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Background: DNA polymerase gamma (POLG)-related disorders are a group of rare neurodegenerative mitochondrial diseases caused by pathogenic variants in POLG, the gene encoding POLG. Patients may experience a range of signs and symptoms, including seizures, vision loss, myopathy, neuropathy, developmental impairment or regression, and liver failure. The diseases follow a progressive, degenerative course, with most affected individuals dying within 3 months-12 years of diagnosis. At present, there are no effective treatments for POLG-related disorders. Methods: In this study we report the interim 6-month data from a long term open-label, single arm phase 2 trial, in which we assessed the safety and efficacy of combination therapy with deoxycytidine and deoxythymidine (dC/dT) in children with POLG-related disorders. dC/dT was given enterally in powder form, dissolved in water. The primary outcome measures included Newcastle Mitochondrial Disease Scale (NMDS) score, serum growth differentiation factor 15 (GDF-15; a biomarker of mitochondrial dysfunction), electroencephalography (EEG), seizure diary, and blood and urine tests to assess end organ and mitochondrial function. Secondary outcome measures included recording of all adverse events to evaluate the safety of the intervention. The trial is registered with ClinicalTrials.gov, NCT04802707 (https://clinicaltrials.gov/ct2/show/NCT04802707). Data were collected from 14 October, 2021 to 13 December, 2023. Findings: We present 6-month interim data from the first ten people with POLG-related disorders enrolled in the trial, six with Alpers-Huttenlocher syndrome, two with ataxia-neuropathy spectrum, and two who do not fit into a classical POLG-related phenotype. During the 6 months of treatment, NMDS score improved from a mean of 27.3 at baseline to 20.7 at 6 months (estimated difference 6.0; 95% CI 2.5-∞). GDF-15 values remained stable or decreased in all patients; the mean decreased from 1031 pg/ml to 729 pg/ml (estimated difference 200; 95% CI 12-∞). 8/10 patients had abnormal baseline EEG; improvement in EEG was seen in 5 of these 8. There were no significant changes in other blood and urine testing. Regarding adverse events, two patients experienced diarrhea that spontaneously resolved. Interpretation: dC/dT is a promising treatment option for people with POLG-related disorders. Further research is needed to assess the long-term safety and efficacy in POLG-related disorders, as well as safety and efficacy in other mitochondrial DNA depletion disorders. Funding: This study was primarily funded by the Liam Foundation, with additional funding from the Savoy Foundation, Grand Défi Pierre Lavoie Foundation, and Fonds de Recherche du Québec - Santé.
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Fabry disease (FD) is a lysosomal storage disorder caused by variants in the GLA gene encoding α-galactosidase A, an enzyme required for catabolism of globotriaosylceramide (Gb3). Accumulation of Gb3 in patients' cells, tissues, and biological fluids causes clinical manifestations including ventricular hypertrophy, renal insufficiency, and strokes. This protocol describes a methodology to analyze urinary Gb3 and creatinine. Samples are diluted with an internal standard solution containing Gb3(C17:0) and creatinine-D3, centrifuged, and directly analyzed by ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) using an 8.7-min method. Eight Gb3 isoforms [C16:0, C18:0, C20:0, C22:1, C22:0, C24:1, C24:0, and (C24:0)OH] are analyzed and the total is normalized to creatinine. Confirmation ions are monitored to detect potential interferences. The Gb3 limit of quantification is 0.023 µg/ml. Its interday coefficients of variation (3 concentrations measured) are ≤15.4%. This method minimizes matrix effects (≤6.5%) and prevents adsorption or precipitation of Gb3. Urine samples are stable (bias <15%) for 2 days at 21°C, 7 days at 4°C, and 4 freeze/thaw cycles, whereas prepared samples are stable for 5 days at 21°C, and 14 days at 4°C. The Gb3/creatinine age-related upper reference limits (mean + 2 standard deviations) are 29 mg/mol creatinine (<7 years) and 14 mg/mol creatinine (≥7 years). This simple, robust protocol has been fully validated (ISO 15189) and provides a valuable tool for diagnosis and monitoring of FD patients. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Analysis of urinary globotriaosylceramide (Gb3) and creatinine by UHPLC-MS/MS Support Protocol 1: Preparation of the urinary quality controls Support Protocol 2: Preparation of the urine matrix used for the Gb3 calibration curve Support Protocol 3: Preparation of the Gb3 calibrators Support Protocol 4: Preparation of the working solution containing the internal standards Support Protocol 5: Preparation of the creatinine calibrators Support Protocol 6: Preparation of the UHPLC solutions and mobile phases.
