RESUMEN
X-ALD is a neurological disorder associated with inherited defects in the ABCD1 (ALD) gene located on Xq28 and with impaired peroxisomal very long-chain fatty acid beta-oxidation. We examined the ABCD1 gene in probands from 11 unrelated X-ALD Czech and Slovak families by the direct sequencing of cDNA or genomic PCR products. In 10 families there were 10 different mutations, eight of which were novel. The spectrum of mutations consists of six point mutations, three microdeletions (1bp, 2bp, 4 bp), and one large deletion (229bp). In the 11th family we detected two novel single-base pair substitutions in exon 1 (c.38 A>C and c.649 A>G), both causing amino acid exchanges (N13T and K217E). 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. The N13T amino acid exchange, on the other hand, did not affect ALDP function. Thus, N13T represents the first polymorphism causing an amino acid exchange in the ABCD1 gene. As this polymorphism was observed neither in 100 control alleles nor in 300 X-ALD patients who have been sequenced so far world-wide, it seems to be very rare or unique. Two additional novel polymorphisms were found by the sequencing of the ABCD1 gene from our patients: c.-59 C/T in the 5'untranslated region and c.2019 C/T (F673F) in exon 10. The frequencies of these two polymorphisms, were 11/150 and 2/150 control alleles, respectively.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Adrenoleucodistrofia/genética , Ligamiento Genético/genética , Proteínas de la Membrana/genética , Mutación Missense/genética , Polimorfismo Genético/genética , Cromosoma X/genética , Regiones no Traducidas 5'/genética , Miembro 1 de la Subfamilia D de Transportador de Casetes de Unión al ATP , Transportadoras de Casetes de Unión a ATP/química , Adolescente , Adulto , Alelos , Secuencias de Aminoácidos , Niño , Secuencia Conservada , República Checa , Análisis Mutacional de ADN , Exones/genética , Ácidos Grasos/metabolismo , Femenino , Humanos , Masculino , Proteínas de la Membrana/química , Datos de Secuencia Molecular , Oxidación-Reducción , Ácido Palmítico/metabolismo , Fenotipo , Polimorfismo de Nucleótido Simple/genética , EslovaquiaRESUMEN
Inherited defects in the X-chromosomal adrenoleukodystrophy (ALD; ABCD1) gene are the genetic cause of the severe neurodegenerative disorder X-linked adrenoleukodystrophy (X-ALD). Biochemically the accumulation of very long-chain fatty acids, caused by impaired peroxisomal beta-oxidation, is the pathognomonic characteristic of the disease. Due to the X-chromosomal inheritance of X-ALD no data are available to clarify the question whether mutated adrenoleukodystrophy proteins (ALDPs) can negatively influence normal ALDP function. Here we show that restoration of beta-oxidation in X-ALD fibroblasts following transient transfection with normal ALD cDNA is more effective in ALDP-deficient fibroblasts compared with fibroblasts expressing normal amounts of mutated ALDP. Furthermore, we utilized the HeLa Tet-on system to construct a stable HeLa cell line expressing a constant level of endogenous ALDP and doxycycline-inducible levels of mutated ALDP. The induction was doxycycline dosage-dependent and the ALDP correctly localized. Interestingly, although mutated ALDP increased >6-fold in a dosage-dependent manner the total amount of ALDP (mutated and normal) remained approximately even as demonstrated by western blot and flow cytometric analyses. Thus, apparently mutated and normal ALDP compete for integration into a limited number of sites in the peroxisomal membrane. Consequently, increased amounts of mutated ALDP resulted in decreased peroxisomal beta-oxidation and accumulation of very long-chain fatty acids. These findings have direct implications on future gene therapy approaches for treatment of X-ALD, since in some patients a non-functional endogenous protein could act in a dominant negative way or displace the introduced, normal protein.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Adrenoleucodistrofia/terapia , Ligamiento Genético/genética , Terapia Genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mutación Puntual/genética , Cromosoma X/genética , Miembro 1 de la Subfamilia D de Transportador de Casetes de Unión al ATP , Transportadoras de Casetes de Unión a ATP/química , Adrenoleucodistrofia/genética , Western Blotting , Doxiciclina/farmacología , Ácidos Grasos/química , Ácidos Grasos/metabolismo , Femenino , Fibroblastos , Citometría de Flujo , Técnica del Anticuerpo Fluorescente Indirecta , Expresión Génica/efectos de los fármacos , Células HeLa , Humanos , Masculino , Proteínas de la Membrana/química , Datos de Secuencia Molecular , Oxidación-Reducción , Peroxisomas/metabolismo , Conformación Proteica , ARN Mensajero/análisis , ARN Mensajero/genética , TransfecciónAsunto(s)
Adrenoleucodistrofia/genética , Sustitución de Aminoácidos/genética , Mutación Missense/genética , Proteínas/análisis , Proteínas/genética , Subfamilia D de Transportadores de Casetes de Unión al ATP , Miembro 1 de la Subfamilia D de Transportador de Casetes de Unión al ATP , Transportadoras de Casetes de Unión a ATP/genética , Insuficiencia Suprarrenal/genética , Adulto , Arginina/genética , Cisteína/genética , Humanos , Isoleucina/genética , Serina/genéticaRESUMEN
Inherited defects in the peroxisomal ATP-binding cassette (ABC) transporter adrenoleukodystrophy protein (ALDP) lead to the lethal peroxisomal disorder X-linked adrenoleukodystrophy (X-ALD), for which no efficient treatment has been established so far. Three other peroxisomal ABC transporters currently are known: adrenoleukodystrophy-related protein (ALDRP), 70 kDa peroxisomal membrane protein (PMP70) and PMP70- related protein. By using transient and stable overexpression of human cDNAs encoding ALDP and its closest relative ALDRP, we could restore the impaired peroxisomal beta-oxidation in fibroblasts of X-ALD patients. The pathognomonic accumulation of very long chain fatty acids could also be prevented by overexpression of ALDRP in immortalized X-ALD cells. Immunofluorescence analysis demonstrated that the functional replacement of ALDP by ALDRP was not due to stabilization of the mutated ALDP itself. Moreover, we were able to restore the peroxisomal beta-oxidation defect in the liver of ALDP-deficient mice by stimulation of ALDRP and PMP70 gene expression through a dietary treatment with the peroxisome proliferator fenofibrate. These results suggest that a correction of the biochemical defect in X-ALD could be possible by drug-induced overexpression or ectopic expression of ALDRP.