RESUMO
Newborn screening (NBS) for Krabbe disease, a rare neurodegenerative disorder caused by deficient galactocerebrosidase (GALC) enzyme activity, has recently been implemented in a number of US states. However, the spectrum of phenotypic manifestations associated with deficient GALC activity complicates the management of screen-positive newborns and underscores the need to identify clinically relevant biomarkers. Earlier studies with a small number of patients identified psychosine, a substrate of the GALC enzyme, as a potential biomarker for Krabbe disease. In this study, we provide, for the first time, longitudinal data on dried blood spot (DBS) psychosine concentrations in different Krabbe disease phenotypes for both untreated patients and those treated with hematopoietic stem cell transplantation (HSCT). Our cohort included patients previously identified by NBS to be at high risk to develop Krabbe disease. Substantially elevated DBS psychosine concentration during the newborn period was found to be a highly specific marker for infantile Krabbe disease. This finding supports the use of DBS psychosine concentration as a second-tier NBS test to aid in the identification of patients who require urgent evaluation for HSCT. In addition, longitudinal assessments showed that both natural disease progression and treatment with HSCT were associated with decreases in DBS psychosine concentrations. Based on these findings we provide recommendations for the interpretation of psychosine concentrations in DBS specimens collected during the first year of life. Future studies should aim to better delineate the relationship between DBS psychosine concentration and disease onset in patients with later-onset forms of Krabbe disease.
Assuntos
Biomarcadores/sangue , Leucodistrofia de Células Globoides/diagnóstico , Psicosina/sangue , Adolescente , Criança , Pré-Escolar , Progressão da Doença , Teste em Amostras de Sangue Seco , Humanos , Lactente , Recém-Nascido , Leucodistrofia de Células Globoides/tratamento farmacológico , Triagem Neonatal , Fenótipo , Espectrometria de Massas em TandemRESUMO
BACKGROUND: In patients treated with azathioprine, deficient thiopurine methyltransferase (TPMT) activity has been associated with haematologic toxicity. OBJECTIVES: To determine how many Saskatchewan rheumatologists routinely pre-screen TPMT activity before prescribing azathioprine. Further, to retrospectively review TPMT activity levels from pre-screening in one patient cohort. METHODS: All rheumatologists practicing in the province of Saskatchewan were surveyed by questionnaire. The questionnaire scrutinized prevalence of routine TPMT screening pre-azathioprine initiation, response to abnormal test results, monitoring frequencies, starting dosages, and attitudes towards potential consensus guidelines or practice standards. For the second objective of this study, health region laboratory database retrieval identified 42 patients who had undergone TPMT phenotype testing within a single rheumatology practice. Chart review of all 42 patients was performed to verify test results. RESULTS: In a survey of all eleven Saskatchewan rheumatologists, 55% reported routinely pre-screening, compared to 45% who never screen pre-azathioprine. Half of those who pre-screen, report avoidance of azathioprine in both patients with deficiency and those with intermediate activity levels. The majority of respondents indicated they would adjust their practice to conform to future national rheumatology clinical guidelines. A retrospective review in one practice revealed TPMT activity values consistent with deficiency, carrier status, and normal ranges in 2.4%, 21.4%, and 76.2% of patients pre-screened, respectively. CONCLUSIONS: Provincial rheumatologists were divided on the practice of pre-screening TPMT status prior to initiation of azathioprine. Azathioprine may be underutilized by half of those who pre-screen. A need for practice guidelines was recognized by the participants. In this patient group, diminished TPMT activity was observed in 23.8% of those who were tested.