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Enfermedad de Fabry , Espectrometría de Masas en Tándem , Trihexosilceramidas , Humanos , Espectrometría de Masas en Tándem/métodos , Trihexosilceramidas/orina , Trihexosilceramidas/metabolismo , Cromatografía Líquida de Alta Presión/métodos , Enfermedad de Fabry/orina , Enfermedad de Fabry/diagnóstico , Creatinina/orinaRESUMEN
Measurement of alpha-glucosidase activity on dried blood spots has been the main method to screen for Pompe disease, but a paradigm shift has been observed in recent years with the incorporation of gene panels and exome sequencing in molecular diagnostic laboratories. An 89-gene panel has been available to Canadian physicians since 2017 and was analyzed in 2030 patients with a suspected muscle disease. Acid alpha-glucosidase activity was measured in parallel in dried blood spots from 1430 patients. Pompe disease was diagnosed in 14 patients, representing 0.69% of our cohort. In 7 other patients, low enzyme activities overlapping those of Pompe disease cases were attributable to the presence of pseudodeficiency alleles. Only two other patients had enzymatic activity in the Pompe disease range, and a single heterozygous pathogenic variant was identified. It is possible that a second variant could have been missed; we suggest that RNA analysis should be considered in such cases. With gene panel testing increasingly being performed as a first-tier analysis of patients with suspected muscle disorders, our study supports the relevance of performing reflex enzymatic activity assay in selected patients, such as those with a single GAA variant identified and those in whom the observed genotype is of uncertain clinical significance.
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OBJECTIVE: To evaluate the diagnostic yield of an 89-gene panel in a large cohort of patients with suspected muscle disorders and to compare the diagnostic yield of gene panel and exome sequencing approaches. METHODS: We tested 1,236 patients from outpatient clinics across Canada using a gene panel and performed exome sequencing for 46 other patients with sequential analysis of 89 genes followed by all mendelian genes. Sequencing and analysis were performed in patients with muscle weakness or symptoms suggestive of a muscle disorder and showing at least 1 supporting clinical laboratory. RESULTS: We identified a molecular diagnosis in 187 (15.1%) of the 1,236 patients tested with the 89-gene panel. Diagnoses were distributed across 40 different genes, but 6 (DMD, RYR1, CAPN3, PYGM, DYSF, and FKRP) explained about half of all cases. Cardiac anomalies, positive family history, age <60 years, and creatine kinase >1,000 IU/L were all associated with increased diagnostic yield. Exome sequencing identified a diagnosis in 10 (21.7%) of the 46 patients tested. Among these, 3 were attributed to genes not included in the 89-gene panel. Despite differences in median coverage, only 1 of the 187 diagnoses that were identified on gene panel in the 1,236 patients could have been potentially missed if exome sequencing had been performed instead. CONCLUSIONS: Our study supports the use of gene panel testing in patients with suspected muscle disorders from outpatient clinics. It also shows that exome sequencing has a low risk of missing diagnoses compared with gene panel, while potentially increasing the diagnostic yield of patients with muscle disorders.
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Targeting definite genomic locations using CRISPR-Cas systems requires a set of enzymes with unique protospacer adjacent motif (PAM) compatibilities. To expand this repertoire, we engineered nucleases, cytosine base editors, and adenine base editors from the archetypal Streptococcus thermophilus CRISPR1-Cas9 (St1Cas9) system. We found that St1Cas9 strain variants enable targeting to five distinct A-rich PAMs and provide a structural basis for their specificities. The small size of this ortholog enables expression of the holoenzyme from a single adeno-associated viral vector for in vivo editing applications. Delivery of St1Cas9 to the neonatal liver efficiently rewired metabolic pathways, leading to phenotypic rescue in a mouse model of hereditary tyrosinemia. These robust enzymes expand and complement current editing platforms available for tailoring mammalian genomes.
