Purpose: To screen superoxide dismutase 1 (SOD1) on chromosome 21 as a possible candidate gene for familial keratoconus (KC).
Methods: Total genomic DNA was extracted from the blood of 15 different KC families and 156 unaffected subjects. All five exons of the SOD1 gene were sequenced. For a rapid screening test, DNA was amplified by polymerase chain reaction (PCR), digested with HpyCH4 III or analyzed by radioactively end-labeled exon PCR. RNA was extracted from leukocytes and reverse transcribed to cDNA, and the PCR was amplified for splice variants. Some samples were cloned and sequenced.
Results: A heterozygous genomic 7-base deletion in intron 2 of the SOD1 gene was identified in two KC families (pedigrees 1 and 6). The deletion segregated within pedigree 1 and was absent in 312 chromosomes from normal individuals. RNA from the proband of pedigree 1 showed that in addition to the wild-type transcript, two other transcripts were expressed for the CuZn SOD (SOD1) gene: lacking entire exon 2 (LE2) and lacking entire exon 2 and entire exon 3 (LE2E3).
Conclusions: A unique genomic deletion within intron 2 close to the 5' splice junction of the SOD1 gene was identified in three patients with KC. Moreover, mRNA from one affected individual also had two transcript splice variants (LE2 and LE2E3) that others have shown to code for proteins lacking the active site of the SOD1 enzyme. Further studies should be conducted to determine whether a causal relationship exists between these two events that may increase oxidative stress and be associated with KC.