ● Hydrophobic Predicted consequence of substitution ● Deleterious, new polarity in hydrophobic environment	See p.Ile499Phe	n/a
p.Ser502Pro c.1504T>C	Diagnosis ● JOAG (Stoilova et al., 1998) Country/Ethnicity ● English Family History of POAG/JOAG? ● Yes (Stoilova et al., 1998) gnomAD?  ○ n/a	Secretion Assay ● 37 °C  ○ - (Gobeil et al., 2006) ● 30 °C  ○ - (Gobeil et al., 2006) Triton X-100 Solubility Assay  ○ n/a	Destabilized  ○ Tm= 41.0 °C (Donegan et al., 2012) or 43.0 °C (Burns et al., 2011), buffer dependent	Location ● Blade E, molecular clasp, surface exposed Interactions ● Main chain to Glu247 Predicted consequence of substitution ● Deleterious, new backbone constraint	n/a	● Adenovirus-injected myocilinS502P into mouse eyes did not result in IOP elevation (Shepard et al., 2007)
C. Likely Pathogenic
Variant	Clinical Data	Cell Assays	Thermal Stability	Structure Analysis	Additional Variants	Other
p.Val251Ala c.752T>C	Diagnosis ● JOAG (Kuchtey, Chowdhury, Uptegraft, Fautsch, & Kuchtey, 2013; Lu et al., 2020; K. Michels-Rautenstrauss et al., 2002) ● POAG (Lu et al., 2020) ● OHT (Lu et al., 2020) Country/Ethnicity ● German ● Chinese ● US/Caucasian Family History of POAG/JOAG? ● Yes (Kuchtey et al., 2013; Lu et al., 2020; K. Michels-Rautenstrauss et al., 2002) gnomAD?  ○ n/a	Secretion Assay ● 37 °C  ○ n/a ● 30 °C  ○ n/a Triton X-100 Solubility Assay  ○ n/a	n/a	Location ● Blade E, molecular clasp, side chain pointed inward toward hydrophobic belt Interactions ● None Predicted consequence of substitution ● Uncertain, Val to Ala substitution is conservative, but weaker hydrophobic packing	● p.Val251Ile (c.751G>A)  ○ gnomAD: allele count 1, allele frequency 4.14e-6  ○ Likely deleterious to structure	● MyocilinV251A was not found in the aqueous humor of a patient with JOAG (Kuchtey et al., 2013)
p.Pro254Arg c.761C>G	Diagnosis ● JOAG (Yang et al., 2015) ● POAG (Yang et al., 2015) Country/Ethnicity ● Chinese Family History of POAG/JOAG? ● Yes (Yang et al., 2015) gnomAD?  ○ n/a	Secretion Assay ● 37 °C  ○ n/a ● 30 °C  ○ n/a Triton X-100 Solubility Assay  ○ n/a	n/a	Location ● Blade E, molecular clasp Interactions ● Main chain carbonyl forms polar interactions with Arg470, located on an opposing loop that connects Blade D and Blade E Predicted consequence of substitution ● Deleterious, would remove backbone restriction in molecular clasp and side chain too large	● p.Pro254Leu (c.761C>T) (Souzeau et al., 2016)  ○ Likely deleterious to structure	n/a
p.Leu255Pro c.764T>C	Diagnosis ● JOAG (Park et al., 2016) Country/Ethnicity ● Korean Family History of POAG/JOAG? ● Yes (Park et al., 2016) gnomAD?  ○ n/a	Secretion Assay ● 37 °C  ○ n/a ● 30 °C  ○ n/a Triton X-100 Solubility Assay  ○ n/a	n/a	Location ● Blade E, outer strand, surface exposed Interactions ● Main chain H-bonds with Thr496 main chain Predicted consequence of substitution ● Deleterious, would introduce di-proline motif, further limiting the polypeptide backbone mobility	● p.Leu255Val (c.763G>C)  ○ gnomAD: allele count 3, allele frequency 1.23e-5  ○ Uncertain	n/a
p.Pro274Arg c.821C>G	Diagnosis ● JOAG (Markandaya et al., 2004) Country/Ethnicity ● Indian Family History of POAG/JOAG? ● Yes (Markandaya et al., 2004) gnomAD?  ○ n/a	Secretion Assay ● 37 °C  ○ n/a ● 30 °C  ○ n/a Triton X-100 Solubility Assay  ○ n/a	n/a	Location ● Blade A, loop connecting innermost strands Interactions ● None Predicted consequence of substitution ● Deleterious, Arg would remove restrictions placed on polypeptide backbone and also disrupt packing	● p.Pro274Leu (c.821C>T)  ○ gnomAD: allele count 1, allele frequency 4e-6  ○ Likely deleterious to structure	● Youngest family member homozygous for mutation(Markandaya et al., 2004)
p.Gln337Arg ● c.1010A>G	Diagnosis ● JOAG (Gupta et al., 2020; Stoilova et al., 1997) Country/Ethnicity ● Scottish ● Indian Family History of POAG/JOAG? ● Yes (Gupta et al., 2020) gnomAD?  ○ n/a	Secretion Assay ● 37 °C  ○ n/a ● 30 °C  ○ n/a Triton X-100 Solubility Assay  ○ n/a	n/a	Location ● Blade B, loop connecting inner strands Interactions ● Main chain carbonyl forms polar contact with Glu323 main chain NH ● Side chain forms polar contact with main chain carbonyl of Ser341 Predicted consequence of substitution ● Deleterious, sterically incompatible	● See p.Gln337* (c.1009delC) ● p.Gln337Glu (c.1009C>G) (Vázquez et al., 2009)  ○ Likely deleterious to structure ● p.Gln337His (c.1011G>T)  ○ gnomAD: allele count 1, allele frequency 3.98e-6  ○ Likely deleterious to structure	● Homozygous individuals identified in two families (Gupta et al., 2020)
p.Ser341Pro c.1021T>C	Diagnosis ● JOAG (X. Huang et al., 2014) ● POAG (X. Huang et al., 2014; Kee & Ahn, 1997; Moon, Kim, & Lee, 2020; F. Wang et al., 2015) ● OHT (F. Wang et al., 2015) Country/Ethnicity ● Chinese ● Korean Family History of POAG/JOAG? ● Ye s(X. Huang et al., 2014; Kee & Ahn, 1997; Moon et al., 2020; F. Wang et al., 2015) ● No (X. Huang et al., 2014) gnomAD?  ○ n/a	Secretion Assay ● 37 °C  ○ n/a ● 30 °C  ○ n/a Triton X-100 Solubility Assay  ○ n/a	n/a	Location ● Blade B, turn between internal strands Interactions ● Main chain carbonyl forms polar contact with Gln337 side chain, main chain NH forms polar contact with carbonyl of Gly338 ● Side chain hydroxyl forms polar interactions with NH Thr343 and CO Gly338 Predicted consequence of substitution ● Deleterious, restrict backbone and reduce available stabilizing interactions	n/a	n/a
