position 132. The proband belonged to a two-generation family in which three members had suffered from motor neuron disease. The proband had a phenotype of predominantly lower motor neuron signs, whereas the two other affected members showed classic signs of ALS. Age of death was 52 years, 61 years, and 44 years, respectively, all within approximately 3 years of onset of disease.
Exons 1,2,3, and 4 were screened for mutations by single-strand conformational polymorphism (SSCP) and showed no mobility shifts. Exon 5 was screened by denaturing gradient gel electrophoresis (DGGE) on a 6% polyacrylamide gel with a 040% denaturant gradient (urea, formamide) parallel to the electric field (Gulberg and Guttler, 1994). Forward primer for exon 5: 5 '-(40-bp GC clamp) -TTGGGAGGAGGTAGTGATTA-3 '; reverse primer: 5 '-GCTAGCAGGATAACAGATCA-3 '. The reverse primer was also used for sequencing. Polymerase chain reaction (PCR) conditions were 94"C, 5YC, 72"C, all for 1 min. Subsequent to ordinary PCR, three additional segments were inserted for heteroduplex formation, i.e., denaturation at 95°C for 5 min, 15 min ramp to 6YC, 60 min followed by a 15-min ramp to 37"C, and also 60 min. The analysis revealed a heterozygous aberrant band. Excision, re-amplification, and cycle sequencing of the exon 5 fragment using Thermosequenase (Amersham, Little Chalfont, England) showed that the mobility shift was due to a 4-bp (ACCC) insertion on one allele at codon 127. The insertion disrupts the reading frame of the protein and puts a termination codon in frame at amino acid position 132, truncating the predicted protein 21 amino acids short of the normal polypeptide. Since these amino acids are involved in dimerization, it would be anticipated that the disruption would lead to reduced enzymatic activity. SOD activity (Marklund and Greenwald, 1985) of skin fibroblasts from the proband was compared to 3 in parallel established control cultures. The CuZn-SOD activity was modestly reduced to 77%, compared to controls (61.5 U/mg protein versus 79.6 U/mg protein). There was no difference between proband and controls in activity of extracellular-SOD (SOD3) and Mn-SOD superoxide dismutase.
Recent observations from transgenic mouse models and knockout mice do not support a loss of function effect of SOD1 mutations, but support a gain of toxic function as mechanism for neuron degenation, probably caused by a conformational change (Siddique and Deng, 1996).