Gene therapy for aromatic L-amino acid decarboxylase deficiency by MR-guided direct delivery of AAV2-AADC to midbrain dopaminergic neurons.

Aromatic L-amino acid decarboxylase (AADC) deficiency is a rare genetic disorder characterized by deficient synthesis of dopamine and serotonin. It presents in early infancy, and causes severe developmental disability and lifelong motor, behavioral, and autonomic symptoms including oculogyric crises (OGC), sleep disorder, and mood disturbance. We investigated the safety and efficacy of delivery of a viral vector expressing AADC (AAV2-hAADC) to the midbrain in children with AADC deficiency (ClinicalTrials.gov Identifier NCT02852213). Seven (7) children, aged 4-9 years underwent convection-enhanced delivery (CED) of AAV2-hAADC to the bilateral substantia nigra (SN) and ventral tegmental area (VTA) (total infusion volume: 80 µL per hemisphere) in 2 dose cohorts: 1.3 × 1011 vg (n = 3), and 4.2 × 1011 vg (n = 4). Primary aims were to demonstrate the safety of the procedure and document biomarker evidence of restoration of brain AADC activity. Secondary aims were to assess clinical improvement in symptoms and motor function. Direct bilateral infusion of AAV2-hAADC was safe, well-tolerated and achieved target coverage of 98% and 70% of the SN and VTA, respectively. Dopamine metabolism was increased in all subjects and FDOPA uptake was enhanced within the midbrain and the striatum. OGC resolved completely in 6 of 7 subjects by Month 3 post-surgery. Twelve (12) months after surgery, 6/7 subjects gained normal head control and 4/7 could sit independently. At 18 months, 2 subjects could walk with 2-hand support. Both the primary and secondary endpoints of the study were met. Midbrain gene delivery in children with AADC deficiency is feasible and safe, and leads to clinical improvements in symptoms and motor function.

Postmortem whole-genome sequencing on a dried blood spot identifies a novel homozygous SUOX variant causing isolated sulfite oxidase deficiency.

Rapid whole-genome sequencing (rWGS) has shown that genetic diseases are a common cause of infant mortality in neonatal intensive care units. Dried blood spots collected for newborn screening allow investigation of causes of infant mortality that were not diagnosed during life. Here, we present a neonate who developed seizures and encephalopathy on the third day of life that was refractory to antiepileptic medications. The patient died on day of life 16 after progressive respiratory failure and sepsis. The parents had lost two prior children after similar presentations, neither of whom had a definitive diagnosis. Postmortem rWGS of a dried blood spot identified a pathogenic homozygous frameshift variant in the SUOX gene associated with isolated sulfite oxidase deficiency (c.1390_1391del, p.Leu464GlyfsTer10). This case highlights that early, accurate molecular diagnosis has the potential to influence prenatal counseling and guide management in rare, genetic disorders and has added importance in cases of a strong family history and risk factors such as consanguinity.

Consensus guideline for the diagnosis and treatment of aromatic l-amino acid decarboxylase (AADC) deficiency.

Aromatic L-amino acid decarboxylase deficiency (AADCD) is a rare, autosomal recessive neurometabolic disorder that leads to a severe combined deficiency of serotonin, dopamine, norepinephrine and epinephrine. Onset is early in life, and key clinical symptoms are hypotonia, movement disorders (oculogyric crisis, dystonia, and hypokinesia), developmental delay, and autonomic symptoms.In this consensus guideline, representatives of the International Working Group on Neurotransmitter Related Disorders (iNTD) and patient representatives evaluated all available evidence for diagnosis and treatment of AADCD and made recommendations using SIGN and GRADE methodology. In the face of limited definitive evidence, we constructed practical recommendations on clinical diagnosis, laboratory diagnosis, imaging and electroencephalograpy, medical treatments and non-medical treatments. Furthermore, we identified topics for further research. We believe this guideline will improve the care for AADCD patients around the world whilst promoting general awareness of this rare disease.

