Aciduria Research Articles

Glutaric aciduria type 1: Genetic and phenotypic spectrum in 53 patients

IntroductionGlutaric aciduria type 1 (GA1) is a rare and inherited autosomal-recessive metabolic disorder that occurs in the deficiency of glutaryl-co-enzyme A dehydrogenase (GCDH) enzyme encoded by GCDH gene. In this study, we aim to retrospectively investigate the clinical, biochemical, and neuroradiological parameters and examine the spectrum of GCDH gene variants in Turkish patients with glutaric aciduria type 1. MethodsThis is a descriptive cross-sectional study. The study was conducted in fifty-three patients from 39 unrelated Turkish families who were diagnosed with GA1 based on their clinical presentation, neuroimaging, and biochemical measurements, at the department of pediatric metabolism of a university hospital between June 1998 and August 2019. Pathogenic variants screening of GCDH gene was performed by direct DNA sequence analysis in forty-six patients with GA1. Pathogenicity of the novel variants was predicted via computational programs. ResultsA total of 53 patients were diagnosed with GA1. Of those, 32 (60.3%) had encephalopathic crisis and 33 (62.3%) had macrocephaly.Twenty different pathogenic variants were detected, 7 of which are novel (p.Glu57Lys, p.Ser145Profs*79, p.Ser246Glyfs*96 p.Ala293Val, p.His348Gln, p.His417Tyr, p.Asp418Val). The p.Arg402Trp, p.Pro248Leu and p.Leu340Phe variants were the most common in Turkish patients, with a frequency of 21.2%, 18.2% and 12.1% respectively. ConclusionThis study is the first comprehensive research from Turkey that provides information about disease-causing variants in the GCDH gene. The identification of common variants and hot spot regions of the GCDH gene is important for genetic counselling and the prenatal diagnosis of Turkish patients with GA1.

Deletion of 2-aminoadipic semialdehyde synthase limits metabolite accumulation in cell and mouse models for glutaric aciduria type 1.

Glutaric aciduria type 1 (GA1) is an inborn error of lysine degradation characterized by acute encephalopathy that is caused by toxic accumulation of lysine degradation intermediates. We investigated the efficacy of substrate reduction through inhibition of 2-aminoadipic semialdehyde synthase (AASS), an enzyme upstream of the defective glutaryl-CoA dehydrogenase (GCDH), in a cell line and mouse model of GA1. We show that loss of AASS function in GCDH-deficient HEK-293 cells leads to an approximately fivefold reduction in the established GA1 clinical biomarker glutarylcarnitine. In the GA1 mouse model, deletion of Aass leads to a 4.3-, 3.8-, and 3.2-fold decrease in the glutaric acid levels in urine, brain, and liver, respectively. Parallel decreases were observed in urine and brain 3-hydroxyglutaric acid levels, and plasma, urine, and brain glutarylcarnitine levels. These in vivo data demonstrate that the saccharopine pathway is the main source of glutaric acid production in the brain and periphery of a mouse model for GA1, and support the notion that pharmacological inhibition of AASS may represent an attractive strategy to treat GA1.

Incidence of newborn screening disorders among 56632 infants in Central Saudi Arabia. A 6-year study.

Objectives:To determine the incidence of newborn screening (NBS) disorders and to study the key performance indicators of the program.Methods:This retrospective single-center study enrolled all infants who underwent NBS from January 2012 to December 2017 at Prince Sultan Military Medical City, Riyadh, Saudi Arabia. We screened 17 NBS disorders. Blood samples were collected 24 hours after birth. If the initial result was positive, a second sample was collected. True positive cases were immediately referred for medical management. Data were extracted from laboratory computerized and non-computerized records using case report forms.Results:During the study period, 56632 infants underwent NBS with a coverage rate of 100%. Thirty-eight cases were confirmed. The incidence of congenital hypothyroidism was 1:3775. The positive predictive value for the detection of congenital hypothyroidism was 11.8%. Propionic aciduria was the most common metabolic disorder, with an incidence of 1:14158. Very long-chain acyl CoA dehydrogenase deficiency and glutaric aciduria type 1 had an incidence of 1:18877 each. Phenylketonuria, biotinidase deficiency, maple syrup urine disease, and citrullinemia had an incidence of 1:28316 each. However, galactosemia and 3-methyl crotonyl carboxylase deficiency had the lowest incidence of 1:56632.Conclusion:The NBS coverage rate at our facility was 100%. Congenital hypothyroidism was the most frequently detected disorder with an incidence that matches worldwide figures. The incidence of other inherited disorders was consistent with regional figures.

