RESUMEN
Isobutyryl-CoA Dehydrogenase Deficiency (IBDD) is an inherited disorder of valine metabolism caused by mutations in ACAD8. Most reported patients have been diagnosed through newborn screening programmes due to elevated C4-carnitine levels and appear clinically asymptomatic. One reported non-screened patient had dilated cardiomyopathy and anaemia at the age of two years. We report a 13 month old girl diagnosed with IBDD after developing hypoglycaemic encephalopathy (blood glucose 1.9 mmol/l) during an episode of rotavirus-induced gastroenteritis. Metabolic investigations demonstrated an appropriate ketotic response (free fatty acids 2594 µmol/l, 3-hydroxybutyrate 3415 µmol/l), mildly elevated plasma lactate (3.4 mmol/l), increased C4-carnitine on blood spot and plasma acylcarnitine analysis and other metabolic abnormalities secondary to ketosis. After recovery, C4-carnitine remained increased and isobutyrylglycine was detected on urine organic acid analysis. Free carnitine was normal in all acylcarnitine samples. IBDD was confirmed by finding a homozygous c.845C > T substitution in ACAD8. The patient was given, but has not used, a glucose polymer emergency regimen and after ten years' follow-up has had no further episodes of hypoglycaemia nor has she developed cardiomyopathy or anaemia. Psychomotor development has been normal to date. Though we suspect IBDD did not contribute to hypoglycaemia in this patient, patients should be followed-up carefully and glucose polymer emergency regimens may be indicated if recurrent episodes of hypoglycaemia occur.
RESUMEN
Neonatal screening of medium-chain acyl-CoA dehydrogenase deficiency (MCADD) is of major importance due to the significant morbidity and mortality in undiagnosed patients. MCADD screening has been performed routinely in Galicia since July 2000, and until now 199,943 newborns have been screened. We identified 11 cases of MCADD, which gives an incidence of 1/18,134. During this period, no false negative screens have been detected. At diagnosis, all identified newborns were asymptomatic. Our data showed that octanoylcarnitine (C8) and C8/C10 ratio are the best markers for screening of MCADD. C8 was increased in all patients and C8/C10 was increased in all but one patient.The common mutation, c.985A > G, was found in homozygosity in seven newborns and in compound heterozygosity in three, while one patient did not carry the common mutation at all. In addition, two novel mutations c.245G > C (p.W82S) and c.542A > G (p.D181G) were identified. Ten of the 11 identified newborns did not experience any episodes of decompensation. The patient with the highest level of medium chain acylcarnitines at diagnosis, who was homozygous for the c.985A > G mutation, died at the age of 2 years due to a severe infection.This is the first report of the results from neonatal screening for MCADD in Spain. Our data provide further evidence of the benefits of MCADD screening and contribute to better understanding of this disease.
RESUMEN
OBJECTIVES: It has been suggested that homozygous c.985A>G medium-chain acyl-CoA dehydrogenase deficiency (MCADD) is a disease of White ethnic origin but little is known regarding its ethnic distribution. We estimated ethnic-specific homozygous c.985A>G MCADD birth prevalence from a large-scale UK newborn screening study. METHODS: Homozygous c.985A>G MCADD cases were ascertained in six English newborn screening centres between 1 March 2004 and 28 February 2007 by screening approximately 1.1 million newborns using tandem mass spectrometry analysis of underivatised blood spot samples to quantitate octanoylcarnitine (C8). Follow-up biochemistry and mutation analyses for cases (mean triplicate C8 value >/=0.5 micromol/L) were reviewed to confirm diagnosis. Ethnicity was ascertained from clinician report and denominators from 2001 UK Census estimates of ethnic group of children less than one year. RESULTS: Sixty-four infants were c.985A>G MCADD homozygotes (overall prevalence 5.8 per 100,000 live births; 95% CI 4.4-7.2). Sixty (93%) were White, two (3%) were mixed/other and two were of unknown ethnic origin. No Asian or Black homozygotes were identified. Proportions of White, mixed/other, Asian and Black births in screening regions were estimated, yielding homozygous c.985A>G MCADD birth prevalence of 6.9 per 100,000 (95% CI 5.2-8.8) in White, and 95% CI estimates of 0-2.7 per 100,000 in Asian and 0-5.8 in Black populations. The c.985A>G carrier frequency in the White group was estimated at one in 65 (95% CI 1/74, 1/61) under Hardy-Weinberg conditions. CONCLUSION: c.985A>G homozygous MCADD is not found in Black and Asian ethnic groups that have been screened at birth in England. This is consistent with the earlier published observations suggesting that MCADD due to the c.985A>G mutation is a disease of White ethnic origin.