p.Ala363Thr c.1087G>A	Diagnosis ● POAG (Ishikawa et al., 2004; R. Kubota et al., 2000; Mengkegale et al., 2008) Country/Ethnicity ● Japanese Family History of POAG/JOAG? ● Yes (R. Kubota et al., 2000; Ryo Kubota et al., 1997; Mengkegale et al., 2008) gnomAD?  ○ n/a	Secretion Assay ● 37 °C  ○ - (Izumi et al., 2003) ● 30 °C  ○ n/a Triton X-100 Solubility Assay  ○ n/a	n/a	Location ● Long loop between Blade B and Blade C, pointed inward Interactions ● Main chain NH forms polar contact with carbonyl Ile360, main chain carbonyl forms polar contact with side chain Arg342 Predicted consequence of substitution ● Uncertain, relatively conservative substitution but introduces new polarity into relatively hydrophobic region	n/a	n/a
p.Phe369Leu c.1105T>C	Diagnosis ● JOAG (Hamanaka et al., 2017) ● POAG (Hamanaka et al., 2017; Ishikawa et al., 2004) Country/Ethnicity ● Japanese Family History of POAG/JOAG? ● Yes (Hamanaka et al., 2017; Ishikawa et al., 2004) gnomAD?  ○ n/a	Secretion Assay ● 37 °C  ○ n/a ● 30 °C  ○ n/a Triton X-100 Solubility Assay  ○ n/a	n/a	Location ● Long loop between Blade B and Blade C, surface exposed Interactions ● Main chain carbonyl makes polar contact with Lys423 Nε Predicted consequence of substitution ● Uncertain, Phe369 protects Lys423 from solvent, unclear if Leu would suffice	n/a	● One patient homozygous for variant (Hamanaka et al., 2017) ● Detailed histochemical analysis (Hamanaka et al., 2017)
p.Tyr371Asp c.1111T>G	Diagnosis ● JOAG (Avisar et al., 2009) Country/Ethnicity ● Uzbek Family History of POAG/JOAG? ● Yes (Avisar et al., 2009) gnomAD?  ○ n/a	Secretion Assay ● 37 °C  ○ n/a ● 30 °C  ○ n/a Triton X-100 Solubility Assay  ○ n/a	n/a	Location ● Long loop between Blade B and Blade C, points inward Interactions ● Main chain NH forms polar contact with side chain hydroxyl of Thr377; carbonyl forms polar contact with main chain NH of Trp373 ● Side chain ring forms cation-π with Lys423 and hydroxyl forms polar interaction with Asp380 Predicted consequence of substitution ● Deleterious, based on analogy with Lys423Glu	n/a	n/a
p.Glu385Lys c.1153G>A	Diagnosis ● JOAG (Criscione et al., 2019) Country/Ethnicity ● Hispanic Family History of POAG/JOAG? ● Ye s(Avisar et al., 2009; Criscione et al., 2019) gnomAD?  ○ n/a	Secretion Assay ● 37 °C  ○ n/a ● 30 °C  ○ n/a Triton X-100 Solubility Assay  ○ n/a	Likely destabilized  ○ Tm= 39.7 °C for OLFE385A (Donegan et al., 2012)	Location ● Blade C, bottom face, on turn between innermost blades Interactions ● Side chain forms polar contacts with main chain NH of Cys433 on Blade D, and with sidechains of Tyr 479 and Lys484 on Blade E Predicted consequence of substitution ● Deleterious, critical interactions would be lost	n/a	n/a
p.Thr438Ile c.1313C>T	Diagnosis ● JOAG (Jansson et al., 2003; Melki, Belmouden, et al., 2003; Souzeau et al., 2015) Country/Ethnicity ● Australian ● French ● Danish Family History of POAG/JOAG? ● Yes (Jansson et al., 2003; Melki, Belmouden, et al., 2003; Souzeau et al., 2015) gnomAD?  ○ n/a	Secretion Assay ● 37 °C  ○ n/a ● 30 °C  ○ n/a Triton X-100 Solubility Assay  ○ n/a	n/a	Location ● Blade D, interior strand Interactions ● Side chain hydroxyl group forms polar interaction with Asn450 sidechain Predicted consequence of substitution ● Deleterious, polar intra-blade contact would be lost	● p.Thr438Pro (c.1312A>C)  ○ gnomAD: allele count 6, allele frequency 2.39e-5  ○ Likely deleterious to structure	n/a
p.Asn450Tyr c.1348A>T	Diagnosis ● JOAG (Zhao et al., 2010) ● OHT (Zhao et al., 2010) Country/Ethnicity ● Chinese Family History of POAG/JOAG? ● Yes (Zhao et al., 2010) gnomAD?  ○ n/a	Secretion Assay ● 37 °C  ○ n/a ● 30 °C  ○ n/a Triton X-100 Solubility Assay  ○ n/a	n/a	Location ● Blade D, interior strand, points to surface Interactions ● Side chain forms polar contact with Thr438 side chain hydroxyl Predicted consequence of substitution ● Deleterious, Tyr would pose a steric challenge with the loop connecting Blade C and Blade D	● p.Asn450Asp (c.1348A>G) (K. Michels-Rautenstrauss et al., 2002)  ○ Likely tolerated in the structure ● p.Asn450Ser (c.1349A>G)  ○ gnomAD: allele count 2, allele frequency 7.95e-6  ○ Likely tolerated in the structure	n/a
p.Leu486Phe ● c.1456C>T	Diagnosis ● JOAG (Liao et al., 2016) ● POAG (X. Huang et al., 2014) Country/Ethnicity ● Chinese Family History of POAG/JOAG? ● Ye s(Liao et al., 2016) ● No (X. Huang et al., 2014) gnomAD?  ○ n/a	Secretion Assay ● 37 °C  ○ n/a ● 30 °C  ○ n/a Triton X-100 Solubility Assay  ○ n/a	n/a	Location ● Blade E, inner strand, pointed inward Interactions ● Main chain NH forms polar interaction with Thr496 sidechain; main chain carbonyl forms polar contact with carbonyl of Tyr497 Predicted consequence of substitution ● Deleterious, would require rearrangements to fit larger side chain	n/a	n/a

