Screening for Novel LOX and SOD1 Variants in Keratoconus Patients from Brazil

Abstract

Purpose: To investigate the presence of the variants of lysyl oxygenase (LOX) and superoxide dismutase 1 (SOD1) genes in Brazilian patients with advanced keratoconus.
 Methods: Donor genomic DNA extracted from blood samples was screened for 5’UTR, exonic LOX, and SOD1 variants in a subset of 26 patients presenting with advanced keratoconus (KISA > 1000% and I–S > 2.0) by Sanger sequencing. The impact of non-synonymous amino acid changes was evaluated by SIFT, PMUT, and PolyPhen algorithms. The Mutation Taster tool was used to evaluate the potential impact of formation of new donor and acceptor splice sites in the promoter region of affected volunteers carrying sequence variants. A 7-base SOD1 deletion (IVS2 + 50del7bp) previously associated with keratoconus was screened in 140 patients presenting classical keratoconus by gel fragment analysis, and positive samples were sequenced for confirmation.
 Results: We found an unreported missense variant in LOX exon 6 in one heterozygous patient, leading to substitution of proline with threonine at residue 392 (p. Thr392Pro) of LOX protein sequence. This mutation was predicted to be potentially damaging to LOX protein. Another LOX variant, Arg158Gln, was also detected in another patient but predicted to be non-pathogenic. Two additional new polymorphisms in LOX 5’UTR region (–116C > T and –58C > T) were found in two patients presenting with advanced keratoconus and were predicted to modulate or create donor/acceptor splice sites in LOX transcripts. Additionally, SOD1 deletion was detected in one patient presenting with severe keratoconus, not in control samples.
 Conclusion: We described three novel LOX polymorphisms identified for the first time in Brazilian patients with advanced keratoconus, as well as a previously described SOD1 deletion strongly associated with keratoconus. A possible role of these variants in modulating transcript levels in the cornea of affected individual requires further investigation.

Further evaluation of differential expression of keratoconus candidate genes in human corneas

“…It is mapped within 5q21 region linked to KTCN in Italian population (Bisceglia et al, 2009). While the first mutational screening of LOX has not confirmed the involvement of LOX variants in KTCN, evidence of a genetic association between KTCN and common SNVs located in the LOX gene has been revealed in Caucasian (Bykhovskaya et al, 2012), Czech (Dudakova et al, 2015), Iranian (Hasanian-Langroudi et al, 2015), Brazilian (Gadelha et al, 2020), and Han Chinese (Xu et al, 2020) KTCN patients. In our study, the level of LOX was decreased in KTCN corneas.…”

Peer Review #1 of "Further evaluation of differential expression of keratoconus candidate genes in human corneas (v0.1)"

“…Manuscript to be reviewed et al, 2012), Czech (Dudakova et al, 2015), Iranian (Hasanian-Langroudi et al, 2015), Brazilian (Gadelha et al, 2020), and Han Chinese (Xu et al, 2020) KTCN patients. In our study, the level of LOX was decreased in KTCN corneas.…”

The Underlying Relationship between Keratoconus and Down Syndrome

“…Superoxide dismutase 1 (SOD1) is another potential gene that has been linked to KC and that directly participates in antioxidative activities [29,[106][107][108]. SOD1 gene consists of five exons located on chromosome 21q22.11.…”

“…The CuZn-SOD enzyme is present in the human cornea and is an essential antioxidant enzyme [109]. Given the substantial buildup of oxidative stress indicators in the corneas of KC patients, there is evidence of a connection between SOD1 mutations and the development of KC [108]. Studies by Udar et al identified a 7-base intronic deletion in the SOD1 gene in two KC families after screening the exons and intron-exon junctions of SOD1 using PCR-based Sanger sequencing [109].…”