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Proteína 9 Asociada a CRISPR/metabolismo , Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Edición Génica , Streptococcus thermophilus/enzimología , Streptococcus thermophilus/genética , Animales , Proteína 9 Asociada a CRISPR/química , Línea Celular , Células Cultivadas , División del ADN , Humanos , Mamíferos , Ratones , Ratones Noqueados , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
BACKGROUND: HSD10 mitochondrial disease (HSD10MD), originally described as a deficiency of 2-methyl-3-hydroxybutyryl-CoA dehydrogenase (MHBD), is a rare X-linked disorder of a moonlighting protein encoded by the HSD17B10. The diagnosis is usually first suspected on finding elevated isoleucine degradation metabolites in urine, reflecting decreased MHBD activity. However, it is now known that clinical disease pathogenesis reflects other independent functions of the HSD10 protein; particularly its essential role in mitochondrial transcript processing and tRNA maturation. The classical phenotype of HSD10MD in affected males is an infantile-onset progressive neurodegenerative disorder associated with severe mitochondrial dysfunction. PATIENTS, METHODS, AND RESULTS: In four unrelated families, we identified index patients with MHBD deficiency, which implied a diagnosis of HSD10MD. Each index patient was independently investigated because of neurological or developmental concerns. All had persistent elevations of urinary 2-methyl-3-hydroxybutyric acid and tiglylglycine. Analysis of HSD17B10 identified a single missense variant, c.364C>G, p.Leu122Val, in each case. This rare variant (1/183336 alleles in gnomAD) was previously reported in one Dutch patient and was described as pathogenic. The geographic origins of our families and results of haplotype analysis together provide evidence of a founder effect for this variant in Quebec. Notably, we identified an asymptomatic hemizygous adult male in one family, while a second independent genetic disorder contributed substantially to the clinical phenotypes observed in probands from two other families. CONCLUSION: The phenotype associated with p.Leu122Val in HSD17B10 currently appears to be attenuated and nonprogressive. This report widens the spectrum of phenotypic severity of HSD10MD and contributes to genotype-phenotype correlation. At present, we consider p.Leu122Val a "variant of uncertain significance." Nonetheless, careful follow-up of our patients remains advisable, to assess long-term clinical course and ensure appropriate management. It will also be important to identify other potential patients in our population and to characterize their phenotype.
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3-Hidroxiacil-CoA Deshidrogenasas/genética , Sustitución de Aminoácidos , Efecto Fundador , Enfermedades Mitocondriales/genética , Mutación Missense , Adulto , Edad de Inicio , Niño , Preescolar , Femenino , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Hemicigoto , Humanos , Lactante , Masculino , Persona de Mediana Edad , Linaje , Quebec , Adulto JovenRESUMEN
BACKGROUND: The clinical significance of combined malonic and methylmalonic aciduria due to ACSF3 deficiency (CMAMMA) is controversial. In most publications, affected patients were identified during the investigation of various complaints. METHODS: Using a cross-sectional multicenter retrospective natural history study, we describe the course of all known CMAMMA individuals in the province of Quebec. RESULTS: We identified 25 CMAMMA patients (6 months to 30 years old) with a favorable outcome regardless of treatment. All but one came to clinical attention through the Provincial Neonatal Urine Screening Program (screening on day 21 of life). Median methylmalonic acid (MMA) levels ranged from 107 to 857 mmol/mol creatinine in urine (<10) and from 8 to 42 µmol/L in plasma (<0.4); median urine malonic acid (MA) levels ranged from 9 to 280 mmol/mol creatinine (<5). MMA was consistently higher than MA. These findings are comparable to those previously reported in CMAMMA. Causal ACSF3 mutations were identified in all patients for whom genotyping was performed (76% of cases). The most common ACSF3 mutations in our cohort were c.1075G > A (p.E359K) and c.1672C > T (p.R558W), representing 38.2 and 20.6% of alleles in genotyped families, respectively; we also report several novel mutations. CONCLUSION: Because our province still performs urine newborn screening, our patient cohort is the only one free of selection bias. Therefore, the favorable clinical course observed suggests that CMAMMA is probably a benign condition, although we cannot exclude the possibility that a small minority of patients may present symptoms attributable to CMAMMA, perhaps as a result of interactions with other genetic or environmental factors.