Asunto(s)
Acil-CoA Deshidrogenasa/deficiencia , Acil-CoA Deshidrogenasa/genética , Errores Innatos del Metabolismo Lipídico/genética , Polimorfismo de Nucleótido Simple , Niño , Etnicidad/genética , Pruebas Genéticas/métodos , Homocigoto , Humanos , Incidencia , Recién Nacido , Errores Innatos del Metabolismo Lipídico/diagnóstico , Errores Innatos del Metabolismo Lipídico/epidemiología , Tamizaje Masivo , Tamizaje Neonatal , Prevalencia , Reino Unido/epidemiologíaRESUMEN
BACKGROUND: Medium Chain Acyl-CoA Dehydrogenase (MCAD) Deficiency is an autosomal recessive disorder of fatty acid oxidation, with potential fatal outcome. MCAD deficiency is diagnosed by acylcarnitine analysis on newborn screening blood spot cards by tandem mass spectrometry. Early diagnosis of MCAD and presymptomatic treatment can potentially reduce morbidity and mortality. OBJECTIVES: To evaluate incidence, clinical outcome, biochemical and molecular phenotype of MCAD cases detected in the first three years of newborn screening in British Columbia (BC). METHODS AND RESULTS: Medium chain length acylcarnitines, octanoylcarnitine (C8) and decanoylcarnitine (C10), were measured on newborn screening blood spot cards. Out of 121,000 live births, 17 newborns had C8 values above the screening cut-off of 0.38 umol/L. Ten newborns had elevated C8 on repeat cards and were investigated further. Both C8 and C8/C10 ratios remained abnormal in all confirmed MCAD cases. Positive predictive value of screening was 58% with no false negative results. Seven patients were homozygous for the common c.985A > G MCAD mutation and three others were compound heterozygous for the c.985A > G and a second mutation. Two novel mutations were identified (c.260T > C and c.382T > A). The estimated incidence of MCAD was approximately 1:12,000 live births. Upon frequent feeding and carnitine supplementation, none of the patients had metabolic crises or adverse outcomes. CONCLUSION: Frequency of MCAD in BC is comparable to reports from other newborn screening programs. Persistence of elevated C8 levels and C8/C10 ratios in confirmed MCAD cases suggest that these are sensitive markers for newborn screening. Early detection and treatment have successfully prevented adverse health outcomes in patients with MCAD.
Asunto(s)
Acil-CoA Deshidrogenasa/deficiencia , Tamizaje Neonatal , Acil-CoA Deshidrogenasa/genética , Colombia Británica/epidemiología , Muerte Súbita Cardíaca/epidemiología , Muerte Súbita Cardíaca/prevención & control , Femenino , Indicadores de Salud , Humanos , Incidencia , Recién Nacido , Errores Innatos del Metabolismo Lipídico/diagnóstico , Errores Innatos del Metabolismo Lipídico/tratamiento farmacológico , Errores Innatos del Metabolismo Lipídico/epidemiología , Errores Innatos del Metabolismo Lipídico/genética , Masculino , Fenotipo , Factores de Tiempo , Resultado del TratamientoRESUMEN
Very-long-chain acyl-coenzyme A dehydrogenase (VLCAD) deficiency is an inborn mitochondrial fatty-acid beta-oxidation (FAO) defect associated with a broad mutational spectrum, with phenotypes ranging from fatal cardiopathy in infancy to adolescent-onset myopathy, and for which there is no established treatment. Recent data suggest that bezafibrate could improve the FAO capacities in beta-oxidation-deficient cells, by enhancing the residual level of mutant enzyme activity via gene-expression stimulation. Since VLCAD-deficient patients frequently harbor missense mutations with unpredictable effects on enzyme activity, we investigated the response to bezafibrate as a function of genotype in 33 VLCAD-deficient fibroblasts representing 45 different mutations. Treatment with bezafibrate (400 microM for 48 h) resulted in a marked increase in FAO capacities, often leading to restoration of normal values, for 21 genotypes that mainly corresponded to patients with the myopathic phenotype. In contrast, bezafibrate induced no changes in FAO for 11 genotypes corresponding to severe neonatal or infantile phenotypes. This pattern of response was not due to differential inductions of VLCAD messenger RNA, as shown by quantitative real-time polymerase chain reaction, but reflected variable increases in measured VLCAD residual enzyme activity in response to bezafibrate. Genotype cross-analysis allowed the identification of alleles carrying missense mutations, which could account for these different pharmacological profiles and, on this basis, led to the characterization of 9 mild and 11 severe missense mutations. Altogether, the responses to bezafibrate reflected the severity of the metabolic blockage in various genotypes, which appeared to be correlated with the phenotype, thus providing a new approach for analysis of genetic heterogeneity. Finally, this study emphasizes the potential of bezafibrate, a widely prescribed hypolipidemic drug, for the correction of VLCAD deficiency and exemplifies the integration of molecular information in a therapeutic strategy.