Assuntos
Azatioprina/uso terapêutico , Doenças Hematológicas/prevenção & controle , Metiltransferases/genética , Padrões de Prática Médica/estatística & dados numéricos , Antirreumáticos/administração & dosagem , Antirreumáticos/efeitos adversos , Antirreumáticos/uso terapêutico , Atitude do Pessoal de Saúde , Azatioprina/administração & dosagem , Azatioprina/efeitos adversos , Relação Dose-Resposta a Droga , Pesquisas sobre Atenção à Saúde , Doenças Hematológicas/induzido quimicamente , Humanos , Programas de Rastreamento/métodos , Projetos Piloto , Guias de Prática Clínica como Assunto , Estudos Retrospectivos , Saskatchewan , Inquéritos e QuestionáriosRESUMO
Newborn screening (NBS) by tandem mass spectrometry (MS/MS) has allowed for early detection and initiation of treatment in many patients with maple syrup urine disease (MSUD) (OMIM 248600), however, a recent report suggests that variants forms may be missed. Information on these patients is limited. We present clinical, biochemical and molecular information on patients with variant forms of MSUD not detected by the California Newborn Screening Program. Between July 2005 and July 2009, 2200,000 newborns were screened in California by MS/MS. Seventeen cases of MSUD were detected and three (two siblings) were missed. Additionally, the NBS cards of two siblings with late onset MSUD, who were born pre-expanded NBS, were retrospectively analyzed. None of the five patients met criteria to be considered presumptive positive for MSUD (leucine>200micromol/L and a ratio of leucine/alanine>or=1.5). Alloisoleucine (allo-ile) was subsequently analyzed in the NBS cards of all five patients, two of whom were found to have elevated levels. The proband in each family was diagnosed following symptoms triggered by an intercurrent illness or increased protein intake. At diagnosis, leucine levels ranged between 561 and >4528micromol/L, and allo-ile ranged from 137 to 239micromol/L. Two affected siblings had normal plasma amino acids when asymptomatic; however, their biochemical profiles were diagnostic of MSUD during intercurrent illnesses. The median age at diagnosis of all patients was one year (range 0.8-6.7). Heterozygous BCKDHB (E1beta) mutations (c.832G>A/c.970C>T) were identified in one family and a homozygous DBT (E2) sequence variant (c.1430 T>G) in another. The third family had one identifiable DBT mutation (c.827T>G), however, a second mutation was not detected. This report provides further evidence that NBS by MS/MS is unable to detect all cases of MSUD. Second-tier testing with allo-ile may improve sensitivity; however, some children with variant forms will invariably be missed.
Assuntos
Doença da Urina de Xarope de Bordo/diagnóstico , Doença da Urina de Xarope de Bordo/genética , Triagem Neonatal , Aminoácidos de Cadeia Ramificada/sangue , Criança , Pré-Escolar , Dieta com Restrição de Proteínas , Humanos , Recém-Nascido , Isoleucina/sangue , Leucina/sangue , Masculino , Triagem Neonatal/métodos , Espectrometria de Massas em TandemRESUMO
We report a 16-month-old asymptomatic male with enzyme confirmed isovaleric acidaemia (IVA; isovaleryl-CoA dehydrogenase deficiency; OMIM 243500) who, upon routine nutritional follow-up, presented evidence of peroxisomal dysfunction. The newborn screen (2 days of life) revealed elevated C(5)-carnitine (2.95 µmol/L; cutoff <0.09 µmol/L) and IVA was subsequently confirmed by metabolic profiling and in vitro enzymology. Plasma essential fatty acid (EFA) analysis, assessed to evaluate nutritional status during protein restriction and L: -carnitine supplementation, revealed elevated C(26:0) (5.0 µmol/L; normal <1.3). Subsequently, metabolic profiling and molecular genetic analysis confirmed X-linked adrenoleukodystrophy (XALD). Identification of co-inherited XALD with IVA in this currently asymptomatic patient holds significant treatment ramifications for the proband prior to the onset of neurological sequelae, and critically important counselling implications for this family.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/diagnóstico , Ácidos Graxos Essenciais/sangue , Avaliação Nutricional , Transtornos Peroxissômicos/diagnóstico , Erros Inatos do Metabolismo dos Aminoácidos/sangue , Erros Inatos do Metabolismo dos Aminoácidos/complicações , Erros Inatos do Metabolismo dos Aminoácidos/genética , Biomarcadores/sangue , Análise Mutacional de DNA , Humanos , Lactente , Recém-Nascido , Isovaleril-CoA Desidrogenase/sangue , Isovaleril-CoA Desidrogenase/deficiência , Isovaleril-CoA Desidrogenase/genética , Masculino , Triagem Neonatal , Transtornos Peroxissômicos/sangue , Transtornos Peroxissômicos/complicações , Transtornos Peroxissômicos/genética , Valor Preditivo dos TestesRESUMO
The medical and neurodevelopmental characteristics of 14 children with short-chain acyl-CoA dehydrogenase deficiency (SCADD) are described. Eight were detected as neonates by newborn screening. Three children diagnosed on the basis of clinical symptoms had normal newborn screening results while three were born in states that did not screen for SCADD. Treatment included frequent feedings and a low fat diet. All children identified by newborn screening demonstrated medical and neuropsychological development within the normative range on follow-up, although one child had a relative weakness in the motor area and another child exhibited mild speech delay. Of the three clinically identified children with newborn screening results below the cut-off value, two were healthy and performed within the normal range on cognitive and motor tests at follow-up. Four clinically identified children with SCADD experienced persistent symptoms and/or developmental delay. However, in each of these cases, there were supplementary or alternative explanations for medical and neuropsychological deficits. Results indicated no genotype-phenotype correlations. These findings suggest that SCADD might be benign and the clinical symptoms ascribed to SCADD reflective of ascertainment bias or that early identification and treatment prevented complications that may have occurred due to interaction between genetic susceptibility and other genetic factors or environmental stressors.