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Coenzima A Ligasas/genética , Errores Innatos del Metabolismo/genética , Mutación/genética , Adolescente , Adulto , Alelos , Niño , Preescolar , Estudios de Cohortes , Creatinina/metabolismo , Estudios Transversales , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Malonatos/metabolismo , Ácido Metilmalónico/metabolismo , Tamizaje Neonatal/métodos , Estudios Retrospectivos , Adulto JovenRESUMEN
Ethylmalonic encephalopathy (EE) is caused by mutations in the ETHE1 gene. ETHE1 is vital for the catabolism of hydrogen sulfide (H2S). Patients with pathogenic mutations in ETHE1 have markedly increased thiosulfate, which is a reliable index of H2S levels. Accumulation of H2S is thought to cause the characteristic metabolic derangement found in EE. Recently introduced treatment strategies in EE, such as combined use of metronidazole (MNZ) and N-acetylcysteine (NAC), are aimed at lowering chronic H2S load. Experience with treatment strategies directed against acute episodes of metabolic decompensation (e.g., hemodialysis) is limited. Here we present an unusually mild, molecularly confirmed, case of EE in a 19-year-old male on chronic treatment with MNZ and NAC. During an acute episode of metabolic decompensation, we employed continuous renal replacement therapy (CRRT) to regain metabolic control. On continuous treatment with NAC and MNZ during the months preceding the acute event, plasma thiosulfate levels ranged from 1.6 to 4 µg/mL (reference range up to 2 µg/mL) and had a mean value of 2.5 µg/mL. During the acute decompensation, thiosulfate levels were 6.7 µg/mL, with hyperlactatemia and perturbed organic acid, acylglycine, and acylcarnitine profiles. CRRT decreased thiosulfate within 24 h to 1.4 µg/mL. Following discontinuation of CRRT, mean thiosulfate levels were 3.2 µg/mL (range, 2.4-3.7 µg/mL) accompanied by clinical improvement with metabolic stabilization of blood gas, acylcarnitine, organic acid, and acylglycine profiles. In conclusion, CRRT may help to regain metabolic control in patients with EE who have an acute metabolic decompensation on chronic treatment with NAC and MNZ.
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PURPOSE: We sought to determine the diagnostic yield of whole-exome sequencing (WES) combined with phenotype-driven analysis of variants in patients with suspected genetic disorders. METHODS: WES was performed on a cohort of 51 patients presenting dysmorphisms with or without neurodevelopmental disorders of undetermined etiology. For each patient, a clinical geneticist reviewed the phenotypes and used the phenotype-driven analysis software PhenoVar (http://phenovar.med.usherbrooke.ca/) to analyze WES variants. The prioritized list of potential diagnoses returned was reviewed by the clinical geneticist, who selected candidate variants to be confirmed by segregation analysis. Conventional analysis of the individual variants was performed in parallel. The resulting candidate variants were subsequently reviewed by the same geneticist, to identify any additional potential diagnoses. RESULTS: A molecular diagnosis was identified in 35% of the patients using the conventional analysis, and 17 of these 18 diagnoses were independently identified using PhenoVar. The only diagnosis initially missed by PhenoVar was rescued when the optional "minimal phenotypic cutoff" filter was omitted. PhenoVar reduced by half the number of potential diagnoses per patient compared with the conventional analysis. CONCLUSION: Phenotype-driven software prioritizes WES variants, provides an efficient diagnostic aid to clinical geneticists and laboratories, and should be incorporated in clinical practice.