Magnetic resonance imaging pattern recognition in childhood bilateral basal ganglia disorders.

Bilateral basal ganglia abnormalities on MRI are observed in a wide variety of childhood disorders. MRI pattern recognition can enable rationalization of investigations and also complement clinical and molecular findings, particularly confirming genomic findings and also enabling new gene discovery. A pattern recognition approach in children with bilateral basal ganglia abnormalities on brain MRI was undertaken in this international multicentre cohort study. Three hundred and five MRI scans belonging to 201 children with 34 different disorders were rated using a standard radiological scoring proforma. In addition, literature review on MRI patterns was undertaken in these 34 disorders and 59 additional disorders reported with bilateral basal ganglia MRI abnormalities. Cluster analysis on first MRI findings from the study cohort grouped them into four clusters: Cluster 1—T2-weighted hyperintensities in the putamen; Cluster 2—T2-weighted hyperintensities or increased MRI susceptibility in the globus pallidus; Cluster 3—T2-weighted hyperintensities in the globus pallidus, brainstem and cerebellum with diffusion restriction; Cluster 4—T1-weighted hyperintensities in the basal ganglia. The 34 diagnostic categories included in this study showed dominant clustering in one of the above four clusters. Inflammatory disorders grouped together in Cluster 1. Mitochondrial and other neurometabolic disorders were distributed across clusters 1, 2 and 3, according to lesions dominantly affecting the striatum (Cluster 1: glutaric aciduria type 1, propionic acidaemia, 3-methylglutaconic aciduria with deafness, encephalopathy and Leigh-like syndrome and thiamine responsive basal ganglia disease associated with SLC19A3), pallidum (Cluster 2: methylmalonic acidaemia, Kearns Sayre syndrome, pyruvate dehydrogenase complex deficiency and succinic semialdehyde dehydrogenase deficiency) or pallidum, brainstem and cerebellum (Cluster 3: vigabatrin toxicity, Krabbe disease). The Cluster 4 pattern was exemplified by distinct T1-weighted hyperintensities in the basal ganglia and other brain regions in genetically determined hypermanganesemia due to SLC39A14 and SLC30A10. Within the clusters, distinctive basal ganglia MRI patterns were noted in acquired disorders such as cerebral palsy due to hypoxic ischaemic encephalopathy in full-term babies, kernicterus and vigabatrin toxicity and in rare genetic disorders such as 3-methylglutaconic aciduria with deafness, encephalopathy and Leigh-like syndrome, thiamine responsive basal ganglia disease, pantothenate kinase-associated neurodegeneration, TUBB4A and hypermanganesemia. Integrated findings from the study cohort and literature review were used to propose a diagnostic algorithm to approach bilateral basal ganglia abnormalities on MRI. After integrating clinical summaries and MRI findings from the literature review, we developed a prototypic decision-making electronic tool to be tested using further cohorts and clinical practice.

Two clinical cases of glutaric aciduria type I in the Republic of Bashkortostan

Glutaric aciduria type I is an autosomal recessive disease caused by mutations in the GCDH gene, which encodes the glutaryl-CoA-dehydrogenase enzyme. A lack of this enzyme leads to the accumulation of glutaric and 3-OH-glutaric (3-hydroxyglutaric) acids in biological fluids and tissues, which have a neurotoxic effect primarily on subcortical brain structures. We present two clinical cases of glutaric aciduria type I in children diagnosed after developing neurological symptoms. One patient had acute disease onset in the form of an encephalopathic crisis, which is typical for most clinical cases of the disease. The other patient developed subacute symptoms. Generalized dystonic hyperkinesia was a key sign of the disease in both patients. Information from the medical history and clinical presentation, together with the neuroimaging results, enabled us to suspect the right diagnosis and to confirm it using biochemical and then molecular genetic tests. In both cases, despite diet therapy using a specialized medicinal product, the patients continued to have motor disturbances.