^aqualitative extent of secretion − = no secretion, −/+ = very little secretion, +, ++, +++ increasing levels of secretion

Relevant data to pathogenicity categories

Clinical metrics

MYOC mutations that segregate with early-onset glaucoma in affected pedigrees provide the best evidence for pathogenicity. The most reliable genetic data are come from pedigrees with sufficient size and structure where autosomal-dominant heritability of OAG is evident (MacArthur et al., 2014; Wiggs, 2007). For the purposes of this study, we did not restrict categories based on the size of the pedigree, but defined early onset diagnosis as occurring at or earlier than the 4^th decade of life, with ocular hypertension (OHT) considered with an IOP greater than ~25 mmHg (Gordon et al., 2002) and visual field abnormality reported by an average cup-disc ratio above ~0.3 (Gordon et al., 2002). Note that some variants were only found in study control groups and thus were not diagnosed with OHT or OAG.

Support for Toxic GOF

The pathogenic mechanism by which mutations in myocilin lead to glaucoma is an active area of research, but the toxic GOF hypothesis due to intracellular mutant protein misfolding is well supported. Neither overexpression of WT myocilin in mice (Gould et al., 2004; Zillig, Wurm, Grehn, Russell, & Tamm, 2005), nor ablating myocilin in mice (Kim et al., 2001), nor humans with homozygous N-terminal truncation mutations (Lam et al., 2000) or heterozygous MYOC deletion (Wiggs & Vollrath, 2001) leads to glaucoma. Early studies of myocilin supported the conclusion that OLF-resident myocilin variants accumulate intracellularly, in the endoplasmic reticulum (ER) (Gobeil et al., 2006; Gobeil et al., 2004; Joe et al., 2003; Liu & Vollrath, 2004; Vollrath & Liu, 2006; Yam, Gaplovska-Kysela, Zuber, & Roth, 2007; Z. Zhou & Vollrath, 1999), instead of being secreted to the TM. Cell stress occurs at least in part because Grp94, the ER-resident Hsp90 molecular chaperone that acts late in the folding process (Marzec, Eletto, & Argon, 2012), recognizes the nearly-folded mutant myocilin and catalyzes aberrant coaggregation (D. J. Huard et al., 2018; Stothert, Fontaine, Sabbagh, & Dickey, 2016; Stothert et al., 2014; A. Suntharalingam et al., 2012); however, Grp94 involvement has only been tested explicitly on a limited number of missense variants and cell types. In selected studies, the general ER stress-relieving compound 4-phenylbutyrate was shown to ameliorate misfolding (Yam et al., 2007; Zode et al., 2011). The downstream pathway leading to glaucoma is still unknown, but TM cell death likely compromises the TM matrix that subsequently obstructs aqueous humor fluid outflow. The resulting fluid imbalance could lead to clinically observed IOP increases.