Asunto(s)
Acil-CoA Deshidrogenasa de Cadena Larga/deficiencia , Acil-CoA Deshidrogenasa de Cadena Larga/genética , Bezafibrato/uso terapéutico , Hipolipemiantes/uso terapéutico , Errores Innatos del Metabolismo Lipídico/genética , Acil-CoA Deshidrogenasa de Cadena Larga/química , Acil-CoA Deshidrogenasa de Cadena Larga/metabolismo , Animales , Células Cultivadas , Ácidos Grasos/metabolismo , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/enzimología , Fibroblastos/patología , Terapia Genética/métodos , Genotipo , Humanos , Errores Innatos del Metabolismo Lipídico/enzimología , Modelos Moleculares , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , Ratas , Piel/citología , Piel/enzimología , Piel/patologíaRESUMEN
Deficiency of the hepatic cytosolic enzyme tyrosine aminotransferase (TAT) causes marked hypertyrosinaemia leading to painful palmoplantar hyperkeratoses, pseudodendritic keratitis and variable mental retardation (oculocutaneous tyrosinaemia type II or Richner-Hanhart syndrome). Parents may therefore seek prenatal diagnosis, but this is not possible by biochemical assays as tyrosine does not accumulate in amniotic fluid and TAT is not expressed in chorionic villi or amniocytes. Molecular analysis is therefore the only possible approach for prenatal diagnosis and carrier detection. To this end, we sought TAT gene mutations in 9 tyrosinaemia II patients from three consanguineous Palestinian kindreds. In two kindreds (7 patients), the only potential abnormality identified after sequencing all 12 exons and exon-intron boundaries was homozygosity for a silent, single-nucleotide transversion c.1224G > T (p.T408T) at the last base of exon 11. This was predicted to disrupt the 5' donor splice site of exon 11 and result in missplicing. However, as TAT is expressed exclusively in liver, patient mRNA could not be obtained for splicing analysis. A minigene approach was therefore used to assess the effect of c.1224G > T on exon 11 splicing. Transfection experiments with wild-type and c.1224G > T mutant minigene constructs demonstrated that c.1224G > T results in complete exon 11 skipping, illustrating the utility of this approach for confirming a putative splicing defect when cDNA is unavailable. Homozygosity for a c.1249C > T (R417X) exon 12 nonsense mutation (previously reported in a French patient) was identified in both patients from the third kindred, enabling successful prenatal diagnosis of an unaffected fetus using chorionic villous tissue.
Asunto(s)
Oftalmopatías/genética , Mutación , Enfermedades de la Piel/genética , Tirosina Transaminasa/genética , Tirosinemias/genética , Adulto , Empalme Alternativo , Niño , Preescolar , Análisis Mutacional de ADN/métodos , Exones , Femenino , Humanos , Lactante , Recién Nacido , Israel , Masculino , Datos de Secuencia Molecular , LinajeRESUMEN
We diagnosed six newborn babies with very long-chain acyl-CoA dehydrogenase deficiency (VLCADD) through newborn screening in three years in Victoria (prevalence rate: 1:31,500). We identified seven known and two new mutations in our patients (2/6 homozygotes; 4/6 compound heterozygotes). Blood samples taken at age 48-72 h were diagnostic whereas repeat samples at an older age were normal in 4/6 babies. Urine analysis was normal in 5/5. We conclude that the timing of blood sampling for newborn screening is important and that it is important to perform mutation analysis to avoid false-negative diagnoses of VLCADD in asymptomatic newborn babies. In view of the emerging genotype-phenotype correlation in this disorder, the information derived from mutational analysis can be helpful in designing the appropriate follow-up and therapeutic regime for these patients.
