A total of eleven missense variants, one frameshift variant and, five synonymous/neutral variants were identified in this study (Table 2)(chromatograms and pedigree included as supplement data). Variants identified in intron and untranslated regions (both 5’ and 3’) have not been considered. Details of all identified variants, associated amino acid variants, predictions of their pathogenicity (only for missense variants) and, other information is given in Table 3. The variants identified included:
1. p.Gln48His (g.171652468G>T):
This variant is observed as a heterozygous variant in JOAG patient P28 but not observed in controls. This variant is present in ClinVar and gnomADv2.1.1 as a pathogenic variant.
2. p.Arg76Lys (g.171652385G>A):
This variant is observed as a homozygous variant in eleven JOAG patients and heterozygous variant in thirteen JOAG patients (Table 2). This variant is also observed in control samples. This variant is present in ClinVar and gnomADv2.1.1 database with conflicting interpretations of pathogenicity.
3. p.Gly122Ala (g.171652247G>C):
This variant is observed as a homozygous variant in one JOAG patient (P12) but not observed in the control samples. This is a novel variant and has been registered in GenBank with accession no. MT126741
4. p.Arg136Ile (g.171652205G>T) :
This variant is observed as a heterozygous variant in JOAG patient P13 but not observed in the control samples. This variant was found to be a novel and has been registered in GenBank with accession no. MT126742.
5. p.Ser173Thr (g.171652094G>C):
This variant was observed as a heterozygous variant JOAG patient P45, and not observed in control samples. This variant was also found to be a novel and registered in GenBank with accession no. MT126743.
6. Compound heterozygous substitution/ p.[(Leu215Pro; Lys216Ile)] (g.171607823T>C,g.171607822G>C, g.171607820A>T, g.171607819G>T):
A compound heterozygous complex substitution was present in a JOAG patient P7 with a positive family history. This variant is also novel and has been registered in GenBank with accession no. MT126744. This variant is not observed in controls and databases.
7. p.Ser238Asn (g.171638614G>A):
This is observed as a homozygous variant in two JOAG patients (P24 and P36) but not observed in the control samples. This variant is not present in ClinVar and gnomADv2.1.1
8. Pro370Leu (g.171605471C>T):
This variant is identified in three JOAG patients (P5, P12, and P42). P5 had a positive history (in supplementary data) of the disease on her paternal side, but P12 and P42 were sporadic cases. This variant is present in ClinVar as a pathogenic variant.
9. Arg422His (g.171636175G>A):
This variant is observed as a heterozygous variant in one JOAG patient (P36) but not present in controls. This variant is present in gnomADv2.1.1 but absent in ClinVar.
10. Frameshift mutation:
One frameshift mutation p.Arg200Lysfs*15 also identified in the patient group. This mutation is predicted to produce a truncated myocilin protein of 214 amino acids with altered amino acids from position 200 to 214 due to the frameshift. This variant is identified as a homozygous change in JOAG patient P45 but is absent in controls. This variant is a novel mutation and has been registered in GenBank with accession no. MT126745.
Following five neutral sequence variants are also identified:
1. p.Glu14= (g.171652570G>A)
This variant is present as a heterozygote in three JOAG patients (P37, P38, and P39) but absent in controls. This variant is present in gnomADv2.1.1 as a synonymous variant.
2. p.Lys156= (g.171652144G>A)
This variant is present as a heterozygote in one JOAG patient P39 but absent in controls. This variant is not present in gnomADv2.1.1
3. p.Tyr347= (g.171636399T>C)
This variant is present as a heterozygote in one JOAG patient P67 but absent in controls. This variant is present in gnomADv2.1.1 as a benign synonymous variant.
4 p.Phe430= (g.171636150C>T)
This variant is present as a heterozygote in one JOAG patient P61 but absent in controls. This variant is present in gnomADv2.1.1 as a synonymous variant.
4. p.Lys484= (g.171635988G>A)
This is present as a homozygous variant in four JOAG patients (P26, P43, P47, P48) and a heterozygous variant in twenty-three JOAG patients (Table 2). This variant was also present in controls. This variant is present in gnomADv2.1.1 as a synonymous variant.
A total of 80 JOAG patients and 100 controls individuals have been enrolled in the current study. Of the 80 patients, 27 were females, 53 were males, and the onset age of glaucoma varied from 5 years to 38 years. The most common presenting complaints were heaviness of the forehead and vision problems. DNA sequencing analysis identified pathogenic MYOC variants in six out of eighty (7.5%) JOAG patients. Three patients of these six patients are positive for more than one sequence variant. These are as follows:
Patient P7 was a male with the onset of glaucoma at the age of 25 years. He had four heterozygous complex substitutions in which (g.171607823T>C (Heterozygous), g.171607822G>C (Heterozygous), g.171607820A>T (Heterozygous), g.171607819G>T (Heterozygous) substituted by other bases. This variant produced codon CTG>CCC and AAG>ATT, which resulted in the amino acid changes p.[(L215P; K216I)]. His IOP at diagnosis was 22mmHg in both eyes (Table 2). He was presented with a headache and diminished vision, having a cup/disc ratio of 0.9:1 in both eyes. He underwent glaucoma surgery for both eyes in 2013, 2014, and 2016. He currently has vision 3/60and, 1/60 in the right and left eyes, respectively, and his current visual field is VF<100. One additional sibling of the proband has bilateral glaucoma, and his mother (now deceased) was also having glaucoma in both eyes. After multiple attempts, we could not get a blood sample from proband’s brother and unable to confirm that he might be harbouring the same variants.
