RESUMEN
Historically, d-glyceric aciduria was thought to cause an uncharacterized blockage to the glycine cleavage enzyme system (GCS) causing nonketotic hyperglycinemia (NKH) as a secondary phenomenon. This inference was reached based on the clinical and biochemical results from the first d-glyceric aciduria patient reported in 1974. Along with elevated glyceric acid excretion, this patient exhibited severe neurological symptoms of myoclonic epilepsy and absent development, and had elevated glycine levels and decreased glycine cleavage system enzyme activity. Mutations in the GLYCTK gene (encoding d-glycerate kinase) causing glyceric aciduria were previously noted. Since glycine changes were not observed in almost all of the subsequently reported cases of d-glyceric aciduria, this theory of NKH as a secondary syndrome of d-glyceric aciduria was revisited in this work. We showed that this historic patient harbored a homozygous missense mutation in AMT c.350C>T, p.Ser117Leu, and enzymatic assay of the expressed mutation confirmed the pathogeneity of the p.Ser117Leu mutation. We conclude that the original d-glyceric aciduria patient also had classic NKH and that this co-occurrence of two inborn errors of metabolism explains the original presentation. We conclude that no evidence remains that d-glyceric aciduria would cause NKH.
Asunto(s)
Ácidos Glicéricos/orina , Hiperglicinemia no Cetósica/complicaciones , Hiperoxaluria Primaria/complicaciones , Hiperoxaluria Primaria/genética , Aminoácido Oxidorreductasas/genética , Aminoácido Oxidorreductasas/metabolismo , Aminometiltransferasa/genética , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Diagnóstico Diferencial , Epilepsia , Ácidos Glicéricos/metabolismo , Glicina/metabolismo , Homocigoto , Humanos , Hiperglicinemia no Cetósica/diagnóstico , Hiperglicinemia no Cetósica/etiología , Hiperglicinemia no Cetósica/genética , Hiperoxaluria Primaria/diagnóstico , Masculino , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/metabolismo , Mutación Missense , Fosfotransferasas (Aceptor de Grupo Alcohol)/deficiencia , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Transferasas/genética , Transferasas/metabolismoRESUMEN
Guanidinoacetate methyltransferase deficiency (GAMT-D) is an autosomal recessively inherited disorder of creatine biosynthesis. Creatine deficiency on cranial proton magnetic resonance spectroscopy, and elevated guanidinoacetate levels in body fluids are the biomarkers of GAMT-D. In 74 patients, 50 different mutations in the GAMT gene have been identified with missense variants being the most common. Clinical and biochemical features of the patients with missense variants were obtained from their physicians using a questionnaire. In 20 patients, 17 missense variants, 25% had a severe, 55% a moderate, and 20% a mild phenotype. The effect of these variants on GAMT enzyme activity was overexpressed using primary GAMT-D fibroblasts: 17 variants retained no significant activity and are therefore considered pathogenic. Two additional variants, c.22C>A (p.Pro8Thr) and c.79T>C (p.Tyr27His) (the latter detected in control cohorts) are in fact not pathogenic as these alleles restored GAMT enzyme activity, although both were predicted to be possibly damaging by in silico analysis. We report 13 new patients with GAMT-D, six novel mutations and functional analysis of 19 missense variants, all being included in our public LOVD database. Our functional assay is important for the confirmation of the pathogenicity of identified missense variants in the GAMT gene.
Asunto(s)
Guanidinoacetato N-Metiltransferasa/deficiencia , Trastornos del Desarrollo del Lenguaje/genética , Trastornos del Desarrollo del Lenguaje/patología , Trastornos del Movimiento/congénito , Adolescente , Adulto , Niño , Preescolar , Femenino , Fibroblastos/enzimología , Predisposición Genética a la Enfermedad , Variación Genética , Guanidinoacetato N-Metiltransferasa/genética , Guanidinoacetato N-Metiltransferasa/metabolismo , Humanos , Masculino , Trastornos del Movimiento/genética , Trastornos del Movimiento/patología , Mutación Missense , Encuestas y Cuestionarios , Adulto JovenRESUMEN
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.