3-Hydroxyisobutyryl-CoA Hydrolase Deficiency in a Turkish Child with a Novel HIBCH Gene Mutation and Literature Review

3-hydroxyisobutyryl-CoA hydrolase (HIBCH) deficiency (OMIM 250620) is an autosomal recessive inborn error of valine catabolism characterized by severely delayed psychomotor development, progressive neurodegeneration, recurrent metabolic attacks with intercurrent illness, increased lactic acid, cerebral atrophy, and brain lesions in the basal ganglia. HIBCH gene defect is a very rare organic aciduria and also might cause secondary mitochondrial dysfunction. We report a 12-month-old severely affected female infant with a novel homozygous c.556C>G; p.R186G variant in the HIBCH gene presenting with axial hypotonia, severe developmental delay, and brain lesions in the basal ganglia and provide an overview of the literature. When suspected, newborn and selective screening with tandem mass analyses should include hydroxy-C4-carnitine to diagnose this disorder. However, in some cases, mostly in those with milder phenotype, diagnosis may be missed due to normal hydroxy-C4 carnitine levels.

The role of nitric oxide in glutaric acid-induced convulsive behavior in pup rats.

Glutaric acidaemia type I (GA-I) is a cerebral organic disorder characterized by the accumulation of glutaric acid (GA) and seizures. As seizures are precipitated in children with GA-I and the mechanisms underlying this disorder are not well established, we decided to investigate the role of nitric oxide (NO) in GA-induced convulsive behaviour in pup rats. Pup male Wistar rats (18-day-old) were anesthetized and placed in stereotaxic apparatus for cannula insertion into the striatum for injection of GA. The experiments were performed 3days after surgery (pup rats 21-day-old). An inhibitor of NO synthesis (N-G-nitro-l-arginine methyl ester-L-NAME, 40mg/kg) or saline (vehicle) was administered intraperitoneally 30min before the intrastriatal injection of GA (1µl, 1.3µmol/striatum) or saline. Immediately after the intrastriatal injections, the latency and duration of seizures were recorded for 20min. The administration of L-NAME significantly increased the latency to the first seizure episode and reduced the duration of seizures induced by GA in pup rats. The administration of the NO precursor l-arginine (L-ARG; 80mg/kg) prevented the effects of L-NAME. Besides, GA significantly increased nitrate and nitrite (NOx) levels in the striatum of pup rats and the preadministration of L-NAME prevented this alteration. L-ARG blocked the reduction of striatal NOx provoked by L-NAME. These results are experimental evidence that NO plays a role in the seizures induced by GA in pup rats, being valuable in understanding the physiopathology of neurological signs observed in children with this organic acidaemia and to develop new therapeutic strategies.

P0006THE DELTA GAP - AN UPDATED AND VALIDATED APPROACH TO MIXED ACID-BASE DISORDERS

Abstract Background and Aims The urea & electrolyte panel (U&E) is a basic test commonly performed in clinical medicine. A quick evaluation can often reveal the presence of acid-base disturbances which in turn flags up potentially serious physiologic disturbances and critical conditions. While there are a few ways to screen for acid-base disorders, evaluation of the anion gap remains popular today. The AG is the difference between measured cations and anions and calculated from the main components of the U&E. For mixed acid-base disorders, the delta gap (ΔGap) provides further information (Table 1). However, every test has a range within tolerance limits. The tolerance limit in turn depends on the precision of the laboratory methodology. Figure 1 illustrates how tolerance intervals are determined in a normal distribution. To date, most clinical texts quote a tolerance interval of ±6 mEq/L for ΔGap, referencing the 1990 article by Dr Kieth Wrenn (Ann Emerg Med. 1990 Nov;19(11):1310). The range of ±6 mEq/L was calculated using the standard deviation (SD) of the tests for Na, Cl and bicarbonate in the 1980s, and an ΔGap > +6 or < –6 mEq/L suggests a mixed disorder. Derivation of tolerance interval of ΔGap: ΔGap = ΔAG – ΔHCO3- ΔGap = (AG –12) – (24 – HCO3-) ΔGap = (Na+ – Cl- – HCO3- –12) – (24 – HCO3-) Variance (SD2) of ΔGap = Sum of variances of component electrolytes Variance (SD2) of ΔGap = SD2Na + SD2Cl + SD2HCO+ SD2HCO Standard deviation (SD) of ΔGap = √( SD2ΔGap) Tolerance interval of ΔGap = ±1.96 x SDΔGap However, laboratory techniques have improved in precision since the 1980s. A ΔGap of ±6 mEq/L may be too wide, inadvertently missing mixed metabolic disorders. The aim of this study was to update the reference range of the ΔGap based on current laboratory precision. Interpretation of the acid-base disorder will be applied using the updated ΔGap range in a validation cohort. Method The current SD for Na, Cl and bicarbonate was obtained from the laboratory system data sheet and the tolerance interval of ΔGap was calculated . A validation cohort was generated retrospectively from electronic medical records according to a pre-determined criteria from 1 Jan – 31 Dec 2018. We identified a group of patients with a strong possibility of mixed HAGMA and MALK in the presence of: 1. High AG 2. Significant organic acidosis (lactic acidosis, ketosis or elevated salicylates) AND 3. A serum bicarbonate ≥19.0 mmol/L despite the presence of organic acids. The ΔGap was computed and interpreted based on the original vs new tolerance range. The Chi Square test was used to compare the interpretations based on the 2 tolerance ranges. Results The current SD of Na, Cl, bicarbonate was 0.9, 0.7, 0.3 respectively and the new tolerance interval of AG was ±2.3 mEq/L (rounded to ±2 mEq/L) The validation cohort comprised of 1,565 patients with HAGMA and MALK, aged 58.0±30.9 years with 873 (55.8%) males. Using the original range of ±6 mEq/L, 165 (10.5%) cases were classified correctly. With the new range of ±2 mEq/L, the 813 (51.9%) cases were classified correctly (P<0.001). Conclusion In summary, based on the precision of current laboratory techiques, the tolerance interval of AG is proposed as ±2 mEq/L. This was validated to significantly increase correct classification of mixed acid-base disorders.