Laboratory diagnosis of creatine deficiency syndromes: a technical standard and guideline of the American College of Medical Genetics and Genomics.

Disclaimer: These ACMG Standards and Guidelines are intended as an educational resource for clinical laboratory geneticists to help them provide quality clinical laboratory genetic services. Adherence to these standards and guidelines is voluntary and does not necessarily assure a successful medical outcome. These Standards and Guidelines should not be considered inclusive of all proper procedures and tests or exclusive of others that are reasonably directed to obtaining the same results. In determining the propriety of any specific procedure or test, clinical laboratory geneticists should apply their professional judgment to the specific circumstances presented by the patient or specimen. Clinical laboratory geneticists are encouraged to document in the patient's record the rationale for the use of a particular procedure or test, whether or not it is in conformance with these Standards and Guidelines. They also are advised to take notice of the date any particular guideline was adopted, and to consider other relevant medical and scientific information that becomes available after that date. It also would be prudent to consider whether intellectual property interests may restrict the performance of certain tests and other procedures.Cerebral creatine deficiency syndromes are neurometabolic conditions characterized by intellectual disability, seizures, speech delay, and behavioral abnormalities. Several laboratory methods are available for preliminary and confirmatory diagnosis of these conditions, including measurement of creatine and related metabolites in biofluids using liquid chromatography-tandem mass spectrometry or gas chromatography-mass spectrometry, enzyme activity assays in cultured cells, and DNA sequence analysis. These guidelines are intended to standardize these procedures to help optimize the diagnosis of creatine deficiency syndromes. While biochemical methods are emphasized, considerations for confirmatory molecular testing are also discussed, along with variables that influence test results and interpretation.Genet Med 19 2, 256-263.

Quantitative acylcarnitine determination by UHPLC-MS/MS--Going beyond tandem MS acylcarnitine "profiles".

Tandem MS "profiling" of acylcarnitines and amino acids was conceived as a first-tier screening method, and its application to expanded newborn screening has been enormously successful. However, unlike amino acid screening (which uses amino acid analysis as its second-tier validation of screening results), acylcarnitine "profiling" also assumed the role of second-tier validation, due to the lack of a generally accepted second-tier acylcarnitine determination method. In this report, we present results from the application of our validated UHPLC-MS/MS second-tier method for the quantification of total carnitine, free carnitine, butyrobetaine, and acylcarnitines to patient samples with known diagnoses: malonic acidemia, short-chain acyl-CoA dehydrogenase deficiency (SCADD) or isobutyryl-CoA dehydrogenase deficiency (IBD), 3-methyl-crotonyl carboxylase deficiency (3-MCC) or ß-ketothiolase deficiency (BKT), and methylmalonic acidemia (MMA). We demonstrate the assay's ability to separate constitutional isomers and diastereomeric acylcarnitines and generate values with a high level of accuracy and precision. These capabilities are unavailable when using tandem MS "profiles". We also show examples of research interest, where separation of acylcarnitine species and accurate and precise acylcarnitine quantification is necessary.

An update on serine deficiency disorders.

Serine deficiency disorders are caused by a defect in one of the three synthesising enzymes of the L-serine biosynthesis pathway. Serine deficiency disorders give rise to a neurological phenotype with psychomotor retardation, microcephaly and seizures in newborns and children or progressive polyneuropathy in adult patients. There are three defects that cause serine deficiency of which 3-phosphoglycerate dehydrogenase (3-PGDH) deficiency, the defect affecting the first step in the pathway, has been reported most frequently. The other two disorders in L-serine biosynthesis phosphoserine aminotransferase (PSAT) deficiency and phosphoserine phosphatase (PSP) deficiency have been reported only in a limited number of patients. The biochemical hallmarks of all three disorders are low concentrations of serine in cerebrospinal fluid and plasma. Prompt recognition of affected patients is important, since serine deficiency disorders are treatable causes of neurometabolic disorders. The use of age-related reference values for serine in CSF and plasma can be of great help in establishing a correct diagnosis of serine deficiency, in particular in newborns and young children.

