RESUMEN
Mitochondrial 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase (mHS) deficiency is an autosomal recessive disorder of ketone body synthesis caused by biallelic pathogenic variants in HMGCS2. Clinical symptoms are precipitated by prolonged fasting and/or intercurrent illness with onset before the first year of life. Clinically, patients may present with hypo-/ non-ketotic hypoglycemia, metabolic acidosis, hyperammonemia, lethargy, hepatomegaly, and encephalopathy. During periods of decompensation, elevations of 4-hydroxy-6-methyl-2-pyrone (4-HMP), several hydroxylated hexanoic and hexenoic acid species, and medium-chain dicarboxylic acids in the absence of significant ketonuria may be observed in the urine organic acid profile. Abnormalities may also be observed in plasma which includes elevated acetylcarnitine (C2) and 3-hydroxybutyryl/3-hydroxyisobutyryl (C4-OH) carnitine. We report a patient who presented to the ED at 13 months of age with an undetectable point-of-care blood glucose level. Continuous infusion of dextrose-containing intravenous (IV) fluids were required to correct the hypoglycemia and routine chemistries were notable for an anion gap metabolic acidosis, transaminasemia, and elevated creatine kinase and lactate dehydrogenase. Urine and blood ketones were undetectable. Qualitative assessment of urine organic acids collected â¼46 and â¼ 99 h post-admission were significant for mild elevations of 4-HMP and hydroxy-hexanoic and hydroxy-hexenoic acid species with a notable absence of ketones. Previously, biochemical abnormalities in urine have been shown to normalize in as few as 27 h after treatment giving providers a narrow window with which to obtain a critical sample. Direct communication of laboratory findings to the ordering provider guided the molecular testing and assisted in results interpretation to confirm the molecular diagnosis. Our case emphasizes the importance of collecting samples for biochemical analysis even if the critical period has been missed and acute metabolic decompensation seems to be resolved, as residual abnormalities observed in our patient greatly narrowed the differential diagnosis.
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Glutaric aciduria type I (GA-1) is an inborn error of metabolism with a severe neurological phenotype caused by the deficiency of glutaryl-coenzyme A dehydrogenase (GCDH), the last enzyme of lysine catabolism. Current literature suggests that toxic catabolites in the brain are produced locally and do not cross the blood-brain barrier. In a series of experiments using knockout mice of the lysine catabolic pathway and liver cell transplantation, we uncovered that toxic GA-1 catabolites in the brain originated from the liver. Moreover, the characteristic brain and lethal phenotype of the GA-1 mouse model was rescued by two different liver-directed gene therapy approaches: Using an adeno-associated virus, we replaced the defective Gcdh gene or we prevented flux through the lysine degradation pathway by CRISPR deletion of the aminoadipate-semialdehyde synthase (Aass) gene. Our findings question the current pathophysiological understanding of GA-1 and reveal a targeted therapy for this devastating disorder.
Asunto(s)
Glutaril-CoA Deshidrogenasa , Lisina , Animales , Ratones , Glutaril-CoA Deshidrogenasa/genética , Glutaril-CoA Deshidrogenasa/metabolismo , Lisina/metabolismo , Ratones Noqueados , Hígado/metabolismoRESUMEN
Historically, disease burden and treatment responses in patients with Gaucher disease (GD) was assessed by monitoring clinical data, laboratory, imaging, chitotriosidase (CHITO), and other biomarkers; however, these biomarkers lack specificity and CHITO is uninformative in patients heterozygous or homozygous for the CHIT1 c.1049_1072dup24 variant. Recently, glucosylsphingosine (lyso-Gb1), a sensitive and specific GD biomarker, has been recommended for patient monitoring. Furthermore, studies measuring lyso-Gb1 and CHITO in patients on long-term treatment with enzyme replacement therapy (ERT) and/or substrate reduction therapy (SRT) reported as group data show a reduction in both analytes, yet individualized patient data are generally unavailable. We describe seven patients on long-term treatment with longitudinal clinical data with monitoring based on current treatment guidelines. We present four patients who exhibit stable disease with normalized CHITO despite elevated lyso-Gb1. We present one patient who transitioned from ERT to SRT due to lack of a clinical response with life-threatening thrombocytopenia who responded with marked improvement in platelets, and normalized levels of both CHITO and lyso-Gb1. Finally, we present two ERT to SRT switch patients with stable disease on ERT who exhibited non-compliance on SRT, one with mirrored marked elevations of CHITO and lyso-Gb1; and another with normal CHITO and platelets, but increasing lyso-Gb1 levels and enlarged spleen. These clinical vignettes highlight the role of lyso-Gb1 as a sensitive biomarker in management of patients with GD, and its further value when CHITO is normal and thus uninformative. We highlight the personalized medicine approach needed to optimize treatment outcomes and recommendations for these patients.
