RESUMO
We describe a novel hemoglobin (Hb) variant, caused by a CCC > TCC transition at codon 77 on the alpha gene. The mutation was found in two unrelated patients, in one patient on the alpha1 gene and in the other patient on the alpha2 gene. Both are anemic patients of African origin. Due to the neutral Pro-->Ser substitution, Hb Nile could not be separated from Hb A with common short-run screening methods for high performance liquid chromatography (HPLC) and capillary electrophoresis, but was evidently present after prolonged cation exchange HPLC or separation by isoelectric focusing (IEF). Reversed phase HPLC separation of the globin chains revealed the normal and abnormal alpha chains with an expression of about 20% for Hb Nile[A1], indicative of normal expression and stability of the mutant protein.
Assuntos
Hemoglobinas Anormais/genética , Mutação de Sentido Incorreto , alfa-Globinas/genética , Anemia/sangue , Anemia/genética , Criança , Cromatografia Líquida de Alta Pressão , Códon/genética , Eletroforese Capilar , Feminino , Hemoglobinas Anormais/análise , Hemoglobinas Anormais/isolamento & purificação , Humanos , Focalização Isoelétrica , Masculino , Gravidez , Prolina/genética , Serina/genética , Adulto JovemRESUMO
Hereditary glutathione reductase (GR) deficiency was found in only 2 cases when testing more than 15 000 blood samples. We have investigated the blood cells of 2 patients (1a and 1b) in a previously described family suffering from favism and cataract and of a novel patient (2) presenting with severe neonatal jaundice. Red blood cells and leukocytes of the patients in family 1 did not contain any GR activity, and the GR protein was undetectable by Western blotting. Owing to a 2246-bp deletion in the patients' DNA, translated GR is expected to lack almost the complete dimerization domain, which results in unstable and inactive enzyme. The red blood cells from patient 2 did not exhibit GR activity either, but the patient's leukocytes contained some residual activity that correlated with a weak protein expression. Patient 2 was found to be a compound heterozygote, with a premature stop codon on one allele and a substitution of glycine 330, a highly conserved residue in the superfamily of NAD(P)H-dependent disulfide reductases, into alanine on the other allele. Studies on recombinant GR G330A revealed a drastically impaired thermostability of the protein. This is the first identification of mutations in the GR gene causing clinical GR deficiency.
Assuntos
Catarata/genética , Favismo/genética , Doenças Genéticas Inatas/genética , Glutationa Redutase/deficiência , Icterícia Neonatal/genética , Deleção de Sequência , Alelos , Substituição de Aminoácidos , Catarata/enzimologia , Pré-Escolar , Códon sem Sentido/genética , Eritrócitos/enzimologia , Favismo/enzimologia , Feminino , Doenças Genéticas Inatas/enzimologia , Glutationa Redutase/química , Heterozigoto , Humanos , Recém-Nascido , Icterícia Neonatal/enzimologia , Leucócitos/enzimologia , Masculino , Pessoa de Meia-Idade , Estrutura Quaternária de Proteína , Estrutura Terciária de ProteínaRESUMO
BACKGROUND AND AIM OF THE WORK: The glutathione system has a key role in the defence against oxidative stress. To function properly, this system needs NADPH to maintain glutathione (GSH) in its reduced form. We hypothesized that the clinical problems associated with sarcoidosis might be related to a decreased anti-oxidant defence and we therefore measured the activity of the NADPH-generating enzyme glucose-6-phosphate dehydrogenase (G6PD), the GSH-regenerating enzyme glutathione reductase (GR) and indirectly the level of NADPH in red blood cells from patients with sarcoidosis. METHODS: In a population of sarcoidosis (n = 88) patients, G6PD, GR and GR activity after incubation with chromate (GR-Cr) were measured in erythrocytes. A decreased concentration of NADPH was revealed by an increased GR-Cr (> 0.6 IU/g Hb). To exclude a mutation in the G6PD gene, sequencing was performed in cases with an abnormal GR-Cr. Sarcoidosis pulmonary disease severity was evaluated by means of laboratory data, radiographic staging, HRCT scoring, pulmonary function and exercise capacity testing. RESULTS: Fourteen (29.2%) females and one (2.5%) male demonstrated an increased GR-Cr test, indicative of a decreased NADPH level. Patients with an abnormal test result demonstrated also significantly increased ACE and GR values (p < 0.05). Only one female case (of 6 tested) appeared to have a mutation in the G6PD gene. CONCLUSION: In a considerable percentage of female patients with sarcoidosis, a decreased level of NADPH in the erythrocytes was found.