Organic acidurias: Major gaps, new challenges, and a yet unfulfilled promise.

Organic acidurias (OADs) comprise a biochemically defined group of inherited metabolic diseases. Increasing awareness, reliable diagnostic work-up, newborn screening programs for some OADs, optimized neonatal and intensive care, and the development of evidence-based recommendations have improved neonatal survival and short-term outcome of affected individuals. However, chronic progression of organ dysfunction in an aging patient population cannot be reliably prevented with traditional therapeutic measures. Evidence is increasing that disease progression might be best explained by mitochondrial dysfunction. Previous studies have demonstrated that some toxic metabolites target mitochondrial proteins inducing synergistic bioenergetic impairment. Although these potentially reversible mechanisms help to understand the development of acute metabolic decompensations during catabolic state, they currently cannot completely explain disease progression with age. Recent studies identified unbalanced autophagy as a novel mechanism in the renal pathology of methylmalonic aciduria, resulting in impaired quality control of organelles, mitochondrial aging and, subsequently, progressive organ dysfunction. In addition, the discovery of post-translational short-chain lysine acylation of histones and mitochondrial enzymes helps to understand how intracellular key metabolites modulate gene expression and enzyme function. While acylation is considered an important mechanism for metabolic adaptation, the chronic accumulation of potential substrates of short-chain lysine acylation in inherited metabolic diseases might exert the opposite effect, in the long run. Recently, changed glutarylation patterns of mitochondrial proteins have been demonstrated in glutaric aciduria type 1. These new insights might bridge the gap between natural history and pathophysiology in OADs, and their exploitation for the development of targeted therapies seems promising.

Animal Models of Metabolic Epilepsy and Epilepsy Associated Metabolic Dysfunction: A Systematic Review.

Epilepsy is a serious neurological disorder affecting around 70 million people globally and is characterized by spontaneous recurrent seizures. Recent evidence indicates that dysfunction in metabolic processes can lead to the alteration of neuronal and network excitability, thereby contributing to epileptogenesis. Developing a suitable animal model that can recapitulate all the clinical phenotypes of human metabolic epilepsy (ME) is crucial yet challenging. The specific environment of many symptoms as well as the primary state of the applicable neurobiology, genetics, and lack of valid biomarkers/diagnostic tests are the key factors that hinder the process of developing a suitable animal model. The present systematic review summarizes the current state of available animal models of metabolic dysfunction associated with epileptic disorders. A systematic search was performed by using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) model. A range of electronic databases, including google scholar, Springer, PubMed, ScienceDirect, and Scopus, were scanned between January 2000 and April 2020. Based on the selection criteria, 23 eligible articles were chosen and are discussed in the current review. Critical analysis of the selected literature delineated several available approaches that have been modeled into metabolic epilepsy and pointed out several drawbacks associated with the currently available models. The result describes available models of metabolic dysfunction associated with epileptic disorder, such as mitochondrial respiration deficits, Lafora disease (LD) model-altered glycogen metabolism, causing epilepsy, glucose transporter 1 (GLUT1) deficiency, adiponectin responsive seizures, phospholipid dysfunction, glutaric aciduria, mitochondrial disorders, pyruvate dehydrogenase (PDH) α-subunit gene (PDHA1), pyridoxine dependent epilepsy (PDE), BCL2-associated agonist of cell death (BAD), Kcna1 knock out (KO), and long noncoding RNAs (lncRNA) cancer susceptibility candidate 2 (lncRNA CASC2). Finally, the review highlights certain focus areas that may increase the possibilities of developing more suitable animal models and underscores the importance of the rationalization of animal models and evaluation methods for studying ME. The review also suggests the pressing need of developing precise robust animal models and evaluation methods for investigating ME.