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Enfermedad de Gaucher , Humanos , Enfermedad de Gaucher/diagnóstico , Enfermedad de Gaucher/tratamiento farmacológico , Enfermedad de Gaucher/genética , Psicosina , Terapia de Reemplazo Enzimático , BiomarcadoresRESUMEN
Gaucher disease (GD), a lysosomal storage disorder caused by ß-glucocerebrosidase deficiency, results in the accumulation of glucosylceramide and glucosylsphingosine. Glucosylsphingosine has emerged as a sensitive and specific biomarker for GD and treatment response. However, limited information exists on its role in guiding treatment decisions in pre-symptomatic patients identified at birth or due to a positive family history. We present two pediatric patients with GD1 and highlight the utility of glucosylsphingosine monitoring in guiding treatment initiation.
RESUMEN
PURPOSE: To develop a method for the combined analysis of plasma and serum glucosylsphingosine (lyso-Gb1) and globotriaosylsphingosine (lyso-Gb3), biomarkers of Gaucher disease (GD) and Fabry disease (FD), respectively. METHODS: Internal standards were added to 100 µL of plasma/serum and glycosphingolipids: lyso-Gb1, lyso-Gb3, and galactosylsphingosine (GalSph) were extracted with dichloromethane/methanol and analyzed by UPLC-MS/MS. Samples from unaffected controls and patients with GD were first analyzed using a HILIC column to separate lyso-Gb1 from its isomer, GalSph. Samples from patients with FD or GD were analyzed using a C18 column to measure lyso-Gb3 and the hexosylsphingosine (HexSph: lyso-Gb1 + GalSph) fraction in a single combined method. RESULTS: Extraction efficiency was between 73% and 87% and day-to-day variability showed a relative standard error of <7.5%. GalSph was determined to have minimal to no contribution to the HexSph fraction in samples from unaffected controls and patients with GD. Lyso-Gb3 and HexSph measurements by the combined method were in good agreement with established methods, with no bias. CONCLUSIONS: HexSph and lyso-Gb3 analysis by reversed-phase chromatography UPLC-MS/MS is a cost-effective, time-efficient approach for evaluating these glycosphingolipid biomarkers in patients with a suspected or confirmed diagnosis of GD and FD.
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Enfermedad de Fabry , Psicosina , Biomarcadores , Cromatografía Líquida de Alta Presión , Cromatografía Liquida , Enfermedad de Fabry/diagnóstico , Glucolípidos , Humanos , Psicosina/análogos & derivados , Esfingolípidos , Espectrometría de Masas en TándemRESUMEN
AIMS: To validate a liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the measurement of glycosaminoglycans (GAGs) in plasma and serum. To establish plasma, cerebrospinal fluid (CSF) and urine reference intervals. To compare GAGs in serum with that in urine and CSF from patients with MPS I. METHODS: Dermatan sulfate (DS), heparan sulfate (HS), and chondroitin sulfate (CS) in serum/plasma, urine and CSF were methanolysed into dimers and analyzed using pseudo isotope dilution UPLC-MS/MS assay. Serum, CSF and urine DS and HS were quantified for 11 patients with mucopolysaccharidosis (MPS) type I before and after treatment with Aldurazyme® (laronidase) enzyme replacement therapy (ERT). RESULTS: The method showed acceptable imprecision and recovery for the quantification of serum/plasma CS, DS, and HS. The serum, urine, and CSF DS and HS concentrations were reduced after 26 weeks of ERT in 4 previously untreated patients. Serum DS and HS concentrations normalized in some patients, and were mildly elevated in others after ERT. In contrast, urine and CSF DS and HS values remained elevated above the reference ranges. Compared with serum GAGs, urine and CSF DS and HS were more sensitive biomarkers for monitoring the ERT treatment of patients with MPS I.
