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1.
Nutrients ; 13(9)2021 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-34578803

RESUMO

Long chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD/MTPD) and medium chain acyl-CoA dehydrogenase deficiency (MCADD) were included in the expanded neonatal screening program (ENBS) in Czechia in 2009, allowing for the presymptomatic diagnosis and nutritional management of these patients. The aim of our study was to assess the nationwide impact of ENBS on clinical outcome. This retrospective study analysed acute events and chronic complications and their severity in pre-ENBS and post-ENBS cohorts. In total, 28 children (12 before, 16 after ENBS) were diagnosed with LCHADD/MTPD (incidence 0.8/100,000 before and 1.2/100,000 after ENBS). In the subgroup detected by ENBS, a significantly longer interval from birth to first acute encephalopathy was observed. In addition, improvement in neuropathy and cardiomyopathy (although statistically non-significant) was demonstrated in the post-ENBS subgroup. In the MCADD cohort, we included 69 patients (15 before, 54 after ENBS). The estimated incidence rose from 0.7/100,000 before to 4.3/100,000 after ENBS. We confirmed a significant decrease in the number of episodes of acute encephalopathy and lower proportion of intellectual disability after ENBS (p < 0.0001). The genotype-phenotype correlations suggest a new association between homozygosity for the c.1528C > G variant and more severe heart involvement in LCHADD patients.

2.
Am J Hum Genet ; 108(7): 1283-1300, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34214447

RESUMO

Most rare clinical missense variants cannot currently be classified as pathogenic or benign. Deficiency in human 5,10-methylenetetrahydrofolate reductase (MTHFR), the most common inherited disorder of folate metabolism, is caused primarily by rare missense variants. Further complicating variant interpretation, variant impacts often depend on environment. An important example of this phenomenon is the MTHFR variant p.Ala222Val (c.665C>T), which is carried by half of all humans and has a phenotypic impact that depends on dietary folate. Here we describe the results of 98,336 variant functional-impact assays, covering nearly all possible MTHFR amino acid substitutions in four folinate environments, each in the presence and absence of p.Ala222Val. The resulting atlas of MTHFR variant effects reveals many complex dependencies on both folinate and p.Ala222Val. MTHFR atlas scores can distinguish pathogenic from benign variants and, among individuals with severe MTHFR deficiency, correlate with age of disease onset. Providing a powerful tool for understanding structure-function relationships, the atlas suggests a role for a disordered loop in retaining cofactor at the active site and identifies variants that enable escape of inhibition by S-adenosylmethionine. Thus, a model based on eight MTHFR variant effect maps illustrates how shifting landscapes of environment- and genetic-background-dependent missense variation can inform our clinical, structural, and functional understanding of MTHFR deficiency.


Assuntos
Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Mutação de Sentido Incorreto , Substituição de Aminoácidos , Análise Mutacional de DNA , Diploide , Biblioteca Gênica , Genótipo , Humanos , Metilenotetra-Hidrofolato Redutase (NADPH2)/deficiência , Metilenotetra-Hidrofolato Redutase (NADPH2)/fisiologia , Saccharomyces cerevisiae/genética
3.
Biochim Biophys Acta Mol Basis Dis ; 1867(10): 166201, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34147638

RESUMO

Combined methylmalonic aciduria with homocystinuria (cblC type) is a rare disease caused by mutations in the MMACHC gene. MMACHC encodes an enzyme crucial for intracellular vitamin B12 metabolism, leading to the accumulation of toxic metabolites e.g. methylmalonic acid (MMA) and homocysteine (Hcy), and secondary disturbances in folate and one-carbon metabolism when not fully functional. Patients with cblC deficiency often present in the neonatal or early childhood period with a severe multisystem pathology, which comprises a broad spectrum of treatment-resistant ophthalmological phenotypes, including retinal degeneration, impaired vision, and vascular changes. To examine the potential function of MMACHC in the retina and how its loss may impact disease, we performed gene expression studies in human and mouse, which showed that local expression of MMACHC in the retina and retinal pigment epithelium is relatively stable over time. To study whether functional MMACHC is required for retinal function and tissue integrity, we generated a transgenic mouse lacking Mmachc expression in cells of the peripheral retina. Characterization of this mouse revealed accumulation of cblC disease related metabolites, including MMA and the folate-dependent purine synthesis intermediates AICA-riboside and SAICA-riboside in the retina. Nevertheless, fundus appearance, morphology, vasculature, and cellular composition of the retina, as well as ocular function, remained normal in mice up to 6 or 12 months of age. Our data indicates that peripheral retinal neurons do not require intrinsic expression of Mmachc for survival and function and questions whether a local MMACHC deficiency is responsible for the retinal phenotypes in patients.