Asunto(s)
Acil-CoA Deshidrogenasa de Cadena Larga/deficiencia , Acil-CoA Deshidrogenasa de Cadena Larga/genética , Errores Innatos del Metabolismo/diagnóstico , Acil-CoA Deshidrogenasa de Cadena Larga/sangre , Sustitución de Aminoácidos/genética , Recolección de Muestras de Sangre/métodos , Carnitina/metabolismo , Carnitina/uso terapéutico , Análisis Mutacional de ADN/métodos , Humanos , Recién Nacido , Espectrometría de Masas , Errores Innatos del Metabolismo/genética , Errores Innatos del Metabolismo/metabolismo , Mutación , Tamizaje Neonatal/instrumentación , Tamizaje Neonatal/métodos , Sitios de Empalme de ARN/genéticaRESUMEN
General mitochondrial trifunctional protein (TFP) deficiency leads to a wide clinical spectrum of disease ranging from severe neonatal/infantile cardiomyopathy and early death to mild chronic progressive sensorimotor poly-neuropathy with episodic rhabdomyolysis. Isolated long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency resulting from the common Glu510Gln mutation usually gives rise to a moderately severe phenotype with multiorgan involvement with high morbidity and mortality. However, isolated LCHAD deficiency can also be consistent with long-term survival in patients identified and treated from an early age. We present biochemical, clinical and mutation data in 9 patients spanning the full spectrum of disease. Fibroblast acylcarnitine profiling shows good correlation with clinical phenotype using the ratio C18(OH)/(C14(OH)+C12(OH)). This ratio shows a gradation of values, from high in four patients with severe neonatal disease (2.5+/-0.8), to low in two neuromyopathic patients (0.35, 0.2). Fibroblast fatty acid oxidation flux assays also show correlation with the patient phenotype, when expressed either as percentage residual activity with palmitate or as a ratio of percentage activity of myristate/oleate (M/O ratio). Fibroblasts from four patients with severe neonatal disease gave an M/O ratio of 4.0+/-0.6 compared to 1.97 and 1.62 in two neuromyopathic patients. Specific enzyme assay of LCHAD and long-chain 3-ketothiolase activity in patient cells shows lack of correlation with phenotype. These results show that measurements in intact cells, which allow all determinative and modifying cellular factors to be present, better reflect patient phenotype. Mutation analysis reveals a number of alpha- and beta-subunit mutations. Peripheral sensorimotor polyneuropathy, often as the initial major presenting feature but usually later accompanied by episodic rhabdomyolysis, is a manifestation of mild TFP protein deficiency. The mild clinical presentation and relative difficulty in diagnosis suggest that this form of TFP is probably underdiagnosed.
Asunto(s)
Acil-CoA Deshidrogenasa de Cadena Larga/deficiencia , Errores Innatos del Metabolismo Lipídico/diagnóstico , Errores Innatos del Metabolismo Lipídico/genética , Mitocondrias/patología , Complejos Multienzimáticos/deficiencia , Cardiomiopatías/diagnóstico , Cardiomiopatías/genética , Carnitina/análogos & derivados , Carnitina/metabolismo , Exones , Ácidos Grasos/metabolismo , Fibroblastos/metabolismo , Homocigoto , Humanos , Masculino , Proteína Trifuncional Mitocondrial , Mutación , Fenotipo , Polineuropatías/diagnóstico , Polineuropatías/genética , Pronóstico , Rabdomiólisis/diagnóstico , Rabdomiólisis/genéticaRESUMEN
We report a patient with lipid-storage myopathy due to multiple acyl-CoA dehydrogenation deficiency (MADD). Molecular genetic analysis showed that she was compound heterozygous for mutations in the gene for electron transfer flavoprotein:ubiquinone oxidoreductase (ETFQO). Despite a good initial response to treatment, she developed respiratory insufficiency at age 14 years and has required long-term overnight ventilation. Thus, MADD is one of the few conditions that can cause a myopathy with weakness of the respiratory muscles out of proportion to the limb muscles.