Patient P12 had compound heterozygous p.[(G122A; P370L)] mutations. Patient P12 was presented at 21 years of age with the heaviness of the forehead, vision problems, and redness of his eyes. He had IOP of 22 and 25 mmHg in the right and left eyes in conjunction with a cup disc ratio of 0.9:1 in the right eye and 0.8:1 in the left eye, respectively. He has no vision in the right eye, and the vision in his left eye is 6/18.
Patient P45 was a female who first presented in the ocular clinic at the age of 26 years. The patient had symptoms of vision loss, watering, and redness of her eyes. Her latest cup/disc ratio was 0.7:1 in both eyes with a vision of 6/36 in the right and 6/18 in the left eye, respectively. She had a deletion of guanine at genomic position g.171652013, predicted to result in p.R200Kfs*15, resulting in an abbreviated protein of 214 amino acids (Figure 1). Since this patient also harbours p.S173T variant, a truncated protein of 215 amino acids produced with threonine at position 173 and 14 novel amino acids (from 200-214) will be different from wild type protein as a result of frameshift (shown in red color in Figure 1). This truncated protein might act as a mutated myocilin protein forming protein aggregates responsible for JOAG.
Patients P5 and P42 are heterozygous for already reported pathogenic p.P370L (g.171605471C>T) variant (Table 2). Patient P5 was a female, who presented at the ocular clinic with complaints of the heaviness of her forehead and vision loss at the age of 30. She had an IOP of 26 and 34 mmHg in the right and left eyes and optic cupping of 0.8:1 and 0.9:1 in the right and left eye, respectively. She had a vision of 6/18 in both eyes. She also underwent glaucoma surgery. Patient P42 was a male who presented at the ocular clinic at the age of 32 years with complaints of halos around light and scattered vision. He had IOPs of 28 and 22 mmHg in the right and left eye, respectively with a cup disc ratio of 0.8:1 in both eyes. He had a vision of 6/36 in both eyes in his most recent clinical examination.
Patient P36 is harbouring p.R422H (g.171636175G>A) variant. She is presented to the ocular clinic at the age of 24 years with the complaints of the heaviness of her forehead, watering in both eyes, with vision problems. She had an IOP of 28 mmHg in both eyes and visual acuities of 6/24 and 6/60 in the right and left eyes, respectively. The pathogenicity of p.R422H is uncertain because it shows an ambiguous pathogenicity prediction in which PolyPhen2 and Mutation taster predict it to be probably damaging and disease-causing. But it is predicted to be tolerated by SIFT and neutral by PROVEAN. Functional analysis of p.R422H mutation essentially required for a final verdict on its pathogenicity.
The presence of the p.Q48H (g.171652468G>T) mutation had already been observed in the JOAG/POAG/PCG cases in earlier glaucoma studies but never reported in controls [19-23].This variant lies in the N-terminal domain of the myocilin protein. A Triton-X-100 assay done using recombinant myocilin containing a histidine residue at the 48th position showed protein aggregation, confirming the deleterious effect of the p.R48H variant [24]. The p.Arg76Lys reported as a non-pathogenic variant in earlier glaucoma studies [19,25,26]. The same message conveyed by its presence in both patients and controls in this study as well [19,25,26].
Myocilin variants predicted to be deleterious/pathogenic by all four programs are p.[(Leu215Pro;Lys216Ile)] and p.Pro370Leu. The position of p.P370L lies in the C-terminal domain of myocilin. It has been reported as a disease-causing variant in several glaucoma studies.[30-32] Transfection of human trabecular meshwork (TM) cells with the p.P370L myocilin mutant resulted in increased levels of endogenous reactive oxygen species (ROS and reduced ATP production, and increased cell death [18]. Sakai et al. (2007) reported that the myocilin protein enters into the mitochondria with a high molecular weight translocator complex in the outer and inner mitochondrial membranes and that myocilin mutations alter the protein structure and its confirmation [31,32]. The p.P370L mutation in the myocilin protein increases the mitochondria's sensitivity to various cellular injuries in the TM cells. It also disables normal TM cell functions and eventually contributes to the TM's failure to control IOP and glaucoma pathogenesis [18].