Asunto(s)
Encefalopatías Metabólicas Innatas/genética , Isocitrato Deshidrogenasa/genética , Mosaicismo , Encefalopatías Metabólicas Innatas/diagnóstico , Células Cultivadas , Niño , Femenino , Frecuencia de los Genes , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Mutación , PadresRESUMEN
Mitochondrial myopathies commonly present with exercise intolerance typified by breathlessness and fatigue on exercise. In contrast, exercise-induced rhabdomyolysis and myoglobinuria occur rarely. We present a 43-year-old man with a lifelong history of exercise intolerance associated with myalgia and recurrent episodes of exercise-induced myoglobinuria. From early childhood, he had weekly episodes of myoglobinuria, which became infrequent (every 3 months) as an adult. Carnitine transporter defect was suspected, because carnitine levels were low in muscle. During childhood, he was treated with carnitine (4-5 g daily), but without effect. With the advent of acylcarnitines, profiles mimicking but not diagnostic for multiple acyl-CoA dehydrogenase deficiency (MADD) were found. This led to treatment with riboflavin (100 mg/day for 3 years), again without effect. Clinical examination, including echocardiography, revealed no signs of involvement from other organs, and all relatives were asymptomatic.
Asunto(s)
Carnitina/análogos & derivados , ADN Mitocondrial/genética , Complejo IV de Transporte de Electrones/genética , Mutación/genética , Mioglobinuria/diagnóstico , Mioglobinuria/genética , Adulto , Carnitina/genética , Humanos , Masculino , RecurrenciaRESUMEN
We have investigated seven patients with the type B form of pyruvate carboxylase (PC) deficiency. Mutation analysis revealed eight mutations, all novel. In a patient with exon skipping on cDNA analysis, we identified a homozygous mutation located in a potential branch point sequence, the first possible branch point mutation in PC. Two patients were homozygous for missense mutations (with normal protein amounts on western blot analysis), and two patients were homozygous for nonsense mutations. In addition, a duplication of one base pair was found in a patient who also harboured a splice site mutation. Another splice site mutation led to the activation of a cryptic splice site, shown by cDNA analysis.All patients reported until now with at least one missense mutation have had the milder type A form of PC deficiency. We thus report for the first time two patients with homozygous missense mutations with the severe type B deficiency, clinically indistinguishable from other patients with type B form of PC deficiency.The mutations found here are novel; it is noteworthy that four Turkish patients did not have any mutations in common, despite the rarity of PC deficiency. There is thus no evidence for recurrent mutations in the Turkish or other populations.
RESUMEN
Succinyl-CoA:3-ketoacid CoA transferase (SCOT) deficiency is a rare inherited metabolic disorder of ketone metabolism, characterized by ketoacidotic episodes and often permanent ketosis. To date there are ~20 disease-associated alleles on the OXCT1 gene that encodes the mitochondrial enzyme SCOT. SCOT catalyzes the first, rate-limiting step of ketone body utilization in peripheral tissues, by transferring a CoA moiety from succinyl-CoA to form acetoacetyl-CoA, for entry into the tricarboxylic acid cycle for energy production. We have determined the crystal structure of human SCOT, providing a molecular understanding of the reported mutations based on their potential structural effects. An interactive version of this manuscript (which may contain additional mutations appended after acceptance of this manuscript) may be found on the web address: http://www.thesgc.org/jimd/SCOT .
Asunto(s)
Acidosis/genética , Coenzima A Transferasas/deficiencia , Análisis Mutacional de ADN/métodos , Mutación Missense , Mapas de Interacción de Proteínas , Coenzima A Transferasas/química , Coenzima A Transferasas/genética , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Dominios y Motivos de Interacción de Proteínas/genética , Mapas de Interacción de Proteínas/genética , Multimerización de Proteína/genética , Estructura Cuaternaria de Proteína/genética , Estructura Secundaria de Proteína/genética , Relación Estructura-ActividadRESUMEN
Expanded newborn screening for selected inborn errors of metabolism (IEM) in Denmark, the Faroe Islands and Greenland was introduced in 2002. We now present clinical, biochemical, and statistical results of expanded screening (excluding PKU) of 504,049 newborns during nine years as well as diagnoses and clinical findings in 82,930 unscreened newborns born in the same period. The frequencies of diagnoses made within the panel of disorders screened for are compared with the frequencies of the disorders in the decade preceding expanded newborn screening. The expanded screening was performed as a pilot study during the first seven years, and the experience obtained during these years was used in the development of the routine neonatal screening program introduced in 2009. Methods for screening included tandem mass spectrometry and an assay for determination of biotinidase activity. A total of 310 samples from 504,049 newborns gave positive screening results. Of the 310 results, 114 were true positive, including results from 12 newborns in which the disease in question was subsequently diagnosed in their mothers. Thus, the overall frequency of an IEM in the screening panel was 1:4942 (mothers excluded) or 1:4421 (mothers included). The false positive rate was 0.038% and positive predictive value 37%. Overall specificity was 99.99%. All patients with true positive results were followed in The Center for Inherited Metabolic Disorders in Copenhagen, and the mean follow-up period was 45 months (range 2109 months). There were no deaths among the 102 children, and 94% had no clinically significant sequelae at last follow-up. Our study confirms the higher frequency of selected IEM after implementation of expanded newborn screening and suggests an improved outcome for several disorders. We argue that newborn screening for these disorders should be standard of care, though unresolved issues remain, e.g. about newborns with a potential for remaining asymptomatic throughout life. Well organized logistics of the screening program from screening laboratory to centralized, clinical management is important.
