Assuntos
Análise Mutacional de DNA/métodos , DNA Complementar/análise , DNA de Cadeia Simples/genética , Reação em Cadeia da Polimerase/métodos , Polimorfismo Conformacional de Fita Simples , Análise de Sequência de DNA/métodos , Membro 1 da Subfamília D de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Adrenoleucodistrofia/genética , Substituição de Aminoácidos , Humanos , Mutação de Sentido Incorreto , Sensibilidade e Especificidade , Coloração pela PrataRESUMO
X-linked Adrenoleukodystrophy (X-ALD) is the most frequent peroxisomal disease. It mainly involves the nervous system white matter, adrenal cortex and testes. Several distinct clinical phenotypes are known. The principal biochemical abnormality is the accumulation of saturated very-long-chain fatty acids (VLCFAs : > C22:0, mainly C26:0), which is due to impaired capacity for beta-oxidation in peroxisomes. Diagnosis is usually based on the VLCFA levels in plasma or cultured skin fibroblasts in both patients and carriers. In 0.1% of affected males, however, the plasma C26:0 level is borderline normal, and 15% of obligate female carriers have normal results. Effective mutation detection in these families is therefore fundamental to unambiguously determine the genetic status of each individual at risk. Of particular concern are female members of kindreds segregating X-ALD mutations, because normal VLCFA levels do not guarantee lack of carrier status. We describe a fast method for detection of X-ALD mutations. The method is based on SSCP analysis of nested PCR fragments followed by sequence-determination reactions. Using this methodology we have found X-ALD mutations in 30 kindreds, including 15 not previously reported.
Assuntos
Adrenoleucodistrofia/genética , Mutação/genética , Análise Mutacional de DNA , Ácidos Graxos/genética , Feminino , Humanos , Masculino , Hibridização de Ácido Nucleico , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita Simples , RNA Mensageiro/genética , Deleção de SequênciaRESUMO
X-linked adrenoleukodystrophy is a serious and often fatal disorder, affecting the white matter of the nervous system, the adrenal cortex, and the testis. The gene mutated in X-ALD encodes a peroxisomal membrane protein, ALDP. The presence of very long chain fatty acids in plasma is highly diagnostic for affected males and carrier females, but exclusion of carrier status biochemically is unreliable. Molecular analysis of the X-ALD gene has the potential to either identify or rule out carrier status accurately, but is complicated by the existence of autosomal paralogs. We have developed and validated a robust DNA diagnostic test for this disorder involving nonnested genomic amplification of the X-ALD gene, followed by fluorescent dye-primer sequencing and analysis. This protocol provides a highly reliable means of determining carrier status in women at risk for transmitting X-ALD and is applicable to a clinical diagnostic laboratory.