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Enfermedades Genéticas Congénitas/diagnóstico , Análisis de Secuencia de ADN/métodos , Adolescente , Adulto , Niño , Preescolar , Estudios de Cohortes , Exoma , Femenino , Enfermedades Genéticas Congénitas/genética , Pruebas Genéticas , Humanos , Masculino , Persona de Mediana Edad , Mutación , Programas Informáticos , Secuenciación del Exoma/instrumentación , Secuenciación del Exoma/métodosRESUMEN
BACKGROUND: A high level of succinylacetone (SA) in blood is a sensitive, specific marker for the screening and diagnosis of hepatorenal tyrosinemia (HT1, MIM 276700). HT1 is caused by mutations in the FAH gene, resulting in deficiency of fumarylacetoacetate hydrolase. HT1 newborns are usually clinically asymptomatic, but have coagulation abnormalities revealing liver dysfunction. Treatment with nitisinone (NTBC) plus dietary restriction of tyrosine and phenylalanine prevents the complications of HT1. OBSERVATIONS: Two newborns screened positive for SA but had normal coagulation testing. Plasma and urine SA levels were 3-5 fold above the reference range but were markedly lower than in typical HT1. Neither individual received nitisinone or dietary therapy. They remain clinically normal, currently aged 9 and 15 years. Each was a compound heterozygote, having a splicing variant in trans with a prevalent "pseudodeficient" FAH allele, c.1021C > T (p.Arg341Trp), which confers partial FAH activity. All newborns identified with mild hypersuccinylacetonemia in Québec have had genetic deficiencies of tyrosine degradation: either deficiency of the enzyme preceding FAH, maleylacetoacetate isomerase, or partial deficiency of FAH itself. CONCLUSION: Compound heterozygotes for c.1021C > T (p.Arg341Trp) and a severely deficient FAH allele have mild hypersuccinylacetonemia and to date they have remained asymptomatic without treatment. It is important to determine the long term outcome of such individuals.
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Glutaric aciduria type 3 (GA3) is associated with decreased conversion of free glutaric acid to glutaryl-coA, reflecting deficiency of succinate-hydroxymethylglutarate coA-transferase, caused by variants in the SUGCT (C7orf10) gene. GA3 remains less well known, characterised and understood than glutaric aciduria types 1 and 2. It is generally considered a likely "non-disease," but this is based on limited supporting information, with only nine individuals with GA3 described in the literature. Clinicians encountering a patient with GA3 therefore still face a dilemma of whether or not this should be dismissed as irrelevant.We have identified three unrelated Canadian patients with GA3. Two came to clinical attention because of symptoms, while the third was identified by a population urine-based newborn screening programme and has so far remained asymptomatic. We describe the clinical histories, biochemical characterisation and genotypes of these individuals. Examination of allele frequencies underlines the fact that GA3 is underdiagnosed. While one probable factor is that some GA3 patients remain asymptomatic, we highlight other plausible reasons whereby this diagnosis might be overlooked.Gastrointestinal disturbances were previously reported in some GA3 patients. In one of our patients, severe episodes of cyclic vomiting were the major problem. A trial of antibiotic treatment, to minimise bacterial GA production, was followed by significant clinical improvement.At present, there is insufficient evidence to define any specific clinical phenotype as attributable to GA3. However, we consider that it would be premature to assume that this condition is completely benign in all individuals at all times.
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BACKGROUND AND AIM: Diagnostic and management guidelines for vitamin B12 (cobalamin, Cbl) deficiency in inflammatory bowel disease (IBD) are lacking. True deficiency is defined as Cbl concentrations below reference range combined with elevated methylmalonic acid (MMA) concentrations. Studies analyzing Cbl status in IBD use only Cbl concentrations without confirmatory MMA. This study aims to determine the proportion of IBD patients with Cbl concentrations below reference range and their predisposing clinical and genetic characteristics. We then compared this to the proportion with true deficiency. PATIENTS AND METHODS: In a prospective observational pilot study of adult IBD outpatients, Cbl concentrations, MMA levels, and fucosyltransferase 2 mutations were measured at clinic visits. RESULTS: A total of 66 Crohn's disease (CD) and 30 ulcerative colitis (UC) patients were recruited. Mean Cbl concentrations (pmol/l) in CD (253.7) were not significantly lower than UC (320.5, P=0.24). Serum Cbl below reference range (<148) was observed in 7.6 and 10% of CD and UC patients, respectively (P=0.70). True deficiency in CD and UC was 3 and 3.3%, respectively (P=1.0). Patients with ileal resections more than 30 cm had lower mean Cbl concentrations (177, P=0.02) and a trend toward higher proportions with Cbl levels below reference range (40%, P=0.06), but not increased deficiency rates (0%, P=1.0). Disease location, severity, and fucosyltransferase 2 mutations were not associated with altered Cbl status. CONCLUSION: True Cbl deficiency was rare in IBD patients in this study. A disparity in Cbl status exists when confirmatory MMA levels are used compared with Cbl concentrations alone. Asymptomatic IBD patients with low serum Cbl require confirmatory tests to guide management and avoid unnecessary treatment.
