Exploring the Genetic Frontier: Advances in Understanding Eye Disorders

         January 30, 2024

Welcome to the exploration of groundbreaking genetic research in ophthalmology. Recent studies have unveiled fascinating insights into the complex interplay between genetics and eye diseases, offering hope and new directions for understanding and treating these conditions. From novel genetic variants linked to microphthalmia and glaucoma to the long-term implications of cataract surgery in childhood, we’re diving into the depths of genetic influences on ocular health.

A De Novo Noncoding RARB Variant Associated with Complex Microphthalmia Alters a Putative Regulatory Element

Retinoic acid receptor beta (RARB) is a transcriptional regulator crucial for coordinating retinoic acid- (RA-) mediated morphogenic movements, cell growth, and differentiation during eye development. Loss- or gain-of-function RARB coding variants have been associated with microphthalmia, coloboma, and anterior segment defects. We identified a de novo variant c.157+1895G>A located within a conserved region (CR1) in the first intron of RARB in an individual with complex microphthalmia and significant global developmental delay. Based on the phenotypic overlap, we further investigated the possible effects of the variant on mRNA splicing and/or transcriptional regulation through in silico and functional studies. In silico analysis identified the possibility of alternative splicing, suggested by one out of three (HSF, SpliceAI, and MaxEntScan) splicing prediction programs, and a strong indication of regulatory function based on publicly available DNase hypersensitivity, histone modification, chromatin folding, and ChIP-seq data sets. Consistent with the predictions of SpliceAI and MaxEntScan, in vitro minigene assays showed no effect on RARB mRNA splicing. Evaluation of CR1 for a regulatory role using luciferase reporter assays in human lens epithelial cells demonstrated a significant increase in the activity of the RARB promoter in the presence of wild-type CR1. This activity was further significantly increased in the presence of CR1 carrying the c.157+1895G>A variant, suggesting that the variant may promote RARB overexpression in human cells. Induction of RARB overexpression in human lens epithelial cells resulted in increased cell proliferation and elevated expression of FOXC1, a known downstream target of RA signaling and a transcription factor whose down- and upregulation is associated with ocular phenotypes overlapping the RARB spectrum. These results support a regulatory role for the CR1 element and suggest that the de novo c.157+1895G>A variant affecting this region may alter the proper regulation of RARB and, as a result, its downstream genes, possibly leading to abnormal development.

“Trio exome sequencing was performed through Psomagen (Rockville, MD) and analyzed using SVS and VarSeq software (Golden Helix, Bozeman, MT) as previously described, including analysis of known ocular genes, OMIM genes, and copy number variation analysis [1718]. General population data was obtained from gnomAD (v2.1.1 and v3.1.2) [19] with variant annotations including Combined Annotation Dependent Depletion (CADD- v1.4) [20] and Genomic Evolutionary Rate Profiling (GERP) scores [2122] obtained through VarSeq or UCSC Genome Browser (http://genome.ucsc.edu) [2324]. Trio analysis of variants with a read , CADD , and  with <3 homozygotes (recessive) or fewer than 5 alleles (de novo, X-linked) in gnomAD v2 or v3 did not identify any strong candidates (Supplemental Table 1). Subsequently, trio genome sequencing was undertaken through Psomagen. Briefly, samples were prepared using the Illumina TruSeq Nano DNA library and sequenced using an Illumina HiSeq X sequencer. VCF and BAM files were generated using the Isaac Aligner and Isaac Variant Caller using human genome build hg19. Data was analyzed using VarSeq for rare variants fitting a de novo, X-linked, homozygous, or compound heterozygous inheritance pattern using the same criteria applied to exome data with the additional requirement of at least one coding variant for compound heterozygous. Variants of interest were manually reviewed, and low-quality or messy regions were excluded. The RARB variant was named based on reference sequence NM_000965.4. Pathogenicity was evaluated using several in silico prediction tools. Effect on splicing was assessed using Human Splicing Finder software (HSF; v3.1) [25], MaxEntScan [26], and SpliceAI (Gencode v36) [27]. Additional in silico analysis of the variant was completed in the UCSC Genome Browser (http://genome.ucsc.edu) [2324]. In order to maximize the additional tracks available for analysis, the coordinates of the variant were converted to hg38. Candidate cis-regulatory elements were identified through the ENCyclopedia of DNA Elements (ENCODE; [28] cCRE track) and confirmed through the H3K27Ac track; regions of chromatin interaction were identified with the Micro-C chromatin structure track [29]; regions associated with transcription factor DNA-binding profiles were identified with the JASPAR CORE collection (predicted) and ReMap (complied from over 7,500 publicly available ChIP-seq data sets) tracks; and sequence conservation was analyzed using PhyloP and Multiz Alignments of 100 Vertebrates tracks.”

Replogle, Maria R., Samuel Thompson, Linda M. Reis, and Elena V. Semina. “A De Novo Noncoding RARB Variant Associated with Complex Microphthalmia Alters a Putative Regulatory Element.” Human Mutation, vol. 2024, 2024, pp. 6619280. Hindawi, doi:10.1155/2024/6619280

Long-term risk of glaucoma after cataract surgery in childhood

Purpose: To examine the long-term risk of glaucoma after cataract surgery in childhood.