Cellular assays

In the laboratory, the extent of secretion has been evaluated by a cellular assay. Secretion assays are typically conducted by transiently transfecting model mammalian cell lines (e.g. HEK293T or CHO) with plasmids encoding myocilin variants and evaluating the extent of secretion and intracellular accumulation after 2-3 days by Western blot (Gobeil et al., 2006; Vollrath & Liu, 2006), fluorescence of the fused corresponding green fluorescence protein (Yam et al., 2007), or more recently, by enzymatic detection of luciferase (Zadoo, Nguyen, Zode, & Hulleman, 2016). Cells are cultured at 37 °C and sometimes at 30 °C (Liu & Vollrath, 2004; Vollrath & Liu, 2006), so-called permissive temperatures in which protein translation and folding is slowed and variants are further away from their melting temperatures (T_m, see biochemical assays below). An additional assay tests the solubility of intracellular aggregates isolated from transfected cells cultured at 37 °C, using Triton X-100, a surfactant that can solubilize all but the most insoluble of aggregates (Z. Zhou & Vollrath, 1999). A tetracycline-inducible HEK cell model expressing Y437H, I477N, or WT myocilin has also been valuable in dissecting cellular defects of mutant myocilins (Joe & Tomarev, 2010; Amirthaa Suntharalingam et al., 2012).

Histochemical analysis and animal models

For a few myocilin variants, ocular tissues from singular affected patient have undergone histochemical analysis (Hamanaka et al., 2017; van der Heide et al., 2018). In terms of animal models, several myocilin glaucoma rodent models have been attempted, using a variety of approaches: inducible models using different promotors, transgenic mice with varying methods, and different genetic backgrounds. Briefly, these models include a myocilin knock out mouse (Kim et al., 2001), mice overexpressing WT myocilin (Gould et al., 2004; Shepard et al., 2007), mice expressing Tyr423His mutant myocilin, equivalent to Tyr437His in humans (Gould et al., 2006; Senatorov et al., 2006), a variety of mice expressing human Tyr437His myocilin (Chou, Tomarev, & Porciatti, 2014; Shepard et al., 2007; Y. Zhou, Grinchuk, & Tomarev, 2008; Zillig et al., 2005; Zode et al., 2011), a mouse expressing human Tyr437His myocilin also deficient in SOD2 (Joe, Nakaya, Abu-Asab, & Tomarev, 2015), a Tyr435His myocilin rat, equivalent to Tyr437His in humans (Lynch et al., 2018), and human myocilin Gly364Val, Ser502Pro, and Gln368X introduced into a mouse eye by adenovirus (Shepard et al., 2007). In general, severity of glaucoma in rodent models expressing mutant myocilins is less than that found in humans. The reasons for this are not yet clear, but genetic background (McDowell et al., 2012), inherent aggregation kinetics of mouse myocilin (Patterson-Orazem et al., 2019), and subtle anatomical differences are possibilities.

Biophysical assays

Biophysical studies are conducted on an isolated, purified OLF domain construct and corresponding point variants introduced by site directed mutagenesis (Burns et al., 2010; Burns, Turnage, Walker, & Lieberman, 2011). To date, nearly 40 variants of the isolated OLF domain have been characterized across different studies. One measure is protein thermal stability, with the T_m defined as the midway point of unfolding, usually measured by differential scanning fluorimetry (Burns et al., 2010; Burns et al., 2011). The T_m of WT OLF is typical of a human protein, 52.7 °C (Burns et al., 2011), with slight variability depending on the buffer (Donegan, Hill, Turnage, Orwig, & Lieberman, 2012). Biophysical studies have clarified that intracellular sequestration arises because glaucoma-associated variants are thermally destabilized (Burns et al., 2011). A second metric is fibrillization of the OLF domain under mild conditions (e.g. pH 7.5, 37 °C), as measured with the amyloid dye thioflavin T (ThT) and corroborated by numerous other experiments (Hill, Donegan, & Lieberman, 2014; Orwig et al., 2012). Disease variants aggregate with faster kinetics than WT OLF (Hill et al., 2014).

Structural data

The OLF crystal structure revealed a β-propeller fold in which five so-called “blades,” (labeled Blades A-E) are composed of four antiparallel β-strands (Donegan et al., 2015) (Fig. 2). β-propellers are unusual in that they do not have the typical stabilizing “hydrophobic core” found in globular proteins. The propeller blades wrap around a central hydrophilic cavity creating a hydrophobic “belt” primarily between the middle hairpins of the β-sheet blades, where packing of hydrophobic residues is observed. The N-and C-terminal strands form part of the final blade in the feature called a molecular clasp. A single disulfide bond between two residues near the start (Cys 245) and end (Cys 433) further stabilize the OLF structure. In the central hydrophilic cavity there is a calcium ion and another metal ion modeled as a sodium, with ligands derived from side chain residues derived from different blades. Connecting Blade B to Blade C is a long loop of 19 residues, and at the interface between Blades C and D there is an unusual stabilizing cation-π interaction formed between a buried Lys423 and Tyr371, which is connected by polar interaction with the calcium site and appears to be stabilized by residues found on the aforementioned long loop.

