Search

Introduction Guanidinoacetate methyltransferase (GAMT) deficiency is an inherited metabolic disorder that impairs the synthesis of creatine (CRE). Lack of CRE in the brain can cause intellectual disability, autistic-like behavior, seizures, and movement disorders. Identification at birth and immediate therapy can prevent intellectual disability and seizures. Here we report the first two cases of GAMT deficiency identified at birth by newborn screening (NBS) in Utah and New York. Methods NBS dried blood spots were analyzed by tandem mass spectrometry (MS/MS) using either derivatized or non-derivatized assays to detect guanidinoacetate (GUAC) and CRE. For any positive samples, a second-tier test using a more selective method, ultra-performance liquid chromatography (UPLC) combined with MS/MS, was performed to separate GUAC from potential isobaric interferences. Results NBS for GAMT deficiency began in Utah on June 1, 2015 using a derivatized method for the detection of GUAC and CRE. In May 2019, the laboratory and method transitioned to a non-derivatized method. GAMT screening was added to the New York State NBS panel on October 1, 2018 using a derivatized method. In New York, a total of 537,408 babies were screened, 23 infants were referred and one newborn was identified with GAMT deficiency. In Utah, a total of 273,902 infants were screened (195,425 with the derivatized method, 78,477 with the non-derivatized method), three infants referred and one was identified with GAMT deficiency. Mean levels of GUAC and CRE were similar between methods (Utah derivatized: GUAC = 1.20 ± 0.43 μmol/L, CRE = 238 ± 96 μmol/L; Utah non-derivatized: GUAC = 1.23 ± 0.61 μmol/L, CRE = 344 ± 150 μmol/L, New York derivatized: GUAC = 1.34 ± 0.57 μmol/L, CRE = 569 ± 155 μmol/L). With either Utah method, similar concentrations of GUAC are observed in first (collected around 1 day of age) and the second NBS specimens (routinely collected at 7–16 days of age), while CRE concentrations decreased in the second NBS specimens. Both infants identified with GAMT deficiency started therapy by 2 weeks of age and are growing and developing normally at 7 (Utah) and 4 (New York) months of age. Conclusions Newborn screening allows for the prospective identification of GAMT deficiency utilizing elevated GUAC concentration as a marker. First-tier screening may be incorporated into existing methods for amino acids and acylcarnitines without the need for new equipment or staff. Newborn screening performed by either derivatized or non-derivatized methods and coupled with second-tier testing, has a very low false positive rate and can prospectively identify affected children. SummaryCerebral creatine deficiency syndromes caused by defects in creatine synthesis can result in intellectual disability, and are preventable if therapy is initiated early in life. This manuscript reports the identification of two infants with GAMT deficiency (one of the cerebral creatine deficiency syndromes) by newborn screening and demonstrates NBS feasibility using a variety of methods.

Newborn screening is an incredibly useful tool for the early identification of many metabolic disorders, including fatty acid oxidation (FAO) disorders. In many cases, molecular tests are necessary to reach a final diagnosis, highlighting the need for a thorough evaluation of genes implicated in FAO disorders. Using the ClinGen (Clinical Genome Resource) clinical validity framework, thirty genes were analyzed for the strength of evidence supporting their association with FAO disorders. Evidence was gathered from the literature by biocurators and presented to disease experts for review in order to assign a clinical validity classification of Definitive, Strong, Moderate, Limited, Disputed, Refuted, or No Reported Evidence. Of the gene-disease relationships evaluated, 22/30 were classified as Definitive, three as Moderate, one as Limited, three as No Reported Evidence and one as Disputed. Gene-disease relationships with a Limited, Disputed, and No Reported Evidence were found on two, six, and up to four panels out of 30 FAO disorder-specific panels, respectively, in the National Institute of Health Genetic Testing Registry, while over 70% of the genes on panels are definitively associated with an FAO disorder. These results highlight the need to systematically assess the clinical relevance of genes implicated in fatty acid oxidation disorders in order to improve the interpretation of genetic testing results and diagnosis of patients with these disorders.

Impaired activity of galactose-1-phosphate uridyltransferase (GALT) causes classic galactosemia (OMIM 230400 ), characterized by the accumulation of galactose-1-phosphate (GAL1P) in patients' red blood cells (RBCs). Our recent study demonstrated a correlation between RBC GAL1P and long-term outcomes in galactosemia patients. Here, we analyze biochemical and molecular results in 77 classic galactosemia patients to evaluate the association between GALT genotypes and GAL1P concentration in RBCs. Experimental data from model organisms were also included to assess the correlation between GAL1P and predicted residual activity of each genotype. Although all individuals in this study showed markedly reduced RBC GALT activity, we observed significant differences in RBC GAL1P concentrations among galactosemia genotypes. While levels of GAL1P on treatment did not correlate with RBC GALT activities (p = 0.166), there was a negative nonlinear correlation between mean GAL1P concentrations and predicted residual enzyme activity of genotype (p = 0.004). These studies suggest that GAL1P levels in RBCs on treatment likely reflect the overall functional impairment of GALT in patients with galactosemia.