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Colitis Ulcerosa/sangre , Enfermedad de Crohn/sangre , Ácido Metilmalónico/sangre , Deficiencia de Vitamina B 12/sangre , Deficiencia de Vitamina B 12/diagnóstico , Vitamina B 12/sangre , Codón sin Sentido , Colitis Ulcerosa/complicaciones , Enfermedad de Crohn/complicaciones , Femenino , Fucosiltransferasas/genética , Humanos , Masculino , Persona de Mediana Edad , Proyectos Piloto , Estudios Prospectivos , Factores de Riesgo , Índice de Severidad de la Enfermedad , Deficiencia de Vitamina B 12/complicaciones , Deficiencia de Vitamina B 12/genética , Galactósido 2-alfa-L-FucosiltransferasaRESUMEN
BACKGROUND: A high level of succinylacetone (SA) in blood is a sensitive, specific newborn screening marker for hepatorenal tyrosinemia type 1 (HT1, MIM 276700) caused by deficiency of fumarylacetoacetate hydrolase (FAH). Newborns with HT1 are usually clinically asymptomatic but show liver dysfunction with coagulation abnormalities (prolonged prothrombin time and/or high international normalised ratio). Early treatment with nitisinone (NTBC) plus dietary restriction of tyrosine and phenylalanine prevents the complications of severe liver disease and neurological crises. METHODS AND RESULTS: Six newborns referred for hypersuccinylacetonaemia but who had normal coagulation testing on initial evaluation had sequence variants in the GSTZ1 gene, encoding maleylacetoacetate isomerase (MAAI), the enzyme preceding FAH in tyrosine degradation. Initial plasma SA levels ranged from 233 to 1282â nmol/L, greater than normal (<24â nmol/L) but less than the initial values of patients with HT1 (16â 944-74â 377â nmol/L, n=15). Four individuals were homozygous for c.449C>T (p.Ala150Val). One was compound heterozygous for c.259C>T (p.Arg87Ter) and an intronic sequence variant. In one, a single heterozygous GSTZ1 sequence variant was identified, c.295G>A (p.Val99Met). Bacterial expression of p.Ala150Val and p.Val99Met revealed low MAAI activity. The six individuals with mild hypersuccinylacetonaemia (MHSA) were not treated with diet or nitisinone. Their clinical course has been normal for up to 13â years. CONCLUSIONS: MHSA can be caused by sequence variants in GSTZ1. Such individuals have thus far remained asymptomatic despite receiving no specific treatment.
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Glutatión Transferasa/genética , Hidrolasas/genética , Hígado/enzimología , Tirosinemias/genética , Adolescente , Niño , Preescolar , Femenino , Variación Genética , Glutatión Transferasa/deficiencia , Heptanoatos/sangre , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Hidrolasas/sangre , Lactante , Recién Nacido , Hígado/patología , Masculino , Tirosina/sangre , Tirosinemias/sangre , Tirosinemias/patologíaRESUMEN
Methylmalonyl-coA epimerase (MCE) follows propionyl-coA carboxylase and precedes methylmalonyl-coA mutase in the pathway converting propionyl-coA to succinyl-coA. MCE deficiency has previously been described in six patients, one presenting with metabolic acidosis, the others with nonspecific neurological symptoms or asymptomatic. The clinical significance and biochemical characteristics of this rare condition have been incompletely defined. We now describe a patient who presented acutely at 5 years of age with vomiting, dehydration, confusion, severe metabolic acidosis and mild hyperammonemia. At presentation, organic acid profiles were dominated by increased ketones and 3-hydroxypropionate, with moderately elevated methylcitrate and propionylglycine, and acylcarnitine profiles showed marked C3 (propionylcarnitine) elevation with normal C4DC (methylmalonylcarnitine + succinylcarnitine). Propionic acidemia was initially suspected, but it was subsequently noted that methylmalonic acid was mildly but persistently elevated in urine, and clearly elevated in plasma and cerebrospinal fluid. The overall biochemical profile prompted consideration of MCE deficiency. Studies on cultured fibroblasts showed moderately decreased propionate incorporation. Complementation analysis permitted assignment to the MCEE group. A heterozygous p.Arg47Ter (p.R47*) mutation in the MCEE gene was identified by sequencing of exons, and RNA studies identified a novel intronic splicing mutation, c.379-644A > G, confirming the diagnosis of MCE deficiency. Following the initial severe presentation, development has been normal and the clinical course over the subsequent six years has remained relatively uneventful on an essentially normal diet. This report contributes to the clinical and biochemical characterisation of this rare disorder, while highlighting potential causes of under-diagnosis or of diagnostic confusion.