4.
J Transl Med ; 19(1): 153, 2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33858441

RESUMO

BACKGROUND: Dietary sulfur amino acid (SAA) restriction is an established animal model for increasing lifespan and improving metabolic health. Data from human studies are limited. In the study outlined in this protocol, we will evaluate if dietary SAA restriction can reduce body weight and improve resting energy expenditure (REE) and parameters related to metabolic health. METHOD/DESIGN: Men and women (calculated sample size = 60), aged 18-45 years, with body mass index of 27-35 kg/m2 will be included in a double-blind 8-week dietary intervention study. The participants will be randomized in a 1:1 manner to a diet with either low or high SAA. Both groups will receive an equal base diet consisting of low-SAA plant-based whole foods and an amino acid supplement free of SAA. Contrasting SAA contents will be achieved using capsules with or without methionine and cysteine (SAAhigh, total diet SAA ~ 50-60 mg/kg body weight/day; SAAlow, total diet SAA ~ 15-25 mg/kg body weight/day). The primary outcome is body weight change. Data and material collection will also include body composition (dual X-ray absorptiometry), resting energy expenditure (whole-room indirect calorimetry) and samples of blood, urine, feces and adipose tissue at baseline, at 4 weeks and at study completion. Measures will be taken to promote and monitor diet adherence. Data will be analyzed using linear mixed model regression to account for the repeated measures design and within-subject correlation. DISCUSSION: The strength of this study is the randomized double-blind design. A limitation is the restrictive nature of the diet which may lead to poor compliance. If this study reveals a beneficial effect of the SAAlow diet on body composition and metabolic health, it opens up for new strategies for prevention and treatment of overweight, obesity and its associated disorders. Trial registration ClinicalTrials.gov: NCT04701346, Registration date: January 8th, 2021.


Assuntos
Aminoácidos Sulfúricos , Obesidade , Adolescente , Adulto , Aminoácidos , Composição Corporal , Metabolismo Energético , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Ensaios Clínicos Controlados Aleatórios como Assunto , Adulto Jovem
5.
Int J Neonatal Screen ; 7(1)2021 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-33808002

RESUMO

Neonatal screening (NBS) was initiated in Europe during the 1960s with the screening for phenylketonuria. The panel of screened disorders ("conditions") then gradually expanded, with a boost in the late 1990s with the introduction of tandem mass spectrometry (MS/MS), making it possible to screen for 40-50 conditions using a single blood spot. The most recent additions to screening programmes (screening for cystic fibrosis, severe combined immunodeficiency and spinal muscular atrophy) were assisted by or realised through the introduction of molecular technologies. For this survey, we collected data from 51 European countries. We report the developments between 2010 and 2020 and highlight the achievements reached with the progress made in this period. We also identify areas where further progress can be made, mainly by exchanging knowledge and learning from experiences in neighbouring countries. Between 2010 and 2020, most NBS programmes in geographical Europe matured considerably, both in terms of methodology (modernised) and with regard to the panel of conditions screened (expanded). These developments indicate that more collaboration in Europe through European organisations is gaining momentum. We can only accomplish the timely detection of newborn infants potentially suffering from one of the many rare diseases and take appropriate action by working together.