Methods: This study took place from January 2022 until December 2022 and included patients from a large family with hereditary childhood cataract who had cataract surgery before 18 years of age. Patients underwent an ophthalmologic examination to determine the presence of glaucoma or ocular hypertension (OHT). Patients who did not want to participate in the examination could contribute with a medical journal from their treating ophthalmologist. The risk of long-term glaucoma was determined using survival analysis, and risk factors were assessed using a Cox proportional hazards regression model.

Results: We included 68 patients (133 eyes) with a median age at cataract surgery of 7years (IQR: 5–10). The median follow-up time after cataract surgery to glaucoma/OHT or the latest ophthalmologic examination was 35years (IQR: 15–48). Twelve patients (18 eyes) had glaucoma, and five patients (eight eyes) had OHT, resulting in 15 patients with glaucoma/OHT. The long-term risk of glaucoma/OHT diagnosed in adulthood was 47.7% (CI: 21.8–70.9) at the age of 70years of patients who were free of glaucoma before their 18th year. We could not confirm or dismiss an association between glaucoma/OHT and sex, age at surgery, number of ocular interventions before 18years of age or glaucoma after cataract surgery in a first-degree relative.

Conclusion: Cataract surgery in childhood is associated with a high risk of lateonset glaucoma. Regular lifelong follow-up is important to ensure early diagnosis and prevent extensive vision loss.

“Individuals were analysed for the HSF4 variant NM_001538.4 (hg19) c.355C>T, p.Arg119Cys using next generation sequencing (NGS). For individuals between 4 and 17 years of age, a panel (SureSelect custom panel, Agilent, Santa Clara, CA, USA) targeted at genes associated with cataract were used, while for individuals above 18 years of age, an exome-based panel (TWIST exome 2.0, TWIST Bioscience, San Francisco, CA, USA) was used. MiSeq or NovaSeq platforms were used for sequencing (Illumina, San Diego, CA, USA), and either SureCall (Agilent) or GATK (Broad Institute) pipeline for alignment and variant calling. VarSeq (Golden Helix, Bozeman, MT, USA) was used for variant annotation and filtering. Data was analysed for the c.355C>T variant in HSF4.”

Schmidt, D.C., Eriksson, F., Bach-Holm, D., Grønskov, K. & Kessel, L. (2024) Long-term risk of glaucoma after cataract surgery in childhood. Acta Ophthalmologica, 00, 1–7. https://doi.org/10.1111/aos.16636

Distribution of TGFBI variants in patients with early onset glaucoma

Purpose: To describe a novel association of TGFBI variants with congenital glaucoma in a family with GAPO (growth retardation, alopecia, pseudoanodontia, and progressive optic atrophy) syndrome, as well as among other unrelated cases of juvenile onset open-angle glaucoma (JOAG) and primary congenital glaucoma (PCG).

Methods: This study of one family of GAPO with congenital glaucoma and three unrelated patients with JOAG analyzed a common link to glaucoma pathogenesis. Three girls with GAPO syndrome born to consanguineous parents in a multi-generation consanguineous family were identified. Two of the girls had congenital glaucoma in both eyes, while the elder sibling (a 10-year-old female) had features of GAPO syndrome without glaucoma.

Results: A genetic evaluation using whole exome sequencing revealed a novel homozygous ANTXR1 mutation in all three affected siblings with GAPO. No other mutations were detected in the genes associated with glaucoma. A rare missense variant in the TGFBI gene was shared in the two siblings with congenital glaucoma and GAPO syndrome. We found three other unrelated patients with JOAG and one patient with primary congenital glaucoma with no known glaucoma causing gene mutations, but having four different missense variants in the TGFBI gene. One of these patients with JOAG had familial granular corneal dystrophy. Molecular dynamic simulations of TGFBI and 3-D structural models of three of its variants showed significant alterations that could influence TGFBI protein function.

Conclusions: The possibility that variations in the TGFBI gene could have a possible role in the pathogenesis of congenital and juvenile onset open-angle glaucomas needs further evaluation.

“Genomic DNA was extracted from peripheral blood for genetic evaluation. 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 generated variant call files (VCFs) 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. Rare mutations were identified using variant frequency databases to remove variants 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), 1000 Genomes Project and gnomAD.”

Gupta, Viney, et al. “Distribution of TGFBI variants in patients with early onset glaucoma.” Molecular Vision, vol. 29, 2023, pp. 365-377.

The discovery of new genetic variants and the use of advanced genetic sequencing tools like VarSeq are opening doors to personalized medicine and targeted therapies. While challenges remain, the future of ophthalmology looks brighter, driven by relentless scientific inquiry and technological advancements.

Each article we have referenced contributes significantly to this rapidly evolving field, providing crucial insights into the genetic underpinnings of eye disorders and paving the way for innovative treatments and diagnostic methods. To learn more about VarSeq’s analysis capabilities, visit us here.

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