Assuntos
Acil-CoA Desidrogenase/deficiência , Erros Inatos do Metabolismo dos Aminoácidos/diagnóstico , Erros Inatos do Metabolismo dos Aminoácidos/enzimologia , Deficiências do Desenvolvimento/diagnóstico , Deficiências do Desenvolvimento/enzimologia , Triagem Neonatal , Acil-CoA Desidrogenase/genética , Adaptação Psicológica , Erros Inatos do Metabolismo dos Aminoácidos/genética , Erros Inatos do Metabolismo dos Aminoácidos/psicologia , Boston , Pré-Escolar , Deficiências do Desenvolvimento/genética , Deficiências do Desenvolvimento/psicologia , Feminino , Seguimentos , Humanos , Lactente , Recém-Nascido , Masculino , Exame NeurológicoRESUMO
The continued expansion of newborn screening programmes to include additional conditions increases the responsibility of newborn screening laboratories to provide testing with the highest sensitivity and specificity to allow for identification of affected patients while minimizing the false-positive rate. Some assays and analytes are particularly problematic. Over recent years, our laboratory tried to improve this situation by developing second-tier tests to reduce false-positive results in the screening for congenital adrenal hyperplasia (CAH), tyrosinaemia type I, methylmalonic acidaemias, homocystinuria, and maple syrup urine disease (MSUD). Beginning in 2004, this approach was applied to Mayo's newborn screening programme and resulted in a false-positive rate of 0.09%, a positive predictive value of 41%, and a positive detection rate of 1 affected case in 1672 babies screened.
Assuntos
Hiperplasia Suprarrenal Congênita/diagnóstico , Homocistinúria/diagnóstico , Doença da Urina de Xarope de Bordo/diagnóstico , Espectrometria de Massas/métodos , Triagem Neonatal/métodos , Tirosinemias/diagnóstico , Deficiência de Vitamina B 12/diagnóstico , Hiperplasia Suprarrenal Congênita/sangue , Reações Falso-Positivas , Homocistinúria/sangue , Humanos , Recém-Nascido , Doença da Urina de Xarope de Bordo/sangue , Valor Preditivo dos Testes , Sensibilidade e Especificidade , Fatores de Tempo , Tirosinemias/sangue , Deficiência de Vitamina B 12/sangueRESUMO
Glutaric acidemia type I (GA-1) is a progressive neurodegenerative inborn error of metabolism that typically manifests acutely in infants during an intercurrent illness. The diagnosis is established biochemically by the detection of glutaric acid and 3-hydroxy glutaric acid in urine and glutarylcarnitine in plasma. However, some patients excrete only small amounts of glutaric acid and may be overlooked, especially if the plasma concentration of glutarylcarnitine is not elevated. To test the hypothesis that measuring the excretion of glutarylcarnitine may improve the recognition of GA-1 patients without significant glutaric aciduria, urine glutarylcarnitine was analyzed in 14 cases. Five of them lacked significant glutaric aciduria, 9 (of 10 available) had a normal plasma glutarylcarnitine concentration. As controls, we also evaluated 54 subjects with glutaric aciduria secondary to other causes (16-7509 mmol/mol creatinine; reference range: <15; no significant amounts of 3-hydroxy glutaric acid detectable). The excretion of glutarylcarnitine was significantly elevated in all GA-1 patients (14-522 mmol/mol creatinine; reference range: <5.2) and in none of the controls with glutaric aciduria. These findings suggest that the urinary excretion of glutarylcarnitine is a specific biochemical marker of GA-1 which could be particularly useful in the work up of patients with suggestive clinical manifestations but without glutaric aciduria and with normal plasma acylcarnitine profiles.