6.
Orphanet J Rare Dis ; 16(1): 95, 2021 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-33602304

RESUMO

BACKGROUND: Following the broad application of new analytical methods, more and more pathophysiological processes in previously unknown diseases have been elucidated. The spectrum of clinical presentation of rare inherited metabolic diseases (IMDs) is broad and ranges from single organ involvement to multisystemic diseases. With the aim of overcoming the limited knowledge about the natural course, current diagnostic and therapeutic approaches, the project has established the first unified patient registry for IMDs that fully meets the requirements of the European Infrastructure for Rare Diseases (ERDRI). RESULTS: In collaboration with the European Reference Network for Rare Hereditary Metabolic Disorders (MetabERN), the Unified European registry for Inherited Metabolic Diseases (U-IMD) was established to collect patient data as an observational, non-interventional natural history study. Following the recommendations of the ERDRI the U-IMD registry uses common data elements to define the IMDs, report the clinical phenotype, describe the biochemical markers and to capture the drug treatment. Until today, more than 1100 IMD patients have been registered. CONCLUSION: The U-IMD registry is the first observational, non-interventional patient registry that encompasses all known IMDs. Full semantic interoperability for other registries has been achieved, as demonstrated by the use of a minimum common core data set for equivalent description of metabolic patients in U-IMD and in the patient registry of the European Rare Kidney Disease Reference Network (ERKNet). In conclusion, the U-IMD registry will contribute to a better understanding of the long-term course of IMDs and improved patients care by understanding the natural disease course and by enabling an optimization of diagnostic and therapeutic strategies.


Assuntos
Doenças Metabólicas , Humanos , Doenças Metabólicas/genética , Doenças Raras/genética , Sistema de Registros
7.
J Inherit Metab Dis ; 44(3): 777-786, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33089527

RESUMO

5,10-Methylenetetrahydrofolate reductase (MTHFR) deficiency usually presents as a severe neonatal disease. This study aimed to characterize natural history, biological and molecular data, and response to treatment of patients with late-onset MTHFR deficiency. The patients were identified through the European Network and Registry for Homocystinuria and Methylation Defects and the Adult group of the French Society for Inherited Metabolic Diseases; data were retrospectively colleted. To identify juvenile to adult-onset forms of the disease, we included patients with a diagnosis established after the age of 10 years. We included 14 patients (median age at diagnosis: 32 years; range: 11-54). At onset (median age: 20 years; range 9-38), they presented with walking difficulties (n = 8), cognitive decline (n = 3) and/or seizures (n = 3), sometimes associated with mild mental retardation (n = 6). During the disease course, symptoms were almost exclusively neurological with cognitive dysfunction (93%), gait disorders (86%), epilepsy (71%), psychiatric symptoms (57%), polyneuropathy (43%), and visual deficit (43%). Mean diagnostic delay was 14 years. Vascular events were observed in 28% and obesity in 36% of the patients. One patient remained asymptomatic at the age of 55 years. Upon treatment, median total homocysteine decreased (from 183 µmol/L, range 69-266, to 90 µmol/L, range 20-142) and symptoms improved (n = 9) or stabilized (n = 4). Missense pathogenic variants in the C-terminal regulatory domain of the protein were over-represented compared to early-onset cases. Residual MTHFR enzymatic activity in skin fibroblasts (n = 4) was rather high (17%-58%). This series of patients with late-onset MTHFR deficiency underlines the still unmet need of a prompt diagnosis of this treatable disease.

8.
Eur J Hum Genet ; 29(1): 67-78, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33040093

RESUMO

Newborn screening (NBS) is an important part of public healthcare systems in many countries. The provision of information to parents about NBS is now recognised as an integral part of the screening process. Informing parents on all aspects of screening helps to achieve the benefits, promote trust and foster support for NBS. Therefore, policies and guidelines should exist to govern how the information about NBS is provided to parents, taking into account evidence-based best practices. The purpose of our survey was to explore whether any legally binding provisions, guidelines or recommendations existed pertaining to the provision of information about NBS to parents across Europe. Questions were designed to determine the regulatory process of when, by whom and how parents should be informed about screening. Twenty-seven countries participated in the survey. The results indicated that most countries had some sort of legal framework or guidelines for the provision of information to parents. However, only 37% indicated that the provision of information was required prenatally. The majority of countries were verbally informing parents with the aid of written materials postnatally, just prior to sample collection. Information was provided by a neonatologist, midwife or nurse. A website dedicated to NBS was available for 67% of countries and 89% had written materials about NBS for parents. The survey showed that there is a lack of harmonisation among European countries in the provision of information about NBS and emphasised the need for more comprehensive guidelines at the European level.