Asunto(s)
Errores Innatos del Metabolismo/diagnóstico , Errores Innatos del Metabolismo/metabolismo , Tamizaje Neonatal/organización & administración , Biotinidasa/metabolismo , Niño , Dinamarca/epidemiología , Reacciones Falso Positivas , Femenino , Groenlandia/epidemiología , Humanos , Recién Nacido , Estudios Longitudinales , Masculino , Errores Innatos del Metabolismo/epidemiología , Proyectos Piloto , Sensibilidad y Especificidad , Espectrometría de Masas en TándemRESUMEN
The Faroe Islands has a high incidence of carnitine transporter deficiency (CTD) and several other autosomal recessive diseases. This article describes the reason for the high frequency, in view of the Faroese history and the diagnosis of CTD. Few individuals founded the Faroese population in the 9th century, and subsequent geographic isolation limited genetic diversity in the Islands.
Asunto(s)
Carnitina/deficiencia , Deficiencia de Vitamina B/genética , Adulto , Carnitina/sangre , Niño , Dinamarca/epidemiología , Efecto Fundador , Variación Genética , Humanos , Proteínas de Transporte de Catión Orgánico/genética , Prevalencia , Miembro 5 de la Familia 22 de Transportadores de Solutos , Deficiencia de Vitamina B/epidemiologíaRESUMEN
Medium-chain acyl-CoA dehydrogenase deficiency (MCADD) is the most common defect of fatty acid oxidation. Many countries have introduced newborn screening for MCADD, because characteristic acylcarnitines can easily be identified in filter paper blood spot samples by tandem mass spectrometry (MS/MS), because MCADD is a frequent disease, and because of the success of early treatment initiated before clinical symptoms have emerged. In Denmark we have screened 519,350 newborns for MCADD by MS/MS and identified 58 affected babies. The diagnosis of MCADD was confirmed in all 58 newborns by mutation analysis. This gives an incidence of MCADD detected by newborn screening in Denmark of 1/8954. In sharp contrast to this we found that the incidence of clinically presenting MCADD in Denmark in the 10 year period preceding introduction of MS/MS-based screening was only 1 in 39,691. This means that four times more newborns with MCADD are detected by screening than what is expected based on the number of children presenting clinically in an unscreened population. The mutation spectrum in the newborns detected by screening is different from that observed in clinically presenting patients with a much lower proportion of newborns being homozygous for the prevalent disease-causing c.985A>G mutation. A significant number of the newborns have genotypes with mutations that have not been observed in patients detected clinically. Some of these mutations, like c.199T>C and c.127G>A, are always associated with a milder biochemical phenotype and may cause a milder form of MCADD with a relatively low risk of disease manifestation, thereby explaining part of the discrepancy between the frequency of clinically manifested MCADD and the frequency of MCADD determined by screening. In addition, our data suggest that some of this discrepancy can be explained by a reduced penetrance of the c.985A>G mutation, with perhaps only 50% of c.985A>G homozygotes presenting with disease manifestations. Interestingly, we also report that the observed number of newborns identified by screening who are homozygous for the c.985A>G mutation is twice that predicted from the estimated carrier frequency. We therefore redetermined the carrier frequency in a new sample of 1946 blood spots using a new assay, but this only confirmed that the c.985A>G carrier frequency in Denmark is approximately 1/105. We conclude that MCADD is much more frequent than expected, has a reduced penetrance and that rapid genotyping using the initial blood spot sample is important for correct diagnosis and counseling.