Assuntos
Carnitina/análogos & derivados , Carnitina/urina , Glutaratos/sangue , Estudos de Casos e Controles , Glutaratos/urina , HumanosRESUMO
Genetic disorders are recognized to play an increasing role in pediatrics. Close to 10% of diseases among hospitalized children have been ascribed to Mendelian traits inherited as single gene defects, not a surprising figure considering that approximately 1000 inborn errors of metabolism (IEM) have been identified to date, primarily through the detection of endogenous metabolites abnormally accumulated in biological fluids and tissues. The laboratory discipline that covers the biochemical diagnosis of IEM is known as clinical biochemical genetics, and is defined as one concerned with the evaluation and diagnosis of patients and families with inherited metabolic disease, monitoring of treatment, and distinguishing heterozygous carriers from non-carriers by metabolite and enzymic analysis of physiological fluids and tissues. The biochemical genetics laboratory differs from the clinical chemistry laboratory in the extent of interpretation necessary to make its results meaningful to the clinician. While dramatic advances in molecular genetics have greatly changed the landscape of diagnostic options for many genetic disorders, a biochemical approach remains the dominant force for the diagnosis and monitoring of IEM. Owing to the stereotypical clinical presentation of many of these disorders, a major role of the biochemical genetics laboratory is to analyze ever more complex metabolic profiles to reach a preliminary diagnosis, which then needs to be confirmed by enzymic and/or molecular studies in vitro. Accordingly, the role of biochemical genetics in the pediatric practice of the 21st century is to provide a multicomponent screening process that can be divided into four major components: (i) at-risk screening (prenatal diagnosis); (ii) newborn screening (testing of presymptomatic patients); (iii) high-risk screening (testing of symptomatic patients); and (iv) postmortem screening (metabolic autopsy). The focus of our laboratory is to apply state-of-the-art technology such as tandem mass spectrometry to bring as many as possible IEM within the boundaries of newborn screening programs, and to investigate the role played by individual disorders in maternal complications of pregnancy, pediatric acute/fulminant liver failure, and sudden and unexpected death in early life.
Assuntos
Testes Genéticos/tendências , Erros Inatos do Metabolismo/diagnóstico , Erros Inatos do Metabolismo/genética , Biologia Molecular/tendências , Triagem Neonatal/tendências , Autopsia , Feminino , Humanos , Recém-Nascido , Sistemas de Informação , Erros Inatos do Metabolismo/patologia , Minnesota , Gravidez , Diagnóstico Pré-Natal , Estados UnidosRESUMO
Newborn screening by tandem mass spectrometry (MS/MS) identifies patients with medium chain acyl-CoA dehydrogenase (MCAD) deficiency the most frequently observed disorder of fatty acid oxidation. Molecular genetic analysis is becoming a common tool to confirm those identified as affected by prospective screening and for carrier detection in family studies. The A985G (K304E) mutation accounts for approximately 80% of mutant alleles in MCAD deficient patients, presenting symptomatically, while greater variability of mutant alleles is observed among cases identified through prospective screening. Aside from A985G, the mutation spectrum in MCAD deficient patients is heterogeneous such that comprehensive gene analysis is required. Traditionally the MCAD gene is assayed by sequencing the entire coding region. Although effective and definitive, this approach is expensive, turn around time is slow, and is poorly amenable to a clinical service molecular genetics laboratory. Dye-binding/high-resolution thermal denaturation is a rapid and homogeneous method by which to scan a PCR product for evidence of sequence aberration. PCR is performed in capillaries in the presence of the dsDNA-binding dye LCGreen I and subsequently the DNA/dye complexes are analyzed by high-resolution thermal denaturation. DNA sequencing was limited to fragments displaying abnormal melting profiles. Of 18 specimens analyzed, 11 have a genotype consistent with MCAD deficiency and seven have a genotype consistent with carrier status. Clinical and biochemical data corroborate that the genotype results identified the affected patients and differentiates them from carriers. The entire process is homogeneous requiring no post-PCR manipulation and is completed in under 3 h.
