RESUMO
Molecular basis of D-bifunctional protein (D-BP) deficiency was studied with wild type and five disease-causing variants of 3R-hydroxyacyl-CoA dehydrogenase fragment of the human MFE-2 (multifunctional enzyme type 2) protein. Complementation analysis in vivo in yeast and in vitro enzyme kinetic and stability determinants as well as in silico stability and structural fluctuation calculations were correlated with clinical data of known patients. Despite variations not affecting the catalytic residues, enzyme kinetic performance (K(m), V(max) and k(cat)) of the recombinant protein variants were compromised to a varying extent and this can be judged as the direct molecular cause for D-BP deficiency. Protein stability plays an additional role in producing non-functionality of MFE-2 in case structural variations affect cofactor or substrate binding sites. Structure-function considerations of the variant proteins matched well with the available data of the patients.
Assuntos
17-Hidroxiesteroide Desidrogenases/química , 17-Hidroxiesteroide Desidrogenases/deficiência , Disgenesia Gonadal 46 XX/genética , Perda Auditiva Neurossensorial/genética , Hidroliases/química , Hidroliases/deficiência , Mutação , Peroxissomos/genética , 17-Hidroxiesteroide Desidrogenases/genética , Domínio Catalítico , Criança , Pré-Escolar , Clonagem Molecular , Estabilidade Enzimática , Escherichia coli/genética , Ácidos Graxos/metabolismo , Feminino , Teste de Complementação Genética , Disgenesia Gonadal 46 XX/enzimologia , Perda Auditiva Neurossensorial/enzimologia , Humanos , Hidroliases/genética , Cinética , Metabolismo dos Lipídeos , Masculino , Modelos Moleculares , Oxirredução , Proteína Multifuncional do Peroxissomo-2 , Peroxissomos/enzimologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/genética , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Perrault syndrome is a genetically heterogeneous recessive disorder characterized by ovarian dysgenesis and sensorineural hearing loss. In a nonconsanguineous family with five affected siblings, linkage analysis and genomic sequencing revealed the genetic basis of Perrault syndrome to be compound heterozygosity for mutations in the mitochondrial histidyl tRNA synthetase HARS2 at two highly conserved amino acids, L200V and V368L. The nucleotide substitution creating HARS2 p.L200V also created an alternate splice leading to deletion of 12 codons from the HARS2 message. Affected family members thus carried three mutant HARS2 transcripts. Aminoacylation activity of HARS2 p.V368L and HARS2 p.L200V was reduced and the deletion mutant was not stably expressed in mammalian mitochondria. In yeast, lethality of deletion of the single essential histydyl tRNA synthetase HTS1 was fully rescued by wild-type HTS1 and by HTS1 p.L198V (orthologous to HARS2 p.L200V), partially rescued by HTS1 p.V381L (orthologous to HARS2 p.V368L), and not rescued by the deletion mutant. In Caenorhabditis elegans, reduced expression by RNAi of the single essential histydyl tRNA synthetase hars-1 severely compromised fertility. Together, these data suggest that Perrault syndrome in this family was caused by reduction of HARS2 activity. These results implicate aberrations of mitochondrial translation in mammalian gonadal dysgenesis. More generally, the relationship between HARS2 and Perrault syndrome illustrates how causality may be demonstrated for extremely rare inherited mutations in essential, highly conserved genes.
Assuntos
Aminoacil-tRNA Sintetases/metabolismo , Proteínas Mitocondriais/metabolismo , Mutação de Sentido Incorreto , Processamento Alternativo/genética , Substituição de Aminoácidos , Aminoacil-tRNA Sintetases/genética , Animais , Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/genética , Disgenesia Gonadal 46 XX/enzimologia , Disgenesia Gonadal 46 XX/genética , Perda Auditiva Neurossensorial/enzimologia , Perda Auditiva Neurossensorial/genética , Histidina-tRNA Ligase/genética , Histidina-tRNA Ligase/metabolismo , Humanos , Masculino , Proteínas Mitocondriais/genética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genéticaAssuntos
17-Hidroxiesteroide Desidrogenases/genética , Disgenesia Gonadal 46 XX/genética , Perda Auditiva Neurossensorial/genética , Feminino , Disgenesia Gonadal 46 XX/enzimologia , Perda Auditiva Neurossensorial/enzimologia , Humanos , Hidroliases/genética , Masculino , Mutação/genética , Proteína Multifuncional do Peroxissomo-2RESUMO
BACKGROUND/AIM: 11-beta-hydroxylase deficiency (11betaOHD) is caused by CYP11B1 gene defects and leads to congenital adrenal hyperplasia associated with hypertension. Recently, a novel L299P mutation has been described in a compound heterozygous male individual. We observed two 46,XX siblings with a homozygous L299P mutation and investigated the functional properties of this CYP11B1 variant. PATIENTS: The index patient from a consanguineous Turkish family showed complete external virilization and was diagnosed incidentally at the age of 19 months during hospital admission for severe combined bacterial (urosepsis) and viral (CMV and EBV) infection. The younger sibling was diagnosed at the age of 5 months. Their genital phenotype was identical and both demonstrated borderline elevated blood pressure. RESULTS: Biochemical findings revealed 11betaOHD. A homozygous L299P mutation of the CYP11B1 gene was detected. In vitro expression studies performed in HCT116 cells showed a markedly decreased CYP11B1 activity in the L299P mutant (1.6 +/- 0.8%) for the conversion of 11-deoxycortisol to cortisol. CONCLUSIONS: Our study provides clear data on the functional properties and clinical phenotype in 46,XX individuals homozygous for this point mutation. Adrenal insufficiency might have contributed to the severe infectious disease that was present in the index patient at diagnosis.