9.
Curr Opin Clin Nutr Metab Care ; 24(1): 62-70, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33060459

RESUMO

PURPOSE OF REVIEW: Metabolism of sulfur amino acids (SAA) provides compounds important for many cellular functions. Inherited disorders of SAA metabolism are typically severe multisystemic diseases affecting brain, liver, connective tissue, or vasculature. The review summarizes the present therapeutic approaches and advances in identifying novel treatment targets, and provides an overview of new therapies. RECENT FINDINGS: Current treatments of genetic disorders of SAA metabolism are primarily based on modulation of affected pathways by dietary measures and provision of lacking products or scavenging of toxic molecules. Recent studies identified additional therapeutic targets distant from the primary defects and explored ideas envisioning novel treatments, such as chaperone and gene therapy. Recombinant protein production and engineering resulted in development and clinical testing of enzyme therapies for cystathionine ß-synthase deficiency, the most common inborn error of SAA metabolism. SUMMARY: Complex regulation of pathways involved in SAA metabolism and cellular consequences of genetic defects in SAA metabolism are only partially understood. There is a pressing need to increase substantially our knowledge of the disease mechanisms to develop more effective therapies for patients suffering from these rare disorders.

10.
J Inherit Metab Dis ; 44(3): 677-692, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33295057

RESUMO

Cystathionine ß-synthase (CBS) deficiency has a wide clinical spectrum, ranging from neurodevelopmental problems, lens dislocation and marfanoid features in early childhood to adult onset disease with predominantly thromboembolic complications. We have analysed clinical and laboratory data at the time of diagnosis in 328 patients with CBS deficiency from the E-HOD (European network and registry for Homocystinurias and methylation Defects) registry. We developed comprehensive criteria to classify patients into four groups of pyridoxine responsivity: non-responders (NR), partial, full and extreme responders (PR, FR and ER, respectively). All groups showed overlapping concentrations of plasma total homocysteine while pyridoxine responsiveness inversely correlated with plasma/serum methionine concentrations. The FR and ER groups had a later age of onset and diagnosis and a longer diagnostic delay than NR and PR patients. Lens dislocation was common in all groups except ER but the age of dislocation increased with increasing responsiveness. Developmental delay was commonest in the NR group while no ER patient had cognitive impairment. Thromboembolism was the commonest presenting feature in ER patients, whereas it was least likely at presentation in the NR group. This probably is due to the differences in ages at presentation: all groups had a similar number of thromboembolic events per 1000 patient-years. Clinical severity of CBS deficiency depends on the degree of pyridoxine responsiveness. Therefore, a standardised pyridoxine-responsiveness test in newly diagnosed patients and a critical review of previous assessments is indispensable to ensure adequate therapy and to prevent or reduce long-term complications.

11.
J Nutr ; 150(Suppl 1): 2506S-2517S, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-33000152

RESUMO

The metabolism of sulfur-containing amino acids (SAAs) requires an orchestrated interplay among several dozen enzymes and transporters, and an adequate dietary intake of methionine (Met), cysteine (Cys), and B vitamins. Known human genetic disorders are due to defects in Met demethylation, homocysteine (Hcy) remethylation, or cobalamin and folate metabolism, in Hcy transsulfuration, and Cys and hydrogen sulfide (H2S) catabolism. These disorders may manifest between the newborn period and late adulthood by a combination of neuropsychiatric abnormalities, thromboembolism, megaloblastic anemia, hepatopathy, myopathy, and bone and connective tissue abnormalities. Biochemical features include metabolite deficiencies (e.g. Met, S-adenosylmethionine (AdoMet), intermediates in 1-carbon metabolism, Cys, or glutathione) and/or their accumulation (e.g. S-adenosylhomocysteine, Hcy, H2S, or sulfite). Treatment should be started as early as possible and may include a low-protein/low-Met diet with Cys-enriched amino acid supplements, pharmacological doses of B vitamins, betaine to stimulate Hcy remethylation, the provision of N-acetylcysteine or AdoMet, or experimental approaches such as liver transplantation or enzyme replacement therapy. In several disorders, patients are exposed to long-term markedly elevated Met concentrations. Although these conditions may inform on Met toxicity, interpretation is difficult due to the presence of additional metabolic changes. Two disorders seem to exhibit Met-associated toxicity in the brain. An increased risk of demyelination in patients with Met adenosyltransferase I/III (MATI/III) deficiency due to biallelic mutations in the MATIA gene has been attributed to very high blood Met concentrations (typically >800 µmol/L) and possibly also to decreased liver AdoMet synthesis. An excessively high Met concentration in some patients with cystathionine ß-synthase deficiency has been associated with encephalopathy and brain edema, and direct toxicity of Met has been postulated. In summary, studies in patients with various disorders of SAA metabolism showed complex metabolic changes with distant cellular consequences, most of which are not attributable to direct Met toxicity.


