SOD1 encodes a 645-bp transcript with a genomic length of 9320-bp and a 154-amino-acid (AA) protein product. In addition to the wild-type transcript consisting of five exons, distinct splice variants have been identified by RT-PCR.
45 48 When the
SOD1 splice donor site sequences were matched with the corresponding consensus sequences, it was found that intron 1 had the lowest degree of match (77.2), and this may reduce the splice donor site usage and lead to alternative splicing.
45 In addition, intron 2 had low splice acceptor site scores that may lead to reduced usage of the splice site and the lack of partial or entire exon 2 and/or exon 3.
45
Although mutations in
SOD1 are known to be associated with familial ALS,
49 50 51 52 we did not find this 7-base deletion in the ALS database either as a mutation or polymorphism. This intronic deletion may influence the usage of the splice site such as that reported for exon 5 with resultant truncation or complete deletion of the protein.
53 Intronic deletions have been reported previously as close to the splice junction or as far as 189 bp from the splice site.
53 54 55 Different
SOD1 isoforms have various levels of carbonylation and susceptibility to oxidative modification,
56 thereby leading to different functions for the isoforms. Moreover, the splice variants that are missing exon 2 or exons 2 and 3, yield proteins that lack the active site of the CuZn (SOD1) enzyme and are likely to have modified
SOD1 function.
45 In a cell-free translation system, the exon 2-skipping and exon 2- and 3-skipping
SOD1 showed weak to no protein expression.
48 In addition, transfection studies using 293T cells showed no expression for either exon-skipping
SOD1.
48 These findings imply that elimination of exon 2 and exons 2 and 3 has significant effects on
SOD1 levels and functions.
Our analysis of RNA transcripts showed that the KC proband from family 1 had LE2 and LE2E3 variants in addition to the wild-type transcript. Previous studies have demonstrated that the LE2 and LE2E3 variants code for inactive proteins and changed the translation reading frames. Based on these findings, we hypothesize that there is a relationship between the genomic change and the RNA variants and there is a relationship between the presence of the inactive variants and KC.
The protein encoded by this
SOD1 gene binds copper and zinc ions (CuZn-SOD) and is one of two isozymes responsible for destroying free superoxide radicals in the body. The encoded isozyme is a soluble cytoplasmic protein, acting as a homodimer to convert naturally occurring but harmful superoxide radicals to molecular oxygen and hydrogen peroxide. Mutations in this gene have been associated with familial ALS and rare transcript variants have been reported for the gene.
43 44 Some patients with familial ALS have mutated
SOD1 gene variants, altered
SOD1 crystallographic structures, and decreased SOD activity.
44 By as yet unknown mechanisms, the mutated
SOD1 variants may cause neurotoxic effects that damage the motor neurons.
43 44
The CuZn-SOD is an essential antioxidant enzyme found within the human cornea. We hypothesize that the predominance of a variant
SOD1 transcript with exon 2-skipping or exon 2- and 3-skipping deletions may lead to a decrease in enzyme activities in KC corneas. This would lead to an inability of the corneal tissue to detoxify harmful superoxide radicals, thereby making them available for interaction with nitric oxide to form peroxynitrite, which is found excessively in KC corneas.
10 Of note, normal CuZn-SOD activities have been reported in sporadic KC
13 57 but have not been studied in familial KC. It is likely that there are multiple pathogenetic factors involved in both familial and sporadic KC so it would not be unexpected to have varied responses.
Associations between KC and other candidate genes have been examined. The transforming growth factor-β induced gene (TGF-βI;
BIGH3), which is associated with various corneal dystrophies,
58 59 60 61 was found not to be associated with KC.
62 VSX1 has been identified in a patient who has both KC and posterior polymorphism dystrophy.
39 Moreover, novel mutations have been reported in the
VSX1 gene in a series of individual patients with KC,
40 but a biological mechanism connecting this gene to KC is not clear. Keratoconus is more common in patients with Down syndrome than in normal individuals. A defect of the
SOD1 gene, with its location on chromosome 21 and association with Down syndrome, could play a role in the increased oxidative damage found in KC corneas. Moreover, recent studies show that oxidative stress elements, such as peroxynitrite and nitric oxide, can increase degradative enzyme activities, which could play a role in the stromal thinning characteristically found in KC.
63 Future studies should include screening of both additional families with KC and sporadic KC patients. In addition, the possible relationships between the IVS2+50del7 genomic deletion and expression of the
SOD1 variants LE2 or LE2LE3 should be explored in vitro.
The authors thank all participating patients and their families.