Asunto(s)
Acil-CoA Deshidrogenasas/genética , Flavoproteínas Transportadoras de Electrones/genética , Proteínas Hierro-Azufre/genética , Errores Innatos del Metabolismo Lipídico/diagnóstico , Errores Innatos del Metabolismo Lipídico/genética , Metabolismo de los Lípidos , Enfermedades Musculares/genética , Mutación , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/genética , Acil-CoA Deshidrogenasas/deficiencia , Adolescente , Edad de Inicio , Secuencia de Bases , Western Blotting , Análisis Mutacional de ADN , ADN Complementario/metabolismo , Complejo I de Transporte de Electrón/deficiencia , Complejo I de Transporte de Electrón/genética , Flavoproteínas Transportadoras de Electrones/metabolismo , Femenino , Fibroblastos/metabolismo , Heterocigoto , Humanos , Modelos Genéticos , Datos de Secuencia Molecular , Enfermedades Musculares/diagnóstico , Fenotipo , Respiración ArtificialRESUMEN
Carnitine palmitoyltransferase type II (CPT II) deficiency has three basic phenotypes, late-onset muscular (mild), infantile/juvenile hepatic (intermediate) and severe neonatal. We have measured fatty acid oxidation and CPT II activity and performed mutation studies in 24 symptomatic patients representing the full clinical spectrum of disease. Severe and intermediate phenotypes show a clear correlation with biochemical indices and genetic analysis revealed causative mutations in most patients. Studies of mild phenotypes suggest a more complex interaction, with higher residual fatty acid oxidation, a wider range of CPT II activity (10-60%) but little evidence of genotype-phenotype correlation. Residual CPT II mutant protein from myopathic patients shows thermal instability at 41 degrees C. The common 'polymorphisms' V3681 and M647V are strikingly overrepresented in the myopathic patients, the implication being that they may significantly influence the manifestation of clinical disease and could therefore potentially be considered as a susceptibility variants. Among myopathic individuals, males comprised 88% of patients, suggesting increased susceptibility to clinical disease. A small number of symptomatic patients appear to have significant residual CPT II activity (42-60%) The synergistic interaction of partial deficiencies of CPT II, muscle adenosine monophosphate deaminase and possibly other enzymes of muscle energy metabolism in the aetiology of episodic myopathy deserves wider consideration.
Asunto(s)
Carnitina O-Palmitoiltransferasa/deficiencia , Carnitina O-Palmitoiltransferasa/genética , Errores Innatos del Metabolismo Lipídico/enzimología , Errores Innatos del Metabolismo Lipídico/genética , AMP Desaminasa/metabolismo , Adolescente , Adulto , Línea Celular , Niño , Preescolar , Femenino , Fibroblastos/metabolismo , Genotipo , Humanos , Lactante , Recién Nacido , Masculino , Mutación/genética , Mutación/fisiología , Oxidación-Reducción , Palmitatos/metabolismo , Polimorfismo Genético/genética , TemperaturaRESUMEN
Multiple acyl-CoA-dehydrogenase deficiency (MADD) or glutaric aciduria type II (GAII) are a group of metabolic disorders due to deficiency of either electron transfer flavoprotein (ETF) or electron transfer flavoprotein ubiquinone oxidoreductase (ETF-QO). We report the clinical features and biochemical and molecular genetic analyses of a patient with a mild late-onset form of GAII due to beta-ETF deficiency. Biochemical data showed an abnormal urine organic acid profile, low levels of free carnitine, increased levels of C(10:1n-6), and C(14:1n-9) in plasma, and decreased oxidation of [9,10-3H]palmitate and [9,10-3H]myristate in fibroblasts, suggesting MAD deficiency. In agreement with these findings, mutational analysis of the ETF/ETFDH genes demonstrated an ETFB missense mutation 124T>C in exon 2 leading to replacement of cysteine-42 with arginine (C42R), and a 604_606AAG deletion in exon 6 in the ETFB gene resulting in the deletion of lysine-202 (K202del). The present report delineates further the phenotype of mild beta-ETF deficiency and illustrates that the differential diagnosis of GAII is readily achieved by mutational analysis.
Asunto(s)
Errores Innatos del Metabolismo de los Aminoácidos/diagnóstico , Errores Innatos del Metabolismo de los Aminoácidos/genética , Flavoproteínas Transportadoras de Electrones/deficiencia , Flavoproteínas Transportadoras de Electrones/genética , Electrones , Proteínas Hierro-Azufre/deficiencia , Proteínas Hierro-Azufre/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/deficiencia , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/genética , Arginina/química , Carnitina/sangre , Cisteína/química , Análisis Mutacional de ADN , Exones , Femenino , Fibroblastos/metabolismo , Cromatografía de Gases y Espectrometría de Masas , Eliminación de Gen , Glutaratos/orina , Humanos , Recién Nacido , Lisina/química , Mutación Missense , Oxígeno/metabolismo , FenotipoRESUMEN
Mutation analysis of metabolic disorders, such as the fatty acid oxidation defects, offers an additional, and often superior, tool for specific diagnosis compared to traditional enzymatic assays. With the advancement of the structural part of the Human Genome Project and the creation of mutation databases, procedures for convenient and reliable genetic analyses are being developed. The most straightforward application of mutation analysis is to specific diagnoses in suspected patients, particularly in the context of family studies and for prenatal/preimplantation analysis. In addition, from these practical uses emerges the possibility to study genotype-phenotype relationships and investigate the molecular pathogenesis resulting from specific mutations or groups of mutations. In the present review we summarize current knowledge regarding genotype-phenotype relationships in three disorders of mitochondrial fatty acid oxidation: very-long chain acyl-CoA dehydrogenase (VLCAD, also ACADVL), medium-chain acyl-CoA dehydrogenase (MCAD, also ACADM), and short-chain acyl-CoA dehydrogenase (SCAD, also ACADS) deficiencies. On the basis of this knowledge we discuss current understanding of the structural implications of mutation type, as well as the modulating effect of the mitochondrial protein quality control systems, composed of molecular chaperones and intracellular proteases. We propose that the unraveling of the genetic and cellular determinants of the modulating effects of protein quality control systems may help to assess the balance between genetic and environmental factors in the clinical expression of a given mutation. The realization that the effect of the monogene, such as disease-causing mutations in the VLCAD, MCAD, and SCAD genes, may be modified by variations in other genes presages the need for profile analyses of additional genetic variations. The rapid development of mutation detection systems, such as the chip technologies, makes such profile analyses feasible. However, it remains to be seen to what extent mutation analysis will be used for diagnosis of fatty acid oxidation defects and other metabolic disorders.
