Women were identified as carriers for the ALD gene based on pedigree analysis and biochemical evidence of increased very long chain fatty acids. Skin fibroblasts were obtained on most of the ALD carriers studied as inpatients and outpatients in whom a modified Kurtzke scale defines the degree of clinical severity. Studies utilizing immunohistochemical techniques to determine X-inactivation had shown promise. Studies to determine X-inactivation by indirect immunofluorescence were conducted using an antibody raised against the peptide corresponding to the C-terminal 18 amino acids of the ALD protein (ALDP). The polyclonal ALDP antibody was used in immunofluorescence experiments with human skin fibroblasts and shown to co-localize with the peroxisomal protein catalase in double-labeled experiments. Immunofluorescence could not be detected in an ALD patient with a known large deletion in the ALD gene. Our initial immunofluorescence studies in a limited number of heterozygotes showed results similar to our previous studies by single cell cloning that the percentage of clones with normal or abnormal beta-oxidation correlated with the degree of neurological involvement. Therefore, this immunofluorescent method appears to be acceptable to determine the fraction of cells in which the normal or abnormal X chromosome is active. In order to determine the extent to which phenotypic variation among the ALD heterozygotes is a consequence of variable X-inactivation of normal/abnormal X chromosome, we extended these observations to 101 females, 80 of whom were obligate ALD heterozygotes in each modified Kurtzke category. Sixty of these obligate heterozygotes were informative in that they showed a mixed population of cells indicating variable X inactivation. As heterozygotes become more symptomatic with age, correlation of immunofluorescence results with clinical severity in women over 40 years was attempted. In those with minimal to no involvement (Kurtzke score 0-1) and those with severe involvement(Kurtzke score 3-4), there was a good correlation. In those with scores of 2, results were scattered and larger numbers of patients need to be studied for statistical conclusions. One young carrier, aged 12 years with Addison's disease and cerebral lesions, showed 100% of her cells carrying the abnormal X chromosome and was therefore, functionally and clinically similar to affected males. These exciting results should prove valuable in predicting severity of neurological involvement in female carriers, and identify those in whom aggressive therapeutic intervention is indicated when it becomes available in the future. Also, in informative kindreds the identification of carriers will be made easier, particularly in those whose very long chain fatty acids are normal in blood or skin fibroblasts.
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