Figure 2. Overview of the OLF propeller structure.

Four antiparallel beta strands form each of five blades, labeled A-E. Hydrophobic “belt” largely between middle strands of each blade is represented by grey taurus. Other features described in text, long loop connecting Blade B to Blade C (Glu359-Asp378), metal site with coordinating ligands, side helix adjacent to Blade A (Asp302-Tyr311), and molecular clasp in Blade E (Gly244-Arg258 and Met494-Ser502), as illustrated separately in best-view orientation.

A starting point for evaluating consequences of OLF mutations in the absence of other data would be based on their likelihood of affecting one of the aforementioned main structural features of the OLF domain. In general, though not always (Hill, Cho, Raut, & Lieberman, 2019; Hill, Kwon, et al., 2019), structure-based physicochemical intuition provides a rationale for a deleterious prediction. For example, modifications to surface loops lacking discrete intermolecular interactions with other residues in the domain are likely to tolerate mutations more readily than changes internal to the structure where the substitution interferes with the native packing and intermolecular interactions. Mutations within OLF residues comprising the β-sheet belt by changing its polarity would be expected to be incompatible with hydrophobic packing and thus compromise the stability of the protein. Substitutions that impair the stability of the molecular clasp, weaken or abolish the disulfide bond, remove a metal ion ligand, disrupt the cation-π interaction, or destabilize the well-ordered long loop are likewise expected to be problematic. Knowledge of the extent of sequence conservation at each position can further support tolerance of other amino acids at a given position. For each variant described herein, we visualized the residue in PyMOL in the context of the overall propeller. Then we inspected the local environment to assess polar and nonpolar non-covalent interactions with neighboring residues. With the aid of the mutation wizard within PyMOL, the residue of interest was then mutated to the documented variant and the process of inspecting prospective interactions was repeated.

Selection of variants

We identified variants that appear in at least one literature report (Supp. Figure S1, Table 1) and focused on the 97 tabulated in Table 2, Supp. Table S1, and Supp. Table S2. GnomAD allele counts and frequency as of the submission of this manuscript are included where available but we did not include detailed analysis of variants that only appear in gnomAD (Supp. Table S3). For some amino acid positions, more than one variant with clinical and laboratory data is listed (Table 2, 3, Supp. Table S2). Those listed as ‘additional variants’ include variants found only in gnomAD (Table 3) or ones that have only clinical data from a single literature source (Table 2, Supp. Table S2). The predicted pathogenicity for such additional variants does not necessarily match that of the primary variant.

Table 3.

gnomAD allele counts and frequency for different categories of myocilin (Refseq NM_000261.2) variants