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Dermatán Sulfato , Mucopolisacaridosis I , Cromatografía Liquida , Terapia de Reemplazo Enzimático , Glicosaminoglicanos , Heparitina Sulfato , Humanos , Mucopolisacaridosis I/tratamiento farmacológico , Espectrometría de Masas en TándemRESUMEN
PURPOSE: Successful diagnosis of Fabry disease is often delayed or missed in patients, especially females, due to clinical heterogeneity and a lack of disease awareness. We present our experience testing for Fabry disease in high risk populations and discuss the relative sensitivities of α-galactosidase A (α-Gal A) enzyme activity in blood, plasma lyso-globotriaosylceramide (lyso-Gb3) biomarker, and GLA gene sequencing as diagnostic tests for Fabry disease in both males and females. METHODS: Patients with a clinical suspicion of Fabry disease were evaluated with enzyme analysis, biomarker analysis, and GLA sequencing. All three assays were performed from a single tube of EDTA blood. α-Gal A activity was determined in dried blood spots using a fluorometric assay, plasma lyso-Gb3 by UPLC-MS/MS, and GLA analysis by Sanger sequencing. RESULTS: Peripheral blood samples were received from 94 males and 200 females, of which 29% of males and 22% of females had a positive family history of Fabry disease. A likely pathogenic or pathogenic variant was identified in 87 (30%) patients (50 males, 37 females), confirming a diagnosis of Fabry disease. Of the remaining patients, 178 (61%) were determined to be unaffected based on normal enzyme activity (males) or normal lyso-Gb3 and negative sequencing results (females). A VUS was identified in 29 (10%) patients. The positive and negative predictive value of plasma lyso-Gb3 was 100% and 97% in males and 100% and 99% in females, respectively. This compares with 84% and 100% in males, and 58% and 50% in females for α-Gal A activity testing, respectively. CONCLUSIONS: Plasma lyso-Gb3 has high sensitivity and specificity for Fabry disease in males and females, and provides supportive diagnostic information when gene sequencing results are negative or inconclusive. α-Gal A activity in dried blood spots (DBS) has high sensitivity, but lower specificity for Fabry disease in males, as not all males with low α-Gal A activities were confirmed to have Fabry disease. Therefore, reflexing to gene sequencing and plasma lyso-Gb3 is useful for disease confirmation in males. For females, we found that first tier testing consisting of GLA sequencing and plasma lyso-Gb3 analysis provided the greatest sensitivity and specificity. Enzyme testing has lower sensitivity in females and is therefore less useful as a first-tier test. Enzyme analysis in females may still be helpful as a second-tier test in cases where molecular testing and plasma lyso-Gb3 analysis are uninformative and in vitro enzyme activity is low. SUMMARY: Sex-specific testing algorithms that prioritize tests with high specificity and sensitivity offer an effective means of identifying individuals with Fabry disease.
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Algoritmos , Biomarcadores/sangre , Enfermedad de Fabry/diagnóstico , Glucolípidos/sangre , Esfingolípidos/sangre , alfa-Galactosidasa/metabolismo , Enfermedad de Fabry/metabolismo , Femenino , Humanos , Recién Nacido , Masculino , Mutación , Estudios Retrospectivos , alfa-Galactosidasa/genéticaRESUMEN
In humans, mitochondrial DNA (mtDNA) depletion syndromes are a group of genetically and clinically heterogeneous autosomal recessive disorders that arise as a consequence of defects in mtDNA replication or nucleotide synthesis. Clinical manifestations are variable and include myopathic, encephalomyopathic, neurogastrointestinal or hepatocerebral phenotypes. Through clinical exome sequencing, we identified a homozygous missense variant (c.533C>T; p.Pro178Leu) in mitochondrial transcription factor A (TFAM) segregating in a consanguineous kindred of Colombian-Basque descent in which two siblings presented with IUGR, elevated transaminases, conjugated hyperbilirubinemia and hypoglycemia with progression to liver failure and death in early infancy. Results of the liver biopsy in the proband revealed cirrhosis, micro- and macrovesicular steatosis, cholestasis and mitochondrial pleomorphism. Electron microscopy of muscle revealed abnormal mitochondrial morphology and distribution while enzyme histochemistry was underwhelming. Electron transport chain testing in muscle showed increased citrate synthase activity suggesting mitochondrial proliferation, while respiratory chain activities were at the lower end of normal. mtDNA content was reduced in liver and muscle (11% and 21% of normal controls respectively). While Tfam mRNA expression was upregulated in primary fibroblasts, Tfam protein level was significantly reduced. Furthermore, functional investigations of the mitochondria revealed reduced basal respiration and spare respiratory capacity, decreased mtDNA copy number and markedly reduced nucleoids. TFAM is essential for transcription, replication and packaging of mtDNA into nucleoids. Tfam knockout mice display embryonic lethality secondary to severe mtDNA depletion. In this report, for the first time, we associate a homozygous variant in TFAM with a novel mtDNA depletion syndrome.
