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Identification of rare genetic variants in keratoconus families via exome sequencing

Laboratory Genetics and Genomics
  • Primary Categories:
    • Basic Research
  • Secondary Categories:
    • Basic Research
Introduction:
Keratoconus (KC)(MONDO:0015486) is the most common corneal ectasia disorder, affecting ~1 in 2,000 people. Characterized by centralized thinning and bulging of the cornea, KC manifests as a cone-like morphology. KC impairs visual acuity in patients, inducing myopia (nearsightedness) and astigmatism. Symptoms arise during adolescence and progress into adulthood. Between 5-23% of patients have a family history of KC, though most cases are sporadic. The precise etiology of KC remains unclear, though evidence points to a non-mendelian, multigenic model with incomplete penetrance. Other conditions including Downs’ syndrome and Ehlers-Danlos syndrome are common comorbidities in KC. Environmental and biomechanical risk factors, like ultraviolet radiation and eye-rubbing, also contribute to development. Candidate genes and variants have been identified, but few have been validated. This study sought to reveal genetic variants associated with KC in a cohort of 90 individuals from 27 KC-affected families through exome sequencing (ES).

 

Methods:
This study was approved by the Institutional Review Boards at Cedars-Sinai Medical Center and King Saud University prior to initiation and adhered to the tenets of the Declaration of Helsinki. Keratoconus status was confirmed by a physician through comprehensive ophthalmic examination. Genomic DNA was extracted from peripheral blood samples and sequenced at the Duke University Sequencing and Genomic Technologies Shared Resource. Variant identification and analysis were conducted with Golden Helix SNP & Variation Suite (SVS) version 8.9.1. Variants were filtered by read depth, genotype quality, and minor allele frequency. Synonymous, intergenic, non-splice intronic, and 5’, 3’-UTR variants were excluded as well as common variants and those without functional consequence. Case-specific variants in pooled samples and individual families with at least 4 members were identified and prioritized by frequency. Variants of interest were validated with qPCR.

 

Results:
Seven KC-specific missense variants were identified and present in at least 11% of cases (n=8/71). The most frequent variants, NM_021098.3: c.4843A>C in CACNA1H and NM_001244949.2: c.1469G>T in GPAM, were present in 21% of cases (n=15/71). The remaining 5 variants were: NM_005915.6: c.2428T>C (n=13/71), NM_015409.5: c.98A>C (n=9/71), NM_002661.5: c.1712A>G (n=9/71), NM_144691.4: c.1237C>G (n=8/71), and NM_015125.4: c.2816G>C (n=8/71).

Family-wise analysis revealed several missense variants: 103 in at least 66% of family C cases (n=2/3), 2 in all family I and K cases (n=2/2), and 5 in at least 66% of family R cases (n=4/6). One variant in PIF1, NM_001286496.2: c.778G>C, was shared by 3 of 4 families. Another variant in ARSD, NM_001669.4: c.661G>A, was shared by 2 of 4 families. Overall, NM_001286496.2: c.778G>C was present in 29 cases and 4 controls and NM_001669.4: c.661G>A was present in 35 cases and 9 controls, indicating that they were more prevalent but not exclusive to KC cases.

 

Conclusion:
In this study, we examined 90 individuals belonging to 27 families with histories of KC using ES. We identified 7 rare, case-specific variants. Only 2 of 7 variants, NM_005915.6: c.2428T>C and NM_002661.5: c.1712A>G, have been reported in ClinVar, both classified as benign/likely benign for unrelated conditions. All 7 variants were associated with KC-relevant human phenotype ontologies, either directly or through downstream pathways. Family-wise analysis revealed 2 rare, case-specific variants in PIF1 and ARSD shared by multiple families. Both were associated with abnormal cornea morphology through downstream pathways. Gene ontology analysis revealed that ARSD is closely associated with metal ion binding. Metal ions are crucial for antioxidant activity, collagen synthesis and crosslinking, which are key contributors to KC pathogenesis in the corneal stroma. Given that keratoconus is a multigenic condition with incomplete penetrance, additional factors are likely required to induce a phenotype. Further assessment of the functional consequences produced by the variants identified in this study will be beneficial to understand their additive contributions to KC etiology.

 

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