Asunto(s)
Errores Innatos del Metabolismo Lipídico/epidemiología , Acil-CoA Deshidrogenasa/deficiencia , Acil-CoA Deshidrogenasa/genética , Alelos , Secuencia de Bases , Carnitina/análogos & derivados , Carnitina/metabolismo , Dinamarca/epidemiología , Familia , Femenino , Estudios de Asociación Genética , Genotipo , Humanos , Incidencia , Lactante , Recién Nacido , Errores Innatos del Metabolismo Lipídico/diagnóstico , Errores Innatos del Metabolismo Lipídico/genética , Masculino , Mutación , Tamizaje Neonatal , Fenotipo , Espectrometría de Masas en TándemRESUMEN
BACKGROUND: Isolated 3-methylcrotonyl-CoA carboxylase (MCC) deficiency is an autosomal recessive disorder of leucine metabolism caused by mutations in MCCC1 or MCCC2 encoding the α and ß subunit of MCC, respectively. The phenotype is highly variable ranging from acute neonatal onset with fatal outcome to asymptomatic adults. METHODS: We report clinical, biochemical, enzymatic and mutation data of 88 MCC deficient individuals, 53 identified by newborn screening, 26 diagnosed due to clinical symptoms or positive family history and 9 mothers, identified following the positive newborn screening result of their baby. RESULTS: Fifty-seven percent of patients were asymptomatic while 43% showed clinical symptoms, many of which were probably not related to MCC deficiency but due to ascertainment bias. However, 12 patients (5 of 53 identified by newborn screening) presented with acute metabolic decompensations. We identified 15 novel MCCC1 and 16 novel MCCC2 mutant alleles. Additionally, we report expression studies on 3 MCCC1 and 8 MCCC2 mutations and show an overview of all 132 MCCC1 and MCCC2 variants known to date. CONCLUSIONS: Our data confirm that MCC deficiency, despite low penetrance, may lead to a severe clinical phenotype resembling classical organic acidurias. However, neither the genotype nor the biochemical phenotype is helpful in predicting the clinical course.
Asunto(s)
Trastornos Innatos del Ciclo de la Urea/metabolismo , Ligasas de Carbono-Carbono/deficiencia , Ligasas de Carbono-Carbono/genética , Ligasas de Carbono-Carbono/metabolismo , Línea Celular , Niño , Preescolar , Femenino , Humanos , Lactante , Masculino , Reacción en Cadena de la Polimerasa , Encuestas y Cuestionarios , Trastornos Innatos del Ciclo de la Urea/genética , Trastornos Innatos del Ciclo de la Urea/patología , Trastornos Innatos del Ciclo de la Urea/fisiopatologíaRESUMEN
A patient with suspected glutaric aciduria type 1 (GA-1) was detected by newborn screening. GA-1 is known as an autosomal recessively inherited disease due to defects in the gene coding for glutaryl-CoA dehydrogenase (GCDH), a mitochondrial enzyme involved in the catabolism of the amino acids hydroxylysine, lysine and tryptophan. DNA and cDNA sequencing revealed a 18 bp deletion (c.553_570del18) resulting in deletion of six amino acids (p.Gly185_Ser190del) in one allele and no sequence changes in the other allele. Confirmatory biochemical analysis of blood, urine and cultured fibroblasts from the proband were consistent with a mild biochemical GA-1 phenotype. Recombinant expression of the mutant variant in E. coli showed that the GCDH-(p.Gly185_Ser190del) protein displayed severely decreased assembly into tetramers and enzyme activity. To discover a potential dominant negative effect of the mutant protein, we engineered a prokaryotic expression system in which expression of a wild type and a mutant GCDH allele is controlled by separately inducible promoters. These cells displayed decreased levels of GCDH tetramer and enzyme activity when expressing both the wild type and the mutant GCDH variant protein compared to the situation when only the wild type allele was expressed. Further experiments suggest that the major impact of the GCDH-(p.Gly185_Ser190del) protein in heterozygous cells consists of hampering the assembly of wild type GCDH into tetramers. Our experimental data are consistent with the hypothesis that heterozygosity for this mutation confers a dominant negative effect resulting in a GCDH enzyme activity that is significantly lower than the expected 50%.
