Fig. 1 Molecular analysis of α-actinin-3 genes and proteins. Indirect immunofluorescence (a− c) and westernblot analysis (d) of human skeletal muscle (quadriceps muscle biopsy with normal histology) using affinity purified antibodies (5B) specific for α-actinin-3 (a, c; refs 3, 8) and mouse myosin heavy chain (fast, MY32) specific for type 2 fibres (b). Methodology as described in North and Beggs3. Normal expression of α-actinin-3 is restricted to type 2 (fast) fibres (a) as indicated by double staining with MY32 (b). Corresponding fibres in each section are indicated by the same symbol. c, Complete deficiency of α-actinin-3 in a patient homozygous for the stop codon in exon 16 (577X). d, Western-blot analysis of α-actinin-3 in skeletal muscle from individuals with normal α-actinin-3 expression (genotype 577R/577X; lanes 1, 3) and α-actinin-3 deficiency (genotype 577X/577X; lanes 2, 4). α-actinin-3 migrates at approximately 100 kD. The ACTN3 577X allele encodes a truncated 66-kD protein, which is thought to be incapable of dimerization10. Since the anti-αactinin-3 5B antibody is directed towards the amino terminus of the protein3, 8, it should detect the truncated protein if it is stable. All individuals homozygous for the stop codon demonstrated complete absence of detectable α-actinin-3 by immunocytochemistry, and there was no evidence of the truncated protein in 577R/X or 577X/X individuals on western blots. e, f, g, h, DNA sequence and restriction endonuclease analysis of ACTN3 exon 16 demonstrating the three possible ACTN3 genotypes at position 577. Products were either directly sequenced (e− g) or subjected to DdeI digestion and agarose gel electrophoresis (h). e f g h d b a c