Asunto(s)
Ácidos Grasos/metabolismo , Errores Innatos del Metabolismo Lipídico/genética , Mitocondrias/metabolismo , Acil-CoA Deshidrogenasa , Acil-CoA Deshidrogenasa de Cadena Larga , Acil-CoA Deshidrogenasas/deficiencia , Acil-CoA Deshidrogenasas/genética , Genotipo , Humanos , Errores Innatos del Metabolismo Lipídico/enzimología , Errores Innatos del Metabolismo Lipídico/metabolismo , Mitocondrias/enzimología , Mutación , Oxidación-Reducción , FenotipoRESUMEN
Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most frequently diagnosed mitochondrial beta-oxidation defect, and it is potentially fatal. Eighty percent of patients are homozygous for a common mutation, 985A-->G, and a further 18% have this mutation in only one disease allele. In addition, a large number of rare disease-causing mutations have been identified and characterized. There is no clear genotype-phenotype correlation. High 985A-->G carrier frequencies in populations of European descent and the usual avoidance of recurrent disease episodes by patients diagnosed with MCAD deficiency who comply with a simple dietary treatment suggest that MCAD deficiency is a candidate in prospective screening of newborns. Therefore, several such screening programs employing analysis of acylcarnitines in blood spots by tandem mass spectrometry (MS/MS) are currently used worldwide. No validation of this method by mutation analysis has yet been reported. We investigated for MCAD mutations in newborns from US populations who had been identified by prospective MS/MS-based screening of 930,078 blood spots. An MCAD-deficiency frequency of 1/15,001 was observed. Our mutation analysis shows that the MS/MS-based method is excellent for detection of MCAD deficiency but that the frequency of the 985A-->G mutant allele in newborns with a positive acylcarnitine profile is much lower than that observed in clinically affected patients. Our identification of a new mutation, 199T-->C, which has never been observed in patients with clinically manifested disease but was present in a large proportion of the acylcarnitine-positive samples, may explain this skewed ratio. Overexpression experiments showed that this is a mild folding mutation that exhibits decreased levels of enzyme activity only under stringent conditions. A carrier frequency of 1/500 in the general population makes the 199T-->C mutation one of the three most prevalent mutations in the enzymes of fatty-acid oxidation.
Asunto(s)
Acil-CoA Deshidrogenasas/deficiencia , Acil-CoA Deshidrogenasas/genética , Carnitina/análogos & derivados , Carnitina/sangre , Pruebas Genéticas/métodos , Mutación Missense/genética , Acil-CoA Deshidrogenasa , Acil-CoA Deshidrogenasas/química , Acil-CoA Deshidrogenasas/metabolismo , Alelos , Chaperonina 10/genética , Chaperonina 10/metabolismo , Chaperonina 60/genética , Chaperonina 60/metabolismo , Análisis Mutacional de ADN , Estabilidad de Enzimas , Escherichia coli/genética , Exones/genética , Haplotipos/genética , Heterocigoto , Homocigoto , Humanos , Recién Nacido , Espectrometría de Masas , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida/genética , Polimorfismo de Nucleótido Simple/genética , Pliegue de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reproducibilidad de los Resultados , TemperaturaRESUMEN
A patient with very long chain acyl-CoA dehydrogenase (VLCAD) deficiency is reported. He had a severe neonatal presentation and cardiomyopathy. He was found to be homozygous for a severe mutation with no residual enzyme activity. Tandem mass spectrometry on dried blood spots revealed increased long chain acylcarnitines. VLCAD enzyme activity was severely decreased to 2% of control levels. Dietary management consisted of skimmed milk supplemented with medium chain triglycerides and L-carnitine. Outcome was good and there was no acute recurrence.