Asunto(s)
Errores Innatos del Metabolismo de los Aminoácidos/diagnóstico , Errores Innatos del Metabolismo de los Aminoácidos/genética , Encefalopatías Metabólicas/diagnóstico , Encefalopatías Metabólicas/genética , Glutaril-CoA Deshidrogenasa/deficiencia , Glutaril-CoA Deshidrogenasa/genética , Eliminación de Secuencia , Alelos , Preescolar , Heterocigoto , Humanos , Recién Nacido , Masculino , Tamizaje Neonatal/métodosRESUMEN
Carnitine palmitoyl transferase (CPT) 1 A deficiency is a rare disorder of hepatic long-chain fatty acid oxidation. CPT1 deficiency is included in newborn screening programs in a number of countries to allow presymptomatic detection and early treatment of affected patients.We present a case of presymptomatic CPT1A deficiency detected through newborn screening in Denmark with diagnostic levels of carnitine and acylcarnitines in the initial dried blood spot. Levels of plasma-free carnitine and acylcarnitines in follow-up samples were normal, but reverted to diagnostic levels when the patient developed clinical symptoms at the age of 8 months. At that time, a diagnosis of CPT1A deficiency was confirmed by sequence analysis of the CPT1A gene revealing homozygosity for a novel c.167C>T variation in exon 3. Enzyme activity measurements showed a relatively mild enzyme defect with a decreased residual enzyme activity of 17-25%. We conclude that CPT1A gene testing and/or enzyme assay is mandatory to confirm an abnormal newborn screen suggesting CPT1A deficiency to avoid delayed diagnoses.
RESUMEN
Aminoacylase 1 is a zinc-binding enzyme which hydrolyzes N-acetyl amino acids into the free amino acid and acetic acid. Deficiency of aminoacylase 1 due to mutations in the aminoacylase 1 (ACY1) gene follows an autosomal-recessive trait of inheritance and is characterized by accumulation of N-acetyl amino acids in the urine. In affected individuals neurological findings such as febrile seizures, delay of psychomotor development and moderate mental retardation have been reported. Except for one missense mutation which has been studied in Escherichia coli, mutations underlying aminoacylase 1 deficiency have not been characterized so far. This has prompted us to approach expression studies of all mutations known to occur in aminoacylase 1 deficient individuals in a human cell line (HEK293), thus providing the authentic human machinery for posttranslational modifications. Mutations were inserted using site directed mutagenesis and aminoacylase 1 enzyme activity was assessed in cells overexpressing aminoacylase 1, using mainly the natural high affinity substrate N-acetyl methionine. Overexpression of the wild type enzyme in HEK293 cells resulted in an approximately 50-fold increase of the aminoacylase 1 activity of homogenized cells. Most mutations resulted in a nearly complete loss of enzyme function. Notably, the two newly discovered mutations p.Arg378Trp, p.Arg378Gln and the mutation p.Arg393His yielded considerable residual activity of the enzyme, which is tentatively explained by their intramolecular localization and molecular characteristics. In contrast to aminoacylase 1 variants which showed no detectable aminoacylase 1 activity, aminoacylase 1 proteins with the mutations p.Arg378Trp, p.Arg378Gln and p.Arg393His were also detected in Western blot analysis. Investigations of the molecular bases of additional cases of aminoacylase 1 deficiency contribute to a better understanding of this inborn error of metabolism whose clinical significance and long-term consequences remain to be elucidated.
Asunto(s)
Amidohidrolasas/genética , Amidohidrolasas/análisis , Amidohidrolasas/química , Amidohidrolasas/deficiencia , Errores Innatos del Metabolismo de los Aminoácidos/genética , Western Blotting , Células Cultivadas , Niño , Preescolar , Femenino , Humanos , Masculino , Modelos Moleculares , Mutación , Polimorfismo de Longitud del Fragmento de Restricción , Estructura Cuaternaria de ProteínaRESUMEN
Glutaric aciduria type I (synonym, glutaric acidemia type I) is a rare organic aciduria. Untreated patients characteristically develop dystonia during infancy resulting in a high morbidity and mortality. The neuropathological correlate is striatal injury which results from encephalopathic crises precipitated by infectious diseases, immunizations and surgery during a finite period of brain development, or develops insidiously without clinically apparent crises. Glutaric aciduria type I is caused by inherited deficiency of glutaryl-CoA dehydrogenase which is involved in the catabolic pathways of L-lysine, L-hydroxylysine and L-tryptophan. This defect gives rise to elevated glutaric acid, 3-hydroxyglutaric acid, glutaconic acid, and glutarylcarnitine which can be detected by gas chromatography/mass spectrometry (organic acids) or tandem mass spectrometry (acylcarnitines). Glutaric aciduria type I is included in the panel of diseases that are identified by expanded newborn screening in some countries. It has been shown that in the majority of neonatally diagnosed patients striatal injury can be prevented by combined metabolic treatment. Metabolic treatment that includes a low lysine diet, carnitine supplementation and intensified emergency treatment during acute episodes of intercurrent illness should be introduced and monitored by an experienced interdisciplinary team. However, initiation of treatment after the onset of symptoms is generally not effective in preventing permanent damage. Secondary dystonia is often difficult to treat, and the efficacy of available drugs cannot be predicted precisely in individual patients. The major aim of this revision is to re-evaluate the previous diagnostic and therapeutic recommendations for patients with this disease and incorporate new research findings into the guideline.
