The role of SIX6 gene in juvenile open-angle glaucoma: a subtle contributor to the mutational landscape.

Purpose: Juvenile onset open-angle glaucoma (JOAG) typically affects individuals under 40 years of age, causing a rise in intraocular pressure that results in considerable damage to the optic nerve. To expand the spectrum of mutations linked to JOAG and explore their structural consequences, we examined the genetic alterations within the SIX6 gene.

Study design: We focused on assessing the whole coding region of the SIX6 gene and the clinical significance of the common SIX6 gene single nucleotide polymorphisms (SNP) rs10483727 and rs33912345, specifically analyzing its association with key clinical factors, including intraocular pressure (IOP), visual acuity, and the vertical cup-to-disc ratio (VCDR).

Method: The study included 111 unrelated patients with confirmed JOAG and 100 healthy adult controls without any ocular or systemic conditions. The patients initially underwent genetic screening for pathogenic variants in the CYP1B1, MYOC, and OPTN genes. Individuals who tested negative for pathogenic variants in these three genes (n=81) were subsequently screened for variants in the SIX6 gene while all 111 patients and 100 control samples were screened for SNPs to strengthen the statistical correlation. Identified sequence variations were searched in the ClinVar databases, HGMD, and dbSNP. Six different online available algorithms including REVEL, SIFT, CADD, Mutation Taster, IMutant2.0, and MetaLR were used for the pathogenicity prediction of missense variations. The Structural consequences of detected possible pathogenic variations were predicted by using PyMol, Chimera and molecular dynamics (MD) simulation of these changes. Statistical analyses assessed the association of rs33912345 with disease phenotypes.

Results: Sanger sequencing identified seven nucleotide variants in the SIX6 gene, including five missense and two synonymous variants. Two missense variants, p.(A99G) and p.(S156R), were predicted to be pathogenic. The novel variant p.(A99G) was absent in controls and demonstrated significant structural disruption, with altered intramolecular interactions and steric clashes. Homology analysis revealed high evolutionary conservation at the mutated positions, highlighting their functional significance. MD simulations confirmed altered stability in the mutant proteins. Logistic regression linked rs33912345 to JOAG, with significant associations observed for IOP (p = 0.01538), and VCDR (p = 0.019).

Conclusion: This study identified novel and known pathogenic variants in the SIX6 gene that may contribute to JOAG pathogenesis. Structural and functional analyses suggest that these mutations disrupt protein function. The findings enhance our understanding of JOAG genetics and may aid in early diagnosis and therapeutic targeting.