Position	Additional variant	Allele Count	Allele Frequency	Polyphen Prediction1	Sift prediction1
Benign
p.Thr293Lys		157	5.60e-04	Benign	Tolerated
	p.Thr293Ala	1	4.02e-6	Benign	Tolerated
	p.Thr293Met	3	1.21e-5	Probably_damaging	Deleterious
p.Val329Met		72	2.55e-04	Possibly_damaging	Tolerated
p.Glu352Lys		337	1.19e-03	Probably_damaging	Deleterious
p.Thr353Ile		191	6.75e-04	Benign	Deleterious
p.Lys398Arg		992	3.51e-03	Benign	Tolerated
p.Ala445Val		57	2.01e-04	Benign	Deleterious
p.Lys500Arg		161	5.69e-04	Benign	Tolerated
	p.Lys500Thr	1	3.98e-6	Benign	Tolerated
Pathogenic
p.Gly252Arg		N/A	N/A
	p.Gly252Glu	2	8.25e-06	Probably damaging	Deleterious
	p.Gly252Ala	3	1.15e-05	Probably damaging	Deleterious
p.Arg272Gly		N/A	N/A
	p.Arg272Gln	2	8.01e-06	Benign	Tolerated
	p.Arg272*	2	8.02e-06	High confidence loss of function
p.Glu323Lys		N/A	N/A
	p.Glu323_Val 329dup	1	3.98e-06
p.Gly364Val		N/A	N/A
	p.Gy364Ser	2	7.95e-06	Probably damaging	Tolerated
p.Pro370Leu		N/A	N/A
	p.Pro370Ser	1	3.19e-05	Probably damaging	Deleterious
p.Thr377Met		5	1.99e-05	Probably_damaging	Deleterious
	p.Thr377Ala	2	7.96e-6	Probably_damaging	Deleterious
p.Pro481Thr		N/A	N/A
	p.Pro481Ser	1	3.98e-06	Possibly damaging	Tolerated
Likely Pathogenic
p.Val251Ala		N/A	N/A
	p.Val251Ile	1	4.14e-06	Possibly damaging	Tolerated
p.Leu255Pro		N/A	N/A
	p.Leu255Val	3	1.23e-05	Benign	Tolerated
p.Pro274Arg		N/A	N/A
	p.Pro274Leu	1	4e-06	Probably damaging	Deleterious
p.Thr438Ile		N/A	N/A
	p.Thr438Pro	6	2.39e-05	Probably damaging	Deleterious
p.Asn450Tyr		N/A	N/A
	p.Asn450Ser	2	7.95e-06	Probably damaging	Deleterious
Uncertain Significance—Lean Pathogenic
p.Gly244Val		6	2.51e-05	Probably_damaging	Deleterious
	p.Gly244Arg	2	7.95e-6	Possibly_damaging	Deleterious
p.Thr285Met		7	2.80e-05	Probably_damaging	Deleterious
	p.Thr285Pro	1	4e-6	Probably_damaging	Deleterious
p.Trp286Arg		2	8.01e-06	Probably_damaging	Deleterious
p.Gly300Lys		10	3.58e-05	Possibly_damaging	Tolerated
p.Gly326Ser		1	3.98e-06	Probably_damaging	Deleterious
p.Leu334Pro		3	1.19e-05	Probably_damaging	Deleterious
	p.Leu334Val	1	3.98e-6	Probably_damaging	Deleterious
p.Ile360Asn		N/A	N/A
	p.Ile360Leu	1	3.98e-06	Benign	Tolerated
p.Gly387Asp		3	1.19e-05	Possibly_damaging	Deleterious
p.Asp395_Glu396insAspPro		N/A	N/A
	p.Asp395Glu	5	1.99e-05	Benign	Tolerated
	p.Asp395Asn	3	1.19e-05	Benign	Tolerated
p.Gly399Asp		N/A	N/A
	p.Gly399Val	4	1.59e-5	Probably_damaging	Deleterious
p.Ser425Pro		N/A	N/A
	p.Ser425*	1	3.98e-06	High confidence loss of function
p.Phe430Leu		N/A	N/A
	p.Phe430Ser	1	3.98e-06	Probably damaging	Deleterious
p.Arg470Cys		6	3.31e-05	Benign	Tolerated
	p.Arg470His	3	1.19e-5	Benign	Tolerated
	p.Arg470Leu	1	3.19e-5	Benign	Deleterious
Uncertain Significance—Lean Benign
p.Thr256Met		23	8.36e-05	Possibly_damaging	Tolerated
	p.Thr256Ala	1	4.11e-6	Benign	Tolerated
	p.Thr256Arg	3	1.23e-05	Probably damaging	Deleterious
p.Arg296Cys		4	1.61e-5	Probably_damaging	Deleterious
	p.Arg296His	17	6.1e-5	Possibly_damaging	Deleterious
	p.Arg296Leu	1	4.04e-6	Possibly_damaging	Deleterious
p.Gln297His		N/A	N/A
	p.Gln297Arg	1	4.03e-06	Benign	Tolerated
p.Leu303Ile		17	6.08e-05	Benign	Tolerated
	p.Leu303Phe	4	1.43e-5	Benign	Tolerated
p.Ser313Phe		17	6.05e-05	Probably_damaging	Tolerated
	p.Ser313Cys	2	7.11e-6	Probably_damaging	Deleterious
p.Ser331Thr		N/A	N/A
	p.Ser331Leu	12	4.24e-05	Benign	Tolerated
p.Ser333Cys		N/A	N/A
	p.Ser333Asn	5	1.99e-05	Possibly damaging	Deleterious
p.Arg342Lys		N/A	N/A
	p.Arg342Gly	3	1.19e-05	Benign	Deleterious
p.Ile345Met		N/A	N/A
	p.Ile345Thr	1	3.98e-06	Benign	Tolerated
p.Thr419Ala		1	3.98e-06	Probably_damaging	Deleterious
p.Arg422Cys		8	3.18e-5	Probably_damaging	Deleterious
	p.Arg422His	8	3.18e-5	Benign	Tolerated
p.Ala427Thr		12	4.77e-05	Probably_damaging	Deleterious
p.Gly434Ser		1	3.98e-06	Possibly_damaging	Deleterious
p.Asp446Tyr		8	2.83e-05	Possibly_damaging	Deleterious
	p.Asp446His	1	3.98e-6	Benign	Tolerated
p.Ala447Thr		N/A	N/A
	p.Ala447Val	2	7.95e-06	Possibly_damaging	Deleterious
p.Tyr471Cys		7	2.78e-05	Probably_damaging	Deleterious
p.Met476Arg		3	1.19e-05	Probably_damaging	Deleterious
	p.Met476Leu	1	3.98e-6	Possibly_damaging	Deleterious
Uncertain Significance—Premature termination
p.Gln337Argfs*9		N/A	N/A
	p.Gln337His	1	3.98e-6	Probably_damaging	Deleterious
p.Gln368*		314	1.11e-03	High confidence loss of function
Tyr453Metsf*11		20	7.07e-5	High confidence loss of function

¹As reported within gnomAD (https://gnomad.broadinstitute.org/gene/ENSG00000034971?dataset=gnomad_r2_1)

Pathogenicity categories

Pathogenicity categories for myocilin variants were defined as recommended by the ACMG- American College of Medical Genetics and Association for Molecular Pathology (Richards et al., 2015)- except for the large category of Uncertain Significance, which we batched further into lean pathogenic, lean benign, and premature termination. The associated criteria for defined categories are presented here, Table 1 additionally lists the variants for each category. For each variant (Table 2, Supp. Table S2), we include detailed available data (Supp. Table S1) that support its inclusion in the given category.