Asunto(s)
Errores Innatos del Metabolismo de los Aminoácidos/diagnóstico , Errores Innatos del Metabolismo de los Aminoácidos/terapia , Encefalopatías Metabólicas/diagnóstico , Encefalopatías Metabólicas/terapia , Guías de Práctica Clínica como Asunto , Algoritmos , Errores Innatos del Metabolismo de los Aminoácidos/complicaciones , Encefalopatías Metabólicas/complicaciones , Servicios Médicos de Urgencia/métodos , Glutaril-CoA Deshidrogenasa/deficiencia , Humanos , Recién Nacido , Tamizaje Masivo/métodos , Monitoreo Fisiológico/métodos , Tamizaje Neonatal/métodos , Enfermedades del Sistema Nervioso/etiología , Enfermedades del Sistema Nervioso/terapiaRESUMEN
Accumulation of glutaric acid (GA) and 3-hydroxyglutaric acid (3HGA) in body fluids is the biochemical hallmark of type 1 glutaric aciduria (GA1), a disorder characterized by acute striatal degeneration and a subsequent dystonia. To date, methods for quantification of 3HGA are mainly based on stable isotope dilution gas chromatography mass spectrometry (GC-MS) and require extensive sample preparation. Here we describe a simple liquid chromatography tandem MS (LC-MS/MS) method to quantify this important metabolite in dried urine spots (DUS). This method is based on derivatization with 4-[2-(N,N-dimethylamino)ethylaminosulfonyl]-7-(2-aminoethylamino)-2,1,3-benzoxadiazole (DAABD-AE). Derivatization was adopted to improve the chromatographic and mass spectrometric properties of the studied analytes. Derivatization was performed directly on a 3.2-mm disc of DUS as a sample without extraction. Sample mixture was heated at 60°C for 45 min, and 5 µl of the reaction solution was analyzed by LC-MS/MS. Reference ranges obtained were in excellent agreement with the literature. The method was applied retrospectively for the analysis of DUS samples from established low- and high-excreter GA1 patients as well as controls (n = 100). Comparison of results obtained versus those obtained by GC-MS was satisfactory (n = 14). In populations with a high risk of GA1, this approach will be useful as a primary screening method for high- or low-excreter variants. In these populations, however, DUS analysis should not be implemented before completing a parallel comparative study with the standard screening method (i.e., molecular testing). In addition, follow-up DUS GA and 3HGA testing of babies with elevated dried blood spot C5DC acylcarnitines will be useful as a first-tier diagnostic test, thus reducing the number of cases requiring enzymatic and molecular analyses to establish or refute the diagnosis of GA1.
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Glutaratos/orina , Espectrometría de Masas en Tándem/métodos , Urinálisis/métodos , Errores Innatos del Metabolismo de los Aminoácidos/diagnóstico , Errores Innatos del Metabolismo de los Aminoácidos/orina , Encefalopatías Metabólicas/diagnóstico , Encefalopatías Metabólicas/orina , Cromatografía Liquida/métodos , Desecación , Glutaratos/análisis , Glutaril-CoA Deshidrogenasa/deficiencia , Glutaril-CoA Deshidrogenasa/orina , Humanos , Recién Nacido , Tamizaje Neonatal/métodosRESUMEN
We present a case with a ten-year-old girl with trimethylaminuria (TMAU). Primary TMAU is caused by a deficiency of flavin monooxygenase 3 (FMO3) due to mutations in the FMO3-gene. Patients suffering from TMAU show an impaired enzymatic oxidation of fish-smelling trimethylamine, and their excretion of this amine in body fluids produces an unpleasant body odour. TMAU is also seen secondary to e.g. liver diseases. It remains unknown if TMAU causes other problems than malodour, and today social and psychological problems are considered the most important consequence. Treatment includes a low-choline diet and antibiotics.