Asunto(s)
Acil-CoA Deshidrogenasa de Cadena Larga/deficiencia , Cardiomiopatías/enzimología , Homocigoto , Acil-CoA Deshidrogenasa de Cadena Larga/genética , Cardiomiopatías/dietoterapia , Cardiomiopatías/genética , Estudios de Seguimiento , Humanos , Recién Nacido , Masculino , Espectrometría de Masas , Mutación , PronósticoRESUMEN
AIMS: To study the distribution of Epstein-Barr virus (EBV) variants containing mutations in the latent membrane protein 1 (LMP-1) oncogene and promoter in EBV associated Hodgkin's disease and infectious mononucleosis compared with previous findings in asymptomatic EBV carriers. METHODS: Sequence analysis of the EBV LMP-1 promoter and gene in isolates from Danish patients with Hodgkin's disease (n = 61) and infectious mononucleosis (n = 10). RESULTS: Viruses (previously designated group D) that contain two mutations in the activating transcription factor/cAMP response element (ATF/CRE) in the LMP-1 promoter, which are known to decrease promoter activity greatly, were significantly less frequent in Hodgkin's disease than in both infectious mononucleosis (p = 0.0081) and asymptomatic EBV carriers (p = 0.0084). In some cases, the LMP-1 gene contained mutations in a recently identified cytotoxic T cell (CTL) epitope. Most viral isolates contained mutations shown to increase nuclear factor kappa B (NF-kappa B) activation and had one of two newly identified C-terminal activation regions 3 (CTAR-3) deleted. The exon 1 Xho-I restriction site in the LMP-1 gene could be lost through a range of different mutations. CONCLUSIONS: These findings indicate selection pressure against EBV strains with weak LMP-1 promoter activity in Hodgkin's disease and thus provide further strong circumstantial evidence for the pathogenic role of EBV (and LMP-1) in this disease. Mutation of the CTL epitope suggests immune selection of EBV strains. Many EBV isolates contain functionally important mutations in the LMP-1 gene. Loss of the Xho-I restriction site should not be used as a marker of specific LMP-1 variants.
Asunto(s)
Proteínas de Unión al ADN , Herpesvirus Humano 4/genética , Enfermedad de Hodgkin/virología , Mononucleosis Infecciosa/virología , Mutación , Regiones Promotoras Genéticas/genética , Proteínas de la Matriz Viral/genética , Factor de Transcripción Activador 1 , Secuencia de Bases , Proteína de Unión a CREB , Epítopos de Linfocito T/genética , Marcadores Genéticos , Enfermedad de Hodgkin/inmunología , Humanos , Mononucleosis Infecciosa/inmunología , Datos de Secuencia Molecular , FN-kappa B/genética , Proteínas Nucleares/genética , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADN , Linfocitos T Citotóxicos/ultraestructura , Transactivadores/genética , Factores de Transcripción/genéticaRESUMEN
Acyl-CoA dehydrogenase (ACAD) defects in isoleucine and valine catabolism have been proposed in clinically diverse patients with an abnormal pattern of metabolites in their urine, but they have not been proved enzymatically or genetically, and it is unknown whether one or two ACADs are involved. We investigated a patient with isolated 2-methylbutyrylglycinuria, suggestive of a defect in isoleucine catabolism. Enzyme assay of the patient's fibroblasts, using 2-methylbutyryl-CoA as substrate, confirmed the defect. Sequence analysis of candidate ACADs revealed heterozygosity for the common short-chain ACAD A625 variant allele and no mutations in ACAD-8 but a 100-bp deletion in short/branched-chain ACAD (SBCAD) cDNA from the patient. Our identification of the SBCAD gene structure (11 exons; >20 kb) enabled analysis of genomic DNA. This showed that the deletion was caused by skipping of exon 10, because of homozygosity for a 1228G-->A mutation in the patient. This mutation was not present in 118 control chromosomes. In vitro transcription/translation experiments and overexpression in COS cells confirmed the disease-causing nature of the mutant SBCAD protein and showed that ACAD-8 is an isobutyryl-CoA dehydrogenase and that both wild-type proteins are imported into mitochondria and form tetramers. In conclusion, we report the first mutation in the SBCAD gene, show that it results in an isolated defect in isoleucine catabolism, and indicate that ACAD-8 is a mitochondrial enzyme that functions in valine catabolism.