Assuntos
Aminoácidos Sulfúricos/metabolismo , Cisteína/metabolismo , Homocisteína/metabolismo , Doenças Metabólicas/genética , Metionina/metabolismo , Compostos de Enxofre/metabolismo , Enxofre/metabolismo , Animais , Encefalopatias/etiologia , Encefalopatias/metabolismo , Glutationa/metabolismo , Homocistinúria/etiologia , Homocistinúria/metabolismo , Humanos , Sulfeto de Hidrogênio/metabolismo , Fígado/metabolismo , Doenças Metabólicas/metabolismo , Doenças Metabólicas/patologia , Doenças Metabólicas/terapia , Erros Inatos do Metabolismo/patologia , Erros Inatos do Metabolismo/terapia , Metionina Adenosiltransferase/metabolismo , Metilação , S-Adenosilmetionina/metabolismo , Sulfitos/metabolismo
12.
Hum Mutat ; 41(9): 1662-1670, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32623804

RESUMO

Classical homocystinuria (HCU) is an inborn error of metabolism caused by loss of cystathionine ß-synthase (CBS) activity with the concomitant buildup of homocysteine. In knockout (KO) mice, a mouse model of HCU, complete lack of CBS is neonatally lethal. Administration of OT-58, an enzyme therapy for HCU, during the first 5 weeks of life rescued KO mice survival by preventing liver disease. Here, we studied the impact of a long-term uninterrupted OT-58 treatment or its absence beyond the neonatal period on liver pathology and metabolism. Plasma and liver metabolites of KO mice on OT-58 treatment were substantially improved or normalized compared with those receiving vehicle. Increased plasma activities of alanine aminotransferase and aspartate aminotransferase of vehicle-injected KO mice suggested the progression of liver damage with age and lack of treatment. At 3 months of age, liver histology showed no signs of hepatopathy in both vehicle- and OT-58-treated KO mice. However, moderate to severe liver disease, characterized by steatosis, hepatocellular necroses, disorganized endoplasmic reticulum, and swollen mitochondria, developed in 6-month-old vehicle-injected KO mice. KO mice on OT-58 treatment remained asymptomatic and were indistinguishable from age-matched healthy controls. Long-term uninterrupted OT-58 treatment was essential to prevent severe liver disease in the KO mouse model of HCU.

14.
Genome Med ; 12(1): 13, 2020 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-32000841

RESUMO

BACKGROUND: For the majority of rare clinical missense variants, pathogenicity status cannot currently be classified. Classical homocystinuria, characterized by elevated homocysteine in plasma and urine, is caused by variants in the cystathionine beta-synthase (CBS) gene, most of which are rare. With early detection, existing therapies are highly effective. METHODS: Damaging CBS variants can be detected based on their failure to restore growth in yeast cells lacking the yeast ortholog CYS4. This assay has only been applied reactively, after first observing a variant in patients. Using saturation codon-mutagenesis, en masse growth selection, and sequencing, we generated a comprehensive, proactive map of CBS missense variant function. RESULTS: Our CBS variant effect map far exceeds the performance of computational predictors of disease variants. Map scores correlated strongly with both disease severity (Spearman's ϱ = 0.9) and human clinical response to vitamin B6 (ϱ = 0.93). CONCLUSIONS: We demonstrate that highly multiplexed cell-based assays can yield proactive maps of variant function and patient response to therapy, even for rare variants not previously seen in the clinic.


