Whole Exome Sequencing (WES)
Blood samples were withdrawn from the four children, the parents, and the paternal grandmother after informed consent was taken from the parents of the children. The study was carried out according to the tenets of the declaration of Helsinki, and Ethics committee approval was obtained from our Institutes ethics committee. A signed informed consent according to the guidelines of the Institute Ethics Committee was obtained.
Genomic DNA was extracted from peripheral blood lymphocytes using PAXgene Blood DNA Kit (Qiagen, Germany) for genetic evaluation. DNA was run on 0.8% agarose gel to check for quality and quantified by Nanodrop. WES capture was performed using the Sure Select Clinical Research Exome V2 kit (Agilent Technologies, Santa Clara, CA). Variant analysis was performed using the GenomeAnalysisTK-3.6 toolkit. The variant call files (VCF), containing the variant call results, generated were analyzed using Golden Helix VarSeq Software v.1.2.1 (Bozeman, MT). VarSeq variants with read depth <15 and genotype quality score <20 were excluded. To identify rare mutations, variant frequency databases were used to remove variants that are present at high frequencies among large population groups. The remaining variants were filtered according to minor allele frequency (MAF) <0.001 in multiple databases, including Exome Aggregation Consortium (ExAC) (http://exac.broadinstitute.org/), 1000 Genomes Project (http://browser.1000genomes.org) and gnomAD (http://gnomad.broadinstitute.org/).
Variants specifically in the two sisters with glaucoma were filtered based on only those exonic variants (Non-synonymous missense variants, frameshift and indels and splice region variants) that were present exclusively in the two sisters and absent in the other family members and 20 healthy controls. Inherited variants that were present in both the affected sisters with congenital glaucoma and one of the parents were also looked at, for any association with the known glaucoma genes. Copy number variant (CNV) analysis was performed to look for CNV in the known genes for glaucoma or others with a strong association with glaucoma pathogenesis. The CNV’s were annotated with RefSeq gene annotations and Database of Genomic Variants v107. The CNV’s were then classified to assess the pathogenicity using classify CNV. The CNV’s common in both affected individuals were visually inspected using IGV.
We used Homozygosity mapping to identify long stretches of homozygous haplotypes in genes associated with glaucoma. Regions of Homozygosity (ROH) were identified by using the AutoMap software (Quinodoz et al., https://automap.iob.ch/) on WES data (Quinodoz et al. 2021).
Further functional impact of the protein was predicted by bioinformatic tools; variant effect predictor (VEP), Mutation Taster, Polyphen, and FATHMM. The identified variants were confirmed by Sanger sequencing.