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ADN Mitocondrial/genética , Proteínas de Unión al ADN/genética , Fallo Hepático/genética , Enfermedades Mitocondriales/genética , Proteínas Mitocondriales/genética , Factores de Transcripción/genética , Animales , Replicación del ADN/genética , ADN Mitocondrial/metabolismo , Femenino , Homocigoto , Humanos , Recién Nacido , Hígado/metabolismo , Hígado/fisiopatología , Fallo Hepático/fisiopatología , Masculino , Ratones , Ratones Noqueados , Enfermedades Mitocondriales/fisiopatología , Mutación Missense , Tamizaje Neonatal , Secuenciación del ExomaRESUMEN
2-Ketoadipic aciduria (OMIM 204750), a defect in the catabolic pathway of tryptophan, lysine, and hydroxylysine, is characterized by elevations in 2-ketoadipic, 2-aminoadipic, and 2-hydroxyadipic acids. Patients with the aforementioned biochemical profile have been described with a wide range of clinical presentations, from early-onset developmental delay, epilepsy, ataxia, and microcephaly to completely normal. This broad range of phenotypes has led some to question whether 2-ketoadipic aciduria represents a true disease state or if the biochemical abnormalities found in these patients merely reflect an ascertainment bias. We present four additional individuals from two families, with 2-ketoadipic aciduria with compound heterozygous or homozygous mutations in DHTKD1, three of which remain asymptomatic.
RESUMEN
PURPOSE: 3-Hydroxyisobutryl-CoA hydrolase (HIBCH) deficiency is a rare disorder of valine metabolism. We present a family with the oldest reported subjects with HIBCH deficiency and provide support that HIBCH deficiency should be included in the differential for elevated hydroxy-C4-carnitine in newborn screening (NBS). METHODS: Whole exome sequencing (WES) was performed on one affected sibling. HIBCH enzymatic activity was measured in patient fibroblasts. Acylcarnitines were measured by electrospray ionization tandem mass spectrometry (ESI-MS/MS). Disease incidence was estimated using a cohort of 61,434 individuals. RESULTS: Two siblings presented with infantile-onset, progressive neurodegenerative disease. WES identified a novel homozygous variant in HIBCH c.196C>T; p.Arg66Trp. HIBCH enzymatic activity was significantly reduced in patients' fibroblasts. Acylcarnitine analysis showed elevated hydroxy-C4-carnitine in blood spots of both affected siblings, including in their NBS cards, while plasma acylcarnitines were normal. Estimates show HIBCH deficiency incidence as high as 1 in ~130,000 individuals. CONCLUSION: We describe a novel family with HIBCH deficiency at the biochemical, enzymatic and molecular level. Disease incidence estimates indicate HIBCH deficiency may be under-diagnosed. This together with the elevated hydroxy-C4-carnitine found in the retrospective analysis of our patient's NBS cards suggests that this disorder could be screened for by NBS programs and should be added to the differential diagnosis for elevated hydroxy-C4-carnitine which is already measured in most NBS programs using MS/MS.