Inborn errors of metabolism detectable by tandem mass spectrometry in Beijing.

Background Individual inborn errors of metabolism (IEMs) are rare disorders. Expanded newborn screening for IEMs by tandem mass spectrometry (TMS) is an efficient approach for early diagnosis. Here we provide the newborn screening program for the application of this approach (between July 2014 and March 2019) to the identification of newborns in Beijing at risk of developing a potentially fatal disease. Methods The amino acids and acylcarnitines in dried blood spots were analyzed by TMS. Diagnoses of newborns with elevated metabolites were confirmed by gas chromatography-mass spectrometry, biochemical studies, and genetic analysis. Results Among the healthy newborns, 16 metabolic disorder cases were confirmed, giving a total birth prevalence of 1:3666 live births. Organic acidemia (OA) was the most common (9/16 patients; 56%), and methylmalonic acidemia was the most frequently observed OA (7/9 patients; 89%). Five infants were diagnosed with methylmalonic acidemia with homocystinuria type CblC, two with isolated methylmalonic acidemia, one with propionic acidemia, and one with isovaleric acidemia. Four patients (4/16, 25%) were diagnosed with hyperphenylalaninemia. One suffered with medium-chain acyl CoA dehydrogenase deficiency, one with carnitine uptake deficiency, and one with citrin deficiency. Eleven cases underwent genetic analysis. Seventeen mutations in eight IEM-associated genes were identified in 11 confirmed cases. Symptoms were already present within 2 days after birth in 44% (7/16) cases. The infant with propionic acidemia died at 7 days after birth. The other cases received timely diagnosis and treatment, and most of them grew well. Conclusions The results illustrate challenges encountered in disease management highlighting the importance of newborn screening for inherited metabolic disorders, which is not yet nationally available in our country. Regional newborn screening programs will provide a better estimation of the incidence of IEM.

PIH20 OUTCOME AND ECONOMIC EVALUATION OF NEONATAL SCREENING USING MS/MS FOR INHERITED METABOLIC DISEASES

Neonatal screening aims to detect asymptomatic infants at early stages to prevent the occurrence of body dysfunction and irreversible damage, so as to reduce the rate of infant disability and mortality. Mass spectrometry makes possible the simultaneous measurement of several metabolites and, consequently, the detection of several diseases in one blood spot and in a unique analysis. This study assessed the outcome and cost-effectiveness of neonatal screening using tandem-mass spectrometry (MS/MS). Patients who underwent MS/MS screening for inherited metabolic diseases (IMD) from 2009Q1-2018Q4 were identified with another MS/MS screening plus specified tests and confirmed by gene sequence analysis. All records were extracted from a screening management system in Neonatal Screening Center of Zhejiang province. Direct costs were estimated from the perspective of the Chinese healthcare system. Of the 3,040,815 neonates screened, 26,297(0.86%) neonates were suspected positive after first-round screening and called back to take another MS/MS screening. 25,670(97.62%) of them followed the latter procedures and finally 574(2.24%) cases were diagnosed through gene sequence analysis . The most frequent cause of IMD is fatty acid oxidation disorders (n=248), in most cases organic acidemias (n=211), followed by amino acid diseases(n=115). The costs of MS/MS was RMB$175 for screening and RMB$390 for follow-up, RMB$60 or 116 for specified tests, RMB$3120 for gene sequence analysis and RMB$80 for genetic counselling. The medication expenditures for detection of one single IMD patient was RMB$952,405.04. Overall the cost is huge. Neonatal screening for IMD using MS/MS in common neonates may not be cost-effective, this needs to be confirmed using data on observed mortality and disability reduction.The results prompt the need of prudent consideration for nationwide promotion of IMD neonatal screening using MS/MS.

