Characterisation of two mutations in the ABCD1 gene leading to low levels of normal ALDP

Abstract

A variety of mutations have been identified in the X-linked adrenoleukodystrophy (X-ALD) gene, none of which is prevalent. In this work we describe a reverse transcription polymerase chain reaction (RT-PCR)-based strategy specially suited to the molecular characterisation of mutations in index cases. After RT-PCR amplification of the X-ALD transcript a conformation-sensitive gel electrophoresis analysis is performed followed by sequencing of the fragments with altered mobility. Two X-ALD patients were studied using this strategy. In both cases, splice site mutations were found. The first patient studied has a single base substitution at the first position of the invariant GT dinucleotide donor splice site of intron 8. In spite of this alteration, small quantities of correctly spliced mRNA molecules were easily detected. In agreement with these data, a small amount of ALDP was found by western blotting analysis. An alteration at the -1 position of the donor splice site of exon 1 was detected in the second patient. This mutation results in the utilisation of a cryptic 5' splice site within intron 1. Nevertheless, this transition also allows for some correct splicing. Western blotting analysis revealed the existence of normal-migrating ALDP. However, as expected, the levels of this protein were greatly decreased. 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. It is proposed that the quantification of ALDP levels in these patients could provide important insights concerning the correlation between clinical phenotype and amount of normal ALDP.

Retracted : Identification of seven novel mutations in ABCD1 by a DHPLC‐based assay in Italian patients with X‐linked adrenoleukodystrophy

“…We identified 11 variants, including seven new changes, in 12 families. Consistent with data from other groups (Guimaraes et al, 2001), no prevalent mutations were identified. However, the p.Ser514Arg mutation was found in two families who denied formal consanguinity.…”

Probing substrate-induced conformational alterations in adrenoleukodystrophy protein by proteolysis

“…The cDNA encoding the human full-length ALDP transporter was amplified by RT-PCR using the F1 and F8 oligonucleotides exactly as described previously (Guimaraes et al 2001) and cloned into the pGEM-T easy vector (Promega). Subsequently, the cDNA was inserted into the EcoRI site of pGEM-4 (Promega).…”

Splicing defects in ABCD1 gene leading to both exon skipping and partial intron retention in X-linked adrenoleukodystrophy Tunisian patient

“…Many ABCD1 intronic variants have been identified during diagnostic screening: they account for about 3% of all variants listed at X-ALD database. For the majority of intronic variations, the consequences on mRNA splicing have been only inferred by in-silico analysis, whereas experimental demonstration of their pathogenicity has been obtained by mRNA studies for only few of them (Chiu et al, 2006;Shi et al, 2003;Guimarães et al, 2001Guimarães et al, , 2002.…”