Benign

Seven variants were identified with a high allele count (>50) and frequency (>2e-4) in gnomAD (ENSG00000034971), all missense. Many have been identified in POAG patients in the literature but given their high prevalence in the general population and laboratory data demonstrating key similarities with WT OLF, these are best annotated as benign (Fig. 3A). All but K500R have confirmed WT-like secretion and stability, suggesting that these variants are not causative for glaucoma despite having been documented among glaucoma patients.

Figure 3. Categories of variants mapped onto OLF structure.

See also Table 1. (A) Benign: sites with high frequency variations from gnomAD (>50 allele count, allele frequency >2e-4) and wild-type like laboratory properties. (B) Pathogenic: sites with strongest clinical and laboratory experimental misfolding support. (C) Likely pathogenic: sites with strong clinical but incomplete laboratory support. (D) Uncertain Significance - Lean pathogenic: sites with limited clinical support and incomplete laboratory support. (E) Uncertain Significance- Lean benign: sites clinical findings in control group, incomplete laboratory support. (F) Uncertain Significance- premature termination: premature stop codons result in incomplete translation of OLF protein.

Pathogenic

Twenty-three missense variants and one indel variant have strong support for pathogenicity (Fig. 3B): a confirmed familial inheritance pattern, additional clinical data indicative of early-onset POAG. Nearly variants all are absent from gnomAD, one or more labs have confirmed cellular defects, and our lab has demonstrated stability defects for all but two. In agreement with these features, structural features are not predicted to be tolerated. For example, for Pro to Leu substitutions, eg. Pro481Leu, prolines are known to be important for loops and turns in proteins (Chothia, Gelfand, & Kister, 1998), and substitutions are unlikely to be well tolerated, as seen in other heritable disorders (Darin et al., 2016).

Likely pathogenic

Thirteen missense variants have clinical data that support early onset, familial POAG or JOAG (Fig. 3C). For these, laboratory analyses supporting a misfolding phenotype are largely missing, but intuition based on effects of mutation on local structure generally support the inference that the effect of mutation would be deleterious, leading to a mutant protein with a pathogenic misfolding phenotype. Pathogenic assignments would be strengthened with laboratory studies.

Uncertain significance

For the remaining ~50% variants we considered (53/97), assigning pathogenicity is not straightforward. For these, clinical and segregation data is limited to one report in a small family, or not available because the variant was only detected in large scale genetics studies, and no biochemical data exists. Sixteen missense and two indel variants of uncertain significance binned as lean pathogenic (Fig. 3D) have clinical data indicative of POAG association, but not necessarily Mendelian inheritance patterns and/or early age of diagnosis. Laboratory data are incomplete. Our structural intuition generally suggests pathogenic. Twenty-eight variants clustered as lean benign (Fig. 3E) have clinical data supporting a benign variant designation, namely, being identified in a control population or having glaucoma onset significantly later than age 40. Biochemical data, where present, do not suggest a strong misfolding phenotype and intuition generally suggests these substitutions are unlikely to affect the OLF structure and stability.

Finally, we aggregate 4 indel and 3 missense variants involving premature termination (Fig. 3F) that are described in the literature. A total of 24 premature termination heterozygous variants within OLF are known (18 in gnomAD, 6 additional listed in the literature), including the well-studied and prevalent Gln368* (~ 0.1% allele frequency) and the relatively common frameshift mutation Tyr453Metfs*11 (0.071% allele frequency) that also leads to premature termination. These variants may prove pathogenic with additional studies, but likely by a different mechanism than pathogenic missense variants that adopt a destabilized but near-native folded state. When premature termination is introduced in the middle of the protein e.g. for Gln337Argfs*9, Gly362Glufs*45, Gln368*, one or more β-strands within the OLF domain propeller cannot form, preventing any native-like folded structure from being adopted. Gln368* and likely other mid-OLF termination variants share laboratory characteristics of missense variants: the intracellular deposits (Gobeil et al., 2006) exhibit a dominant negative effect with respect to WT myocilin (Gobeil et al., 2004) and are insoluble in Triton X-100 (Shimizu et al., 2000). Such non-folding termination variants would likely not reach the stage of protein quality control involving Grp94 (D. J. E. Huard et al., 2018; Huard, Jonke, Torres, & Lieberman, 2019; Marzec et al., 2012; Stothert et al., 2014; Amirthaa Suntharalingam et al., 2012) and thus be identified by different ER chaperones (Ellgaard, McCaul, Chatsisvili, & Braakman, 2016), leading to different consequences. Further laboratory investigations of termination variants would be informative. In addition, recent finding that Gln368* is pathogenic in the presence of other age-onset glaucoma susceptibility genes (Craig et al., 2020) suggests that pathogenicity of other premature stop or indel variants could be further modulated by considering the broader context of a given individual.

