title: Exploration of molecular factors impairing superoxide dismutase isoforms activity in human senile cataractous lenses.
abstract: OBJECTIVE
  To explore different molecular factors impairing the activities of superoxide dismutase (SOD) isoforms in senile cataractous lenses.
  
  
  METHODS
  Enzyme activity of SOD isoforms, levels of their corresponding cofactors copper (Cu), manganese (Mn), zinc (Zn), and expression of mRNA transcripts and proteins were determined in the lenses of human subjects with and without cataract. DNA from lens epithelium (LE) and peripheral blood was isolated. Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) followed by sequencing was carried out to screen somatic mutations. The impact of intronic insertion/deletion (INDEL) variations on the splicing process and on the resultant transcript was evaluated. Genotyping of IVS4+42delG polymorphism of SOD1 gene was done by PCR-restriction fragment length polymorphism (RFLP).
  
  
  RESULTS
  A significant decrease in Cu/Zn- and Mn-SOD activity (P < 0.001) and in Cu/Zn-SOD transcript (P < 0.001) and its protein (P < 0.05) were found in cataractous lenses. No significant change in the level of copper (P = 0.36) and an increase in the level of manganese (P = 0.01) and zinc (P = 0.02) were observed in cataractous lenses. A significant positive correlation between the level of Cu/Zn-SOD activity and the levels of Cu (P = 0.003) and Zn (P = 0.005) was found in the cataractous lenses. DNA sequencing revealed three intronic INDEL variations in exon4 of SOD1 gene. Splice-junction analysis showed the potential of IVS4+42delG in creating a new cryptic acceptor site. If it is involved in alternate splicing, it could result in generation of SOD1 mRNA transcripts lacking exon4 region. Transcript analysis revealed the presence of complete SOD1 mRNA transcripts. Genotyping revealed the presence of IVS4+42delG polymorphism in all subjects.
  
  
  CONCLUSIONS
  The decrease in the activity of SOD1 isoform in cataractous lenses was associated with the decreased level of mRNA transcripts and their protein expression and was not associated with either modulation in the level of enzyme cofactors or with INDEL variations.
authors: Department of Molecular Genetics and Biochemistry, Iladevi Cataract and IOL Research Centre, Memnagar, Ahmedabad, Gujarat, India.
authors: Sankaranarayanan
authors: S
authors: Rajkumar
authors: Abhay R
authors: AR
authors: Vasavada
authors: Mamidipudi R
authors: MR
authors: Praveen
authors: Rajendran
authors: R
authors: Ananthan
authors: Geereddy B
authors: GB
authors: Reddy
authors: Harsha
authors: H
authors: Tripathi
authors: Darshini A
authors: DA
authors: Ganatra
authors: Anshul I
authors: AI
authors: Arora
authors: Alpesh R
authors: AR
authors: Patel
mesh_terms: D000368:Aged; D015153:Blotting, Western; D002386:Cataract / Q000201:enzymology* / Q000235:genetics; D003067:Coenzymes / Q000378:metabolism*; D003300:Copper / Q000378:metabolism; D004252:DNA Mutational Analysis; D019573:Epithelium, Corneal / Q000201:enzymology; D005260:Female; D015971:Gene Expression Regulation, Enzymologic; D005838:Genotype; D006801:Humans; D054643:INDEL Mutation; D008297:Male; D008345:Manganese / Q000378:metabolism; D008875:Middle Aged; D020641:Polymorphism, Single Nucleotide; D020033:Protein Isoforms / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D013482:Superoxide Dismutase / Q000378:metabolism*; D000072105:Superoxide Dismutase-1; D015032:Zinc / Q000378:metabolism
chemical_list: D003067:Coenzymes; D020033:Protein Isoforms; D012333:RNA, Messenger; C000606290:SOD1 protein, human; D008345:Manganese; D003300:Copper; D013482:Superoxide Dismutase; D000072105:Superoxide Dismutase-1; D015032:Zinc
keywords: cofactors; senile cataract; somatic mutation; splice-transcript variant; superoxide dismutase
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