2-methylacetoacetyl-coenzyme A thiolase (beta-ketothiolase) deficiency: one disease - two pathways

Background2-methylacetoacetyl-coenzyme A thiolase deficiency (MATD; deficiency of mitochondrial acetoacetyl-coenzyme A thiolase T2/ “beta-ketothiolase”) is an autosomal recessive disorder of ketone body utilization and isoleucine degradation due to mutations in ACAT1.MethodsWe performed a systematic literature search for all available clinical descriptions of patients with MATD. Two hundred forty-four patients were identified and included in this analysis. Clinical course and biochemical data are presented and discussed.ResultsFor 89.6% of patients at least one acute metabolic decompensation was reported. Age at first symptoms ranged from 2 days to 8 years (median 12 months). More than 82% of patients presented in the first 2 years of life, while manifestation in the neonatal period was the exception (3.4%). 77.0% (157 of 204 patients) of patients showed normal psychomotor development without neurologic abnormalities.ConclusionThis comprehensive data analysis provides a systematic overview on all cases with MATD identified in the literature. It demonstrates that MATD is a rather benign disorder with often favourable outcome, when compared with many other organic acidurias.

Practical management of organic acidemias

Organic acidemias or acidurias (OAs) are a group of metabolic disorders which result in organic acids detected in the urine (or plasma). They include the systemic OAs, the cerebral OAs, and the ketogenic/ketolytic OAs. In general, all types of OA can

Cardiometabolic risk factor clustering in patients with deficient branched‐chain amino acid catabolism: A case‐control study

Classical organic acidemias (OAs) result from defective mitochondrial catabolism of branched-chain amino acids (BCAAs). Abnormal mitochondrial function relates to oxidative stress, ectopic lipids and insulin resistance (IR). We investigated whether genetically impaired function of mitochondrial BCAA catabolism associates with cardiometabolic risk factors, altered liver and muscle energy metabolism, and IR. In this case-control study, 31 children and young adults with propionic acidemia (PA), methylmalonic acidemia (MMA) or isovaleric acidemia (IVA) were compared with 30 healthy young humans using comprehensive metabolic phenotyping including in vivo 31 P/1 H magnetic resonance spectroscopy of liver and skeletal muscle. Among all OAs, patients with PA exhibited abdominal adiposity, IR, fasting hyperglycaemia and hypertriglyceridemia as well as increased liver fat accumulation, despite dietary energy intake within recommendations for age and sex. In contrast, patients with MMA more frequently featured higher energy intake than recommended and had a different phenotype including hepatomegaly and mildly lower skeletal muscle ATP content. In skeletal muscle of patients with PA, slightly lower inorganic phosphate levels were found. However, hepatic ATP and inorganic phosphate concentrations were not different between all OA patients and controls. In patients with IVA, no abnormalities were detected. Impaired BCAA catabolism in PA, but not in MMA or IVA, was associated with a previously unrecognised, metabolic syndrome-like phenotype with abdominal adiposity potentially resulting from ectopic lipid storage. These findings suggest the need for early cardiometabolic risk factor screening in PA.

Molecular and biochemical study of glutaric aciduria type 1 in 49 Russian families: nine novel mutations in the GCDH gene.

Glutaric aciduria type 1 (GA1, deficiency of glutaryl CoA dehydrogenase, glutaric acidemia type 1) (ICD-10 code: E72.3; MIM 231670) is an autosomal recessive disease caused by mutations in the gene encoding the enzyme glutaryl CoA dehydrogenase (GCDH). Herein, we present the biochemical and molecular genetic characteristics of 51 patients diagnosed with GA1 from 49 unrelated families in Russia. We identified a total of 21 variants, 9 of which were novel: c.127 + 1G > T, с.471_473delCGA, c.161T > C (p.Leu54Pro), c.531C > A (р.Phe177Leu), c.647C > T (p.Ser216Leu), c.705G > A (р.Gly235Asp), c.898G > A (р.Gly300Ser), c.1205G > C (р.Arg402Pro), c.1178G > A (р.Gly393Glu). The most commonly detected missense variants were c.1204C > T (p.Arg402Trp) and с.1262C > T (р.Ala421Val), which were identified in 56.38% and 11.7% of mutated alleles. A heterozygous microdeletion of the short arm (p) of chromosome 19 from position 12,994,984-13,003,217 (8233 b.p.) and from position 12,991,506-13,003,217 (11,711 b.p.) were detected in two patients. Genes located in the area of imbalance were KLF1, DNASE2, and GCDH. Patients presented typical GA1 biochemical changes in the biological fluids, except one patient with the homozygous mutation p.Val400Met. No correlation was found between the GCDH genotype and glutaric acid (GA) concentration in the cohort of our patients.