Gene therapy for aromatic L-amino acid decarboxylase deficiency.

Aromatic L-amino acid decarboxylase (AADC) is required for the synthesis of the neurotransmitters dopamine and serotonin. Children with defects in the AADC gene show compromised development, particularly in motor function. Drug therapy has only marginal effects on some of the symptoms and does not change early childhood mortality. Here, we performed adeno-associated viral vector-mediated gene transfer of the human AADC gene bilaterally into the putamen of four patients 4 to 6 years of age. All of the patients showed improvements in motor performance: One patient was able to stand 16 months after gene transfer, and the other three patients achieved supported sitting 6 to 15 months after gene transfer. Choreic dyskinesia was observed in all patients, but this resolved after several months. Positron emission tomography revealed increased uptake by the putamen of 6-[(18)F]fluorodopa, a tracer for AADC. Cerebrospinal fluid analysis showed increased dopamine and serotonin levels after gene transfer. Thus, gene therapy targeting primary AADC deficiency is well tolerated and leads to improved motor function.

Amino acid disorders detected by quantitative amino acid HPLC analysis in Thailand: an eight-year experience.

BACKGROUND: Amino acid disorders are a major group of inborn errors of metabolism (IEM) with variable clinical presentations. This study was aimed to provide the data of amino acid disorders detected in high-risk Thai patients referred to our metabolic lab from all over the country. METHODS: From 2001 to 2009, we analyzed amino acids by HPLC in 1214 plasma and cerebrospinal fluid specimens. These specimens were obtained from patients with clinical suspicion of IEM or with positive newborn screening. The clinical data of the patients with confirmed diagnoses of amino acid disorders were also analyzed. RESULTS: Fifty-eight patients were diagnosed with amino acid disorders, including 20 cases (34.5%) with maple syrup urine disease, 13 (22.4%) with phenylketonuria and hyperphenylalaninemia, 13 (22.4%) with nonketotic hyperglycinemia, 9 (15.5%) with urea cycle defects, 2 (3.4%) with classical homocystinuria, and 1 (1.7%) with ornithine aminotransferase deficiency. There was considerable delay in diagnoses which led to poor outcomes in most patients. CONCLUSION: The prevalence of amino acid disorders in Thailand is distinct from other countries. This will guide the selection of the prevalent IEM for the future expansion of newborn screening program in this country.

A serine synthesis defect presenting with a Charcot-Marie-Tooth-like polyneuropathy.

BACKGROUND: Serine synthesis defects, characterized by developmental delay and seizures, have been described in children. OBJECTIVE: To describe a case of serine synthesis defect due to 3-phosphoglycerate dehydrogenase deficiency in an adult with prominent chronic polyneuropathy. DESIGN: Case report. SETTING: Neurologic referral center. PATIENT: A 31-year-old man with congenital cataracts, mild psychomotor retardation, slight cerebellar ataxia, and chronic axonal sensorimotor polyneuropathy. INTERVENTIONS: Electrophysiologic, metabolic, and genetic testing and treatment with oral L-serine. MAIN OUTCOME MEASURES: Serine values in plasma and cerebrospinal fluid and clinical examination. RESULTS: Amino acid analysis showed low serine levels in plasma and cerebrospinal fluid, and genetic analysis revealed 2 heterozygous mutations in the PGDH gene. Treatment with high-dose serine resulted in normalization of plasma serine values and subjective functional improvement. CONCLUSIONS: This case expands the phenotypic spectrum of 3-phosphoglycerate dehydrogenase deficiency. Plasma amino acid chromatography should be added to the list of investigations performed in patients with Charcot-Marie-Tooth­like polyneuropathy, especially if it is associated with psychomotor delay and congenital cataracts.