Assuntos
Cistationina beta-Sintase/genética , Teste de Complementação Genética/métodos , Testes Genéticos/métodos , Homocistinúria/genética , Mutação de Sentido Incorreto , Cistationina beta-Sintase/metabolismo , Genótipo , Humanos , Fenótipo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética
15.
Cent Eur J Public Health ; 27(2): 153-159, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31241292

RESUMO

OBJECTIVES: Presymptomatic detection of patients with rare diseases (RD), defined by a population frequency less than 1 : 2,000, is the task of newborn screening (NBS). In the Czech Republic (CZ), currently eighteen RD are screened: phenylketonuria/hyperphenylalaninemia (PKU/HPA), congenital hypothyroidism (CH), congenital adrenal hyperplasia (CAH), cystic fibrosis (CF), medium chain acyl-CoA dehydrogenase deficiency (MCADD), long chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD), very long chain acyl-CoA dehydrogenase deficiency (VLCADD), carnitine palmitoyl transferase I and II deficiency (CPTID, CPTIID), carnitine-acylcarnitine translocase deficiency (CACTD), maple syrup urine disease (MSUD), glutaric aciduria type I (GA I), isovaleryl-CoA dehydrogenase deficiency (IVA), argininemia (ARG), citrullinemia (CIT), biotinidase deficiency (BTD), cystathionine beta-synthase-deficient homocystinuria (CBSD HCU), and methylenetetrahydrofolate reductase deficiency homocystinuria (MTHFRD HCU). The aim was to analyze the prevalence of RD screened by NBS in CZ. METHODS: We examined the NBS programme in CZ from 1 January 2010 to 31 December 2017, which covered 888,891 neonates. Dried blood spots were primarily analyzed using fluorescence immuno-assay, tandem mass spectrometry and fluorimetry. RESULTS: The overall prevalence of RD among the neonate cohort was 1 : 1,043. Individually, 1 : 2,877 for CH, 1 : 5,521 for PKU/HPA, 1 : 6,536 for CF (1 : 5,887 including false negative patients), 1 : 12,520 for CAH, 1 : 22,222 for MCADD, 1 : 80,808 for LCHADD, 1 : 177,778 for GA I, 1 : 177,778 for IVA, 1 : 222,223 for VLCADD, 1 : 296,297 for MSUD, 1 : 8,638 for BTD, and 1 : 181,396 for CBSD HCU. CONCLUSIONS: The observed prevalence of RD, based on NBS, corresponds to that expected, more precisely it was higher for BTD and lower for MSUD, IVA, CBSD HCU, MCADD and VLCADD. Early detection of rare diseases by means of NBS is an effective secondary prevention tool.


Assuntos
Triagem Neonatal/métodos , Doenças Raras/epidemiologia , Biomarcadores/sangue , República Tcheca/epidemiologia , Fluorometria , Humanos , Recém-Nascido , Doenças Raras/sangue , Espectrometria de Massas em Tandem
16.
Free Radic Biol Med ; 136: 146-158, 2019 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-30970274

RESUMO

A rapidly increasing number of studies report on widespread biological functions for endogenous hydrogen sulfide. However, the use of multiple, chemically distinct analytical methods to measure free hydrogen sulfide levels in biological samples accumulate data that are not in agreement with each other. In this work a widely appreciated technique, the monobromobimane method, was thoroughly investigated with the overall aims i) to demonstrate how results obtained by different versions of the method should be interpreted and ii) to provide an easy protocol for the community in order to obtain reliable and comparable results. We demonstrate that none of the previously published versions of the method measure free sulfide concentrations in blood serum or plasma samples due to significant interferences with the biomolecule-bound sulfide pool. On the other hand, we stress the biological relevance of these measurements in cases in which they are carefully conducted. To aid future studies, we extensively investigated the entire procedure from sample withdrawal through handling and storing of injection-ready samples until the detection protocol in order to pinpoint all parameters that can affect the final readouts. Based on our rigorous analytical investigations a set of recommendations were compiled that are necessary to ensure reliable, reproducible and comparable results in the field and a detailed standardized protocol is provided.