Assuntos
Acil-CoA Desidrogenase/deficiência , Acil-CoA Desidrogenase/genética , Testes Genéticos/métodos , Humanos , Recém-Nascido , Desnaturação de Ácido Nucleico , Reação em Cadeia da Polimerase , Fatores de TempoRESUMO
Although many patients have been found to have very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency, none have been documented with long-chain acyl-CoA dehydrogenase (LCAD) deficiency. In order to understand the metabolic pathogenesis of long-chain fatty acid oxidation disorders, we generated mice with VLCAD deficiency (VLCAD(-/-)) and compared their pathologic and biochemical phenotypes of mice with LCAD deficiency (LCAD(-/-)) and wild-type mice. VLCAD(-/-) mice had milder fatty change in liver and heart. Dehydrogenation of various acyl-CoA substrates by liver, heart and skeletal muscle mitochondria differed among the three genotypes. The results for liver were most informative as VLCAD(-/-) mice had a reduction in activity toward palmitoyl-CoA and oleoyl-CoA (58 and 64% of wild-type, respectively), whereas LCAD(-/-) mice showed a more profoundly reduced activity toward these substrates (35 and 32% of wild-type, respectively), with a significant reduction of activity toward the branched chain substrate 2,6-dimethylheptanoyl-CoA. C(16) and C(18) acylcarnitines were elevated in bile, blood and serum of fasted VLCAD(-/-) mice, whereas abnormally elevated C(12) and C(14) acylcarnitines were prominent in LCAD(-/-) mice. Progeny with the combined LCAD(+/+)//VLCAD(+/-) genotype were over-represented in offspring from sires and dams heterozygous for both LCAD and VLCAD mutations. In contrast, no live mice with a compound LCAD(-/-)//VLCAD(-/-) genotype were detected.
Assuntos
Acil-CoA Desidrogenase de Cadeia Longa/deficiência , Carnitina/análogos & derivados , Erros Inatos do Metabolismo Lipídico/enzimologia , Acil-CoA Desidrogenase de Cadeia Longa/metabolismo , Animais , Carnitina/metabolismo , Ácidos Graxos/metabolismo , Feminino , Humanos , Erros Inatos do Metabolismo Lipídico/patologia , Tamanho da Ninhada de Vivíparos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias Cardíacas/enzimologia , Mitocôndrias Hepáticas/enzimologia , Mitocôndrias Musculares/enzimologiaRESUMO
Mitochondrial trifunctional protein (MTP) is a hetero-octamer of four alpha and four beta subunits that catalyzes the final three steps of mitochondrial long chain fatty acid beta-oxidation. Human MTP deficiency causes Reye-like syndrome, cardiomyopathy, or sudden unexpected death. We used gene targeting to generate an MTP alpha subunit null allele and to produce mice that lack MTP alpha and beta subunits. The Mtpa(-/-) fetuses accumulate long chain fatty acid metabolites and have low birth weight compared with the Mtpa(+/-) and Mtpa(+/+) littermates. Mtpa(-/-) mice suffer neonatal hypoglycemia and sudden death 6-36 hours after birth. Analysis of the histopathological changes in the Mtpa(-/-) pups revealed rapid development of hepatic steatosis after birth and, later, significant necrosis and acute degeneration of the cardiac and diaphragmatic myocytes. This mouse model documents that intact mitochondrial long chain fatty acid oxidation is essential for fetal development and for survival after birth. Deficiency of MTP causes fetal growth retardation, neonatal hypoglycemia, and sudden death.