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BACKGROUND: Late-onset Pompe disease (LOPD) is a rare treatable lysosomal storage disorder characterized by progressive lysosomal glycogen accumulation and muscle weakness, with often a limb-girdle pattern. Despite published guidelines, testing for LOPD is often overlooked or delayed in adults, owing to its low frequency compared to other muscle disorders with similar muscle patterns. Next-generation sequencing has the capability to test concurrently for several muscle disorders. This could potentially lead to increased diagnosis of LOPD, disorders with non-specific muscle weakness or atypical patients. METHODS: We developed a gene panel to further study its clinical utility in a cohort of patients with suspected muscle disorders. We designed a gene panel to analyze the coding sequences and splice site junctions of GAA causing LOPD, along with 77 other genes causing muscle disorders with overlapping phenotypes. RESULTS: At a median coverage of ~200X (sequences per base), all GAA exons were successfully covered with >20X and only 0.3 % of exons across all genes were <20X. The panel showed an excellent sensitivity (100 %) and specificity (98 %) across all selected genes, using known variations in Pompe patients and controls. We determined its clinical utility by analyzing 34 patients with suspected muscle disorders of undetermined etiology and various muscle patterns, who were referred or followed in neuromuscular and genetics clinics. A putative diagnosis was found in up to 32 % of patients. The gene panel was instrumental in reaching a diagnosis in atypical patients, including one LOPD case. Acid alpha-glucosidase activity was used to confirm the molecular results in all patients. CONCLUSION: This work highlights the high clinical utility of gene panels in patients with suspected muscle disorders and its potential to facilitate the diagnosis of patients showing non-specific muscle weakness or atypical phenotypes. We propose that gene panels should be used as a first-tier test in patients with suspected muscle disorders of undetermined etiology, which could further increase overall diagnosis of muscle conditions, and potentially reduce diagnostic delay. Further studies are necessary to determine the impact of first-tier gene panels on diagnostic delay and on treatment outcome for LOPD.
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Enfermedad del Almacenamiento de Glucógeno Tipo II/diagnóstico , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Enfermedades Musculares/diagnóstico , Adolescente , Adulto , Edad de Inicio , Niño , Preescolar , Femenino , Humanos , Lactante , Masculino , Adulto JovenRESUMEN
A decline in mitochondrial respiration represents the root cause of a large number of inborn errors of metabolism. It is also associated with common age-associated diseases and the aging process. To gain insight into the systemic, biochemical consequences of respiratory chain dysfunction, we performed a case-control, prospective metabolic profiling study in a genetically homogenous cohort of patients with Leigh syndrome French Canadian variant, a mitochondrial respiratory chain disease due to loss-of-function mutations in LRPPRC. We discovered 45 plasma and urinary analytes discriminating patients from controls, including classic markers of mitochondrial metabolic dysfunction (lactate and acylcarnitines), as well as unexpected markers of cardiometabolic risk (insulin and adiponectin), amino acid catabolism linked to NADH status (α-hydroxybutyrate), and NAD(+) biosynthesis (kynurenine and 3-hydroxyanthranilic acid). Our study identifies systemic, metabolic pathway derangements that can lie downstream of primary mitochondrial lesions, with implications for understanding how the organelle contributes to rare and common diseases.