Asunto(s)
Errores Innatos del Metabolismo de los Aminoácidos/enzimología , Errores Innatos del Metabolismo de los Aminoácidos/genética , Isoleucina/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , Oxidorreductasas/deficiencia , Valina/metabolismo , Empalme Alternativo/genética , Errores Innatos del Metabolismo de los Aminoácidos/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células COS , Preescolar , Consanguinidad , Análisis Mutacional de ADN , Estabilidad de Enzimas , Exones , Femenino , Heterocigoto , Humanos , Intrones , Masculino , Mitocondrias/enzimología , Mitocondrias/metabolismo , Mutación/genética , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Pakistán , Transporte de Proteínas , Eliminación de Secuencia/genética , TransfecciónRESUMEN
We have determined the cDNA sequence and exon/intron structure of the human CLPX gene encoding a human ortholog of the E. coli ClpX chaperone and protease subunit. The CLPX gene comprises 14 exons and encodes a 633-amino acid-long precursor polypeptide. The polypeptide contains an N-terminal putative mitochondrial transit peptide, and expression of a full-length ClpX cDNA tagged at its C-terminus (Myc-His) shows that the polypeptide is transported into mitochondria. FISH analysis localized the CLPX gene to human Chromosome (Chr) 15q22.1-22.32. This localization was refined by radiation hybrid mapping placing the CLPX gene 4.6 cR distal to D15S159. Murine ClpX cDNA was sequenced, and the mouse Clpx locus was mapped to a position between 31 and 42 cM offset from the centromere on mouse Chr 9. Experimental observations indicate the presence of a pseudogene in the mouse genome and sequence variability between mouse ClpX cDNAs from different strains. Alignment of the human and mouse ClpX amino acid sequences with ClpX sequences from other organisms shows that they display the typical modular organization of domains with one AAA(+) domain common to a large group of ATPases and several other domains conserved in ClpX orthologs linked by non-conserved sequences. Notably, a C-4 zinc finger type motif is recognized in human and mouse ClpX. This motif of so far unknown function is present only in a subset of the known ClpX sequences.
Asunto(s)
Adenosina Trifosfatasas/genética , Cromosomas Humanos Par 15 , Mitocondrias/genética , ATPasas Asociadas con Actividades Celulares Diversas , Adenosina Trifosfatasas/fisiología , Secuencia de Aminoácidos , Animales , Mapeo Cromosómico , ADN/análisis , Endopeptidasa Clp , Proteínas de Escherichia coli , Expresión Génica , Genoma , Humanos , Ratones , Ratones Endogámicos C57BL , Chaperonas Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Homología de Secuencia de Aminoácido , Fracciones SubcelularesRESUMEN
Many disease-causing point mutations do not seriously compromise synthesis of the affected polypeptide but rather exert their effects by impairing subsequent protein folding or stability of the folded protein. This often results in rapid degradation of the affected protein. The concepts of such 'conformational disease' are illustrated by reference to cystic fibrosis, phenylketonuria and short-chain acyl-CoA dehydrogenase deficiency. Other cellular components such as chaperones and proteases, as well as environmental factors, may combine to modulate the phenotype of such disorders and this may open up new therapeutic approaches.
Asunto(s)
Enfermedades Genéticas Congénitas/metabolismo , Proteínas/química , Proteínas/metabolismo , Acil-CoA Deshidrogenasa , Acil-CoA Deshidrogenasas/deficiencia , Fibrosis Quística/genética , Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Citosol/metabolismo , Retículo Endoplásmico/metabolismo , Enfermedades Genéticas Congénitas/genética , Humanos , Mitocondrias/metabolismo , Mutación , Fenilcetonurias/genética , Fenilcetonurias/metabolismo , Conformación Proteica , Pliegue de Proteína , Proteínas/genéticaRESUMEN
The rapid development in human genome research has resulted in a tremendous increase in our understanding of the molecular basis of many genetic skin diseases. One outstanding example of this is diseases caused by mutations in keratin genes, which comprise several disorders of the epidermis, as for example the different types of epidermolysis bullosa simplex. In this respect, the most important questions have been to 1. Define the molecular defect. 2. Unravel the pathophysiological mechanisms that lead to the characteristic phenotype and 3. Design of new therapeutic strategies. Molecular research has contributed significantly to the first two issues whereas a therapeutic break-through has yet to appear.