Assuntos
Análise Química do Sangue/métodos , Análise Química do Sangue/normas , Compostos Bicíclicos com Pontes , Sulfeto de Hidrogênio/sangue , Humanos , Manejo de Espécimes/métodos
17.
J Inherit Metab Dis ; 42(2): 333-352, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30773687

RESUMO

AIM: To explore the clinical presentation, course, treatment and impact of early treatment in patients with remethylation disorders from the European Network and Registry for Homocystinurias and Methylation Defects (E-HOD) international web-based registry. RESULTS: This review comprises 238 patients (cobalamin C defect n = 161; methylenetetrahydrofolate reductase deficiency n = 50; cobalamin G defect n = 11; cobalamin E defect n = 10; cobalamin D defect n = 5; and cobalamin J defect n = 1) from 47 centres for whom the E-HOD registry includes, as a minimum, data on medical history and enrolment visit. The duration of observation was 127 patient years. In 181 clinically diagnosed patients, the median age at presentation was 30 days (range 1 day to 42 years) and the median age at diagnosis was 3.7 months (range 3 days to 56 years). Seventy-five percent of pre-clinically diagnosed patients with cobalamin C disease became symptomatic within the first 15 days of life. Total homocysteine (tHcy), amino acids and urinary methylmalonic acid (MMA) were the most frequently assessed disease markers; confirmatory diagnostics were mainly molecular genetic studies. Remethylation disorders are multisystem diseases dominated by neurological and eye disease and failure to thrive. In this cohort, mortality, thromboembolic, psychiatric and renal disease were rarer than reported elsewhere. Early treatment correlates with lower overall morbidity but is less effective in preventing eye disease and cognitive impairment. The wide variation in treatment hampers the evaluation of particular therapeutic modalities. CONCLUSION: Treatment improves the clinical course of remethylation disorders and reduces morbidity, especially if started early, but neurocognitive and eye symptoms are less responsive. Current treatment is highly variable. This study has the inevitable limitations of a retrospective, registry-based design.


Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/diagnóstico , Erros Inatos do Metabolismo dos Aminoácidos/terapia , Homocistinúria/metabolismo , Metilenotetra-Hidrofolato Redutase (NADPH2)/deficiência , Espasticidade Muscular/metabolismo , Vitamina B 12/metabolismo , Adolescente , Adulto , Idade de Início , Criança , Pré-Escolar , Estudos Transversais , Progressão da Doença , Europa (Continente) , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Metilação , Metilenotetra-Hidrofolato Redutase (NADPH2)/metabolismo , Ácido Metilmalônico/urina , Fenótipo , Gravidez , Transtornos Psicóticos/metabolismo , Sistema de Registros , Estudos Retrospectivos , Adulto Jovem
18.
J Inherit Metab Dis ; 42(6): 1064-1076, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-30714172

RESUMO

The most common ureagenesis defect is X-linked ornithine transcarbamylase (OTC) deficiency which is a main target for novel therapeutic interventions. The spf ash mouse model carries a variant (c.386G>A, p.Arg129His) that is also found in patients. Male spf ash mice have a mild biochemical phenotype with low OTC activity (5%-10% of wild-type), resulting in elevated urinary orotic acid but no hyperammonemia. We recently established a dried blood spot method for in vivo quantification of ureagenesis by Gas chromatography-mass spectrometry (GC-MS) using stable isotopes. Here, we applied this assay to wild-type and spf ash mice to assess ureagenesis at different ages. Unexpectedly, we found an age-dependency with a higher capacity for ammonia detoxification in young mice after weaning. A parallel pattern was observed for carbamoylphosphate synthetase 1 and OTC enzyme expression and activities, which may act as pacemaker of this ammonia detoxification pathway. Moreover, high ureagenesis in younger mice was accompanied by elevated periportal expression of hepatic glutamine synthetase, another main enzyme required for ammonia detoxification. These observations led us to perform a more extensive analysis of the spf ash mouse in comparison to the wild-type, including characterization of the corresponding metabolites, enzyme activities in the liver and plasma and the gut microbiota. In conclusion, the comprehensive enzymatic and metabolic analysis of ureagenesis performed in the presented depth was only possible in animals. Our findings suggest such analyses being essential when using the mouse as a model and revealed age-dependent activity of ammonia detoxification.


