Adding new variants to the repertoire of ABCD1 mutations in X-ALD, our data provide an efficient, cost-effective, and reliable DHPLC detection protocol for mutation screening of X-ALD families.
To determine whether mutations occur in the putative NBF of ALD protein, we analyzed by denaturing gradient gel electrophoresis (DGGE) exon 6 and 8 that encode most part of this domain in 50 ALD patients.
Taken together, our data suggest that some less severe or late-onset forms of X-ALD associated with splice mutations result from the production of small amounts of normal ALDP.
Twenty-five of the ALD probands whose ALD genes appeared normal by Southern blot analysis were surveyed for mutations by Single Strand Conformation Polymorphism (SSCP) procedures and DNA sequence analysis.
Amongst 489 X-ALD families tested at Kennedy Krieger Institute, we identified 20 cases in which the ABCD1 mutation was de novo in the index case, indicating that the mutation arose in the maternal germ line and supporting a new mutation rate of at least 4.1% for this group.
These results provide the first evidence of both homo- and heterodimerization of mammalian ABC half-transporters and suggest that the loss of ALDP dimerization plays a role in X-ALD pathogenesis.
Expression studies revealed that only K217E is a deleterious mutation, because a plasmid encoding ALDP with K217E was ineffective in the restoration of defective beta-oxidation in X-ALD fibroblasts.
Since 5%-15% of heterozygous women have normal VLCFA levels, the immunodetection of ALDP in white blood cells can be applicable in a majority of ALD kindred, to identify heterozygous women, particularly when the ALD gene mutation has not yet been identified.
ALD shows a highly variable phenotypic expression and extensive mutation analysis in ALD patients has failed to establish a genotype-phenotype correlation, even in the presence of the same ALD-gene defect.
The disruptive nature of two mutations (i.e., the frameshift and the nonsense mutation) in patients with biochemically proved childhood ALD and AMN further strongly supports the hypothesis that alterations in this gene play a crucial role in the pathogenesis of X-ALD.