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Enfermedad de Leigh/metabolismo , Metaboloma , Mitocondrias/metabolismo , Adiponectina/sangre , Adolescente , Adulto , Aminas/metabolismo , Biomarcadores/sangre , Biomarcadores/orina , Estudios de Casos y Controles , Niño , Femenino , Humanos , Insulina/sangre , Enfermedad de Leigh/sangre , Enfermedad de Leigh/genética , Enfermedad de Leigh/orina , Metabolismo de los Lípidos , Masculino , NAD/metabolismo , Proteínas de Neoplasias/genéticaRESUMEN
BACKGROUND: The pathophysiology of hypertension in patients with mitochondrial diseases is different from that of the general population. Growing evidence exists linking mtDNA, its mutations, and mitochondrial dysfunction to the pathogenesis of hypertension. No reports on the prevalence of hypertension in late-onset mtDNA diseases have been described. METHODS: We performed a retrospective chart review of adult patients with late-onset mtDNA diseases between January 1999 and January 2012 at our center. We grouped them into age categories to allow comparison with previously reported Canadian Health Measures Survey (CHMS) prevalence data. RESULTS: Twenty-three subjects with hypertension were identified for a crude prevalence of 39.7 % (95 % CI 27-53 %) as compared to the CHMS age-predicted prevalence of 30.5 %. When analyzed by individual age group, there were no significant differences between the observed and the CHMS predicted prevalence rates in the 40 years and older cohorts (age category 40-59, p = 0.63; age category 60-79, p = 0.85). However, hypertension rates were significantly higher than predicted in the under 40 years cohort (55.6 vs. 2.8 %, p < 0.001, CI 21-86 %), in which hypertensive patients with the MELAS m.3243A>G mutation were significantly clustered (p < 0.01). This younger MELAS cohort (n = 4, mean age = 24 years) with hypertension had heteroplasmy levels (mean = 68 %) that were significantly higher than the levels found in the older non-hypertensive MELAS cohort (n = 8, mean age = 52 years, mean = 33 %) (p = 0.04). CONCLUSION: Relative to age, gender, and mtDNA disease subtype, young adults with high heteroplasmy levels of the MELAS m.3243A>G mutation demonstrate an increased prevalence of hypertension. Further prospective data are needed to confirm this initial finding, which has potentially important treatment implications.
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AIMS: We wished to demonstrate the feasibility of performing diagnostic mitochondrial DNA (mtDNA) analysis on biopsies of the orbicularis oculi muscle in patients with a chronic progressive external ophthalmoplegia (CPEO) phenotype and suspicion of an underlying mitochondrial disorder. METHOD: Case series of three patients who underwent ptosis surgery and had simultaneous biopsy of the orbicularis oculi muscle because of a suspicion of a mitochondrial disorder. Orbicularis muscle samples were divided into two pieces at the time of biopsy. The first was snap-frozen in liquid nitrogen, DNA was extracted and mtDNA deletion analysis was performed by two complementary methods (long PCR and Southern blot analysis). The second piece of muscle was assessed using routine histopathology, electron microscopy and immuno-histochemical analysis. RESULTS: Three patients with clinical features of CPEO, without any positive family history, underwent orbicularis muscle biopsies at time of eyelid ptosis surgery. All biopsies were adequate to conduct histopathological and immuno-histochemical analysis, which showed evidence of abnormal muscle structure and function. mtDNA was successfully extracted from all biopsies, and long PCR and Southern blot analysis confirmed diagnostic large single mtDNA deletions in all three cases. CONCLUSIONS: Orbicularis oculi muscle biopsies are useful in patients with CPEO to perform mtDNA analysis, thus avoiding a separate biopsy of skeletal muscle elsewhere.
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Biopsia/métodos , ADN Mitocondrial/genética , Oftalmoplejía Externa Progresiva Crónica/genética , Oftalmoplejía Externa Progresiva Crónica/patología , Blefaroptosis/genética , Blefaroptosis/patología , Blefaroptosis/cirugía , Femenino , Asesoramiento Genético , Humanos , Persona de Mediana Edad , Oftalmoplejía Externa Progresiva Crónica/cirugía , Estudios RetrospectivosRESUMEN
Pyridoxine dependent epilepsy is an autosomal recessive disorder characterized by early onset seizures responsive to pyridoxine and caused by a defect in the α-aminoadipic semialdehyde dehydrogenase (antiquitin) gene (ALDH7A1). In order to characterize the effects of a series of twelve disease-associated ALDH7A1 missense mutations on antiquitin activity, we generated the mutations in a recombinant human antiquitin cDNA and expressed them in Escherichia coli. We developed an automated spectrophotometric assay of antiquitin enzymatic activity using the natural substrate α-aminoadipic semialdehyde. The substrate was generated using a recombinant lysine aminotransferase gene (lat) from Streptomyces clavuligerus. In the E. coli expression system all the mutants were stably expressed but lacked enzymatic activity. This is consistent with pathogenicity of these mutations in vivo.