Methylmalonic Acid Compromises Energy Metabolism and Decreases the Expression of Neuronal Differentiation Genes in SH‐SY5Y Human Neuroblastoma Cells

Methylmalonic acidemia is an organic acidemia caused by deficient activity of L‐methylmalonyl‐CoA mutase or its cofactor cyanocobalamin and it is biochemically characterized by an accumulation of methylmalonic acid (MMA) in tissue and body fluids of patients. The main clinical manifestations of this disease are neurological and symptoms during metabolic decompensation commonly appear in newborns. Considering that the mechanisms underlying neurological alterations of this disorder are not fully understood, this study aimed to investigate the toxic effects of MMA in a neuroblastoma cell line presenting neuronal features. Undifferentiated and retinoic acid (RA)‐differentiated human neuroblastoma SH‐SY5Y cells were exposed to MMA (1–10mM) for 24 or 48 hours and cell viability, glucose consumption and mitochondrial metabolic function were analyzed. Additionally, the expression of some molecular markers of neuronal differentiation was also quantified after co‐exposition with 10 μM RA and 1 mM of MMA for 3 and 7 days.MMA significantly reduced cell viability after 48‐hour period exposition in undifferentiated and RA differentiated SH‐SY5Y cells. It was also observed an increase in glucose consumption in cells exposed to MMA. Regarding oxygen consumption, there was an increase in respiratory parameters in RA differentiated SH‐SY5Y cells when compared to undifferentiated cells. MMA significantly reduced basal, ATP‐linked and maximal respiration, as well as the reserve respiratory capacity of cells after 48‐hour exposition in both undifferentiated and RA differentiated SH‐SY5Y cells. Regarding the effects of MMA in the neuronal differentiation process, it was observed that co‐exposition to RA and MMA significantly decreased the expression of enolase 2 (ENO2) and synaptophysin (SYP) genes after 7 days of exposition.In summary, MMA cell viability and oxygen consumption in SH‐SY5Y neuronal cells, suggesting that this organic acid compromises cell respiration, at resting and reserve capacity states, which might influence the cell capacity to meet energetic demands. In addition, glucose uptake was increased after exposition to MMA, corroborating that aerobic metabolism is compromised. The expression of mature neuronal genes was also reduced, suggesting that MMA possibly compromises neuronal differentiation, which can be involved in symptoms presented by affected patients during brain development. Finally, our findings suggest that these effects could be involved in the pathophysiology of neurological dysfunction of this disease.Support or Funding InformationThis study was supported by grants from FAPESP (2015/25541‐0), CAPES and UFABC.

Impact of enteral arginine supplementation on lysine metabolism in humans: A proof-of-concept for lysine-related inborn errors of metabolism.

Patients with lysine-related inborn errors of metabolism (pyridoxine-dependent epilepsy [PDE] and glutaric aciduria type 1 [GA1]), follow a lysine-restricted diet with arginine-fortified lysine-free amino acid formula and additional oral arginine supplementation as a newer therapy for PDE. The rationale of arginine supplementation is based on arginine's ability to compete with lysine transport across cell membranes via shared transporter systems. Adequate doses of arginine required to competitively inhibit enteral lysine uptake has not been studied in humans This proof-of-concept study investigates the effect of incremental enteral arginine doses on whole-body lysine oxidation using an in vivo stable isotope tracer, L-[1-13 C] lysine, in healthy humans. Five healthy men completed six study days each consuming one dose of l-arginine HCl per study day; range = 50-600 mg/kg/d. Lysine intake was at DRI (30 mg/kg/d). Breath samples were analysed for L-[1-13 C] lysine oxidation to 13 CO2 using an isotope ratio mass spectrometer. Plasma amino acid concentrations were analysed using an amino acid analyser. Increasing doses of l-arginine HCl caused a linear decrease in whole-body lysine oxidation. Plasma arginine concentration increased, and plasma lysine concentration decreased below normal range with high arginine intakes. We provide the first empirical evidence of arginine-lysine antagonism in response to increasing oral arginine doses. Results suggest 300-600 mg/kg/d of l-arginine HCl and lysine intake restricted to DRI is needed to reduce enteral lysine uptake and systemic lysine oxidation. This could potentially lead to a recommended dose for arginine in lysine-related inborn errors of metabolism.

