RESUMEN
Guanidinoacetate methyltransferase (GAMT) deficiency (MIM 601240), an autosomal recessive disorder of creatine biosynthesis, presents with mental retardation, extrapyramidal symptoms, autistic-like behavior and epilepsy. Other hallmarks are cerebral creatine deficiency, increased levels of guanidinoacetate in body fluids and mutations in the GAMT gene. Creatine supplementation partially restores cerebral creatine content. Worldwide, 29 patients have been identified and 15 different mutations have been reported in the GAMT gene. Ten out of these 29 patients are of Portuguese origin. Likely, a founder effect and a high carrier rate in Portugal exist, since in 17 out of the 20 Portuguese alleles the c.59G>C; p.Trp20Ser mutation was found. We investigated the carrier rate of the c.59G>C; p.Trp20Ser mutation in different regions of Portugal and confirmed the pathogenic nature of this missense mutation by transient transfections. Anonymous bloodspots (1002) were screened for the presence of the c.59G>C; p.Trp20Ser mutation by SNaPshot (Single Nucleotide Polymorphism Multiplex Kit). Eight carriers of c.59G>C; p.Trp20Ser were detected of which four are derived from the Archipelagos. This suggests that the carrier rate of the c.59G>C; p.Trp20Ser mutation is relatively high in these islands, as well as in other parts of Portugal. It also implies that newborn screening in these regions is warranted for this treatable disorder.
Asunto(s)
Guanidinoacetato N-Metiltransferasa/genética , Mutación Missense , Secuencia de Aminoácidos , Secuencia de Bases , Análisis Mutacional de ADN , Enfermedades Carenciales/diagnóstico , Enfermedades Carenciales/genética , Geografía , Guanidinoacetato N-Metiltransferasa/deficiencia , Células HeLa , Humanos , Datos de Secuencia Molecular , Polimorfismo de Nucleótido Simple , Portugal , Prevalencia , Alineación de Secuencia , Serina/genética , Serina/metabolismo , Transfección , Triptófano/genética , Triptófano/metabolismoRESUMEN
Guanidinoacetate methyltransferase deficiency (MIM 601240) is an autosomal recessive disorder of creatine biosynthesis. Patients present with mental retardation, extrapyramidal symptoms, autistic-like behavior, epilepsy, cerebral creatine deficiency and increased levels of guanidinoacetate. So far 15 mutations have been reported, including six missense variants that are highly likely to be pathogenic mutations. To prove that mutations in the GAMT gene are responsible for GAMT deficiency we overexpressed the GAMT open reading frame in GAMT-deficient fibroblasts by stable transfection. In addition, HeLa cells were transiently transfected with the same expression vector. In contrast to mock transfectants transfection of primary GAMT-deficient fibroblasts with wild-type GAMT results in the restoration of GAMT activity as measured by GC-MS using stable isotope labeled substrates. Moreover, the expression of the GAMT-EGFP fusion protein was analyzed by Western blot, confirming the presence of GAMT fusion protein, both in the stable as well as in the transient transfectants. Here, we prove that mutations in the GAMT gene are responsible for GAMT deficiency, since overexpression of the GAMT open reading frame restores GAMT activity in GAMT-deficient fibroblasts. Furthermore, the transient transfection of HeLa cells will be important for functional analysis of variants of unknown consequence (i.e., missense mutations).
Asunto(s)
Errores Innatos del Metabolismo de los Aminoácidos/enzimología , Errores Innatos del Metabolismo de los Aminoácidos/genética , Creatina/deficiencia , Guanidinoacetato N-Metiltransferasa/deficiencia , Guanidinoacetato N-Metiltransferasa/genética , Fibroblastos/enzimología , Células HeLa , Humanos , Mutación , TransfecciónRESUMEN
Activation of mitogen-activated protein (MAP) kinase is essential for cyclin D1 expression and provides a link between mitogenic signalling and cell cycle progression. Hydrogen peroxide (H2O2) activates MAP kinase; however, it is not known whether this leads to cyclin D expression. Sustained expression of cyclin D1 and D2 was observed when Her14 fibroblasts were incubated with 3 mM or higher H2O2 concentrations. Similar results were obtained when cells were incubated in the presence of serum (FCS). However, the sustained expression of cyclin D1 and D2 upon H2O2 treatment was not due to the MAP kinase pathway, because MAP kinase kinase inhibitors did not inhibit cyclin D expression. Furthermore, cyclin D1 and D2 levels remained constant even after addition of a protein synthesis inhibitor, indicating that the effect of H2O2 was not due to induction of protein synthesis. These results indicate that H2O2 reversibly inhibits the ubiquitin-proteasome dependent degradation of cyclin D1 and D2, probably by transiently inhibiting ubiquitination and/or the proteasome.
Asunto(s)
Ciclina D1/metabolismo , Ciclinas/metabolismo , Peróxido de Hidrógeno/metabolismo , Complejos Multienzimáticos/antagonistas & inhibidores , Transducción de Señal , Células 3T3 , Animales , Ciclina D1/biosíntesis , Ciclina D2 , Ciclinas/biosíntesis , Cisteína Endopeptidasas , Inhibidores de Cisteína Proteinasa/farmacología , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Peróxido de Hidrógeno/farmacología , Leupeptinas/farmacología , Ratones , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/fisiología , Complejo de la Endopetidasa Proteasomal , Inhibidores de la Síntesis de la Proteína/farmacología , Albúmina Sérica Bovina/farmacologíaRESUMEN
In childhood acute lymphoblastic leukaemia there are large interpatient variations in levels of the apoptosis-regulating proteins Bax and Bcl-2, but the molecular basis for this variation is unknown. Point-mutations in bax have been reported in cell lines derived from haematological malignancies. Frameshift mutations, which result in reduced Bax levels, have also been found in colon cancer of the microsatellite mutator phenotype. Bcl-2 overexpression, or gain of function mutations in the open reading frame (ORF) or in the translational repressor, the upstream ORF(uORF) of bcl-2, might also be important in deregulating its function or expression. We have therefore analyzed 21 bone marrow aspirates from untreated childhood acute lymphoblastic leukaemia and 2 from myeloid leukaemia for mutations in box and bcl-2. DNA sequence analysis of the ORFs of bax and bcl-2 and of the uORF of bcl-2 revealed no mutations, despite the large range in expression levels. Thus, mutations within the (u)ORFs of bax and bcl-2 that (in)activate or deregulate Bax and Bcl-2 are infrequent in primary childhood acute leukaemia and do not play a major role in regulation of the encoded proteins in this disease.