When do we need to perform a diagnostic lumbar puncture for neurometabolic diseases? Positive yield and retrospective analysis from a tertiary center.

Neurometabolic diseases diagnosed by cerebrospinal fluid (CSF) examination are GLUT1 deficiency, serine-deficiency syndromes, glycine encephalopathy, cerebral folate deficiency, neonatal vitamin-responsive epileptic encephalopathies, disorders of monoamine metabolism, and y-amino butyric acid (GABA) metabolism. We retrospectively analyzed and compared the demographic, clinical, laboratory, and neuroimaging features of 62 patients in whom CSF examination was performed. Of the 62 patients, 16 (25.8%) had a final diagnosis, including succinic semialdehyde dehydrogenase (SSADH) deficiency (n=4), aromatic amino acid decarboxylase (AADC) deficiency (n=4), L-dopa-responsive dystonia (n=3), glycine encephalopathy (n=2), pyridoxal-phosphate-dependent seizures (n=l), cerebral folate deficiency (n=1), and serine biosynthesi defect (n=1). Parental consanguinity was present in all patients except one Positive yield of a diagnostic lumbar puncture (LP) for the diagnosis of inherited neurotransmitter metabolism disorder was 25.8% overall. Oculogyric crisis (50%), diurnal variation (81.8%) and consanguinity (93.8%) were the only statistically significant variables between patients with and without a specific diagnosis. It is challenging to diagnose neurotransmitter defects, since there is no ideal set of clinical symptoms. In our cohort, consanguinity, diurnal variation and abnormal ocular movements were the most significant findings associated with a diagnosis of a specific neurometabolic disorder based on CSF examination. Early diagnosis is of great importance not only for specific treatment, but also for genetic counseling and prenatal diagnosis.

Multicentre age-related reference intervals for cerebrospinal fluid serine concentrations: implications for the diagnosis and follow-up of serine biosynthesis disorders.

The disorders of serine biosynthesis are a group of inborn errors of metabolism characterised by congenital microcephaly, seizures and severe psychomotor retardation. Although these disorders are rare the prompt recognition of serine deficiency is important as these disorders are treatable. The diagnosis is based on decreased concentrations of serine in cerebrospinal fluid (CSF). It has previously been reported that CSF serine concentrations are inversely associated with age. However, accurate age-related reference intervals have not been generated which has contributed to cases not being identified. In a multicentre study involving 9 different laboratories a total of 424 CSF serine results were obtained. Regression based analyses were performed to calculate age-specific reference intervals. Lower reference intervals for subjects aged 1week, 1month, 6months, 1year, 3years and 15years were 35.0, 31.0, 26.0, 24.0, 21.0 and 17.0µmol/L respectively. Assessment of CSF serine concentrations in 11 patients (aged 1day to 13years) previously diagnosed with disorders of serine biosynthesis (serine concentrations ranging from 5 to 18µmol/L) were clearly decreased compared to our age-related reference intervals and would have correctly identified all cases, thus enabling prompt treatment. However, if age had not been taken into consideration a reference interval of 12.6-69.4µmol/L would be obtained for the combined data set and would have resulted in 2 cases being missed. In conclusion, appropriate age-related reference intervals for CSF serine should be used to diagnose patients with inborn errors of serine biosynthesis.

Reference data for cerebrospinal fluid and the utility of amino acid measurement for the diagnosis of inborn errors of metabolism.