Rapid screening of MMACHC gene mutations by high-resolution melting curve analysis.

BackgroundCobalamin (cbl) C is a treatable rare hereditary disorder of cbl metabolism with autosomal recessive inheritance. It is the most common organic acidemia, manifested as methylmalonic academia combined with homocysteinemia. Early screening and diagnosis are important. The mutation spectrum of the MMACHC gene causing cblC varies among populations. The mutation spectrum in Chinese population is notably different from that in other populations.MethodsA PCR followed by high‐resolution melting curve analysis (PCR‐HRM) method covering all coding exons of MMACHC gene was designed to verify 14 pathogenic MMACHC gene variants found in patients with cblC, including all common mutations in Chinese patients with cblC.ResultBy PCR‐HRM analysis, 14 pathogenic variants of MMACHC showed distinctly different melting curves, which were consistent with Sanger sequencing. The homozygous type of the most common mutation c.609G > A (p.Trp203Ter) can also be analyzed by specially designed PCR‐HRM.ConclusionThe established PCR‐HRM method for screening common pathogenic MMACHC variants in Chinese patients with cblC has the advantages of high accuracy, high throughput, low cost, and high speed. It is suitable for the large‐sample screening of suspected children with methylmalonic acidemia and carriers in population.

PRELIMINARY RESULTS ON THE USING TANDEM MASS SPECTROMETRY IN DIAGNOSIS OF INHERITED METABOLIC DISEASES

BACKGROUND According to the results of the researches common indexes of the prevalence of inherited metabolic diseases (IMD) varies from 1 to 800 on 1 to 2500 alive newborns. IMD are taking one of the first places among children pathology, early children death (40%) and disability[1]. According to systematic review of the 43 forms of the inborn errors of metabolism are related to unexpected death of newborns. For IMD it is common to have a wide spectrum of the unusual clinical manifestation, often they are not diagnosed, while well timed diagnoses and proper treatment are able to prevent severe systematic lesions, which lead to death and disability[2]. For that reason one of the most significant problems of the modern pediatrics is to early diagnosis of IMD. The only way to diagnosis of orphan metabolic diseases is the tandem mass spectrometry (TMS) [3]. AIM Scientifically substantiate the need for implementation of selective screening IMD of children using TMS method in Republic of Kazakhstan (RK) for early diagnosis, therapy of the inherited metabolic diseases, to reduce disability and death rate. MATERIALS AND METHODS Material of the research – dry blood spots, taken using standard methodology on filtered DBS papers, which are used in RK in the program of neonatal screening (for retrospective research – archived samples of the dry blood spots of the children dead during first year of life). Method of the research is tandem mass spectometry (QSight Perkin Elmer). RESULTS Analysis of the archived dry blood spot samples showed metabolic deviations in 20.4% of the cases. The detected changes are related to amino acids metabolic disorders, defects of -oxidation of the fat acids, decrease activity of the glucocerebrosidase (Gaucher’s disease) and sphingomyelinase (Nimman – Pick disease). Results of the selective screening have shown metabolic disorders in 5% of the cases (defects of -oxidation of the fat acids, aminoacidopathy, organic aciduria). CONCLUSIONS The preliminary results of the using TMS for the diagnosis of IMD have shown the need for implementation of selective screening IMD using TMS, which is able to conduct diagnosis of 75 metabolites of 49 IMD in single blood spot, which were not detected in RK previously. Taking into the consideration economic expenses of the government, related to the costs of the systematic treatment, medical service, life expectancy and lifelong support of the disabled children with IMD, early detection of orphan metabolic diseases is the vital condition of the decrease of newborn and children death rate, sickness rate and disability. This research study was carried out as a part of a scientific project funded by West Kazakhstan Marat Ospanov Medical University.