Asunto(s)
Enfermedades Metabólicas/diagnóstico , Metilaminas/orina , Odorantes , Oxigenasas/genética , Niño , Diagnóstico Diferencial , Femenino , Humanos , Enfermedades Metabólicas/genética , Enfermedades Metabólicas/terapia , Mutación , SíndromeRESUMEN
D-glyceric aciduria is a rare inborn error of serine and fructose metabolism that was first described in 1974. Most affected individuals have presented with neurological symptoms. The molecular basis of D-glyceric aciduria is largely unknown; possible causes that have been discussed are deficiencies of D-glycerate dehydrogenase, triokinase, and D-glycerate kinase. In 1989, van Schaftingen has reported decreased D-glycerate kinase activity in the liver of a single patient with D-glyceric aciduria. However, this analysis has not been performed in other affected individuals, and the underlying defect has remained unknown on the gene level until now. We report three patients with deficiency of D-glycerate kinase. They are of Serbian, Mexican, and Turkish origin and include the patient initially reported in 1974. All had homozygous mutations in exon 5 of the GLYCTK gene encoding D-glycerate kinase: c.1448delT (p.Phe483SerfsX2), c.1478T>G (p.Phe493Cys), or c.1558delC (p.Leu520CysfsX108). Transient overexpression of the variant GLYCTK genes in HEK293 cells clearly showed loss of enzyme activity and immunoreactivity when compared to the reference enzyme. Our work has revealed mutations in the GLYCTK gene as the cause of D-glycerate kinase deficiency and D-glyceric aciduria and provides a noninvasive approach for further diagnostic workup and research.
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Fosfotransferasas (Aceptor de Grupo Alcohol)/deficiencia , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Secuencia de Aminoácidos , Secuencia de Bases , Líquidos Corporales , Niño , Preescolar , Análisis Mutacional de ADN , Resultado Fatal , Femenino , Regulación de la Expresión Génica , Ácidos Glicéricos/metabolismo , Células HEK293 , Humanos , Hiperoxaluria Primaria/enzimología , Hiperoxaluria Primaria/genética , Lactante , Recién Nacido , Masculino , Datos de Secuencia Molecular , Mutación/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Embarazo , Alineación de Secuencia , TransfecciónRESUMEN
Pyridoxine-dependent epilepsy was recently shown to be due to mutations in the ALDH7A1 gene, which encodes antiquitin, an enzyme that catalyses the nicotinamide adenine dinucleotide-dependent dehydrogenation of l-alpha-aminoadipic semialdehyde/L-Delta1-piperideine 6-carboxylate. However, whilst this is a highly treatable disorder, there is general uncertainty about when to consider this diagnosis and how to test for it. This study aimed to evaluate the use of measurement of urine L-alpha-aminoadipic semialdehyde/creatinine ratio and mutation analysis of ALDH7A1 (antiquitin) in investigation of patients with suspected or clinically proven pyridoxine-dependent epilepsy and to characterize further the phenotypic spectrum of antiquitin deficiency. Urinary L-alpha-aminoadipic semialdehyde concentration was determined by liquid chromatography tandem mass spectrometry. When this was above the normal range, DNA sequencing of the ALDH7A1 gene was performed. Clinicians were asked to complete questionnaires on clinical, biochemical, magnetic resonance imaging and electroencephalography features of patients. The clinical spectrum of antiquitin deficiency extended from ventriculomegaly detected on foetal ultrasound, through abnormal foetal movements and a multisystem neonatal disorder, to the onset of seizures and autistic features after the first year of life. Our relatively large series suggested that clinical diagnosis of pyridoxine dependent epilepsy can be challenging because: (i) there may be some response to antiepileptic drugs; (ii) in infants with multisystem pathology, the response to pyridoxine may not be instant and obvious; and (iii) structural brain abnormalities may co-exist and be considered sufficient cause of epilepsy, whereas the fits may be a consequence of antiquitin deficiency and are then responsive to pyridoxine. These findings support the use of biochemical and DNA tests for antiquitin deficiency and a clinical trial of pyridoxine in infants and children with epilepsy across a broad range of clinical scenarios.
