RESUMO
Combined D-2- and L-2-hydroxyglutaric aciduria (D/L-2-HGA) is a devastating neurometabolic disorder, usually lethal in the first years of life. Autosomal recessive mutations in the SLC25A1 gene, which encodes the mitochondrial citrate carrier (CIC), were previously detected in patients affected with combined D/L-2-HGA. We showed that transfection of deficient fibroblasts with wild-type SLC25A1 restored citrate efflux and decreased intracellular 2-hydroxyglutarate levels, confirming that deficient CIC is the cause of D/L-2-HGA. We developed and implemented a functional assay and applied it to all 17 missense variants detected in a total of 26 CIC-deficient patients, including eight novel cases, showing reduced activities of varying degrees. In addition, we analyzed the importance of residues affected by these missense variants using our existing scoring system. This allowed not only a clinical and biochemical overview of the D/L-2-HGA patients but also phenotype-genotype correlation studies.
Assuntos
Proteínas de Transporte de Ânions/metabolismo , Encefalopatias Metabólicas Congênitas/metabolismo , Ácido Cítrico/metabolismo , Glutaratos/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas de Transporte de Ânions/química , Proteínas de Transporte de Ânions/genética , Bioensaio/métodos , Encefalopatias Metabólicas Congênitas/genética , Células Cultivadas , Pré-Escolar , Análise Mutacional de DNA , Feminino , Fibroblastos , Predisposição Genética para Doença , Humanos , Lactente , Recém-Nascido , Masculino , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Modelos Moleculares , Mutação de Sentido Incorreto , Transportadores de Ânions Orgânicos , Fenótipo , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Mosaic IDH1 mutations are described as the cause of metaphyseal chondromatosis with increased urinary excretion of D-2-hydroxyglutarate (MC-HGA), and mutations in IDH2 as the cause of D-2-hydroxyglutaric aciduria (D-2HGA) type II. Mosaicism for IDH2 mutations has not previously been reported as a cause of D-2HGA. Here we describe three cases: one MC-HGA case with IDH1 mosaic mutations, and two D-2HGA type II cases. In one D-2HGA case we identified mosaicism for an IDH2 mutation as the genetic cause of this disorder; the other D-2HGA case was caused by a heterozygous IDH2 mutation, while the unaffected mother was a mosaic carrier. METHODS: We performed amplicon deep sequencing using the 454 GS Junior platform, next to Sanger sequencing, to identify and confirm mosaicism of IDH1 or IDH2 mutations in MC-HGA or D-2HGA, respectively. RESULTS AND CONCLUSIONS: We identified different mutant allele percentages in DNA samples derived from different tissues (blood vs fibroblasts). Furthermore, we found that mutant allele percentages of IDH1 decreased after more passages had occurred in fibroblast cell cultures. We describe a method for the detection and validation of mosaic mutations in IDH1 and IDH2, making quantification with laborious cloning techniques obsolete.