A new method avoids interference of ABCD1 pseudogenes in molecular diagnosis of X-linked adrenoleukodystrophy / 第二军医大学学报

ABSTRACT

Objective: To introduce a new method which can avoid the interference of ABCD1 pseudogenes in the molecular diagnosis of X-linked adrenoleukodystrophy. Methods: The coding regions of ABCD1 gene of 3 unrelated Chinese patients with X-linked adrenoleukodystrophy were amplified from the total RNA of peripheral blood by long distance RT-PCR; the product was further amplified in 4 segments in a second round PCR; and the PCR products were purified and directly sequenced. To confirm the mutations, the genomic DNA from peripheral blood cells of the patients was analyzed by direct sequencing after amplification of the ABCD1 genes by nested PCR, in which the product of the first round PCR covered the fragment starting from exon 6 and ending at 3′UTR of the ABCD1 gene. Results: The 3 Chinese patients with X-linked adrenoleukodystrophy had 3 different base substitutions(2235C>T,2065C>T and 2190A>T)in the ABCD1 genes of the 3 probands and their mothers, which resulted in 2 missense mutations (R617C and P560L) and one nonsense mutation (K602X). Conclusion: Nested PCR can rapidly and efficiently avoid the interference of ABCD1 pseudogenes in the molecular diagnosis of X-ALD.