The complex structure-misfolding relationship for OLF missense variants

Pathogenic variants are destabilized (Burns et al., 2010; Burns et al., 2011; Donegan et al., 2015), a feature that explains their misfolding propensity and downstream GOF pathogenic mechanism. However, as underscored by the numerous surprises we encountered in the lab (Lieberman & Ma, 2021), the effect of a mutation on OLF structure and stability cannot be predicted with a high degree of confidence, despite applying reasonable physicochemical intuition. Sequence conservation mapped onto the structure demonstrates that across the OLF sequence, the surface residues are least conserved and internal sequences are largely conserved (Donegan et al., 2015). Based on this observation, substitution of residues found on the interior of the OLF propeller would be expected to be poorly tolerated. Unexpectedly, we increased OLF stability by introducing single or double-point variants at relatively conserved internal positions corresponding to calcium ligands, which should not have been tolerated (Hill, Cho, et al., 2019; Hill, Kwon, et al., 2019). One variant we did not study in the lab but has clinically been found only in control subjects, N428S, may be similarly stabilizing, or perhaps additional functional insights will reveal a loss of function relevant to glaucoma. Finally, we computationally engineered the OLF domain to be significantly higher stability by changing ~10% of its sequence with a range of extent of conservation (Hill, Kwon, et al., 2019) and the structure of this metallated, 21-variant of OLF was unchanged compared to wild-type (Hill, Kwon, et al., 2019).

Related, GOF is not readily inferred from sequence conservation and protein structure considerations used traditionally to computationally predict the consequence of a missense change (Eilbeck, Quinlan, & Yandell, 2017) and OLF is no exception. Even if loss of function for myocilin plays a role in glaucoma, the role of WT myocilin remains unknown and is apparently not essential (Gould et al., 2006; Kim et al., 2001; Lam et al., 2000). Newer algorithms like SIFT (Vaser, Adusumalli, Leng, Sikic, & Ng, 2016) and Polyphen (Adzhubei, Jordan, & Sunyaev, 2013), whose analyses are conveniently included in gnomAD and integrate population allele frequency data along with gene conservation and other constraints, predict more missense OLF variants to be damaging compared to our analysis (Table 3), as for other GOF diseases (Flanagan, Patch, & Ellard, 2010). Five of our benign variants were predicted to be damaging by one or both prediction programs. The jury is still out for the effect of most rare variants lacking complete data, but Glu352Lys, which is predicted to be damaging by both programs, has the second highest allele frequency within the OLF domain plus clinical and laboratory data that do not align with a misfolding phenotype. OLF may be an especially challenging case for predicting destabilization leading to a GOF.

Summary and Future Directions

Myocilin-associated glaucoma is an excellent example of how the acceleration of large scale sequencing data resulting in a catalog of genetic variation, in combination with recent laboratory and clinical data, adds new complexities in interpreting mutations in the context of a disease with a high burden throughout the world. Glaucoma is an age-onset, multifactorial disease and myocilin variants are not pathogenic without significant accompanying data and analysis. Given the importance of myocilin in the TM tissue that underlies IOP maintenance, and the potential for new medicines once a solid pathophysiological understanding of myocilin-associated glaucoma is established, there is a need to confidently classify myocilin variants.

New variants are expected to arise as additional genome sequencing data becomes available. Here we have considered the current exhaustive list of clinically characterized OLF variants and associated laboratory data, yet these are just ~10% of the total theoretical number of possible single nucleotide variants of the OLF domain, which is closer to ~1500. Research over the past 20 years have made significant inroads correlating clinical findings and biological attributes for a particular variant that can be used to differentiate likely benign and misfolding variants. Going forward, laboratory approaches yielding large datasets may prove effective. For example, one-pot mutagenesis library methods coupled to high throughput secretion assays could provide intracellular sequestration and thermal stability data for the remaining single point variants. In turn, such variants could be properly flagged for potential follow up, if detected genetically.

In the long term, it is tantalizing to think about how complete data could be used with machine learning algorithms to triangulate information for confident variant assessment, and then to tailor effective treatments as being considered for other systems (C. Wang & Balch, 2018). To this end, mechanistic studies are needed to clarify the extent to which different rare GOF myocilin mutations lead to POAG by a similar mechanism, whether there is a specific auxiliary pathogenic role for premature termination variants, why animal models generated thus far do not faithfully replicate disease severity observed in humans, reconcile occasional clinical observations that do not follow expected pathogenicity e.g. a 52 year old patient carrying Asp380Ala (Stoilova et al., 1998), or homozygous carriers of Lys423Glu (Morissette et al., 1998) who do not display glaucoma symptoms, and to what extent myocilin dysfunction plays a role in idiopathic glaucoma. The multidisciplinary approach combining large scale gene sequencing, clinical data, and biological studies will reveal, and hopefully resolve, complexities of this fascinating misfolding-prone protein and its contribution to glaucoma pathogenesis.

Data availability

Data sharing not applicable – no new data generated. Clinical data collected in Supplemental Data Table S1.