BACKGROUND. Pyruvoyl Tetrahydropterin Synthase (PTPS) Deficiency is the most common form of BH4 deficiency resulting in hyperphenylalaninemia. It can have variable clinical severity and there is limited information on the clinical presentation, natural history and effectiveness of newborn screening for this condition. METHODS. Retrospective data (growth and clinical parameters, biochemical and genetic testing results, treatment) were collected from 19 patients with PTPS deficiency in different centers, to evaluate biochemical and clinical outcomes. Descriptive statistics was used for qualitative variables, while linear regression analysis was used to correlate quantitative variables. RESULTS. Patients with PTPS deficiency had an increased incidence of prematurity (4/18) with an average gestational age only mildly reduced (37.8±2.4 weeks) and low birth weight (−1.14±0.97 SD below that predicted for gestational age). With time, weight and height approached normal. values. All patients were identified by newborn screening for an elevated phenylalanine level. However, phenylalanine levels were normal in two whose testing was performed at or before 24 h of age. Sapropterin dihydrochloride treatment normalized phenylalanine levels. Molecular testing identified novel variants in the PTS gene, some of which present in more than one affected family. The neurotransmitter derivatives 5-hydroxyindoleacetic acid (5HIAA) and homovanillic acid (HVA) in the CSF were decreased in most cases except in 2 families with the peripheral form of PTPS deficiency. With time, HVA and 5HIAA became abnormally low in two of these patients requiring therapy. Prolactin (whose secretion is inhibited by dopamine) levels were elevated in several patients with PTPS deficiency and inversely correlated with the z-scores for height (p

Medium-Chain Acyl-CoA Dehydrogenase (MCAD) deficiency is a fatty acid oxidation disorder that can have variable clinical severity. There is still limited information on its clinical presentation and longitudinal history by genotype, and effectiveness of newborn screening (NBS).Retrospective data were collected from 90 patients (44 female, 46 male) to compare biochemical data with clinical outcomes. The frequency of adverse events (number of hypoglycemia-related ER visits and admissions) was assessed by genotype (homozygosity or not for the common pathogenic variant, p.Lys329Glu, in the ACADM gene), and method of diagnosis (NBS vs. clinical).MCAD deficiency in Utah was more frequent compared to the United States average (1: 9266 versus 1:17,759 newborns). With age, C8-carnitine did not change significantly whereas C2-carnitine decreased (p .001), possibly reflecting reduced carnitine supplementation typically seen with age. Children with MCAD deficiency had normal growth. p.Lys329Glu homozygotes had higher NBS C8-carnitine (23.4 ± 19.6 vs. 6.6 ± 3.0 μmol/L) and lifetime plasma C8-carnitine levels (6.2 ± 5 vs. 3.6 ± 1.9 μmol/L) compared to patients with at least one other pathogenic variant (p .001 for both) and higher transaminases compared to compound heterozygotes (ALT 41.9 ± 6.2 vs. 31.5 ± 3.7 U/L, AST 63.9 ± 5.8 vs. 45.7 ± 1.8 U/L, p .05 for both). On average, p.Lys329Glu homozygotes had more hypoglycemic events than compound heterozygotes (1.44 versus 0.49 events/patient) as did patients diagnosed clinically compared to those diagnosed by NBS (2.15 versus 0.62 events/patient), though these differences were not statistically significant. Neonatal death was observed before results of newborn screening were available in one patient homozygous for the common p.Lys329Glu pathogenic variant, but severe neonatal complications (hypoglycemia, cardiac arrhythmia) were also seen in patients with other mutations. No irreversible complications were observed after diagnosis in any patient with MCAD deficiency.Homozygosity for the common ACADM p.Lys329Glu pathogenic variant was associated with increased levels of C8-carnitine and transaminases. Newborn screening provides the opportunity to reduce morbidity and post-neonatal mortality in all patients with MCAD deficiency, regardless of genotype.