Assuntos
Modelos Animais de Doenças , Desenvolvimento Embrionário e Fetal , Retardo do Crescimento Fetal/etiologia , Marcação de Genes , Hipoglicemia/metabolismo , Complexos Multienzimáticos/metabolismo , Animais , Animais Recém-Nascidos , Análise Química do Sangue , Cruzamentos Genéticos , Morte Súbita , Diafragma/patologia , Feminino , Retardo do Crescimento Fetal/metabolismo , Humanos , Immunoblotting , Fígado/patologia , Fígado/ultraestrutura , Masculino , Camundongos , Camundongos Knockout , Proteína Mitocondrial Trifuncional , Complexos Multienzimáticos/genética , Miocárdio/patologiaRESUMO
Beginning in the mid 1960s, mass spectrometry was introduced in a few academic laboratories for the analysis of organic acids by gas chromatography-mass spectrometry. Since then, multiple-stage mass spectrometers have become available and many new applications have been developed. Major advantages of these new techniques include their ability to rapidly determine many different compounds in complex biological matrices with high sensitivity and in sample volumes of usually < 100 microL. A high sample throughput is further realized because extensive sample preparations are often not necessary. However, because the technical know-how is not yet widely available and significant experience is required for correct interpretation of results, these methods are being implemented slowly in routine clinical laboratories as opposed to research laboratories. Several of these new applications are considered with regard to clinical medicine.
Assuntos
Deficiência de Vitaminas/diagnóstico , Espectrometria de Massas , Erros Inatos do Metabolismo/diagnóstico , Humanos , Espectrometria de Massas/instrumentação , Espectrometria de Massas/métodos , Espectrometria de Massas/tendências , Erros Inatos do Metabolismo/sangueRESUMO
We have developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the analysis of homovanillic acid (HVA), a biochemical marker for catecholamine and neurotransmitter metabolism. Urine specimens are spiked with 5 microg of a stable-isotope labeled internal standard, 13C(6)18O-HVA, and prepared by automated solid phase extraction. Residues were dissolved in acetonitrile: 0.05% aqueous acetic acid and analyzed by MS/MS in the selected reaction monitoring mode (HVA: m/z 181 to m/z 137; 13C(6)18O-HVA: m/z 189 to m/z 145) after separation using a Discovery RP Amide C16 column. Consecutive calibrations (n=7) between 0.52 and 16.7 mg/l exhibited consistent linearity and reproducibility. At a urine concentration of 0.51 mg/l, the signal-to-noise ratio for HVA was 21:1. Inter- and intra-assay CVs ranged from 0.3% to 11.4%, at mean concentrations ranging 1.8 to 22.7 mg/l. Recovery of HVA added to urine ranged between 94.7% and 105% (1.25 mg/l added), 92.0% and 102% (5.0 mg/l), and 96.0% and 104% (10 mg/l). LC-MS/MS is well suited to replace an HPLC method for routine HVA determination, by providing positive identification, faster turn around time, virtually no repeat analyses and a 44% reduction of personnel necessary to perform the testing.
Assuntos
Ácido Homovanílico/urina , Espectrometria de Massas por Ionização por Electrospray/métodos , Humanos , Isótopos , Padrões de Referência , Reprodutibilidade dos TestesRESUMO
There is a correlation between pregnancy complications such as acute fatty liver of pregnancy and long-chain 3-hydroxyacyl-coenzyme A dehydrogenase (LCHAD) deficiency. We diagnosed another fatty acid beta-oxidation defect, short-chain acyl-coenzyme A dehydrogenase deficiency, in an infant when evaluating him because his mother had acute fatty liver of pregnancy. Other beta-oxidation defects, in addition to LCHAD deficiency, should be considered in children born after pregnancies complicated by liver disease.
Assuntos
Ácidos Graxos Dessaturases/deficiência , Fígado Gorduroso/complicações , Complicações na Gravidez , Adulto , Ácidos Graxos Dessaturases/genética , Fígado Gorduroso/diagnóstico , Feminino , Idade Gestacional , Humanos , Recém-Nascido , Masculino , Gravidez , Complicações na Gravidez/diagnósticoRESUMO
By postmortem biochemical and molecular genetic analyses, an 8-month-old infant was diagnosed with long-chain 3-hydroxyacyl-coenzyme A dehydrogenase deficiency, an inborn error of mitochondrial fatty acid beta-oxidation. He was born following a pregnancy complicated by a maternal floor infarction of the placenta, a disorder of unknown etiology. We speculate that the child's autosomal recessive fatty acid beta-oxidation disorder and the pregnancy complication are causally related.
