As several age-related diseases result from the accumulation of somatic mutations, we presumed that similar genetic alteration would also have occurred in LE, which would have impaired the tissue function. Since LE is important for the maintenance of lens homeostasis, we performed mutation screening in
SOD1 from DNA isolated from the LE samples to understand the molecular mechanisms responsible for the impairment of
SOD1 activity. A few samples, upon sequencing, revealed three INDEL variations (IVS4+13_14insA, IVS4+42delG, and IVS4+57_58insT) at 3′ exon-intron4 boundary rather than in the coding region. A similar observation was reported in ALS, in which mutation screening in coding regions of all five exons of
SOD1 revealed no aberrations but revealed an intronic variation and up to 50% decreased activity.
28 So far, three splice-site mutations have been identified in the fourth exon of the
SOD1 gene in ALS patients
48–51 and 7-bp deletion mutations in the second intron (IVS2+50del7 and c.16950delTAAACAG) in keratoconus patients.
18,45 The INDEL variations that we observed in the present study, however, did not match with the previous studies. Intronic polymorphisms of any gene can cause splice-site aberrations and result in the deletion or insertion of amino acids as well as truncations. Such perturbations will significantly disrupt the structure and stability of the protein, and the result will be a major loss of function.
28 As the observed INDEL variations in our study existed at the splice region, we believed they might have some effect on the alternate splicing mechanism. Therefore, we performed in silico splice-junction analysis using the Sequence Walker Program, which calculates the strength of each of the native and cryptic splice sites.
29,30 Splice-junction analysis of IVS4+13_14insA and IVS4+57_58insT polymorphisms revealed that they do not have much impact on the splicing mechanism as they do not affect the strength of either the native or cryptic splice sites. However, IVS4+42delG polymorphism was found to enhance the strength of a cryptic acceptor splice site 2 (CASS2) from 2.2 to 4.0 bits, which is strong enough to generate a
SOD1 transcript without exon4 during alternate splicing. We performed transcript profiling to check the presence of
SOD1 transcripts lacking exon4 as a result of IVS4+42delG polymorphism. Of interest, our results did not find such transcripts in both clear and cataractous lenses. This finding indicates that the newly created CASS2, owing to IVS4+42delG polymorphism, does not have any direct influence on the
SOD1 transcript splicing process. Further, we evaluated the prevalence of IVS4+42delG polymorphism in the cataractous and control populations and found a loss of restriction sites for
TatI restriction enzyme in all cataractous and control samples. This indicates that the variation IVS4+42delG might be common in the Western Indian population. Hence, the mechanisms other than genetic mutations and/or polymorphisms might play a major role in the impairment of
SOD1 expression and/or subsequent activity.