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Anomalías Múltiples/diagnóstico , Errores Innatos del Metabolismo de los Aminoácidos/diagnóstico , Enfermedad de Leigh/metabolismo , Tamizaje Neonatal , Tioléster Hidrolasas/deficiencia , Anomalías Múltiples/metabolismo , Adolescente , Errores Innatos del Metabolismo de los Aminoácidos/metabolismo , Carnitina/análogos & derivados , Carnitina/metabolismo , Niño , Preescolar , Estudios de Cohortes , Exoma , Femenino , Fibroblastos/enzimología , Humanos , Lactante , Recién Nacido , Enfermedad de Leigh/enzimología , Masculino , Espectrometría de Masas , Pronóstico , Estudios Retrospectivos , Análisis de Secuencia de ADN , Hermanos , Tioléster Hidrolasas/química , Tioléster Hidrolasas/metabolismoRESUMEN
An unknown fraction of the genome participates in the metabolism of sterols and vitamin D, two classes of lipids with diverse physiological and pathophysiological roles. Here, we used mass spectrometry to measure the abundance of >60 sterol and vitamin D derivatives in 3,230 serum samples from a well-phenotyped patient population. Twenty-nine of these lipids were detected in a majority of samples at levels that varied over thousands of fold in different individuals. Pairwise correlations between sterol and vitamin D levels revealed evidence for shared metabolic pathways, additional substrates for known enzymes, and transcriptional regulatory networks. Serum levels of multiple sterols and vitamin D metabolites varied significantly by sex, ethnicity, and age. A genome-wide association study identified 16 loci that were associated with levels of 19 sterols and 25-hydroxylated derivatives of vitamin D (P < 10(-7)). Resequencing, expression analysis, and biochemical experiments focused on one such locus (CYP39A1), revealed multiple loss-of-function alleles with additive effects on serum levels of the oxysterol, 24S-hydroxycholesterol, a substrate of the encoded enzyme. Body mass index, serum lipid levels, and hematocrit were strong phenotypic correlates of interindividual variation in multiple sterols and vitamin D metabolites. We conclude that correlating population-based analytical measurements with genotype and phenotype provides productive insight into human intermediary metabolism.
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Índice de Masa Corporal , Sitios Genéticos/fisiología , Genotipo , Hidroxicolesteroles/sangre , Esteroide Hidroxilasas , Vitamina D/sangre , Femenino , Humanos , Masculino , Esteroide Hidroxilasas/genética , Esteroide Hidroxilasas/metabolismo , Vitamina D/genéticaRESUMEN
We describe the development of a method for the extraction and analysis of 62 sterols, oxysterols, and secosteroids from human plasma using a combination of HPLC-MS and GC-MS. Deuterated standards are added to 200 µl of human plasma. Bulk lipids are extracted with methanol:dichloromethane, the sample is hydrolyzed using a novel procedure, and sterols and secosteroids are isolated using solid-phase extraction (SPE). Compounds are resolved on C18 core-shell HPLC columns and by GC. Sterols and oxysterols are measured using triple quadrupole mass spectrometers, and lathosterol is measured using GC-MS. Detection for each compound measured by HPLC-MS was ⪠1 ng/ml of plasma. Extraction efficiency was between 85 and 110%; day-to-day variability showed a relative standard error of <10%. Numerous oxysterols were detected, including the side chain oxysterols 22-, 24-, 25-, and 27-hydroxycholesterol, as well as ring-structure oxysterols 7α- and 4ß-hydroxycholesterol. Intermediates from the cholesterol biosynthetic pathway were also detected, including zymosterol, desmosterol, and lanosterol. This method also allowed the quantification of six secosteroids, including the 25-hydroxylated species of vitamins D2 and D3. Application of this method to plasma samples revealed that at least 50 samples could be extracted in a routine day.
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Secoesteroides/sangre , Secoesteroides/aislamiento & purificación , Esteroles/sangre , Esteroles/aislamiento & purificación , Cromatografía Líquida de Alta Presión , Cromatografía de Gases y Espectrometría de Masas , Humanos , Espectrometría de Masas , Secoesteroides/química , Esteroles/químicaRESUMEN
The steroid 5alpha-reductase (SRD5A) family of enzymes produces steroid hormones that regulate male sexual development. Now, Cantagrel et al. (2010) identify a member of this family, SRD5A3, as a polyprenol reductase with a crucial role in N-linked protein glycosylation and pinpoint SRD5A3 mutations as the cause of a rare Mendelian disease.
RESUMEN
The CYP7B1 cytochrome P450 enzyme hydroxylates carbons 6 and 7 of the B ring of oxysterols and steroids. Hydroxylation reduces the biological activity of these substrates and facilitates their conversion to end products that are readily excreted from the body. CYP7B1 is expressed in the liver, reproductive tract, and brain and performs different physiological functions in each tissue. Hepatic CYP7B1 activity is crucial for the inactivation of oxysterols and their subsequent conversion into bile salts. Loss of CYP7B1 activity is associated with liver failure in children. In the reproductive tract, the enzyme metabolizes androgens that antagonize estrogen action; mice without CYP7B1 have abnormal prostates and ovaries. The role of CYP7B1 in brain is under investigation; recent studies show that spastic paraplegia type 5, a progressive neuropathy, is caused by loss-of-function mutations in the human gene.