Assuntos
3-Hidroxiacil-CoA Desidrogenases/deficiência , Mutação da Fase de Leitura , Mutação de Sentido Incorreto , Doenças Placentárias/etiologia , Placenta/irrigação sanguínea , Gravidez/genética , Gravidez/metabolismo , Feminino , Humanos , Lactente , Infarto/patologia , 3-Hidroxiacil-CoA Desidrogenase de Cadeia Longa , Masculino , Doenças Placentárias/patologia , Polimorfismo Conformacional de Fita SimplesAssuntos
Ácidos Graxos/metabolismo , Erros Inatos do Metabolismo Lipídico/diagnóstico , Mitocôndrias/enzimologia , Diagnóstico Pré-Natal , Transporte Biológico , Análise Mutacional de DNA , Feminino , Humanos , Erros Inatos do Metabolismo Lipídico/enzimologia , Erros Inatos do Metabolismo Lipídico/genética , Oxirredução , GravidezRESUMO
ABSTRACT Short-chain acyl-CoA dehydrogenase (SCAD) deficiency is considered a rare inherited mitochondrial fatty acid oxidation disorder. Less than 10 patients have been reported, diagnosed on the basis of ethylmalonic aciduria and low SCAD activity in cultured fibroblast. However, mild ethylmalonic aciduria, a biochemical marker of functional SCAD deficiency in vivo, is a common finding in patients suspected of having metabolic disorders. Based on previous observations, we have proposed that ethylmalonic aciduria in a small proportion of cases is caused by pathogenic SCAD gene mutations, and SCAD deficiency can be demonstrated in fibroblasts. Another - much more frequent - group of patients with mild ethylmalonic aciduria has functional SCAD deficiency due to the presence of susceptibility SCAD gene variations, i.e. 625G>A and 511C>T, in whom a variable or moderately reduced SCAD activity in fibroblasts may still be clinically relevant. To substantiate this notion we performed sequence analysis of the SCAD gene in 10 patients with ethylmalonic aciduria and diagnosed with SCAD deficiency in fibroblasts. Surprisingly, only one of the 10 patients carried pathogenic mutations in both alleles, while five were double heterozygotes for a pathogenic mutation in one allele and the 625G>A susceptibility variation in the other. The remaining four patients carried only either the 511C>T or the 625G>A variations in each allele. Our findings document that patients carrying these SCAD gene variations may develop clinically relevant SCAD deficiency, and that patients with even mild ethylmalonic aciduria should be tested for these variations.
Assuntos
Acil-CoA Desidrogenases/genética , Variação Genética , Erros Inatos do Metabolismo Lipídico/genética , Acil-CoA Desidrogenase , DNA Complementar , Feminino , Haplótipos , Humanos , Lactente , Recém-Nascido , Masculino , Mutagênese Sítio-Dirigida , MutaçãoRESUMO
BACKGROUND: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) with electrospray ionization is robust and allows accurate measurement of both low- and high-molecular weight components of complex mixtures. We developed a LC-MS/MS method for the analysis of methylmalonic acid (MMA), a biochemical marker for inherited disorders of propionate metabolism and acquired vitamin B(12) deficiency. METHODS: We added 1 nmol of the internal standard MMA-d(3) to 500 microL of plasma or 100 microL of urine before solid-phase extraction. After elution with 18 mol/L formic acid, the eluate was evaporated, and butyl ester derivatives were prepared with 3 mol/L HCl in n-butanol at 65 degrees C for 15 min. For separation, we used a Supelcosil LC-18, 33 x 4.6 mm column with 60:40 (by volume) acetonitrile:aqueous formic acid (1 g/L) as mobile phase. The transitions m/z 231 to m/z 119 and m/z 234 to m/z 122 were used in the selected reaction monitoring mode for MMA and MMA-d(3,) respectively. The retention time of MMA was 2.2 min in a 3.0-min analysis, without interference of a physiologically more abundant isomer, succinic acid. RESULTS: Daily calibrations between 0.25 and 8.33 nmol in 0.5 mL exhibited consistent linearity and reproducibility. At a plasma concentration of 0.12 micromol/L, the signal-to-noise ratio for MMA was 40:1. The regression equation for our previous gas chromatography-mass spectrometry (GC-MS) method (y) and the LC-MS/MS method (x) was: y = 1.030 x -0.032 (S(y|x) = 1.03 micromol/L; n = 106; r = 0.994). Inter- and intraassay CVs were 3. 