Asunto(s)
Aldehído Deshidrogenasa/deficiencia , Aldehído Deshidrogenasa/genética , Epilepsia/genética , Fenotipo , Piridoxina/uso terapéutico , Ácido 2-Aminoadípico/análogos & derivados , Ácido 2-Aminoadípico/orina , Aldehído Deshidrogenasa/orina , Biomarcadores/orina , Epilepsia/tratamiento farmacológico , Epilepsia/orina , Femenino , Genotipo , Humanos , Lactante , Masculino , Mutación MissenseRESUMEN
Evidence-based guidelines for monitoring patients with disorders in fatty acid oxidation (FAO) are lacking, and most protocols are based on expert statements. Here, we describe our protocol for Danish patients. Clinical monitoring is the most important measure and has the main aims of checking growth, development and diet and of bringing families to the clinic regularly to remind them of their child's risk and review how they cope and adjust, e.g. to an acute intercurrent illness. Most of these measures are simple and can be carried out during a routine out-patient visit; we seldom do more complicated assessments by a neuropsychologist, speech therapist, or physical and occupational therapists. Paraclinical measurements are not used for short-chain and medium-chain disorders; electrocardiography (including 24 h monitoring) and echocardiography are done for most patients with long-chain and carnitine transporter deficiencies. Eye examination is done in all, and liver ultrasonography in some patients with long-chain 3-hydroxyacyl-coenzyme A dehydrogenase/tri-functional protein (LCHAD/TFP) deficiencies. Biochemical follow-up includes determination of free carnitine and acylcarnitines. Free carnitine is measured to monitor carnitine supplementation in patients with multiple acyl-coenzyme A dehydrogenase deficiency (MADD) and carnitine transporter deficiency (CTD) and to follow metabolic control and disclose deficiency states in other FAO disorders. We are evaluating long-chain acylcarnitines in patients with long-chain disorders; so far there does not seem to be any clear-cut benefit in following these levels. An erythrocyte fatty acid profile is done in patients with long-chain disorders to test for essential fatty acid and docosahexanoic acid (DHA) deficiencies. The measurement of creatine kinase is helpful in long-chain disorders. Ongoing follow-up and education of the patient is important throughout life to prevent disease morbidity or death from metabolic crises.
Asunto(s)
Metabolismo Energético , Ácidos Grasos/metabolismo , Errores Innatos del Metabolismo Lipídico/diagnóstico , Mitocondrias/enzimología , Enfermedades Mitocondriales/diagnóstico , Adolescente , Adulto , Biomarcadores/sangre , Niño , Preescolar , Continuidad de la Atención al Paciente , Metabolismo Energético/genética , Genotipo , Conocimientos, Actitudes y Práctica en Salud , Humanos , Lactante , Recién Nacido , Errores Innatos del Metabolismo Lipídico/enzimología , Errores Innatos del Metabolismo Lipídico/genética , Errores Innatos del Metabolismo Lipídico/terapia , Enfermedades Mitocondriales/enzimología , Enfermedades Mitocondriales/genética , Enfermedades Mitocondriales/terapia , Oxidación-Reducción , Educación del Paciente como Asunto , Fenotipo , Valor Predictivo de las Pruebas , Pronóstico , Factores de Tiempo , Adulto JovenRESUMEN
We performed molecular, enzyme, and metabolic studies in 50 patients with D-2-hydroxyglutaric aciduria (D-2-HGA) who accumulated D-2-hydroxyglutarate (D-2-HG) in physiological fluids. Presumed pathogenic mutations were detected in 24 of 50 patients in the D-2-hydroxyglutarate dehydrogenase (D2HGDH) gene, which encodes D-2-hydroxyglutarate dehydrogenase (D-2-HGDH). Enzyme assay of D-2-HGDH confirmed that all patients with mutations had impaired enzyme activity, whereas patients with D-2-HGA whose enzyme activity was normal did not have mutations. Significantly lower D-2-HG concentrations in body fluids were observed in mutation-positive D-2-HGA patients than in mutation-negative patients. These results imply that multiple genetic loci may be associated with hyperexcretion of D-2-HG. Accordingly, we suggest a new classification: D-2-HGA Type I associates with D-2-HGDH deficiency, whereas idiopathic D-2-HGA manifests with normal D-2-HGDH activity and higher D-2-HG levels in body fluids compared with Type I patients. It remains possible that several classifications for idiopathic D-2-HGA patients with diverse genetic loci will be revealed in future studies.