Assuntos
Envelhecimento/fisiologia , Amônia/metabolismo , Doença da Deficiência de Ornitina Carbomoiltransferase/metabolismo , Doença da Deficiência de Ornitina Carbomoiltransferase/patologia , Ornitina Carbamoiltransferase/genética , Ureia/metabolismo , Fatores Etários , Animais , Modelos Animais de Doenças , Humanos , Hiperamonemia/genética , Hiperamonemia/metabolismo , Hiperamonemia/patologia , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Transgênicos , Doença da Deficiência de Ornitina Carbomoiltransferase/genética
19.
J Inherit Metab Dis ; 42(1): 128-139, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30740731

RESUMO

PURPOSE: To assess how the current practice of newborn screening (NBS) for homocystinurias compares with published recommendations. METHODS: Twenty-two of 32 NBS programmes from 18 countries screened for at least one form of homocystinuria. Centres provided pseudonymised NBS data from patients with cystathionine beta-synthase deficiency (CBSD, n = 19), methionine adenosyltransferase I/III deficiency (MATI/IIID, n = 28), combined remethylation disorder (cRMD, n = 56) and isolated remethylation disorder (iRMD), including methylenetetrahydrofolate reductase deficiency (MTHFRD) (n = 8). Markers and decision limits were converted to multiples of the median (MoM) to allow comparison between centres. RESULTS: NBS programmes, algorithms and decision limits varied considerably. Only nine centres used the recommended second-tier marker total homocysteine (tHcy). The median decision limits of all centres were ≥ 2.35 for high and ≤ 0.44 MoM for low methionine, ≥ 1.95 for high and ≤ 0.47 MoM for low methionine/phenylalanine, ≥ 2.54 for high propionylcarnitine and ≥ 2.78 MoM for propionylcarnitine/acetylcarnitine. These decision limits alone had a 100%, 100%, 86% and 84% sensitivity for the detection of CBSD, MATI/IIID, iRMD and cRMD, respectively, but failed to detect six individuals with cRMD. To enhance sensitivity and decrease second-tier testing costs, we further adapted these decision limits using the data of 15 000 healthy newborns. CONCLUSIONS: Due to the favorable outcome of early treated patients, NBS for homocystinurias is recommended. To improve NBS, decision limits should be revised considering the population median. Relevant markers should be combined; use of the postanalytical tools offered by the CLIR project (Collaborative Laboratory Integrated Reports, which considers, for example, birth weight and gestational age) is recommended. tHcy and methylmalonic acid should be implemented as second-tier markers.


Assuntos
Homocistinúria/diagnóstico , Acetilcarnitina/metabolismo , Erros Inatos do Metabolismo dos Aminoácidos/diagnóstico , Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Carnitina/análogos & derivados , Carnitina/metabolismo , Feminino , Glicina N-Metiltransferase/deficiência , Glicina N-Metiltransferase/metabolismo , Homocisteína/metabolismo , Homocistinúria/metabolismo , Humanos , Recém-Nascido , Masculino , Metionina/metabolismo , Metilenotetra-Hidrofolato Redutase (NADPH2)/deficiência , Metilenotetra-Hidrofolato Redutase (NADPH2)/metabolismo , Ácido Metilmalônico/metabolismo , Espasticidade Muscular/diagnóstico , Espasticidade Muscular/metabolismo , Triagem Neonatal/métodos , Fenilalanina/metabolismo , Transtornos Psicóticos/diagnóstico , Transtornos Psicóticos/metabolismo
20.
Int J Neonatal Screen ; 5(3): 35, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-33072994

RESUMO

Appropriate and timely education about newborn screening (NBS) helps to foster benefits such as prompt follow up, to promote parents' autonomy via informed consent and minimize the harms such as reducing the impact of NBS false-positive results. The aim of this study was to ascertain how mothers are informed about NBS in the Czech Republic and to identify the variables associated with awareness about NBS. The questionnaires evaluating awareness and its determinants were mailed to a random sample of 3000 mothers 3 months post-delivery. The overall response rate was 42%. We analysed 1100 questionnaires and observed that better awareness about NBS was significantly associated with age, parity, number of information sources, child health status, size of maternity hospital and an obstetrician as the source of prenatally obtained information. Although the majority of mothers (77%) in our study recalled being informed by a physician or nurse in the neonatal ward, results have revealed that over 40% of participants did not have sufficient awareness about the principal aspects of NBS. Several measures including seminars for healthcare providers and the development and distribution of new educational materials were adopted to improve parental education about NBS in the Czech Republic.

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