8-8.5% and 1.3-3.4%, respectively, at mean concentrations of 0.13, 0.25, 0.60, and 2.02 micromol/L. Mean recoveries of MMA added to plasma were 96.9% (0.25 micromol/L), 96.0% (0.60 micromol/L), and 94.8% (2.02 micromol/L). One MS/MS system used only overnight (7.5 h) replaced two GC-MS systems (30 instrument-hours/day) to run 100-150 samples per day, with reductions of total cost (supplies plus equipment), personnel, and instrument time of 59%, 14%, and 75%, respectively. CONCLUSIONS: This method is well suited for large-scale MMA testing (> or =100 samples per day) where a shorter analytical time is highly desirable. Reagents are less expensive than the anion-exchange/cyclohexanol-HCl method, and sample preparation of batches up to 100 specimens is completed in less than 8 h and is automated.
Assuntos
Ácido Metilmalônico/sangue , Ácido Metilmalônico/urina , Cromatografia Líquida , Deutério , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Técnicas de Diluição do Indicador , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
The acylcarnitines in plasma and blood spots of 23 patients with proven deficiency of long-chain 3-hydroxyacylcoenzyme A dehydrogenase were reviewed. Long-chain 3-hydroxyacylcarnitines of C14:1, C14, C16 and C18:1 chain length, and long-chain acylcarnitines of C12, C14:1, C14, C16, C18:2 and C18:1 chain length were elevated. Acetylcarnitine was decreased. In plasma, elevation of hydroxy-C18:1 acylcarnitine over the 95th centile of controls, in combination with an elevation of two of the three acylcarnitines C14, C14:1 and hydroxy-C16, identified over 85% of patients with high specificity (less than 0.1% false positive rate). High endogenous levels of long-chain acylcarnitines in normal erythrocytes reduced the diagnostic specificity in blood spots compared with plasma samples. The results were also diagnostic in asymptomatic patients, and were not influenced by genotype. Treatment with diet low in fat and high in medium-chain triglyceride decreased all disease-specific acylcarnitines, often to normal, suggesting that this assay is useful in treatment monitoring.
Assuntos
3-Hidroxiacil-CoA Desidrogenases/deficiência , Carnitina/análogos & derivados , Carnitina/sangue , Plasma/química , 3-Hidroxiacil-CoA Desidrogenases/genética , Eritrócitos/química , Reações Falso-Negativas , Feminino , Genótipo , Heterozigoto , Homozigoto , Humanos , Masculino , Mutação , Sensibilidade e EspecificidadeRESUMO
Inborn errors of metabolism show considerable variation in the severity of symptoms. This is often ascribed to the differential effects of specific mutations on gene/enzyme function; however, such genotype/phenotype correlations are usually imprecise. In addition, in some patients with clinical and biochemical findings consistent with a defect in a particular metabolic pathway, it is ultimately impossible to arrive at a precise enzymatic diagnosis. In this situation, we have increasingly been identifying concurrent partial defects in more than one pathway, or at multiple steps in one pathway. In this study, we present the clinical, biochemical, and molecular findings from several patients showing multiple partial defects in energy metabolism. These patients show clinical symptoms consistent with a defect in the affected pathways even though they do not have a complete deficiency in any one enzyme. We hypothesize that such patients are exhibiting clinically significant reductions in energy metabolism related to the compound effects of these partial defects, a phenomenon we term "synergistic heterozygosity." Based on the frequencies of known disorders of energy metabolism, we propose that this may represent a previously unrecognized, relatively common mechanism of disease of potentially great clinical relevance.