Background Very-Long-Chain Acyl-CoA Dehydrogenase (VLCAD) deficiency is a disorder of fatty acid oxidation included in the recommended uniform newborn screening (NBS) panel in the USA. It can have variable clinical severity and there is limited information on the natural history of this condition, clinical presentation according to genotype and effectiveness of newborn screening. Methods Retrospective data (growth parameters, morbidity, biochemical and genetic testing results) were collected from patients with VLCAD deficiency, to evaluate biochemical and clinical outcomes. Descriptive statistics was used for qualitative variables, while linear regression analysis was used to correlate continuous variables. Results VLCAD deficiency (screened by measuring elevated levels of C14:1-carnitine in blood spots) was more frequent in Utah than the national average (1:27,617 versus 1:63,481) in the first ten years of screening. Twenty-six patients had a confirmed diagnosis of VLCAD deficiency using DNA testing or functional studies. The c.848T>C (p.V283A) variant in the ACADVL gene was the most frequent in our population. Novel variants (c.623-21A>G (IVS7-21A>G); c.1052C>T (p.T351I); c.1183-7A>G (IVS11-7A>G); c.1281G>C (p.W427C); c.1923G>C (p.L641F); c.1924G>A (p.V642M)) were identified in this study, with their pathogenicity remaining unclear in most cases. C14:1-carnitine levels decreased with age and significantly correlated with CK levels as index of muscle involvement. There were no cases of HELLP syndrome nor liver disease during pregnancies in the mothers of VLCAD patients. None of our patients developed cardiac involvement after birth and all patients had normal growth parameters while on treatment. Clinical manifestations were related to concomitant infections and altered biochemical parameters. Discussion VLCAD deficiency can be identified by neonatal screening. Most patients compliant with therapy normalized biochemical parameters and had no major clinical manifestations. Complications were completely prevented with a relatively low number of pre-emptive ER visits or hospital admissions. It remains unclear whether neonatal screening is now identifying less severely affected patient or if complications will arise as subjects become older. Observation beyond puberty is necessary to fully understand the impact of VLCAD deficiency on morbidity in patients with VLCAD deficiency.

Pyridoxine-Dependent Epilepsy (PDE) is a recessive disorder caused by deficiency of α-aminoadipic semialdehyde dehydrogenase in the catabolic pathway of lysine. It is characterized by intractable seizures controlled by the administration of pharmacological doses of vitamin B6. Despite seizure control with pyridoxine, intellectual disability and developmental delays are still observed in some patients with PDE, likely due to the accumulation of toxic intermediates in the lysine catabolic pathway: alpha-aminoadipic semialdehyde (AASA), delta-1-piperideine-6-carboxylate (P6C), and pipecolic acid. Here we evaluate biochemical and clinical parameters in two PDE patients treated with a lysine-restricted diet and arginine supplementation (100-150mg/kg), aimed at reducing the levels of PDE biomarkers. Lysine restriction resulted in decreased accumulation of PDE biomarkers and improved development. Plasma lysine but not plasma arginine, directly correlated with plasma levels of AASA-P6C (p

Background Guanidinoacetate methyltransferase (GAMT) deficiency causes cerebral creatine deficiency. Patients can have autistic behavior, seizures, intellectual disability, and severe speech delay. The goal of therapy is to increase creatine while reducing potentially neurotoxic guanidinoacetate concentrations. Here we evaluate how different therapies affect plasma guanidinoacetate levels in patients with GAMT deficiency. Methods Retrospective analysis of data from five new patients with GAMT deficiency (four with delays and seizures, one diagnosed at birth). Results The four symptomatic patients had decreased brain creatine by magnetic resonance spectroscopy and three also had abnormal globi pallidi by MRI. GAMT sequencing identified four previously reported mutations and one novel missense mutation (c.233T>A/p.V78E). Treatment with creatine (250–1000 mg/kg/day), ornithine (100–800 mg/kg/day), and sodium benzoate (50–135 mg/kg/day) supplements along with dietary protein restriction (0.8–1.5 g/kg/day) improved seizures and development with all patients becoming verbal. The patient treated at birth remains developmentally normal. Reduction in glycine and increase in ornithine levels significantly decreased plasma guanidinoacetate, with glycine levels being the best predictor of guanidinoacetate levels. In contrast, arginine levels were not significantly correlated with plasma guanidinoacetate. Conclusions Our results show that supplements of creatine, sodium benzoate (to reduce glycine) and ornithine reduce guanidinoacetate levels in patients with GAMT deficiency (dietary therapy was not evaluated in our study). Normal development with early therapy renders GAMT deficiency an ideal candidate for inclusion in newborn screening panels.

Primary carnitine deficiency impairs fatty acid oxidation and can result in hypoglycemia, hepatic encephalopathy, cardiomyopathy and sudden death. We diagnosed primary carnitine deficiency in six unrelated women whose unaffected infants were identified with low free carnitine levels (C0) by newborn screening using tandem mass spectrometry. Given the lifetime risk of morbidity or sudden death, identification of adult patients with primary carnitine deficiency is an added benefit of expanded newborn screening programs.

Propionic acidemia is an autosomal recessive disorder caused by deficiency of propionyl CoA carboxylase. Affected patients can develop severe hyperammonemia, whose causative mechanism is unknown. In this study, we monitored changes in metabolic parameters associated with hyperammonemia in patients with propionic acidemia. Levels of ammonia were correlated with plasma levels of individual amino acids and carnitine and with urinary organic acids. Significance of correlations was determined with analysis of variance. Hyperammonemia positively correlated with an increase in branched-chain amino acids (leucine and isoleucine) and a decrease in glutamine/glutamate and esterified carnitine. The urinary excretion of methylcitric acid, formed by the combination of propionic acid with oxaloacetate from the Krebs cycle, increased while that of citric acid decreased with hyperammonemia. These results suggest that in propionic acidemia, hyperammonemia is triggered by catabolism with the accumulation of propionic acid derivatives. The decrease of the plasma levels of glutamine/glutamate with hyperammonemia in patients with propionic acidemia indicates that the mechanism producing hyperammonemia differs from that in urea cycle defects. The increase in methylcitric acid and decline in citric acid urinary excretion suggest that hyperammonemia in propionic acidemia might be related to inability to maintain adequate levels of glutamine precursors through a dysfunctional Krebs cycle.

Glutaric acidemia type 1 (GA-1) is an autosomal recessive disorder of lysine, hydroxylysine, and tryptophan metabolism. Patients may present with brain atrophy, macrocephaly, and acute dystonia secondary to striatal degeneration typically triggered by an infection, fever, and/or dehydration. This disorder is identified on expanded newborn screening by increased glutarylcarnitine. We evaluated the outcome of 19 patients with GA-1. Ten patients were diagnosed by newborn screening and 9 were diagnosed clinically. DNA testing in 12 patients identified 15 different mutations in the glutaryl-CoA dehydrogenase gene. Plasma glutarylcarnitine and urinary 3-hydroxyglutaric acid were elevated in all patients. However, only 10 of 17 patients who underwent urine organic acid analysis were high excretors of glutaric acid. Levels of glutarylcarnitine in plasma correlated with the urinary excretion of glutaric and 3-hydroxyglutaric acid, but not with clinical outcome. Plasma lysine was also significantly correlated with urinary glutaric acid, but not with urinary 3-hydroxyglutaric acid. Brain magnetic resonance imaging in all patients showed wide Sylvian fissures before treatment, which normalized by 4 years of age in treated patients. The occurrence of three adverse outcomes (oral motor function, ambulatory capability, and dystonic movements) was on average reduced by 75% (relative risk 0.25 to 0.28) in patients identified by newborn screening compared to patients diagnosed before newborn screening (Fisher's exact test; p = 0.0055 for oral motor function and ambulatory capability; p = 0.023 for dystonic movements). Newborn screening is effective in the prevention of complications in patients with GA-1 when coupled with treatment strategies.

Mucopolysaccharidosis VI (MPS VI) is a lysosomal storage disease caused by a deficiency of N-acetylgalactosamine 4-sulfatase (arylsulfatase B, ASB). This enzyme is required for the degradation of dermatan sulfate. In its absence, dermatan sulfate accumulates in cells and is excreted in large quantities in urine. Specific therapeutic intervention is available; however, accurate and timely diagnosis is crucial for maximal benefit. To better understand the current practices for diagnosis and to establish diagnostic guidelines, an international MPS VI laboratory diagnostics scientific summit was held in February of 2011 in Miami, Florida. The various steps in the diagnosis of MPS VI were discussed including urinary glycosaminoglycan (uGAG) analysis, enzyme activity analysis, and molecular analysis. The following conclusions were reached. Dilute urine samples pose a significant problem for uGAG analysis and MPS VI patients can be missed by quantitative uGAG testing alone as dermatan sulfate may not always be excreted in large quantities. Enzyme activity analysis is universally acknowledged as a key component of diagnosis; however, several caveats must be considered and the appropriate use of reference enzymes is essential. Molecular analysis supports enzyme activity test results and is essential for carrier testing, subsequent genetic counseling, and prenatal testing. Overall the expert panel recommends caution in the use of uGAG screening alone to rule out or confirm the diagnosis of MPS VI and acknowledges enzyme activity analysis as a critical component of diagnosis. Measurement of another sulfatase enzyme to exclude multiple sulfatase deficiency was recommended prior to the initiation of therapy. When feasible, the use of molecular testing as part of the diagnosis is encouraged. A diagnostic algorithm for MPS VI is provided.