BACKGROUND: Cerebrospinal fluid (CSF) amino acid analysis is fundamental to the investigation of several inherited metabolic diseases, particularly those presenting with unexplained seizures. CSF glycine measurement is often crucial to the diagnosis of glycine encephalopathy (GE), low CSF serine concentrations are characteristic of 3-phosphoglycerate dehydrogenase deficiency (3-PGDD) and the presence of sulphocysteine is pathognomonic of sulphite oxidase deficiency (SOD), and a vital clue to molybdenum cofactor deficiency (MCD). Limited information is available in the literature on reference values of amino acids in CSF during infancy and CSF samples from healthy individuals are not easily obtained. METHODS: In order to establish paediatric CSF amino acid reference ranges, we performed a retrospective analysis of all quantitative CSF amino acid data collected in our laboratory over a five-year period. Amino acid analysis was performed using ion-exchange chromatography on a Biochrom-20 amino acid analyser with ninhydrin detection. CSF samples were collected from infants undergoing investigation for unexplained seizures. RESULTS: About 18 of the 95 samples received were excluded from the reference data-set; one was from a patient in whom a diagnosis of GE was confirmed by enzyme analysis, one was from a patient with CSF sulphocysteine of 19 micromol/L in whom a diagnosis of SOD was confirmed by enzyme analysis; the remaining 16 were clearly bloodstained (n = 4) or xanthochromic (n = 12). Frequency of distribution analysis revealed that concentration values for each amino acid demonstrated a right-skewed distribution which was not normalized by log transformation. Data were therefore analysed using non-parametric descriptive statistics and reference ranges were defined by the 2.5th and 97.5th centile limits. CONCLUSIONS: Our reference data were derived from 77 CSF samples taken from 77 infants. Median CSF glycine concentration was 9 micromol/L with a reference range of 3-19 micromol/L. For serine, the median CSF concentration was 52 micromol/L with a reference range of 25-105 micromol/L. Sulphocysteine was not normally present in detectable quantities (<1 micromol/L).

One-methyl group metabolism in non-ketotic hyperglycinaemia: mildly elevated cerebrospinal fluid homocysteine levels.

Non-ketotic hyperglycinaemia (NKH) is a rare, severe brain disease caused by deficient glycine cleavage enzyme complex activity resulting in elevated glycine concentrations. Recent experience suggests that factors in addition to glycine kinetics are involved in its pathogenesis. The glycine cleavage reaction through the formation of methylenetetrahydrofolate is an important one-methyl group donor. A deficiency in one-methyl group metabolites, in particular of choline, has been hypothesized in NKH. We investigated metabolites involved in one-methyl group metabolism in plasma and CSF of 8 patients with NKH, and monitored the effect of treatment with choline in one patient. Plasma and CSF choline and phosphatidylcholine concentrations were normal, except for a low plasma choline in the single neonate studied. Choline treatment did not change brain choline content, and was not associated with clinical or radiological improvement. Methionine concentrations and, in one-patient, S-adenosylmethionine and 5-methyltetrahydrofolate concentrations were normal in CSF. Homocysteine concentrations in CSF, however, were slightly but consistently elevated in all four patients examined, but cysteine, cysteinylglycine and glutathione were normal. Serine is important in the transfer of one-methyl groups from mitochondria to cytosol. Serine concentrations were normal in plasma and CSF, but dropped to below normal in CSF in three patients on benzoate treatment. These observations add to our understanding of the complex metabolic disturbances in NKH.

3-Phosphoglycerate dehydrogenase deficiency: an inborn error of serine biosynthesis.

Serine concentrations were markedly decreased in the cerebrospinal fluid of two brothers with congenital microcephaly, profound psychomotor retardation, hypertonia, epilepsy, growth retardation, and hypogonadism. The youngest boy also had congenital bilateral cataract. Magnetic resonance imaging of the brain showed evidence of dysmyelination. Plasma serine as well as plasma and cerebrospinal fluid glycine concentrations were also decreased but to a lesser extent. Treatment with oral serine in the youngest patient significantly increased cerebrospinal fluid serine and abolished the convulsions. In fibroblasts of both patients, a decreased activity was demonstrated of 3-phosphoglycerate dehydrogenase, the first step of serine biosynthesis (22% and 13% of the mean control value). This is an unusual disorder as the great majority of aminoacidopathies are catabolic defects. It is a severe but potentially treatable inborn error of metabolism that has not been previously reported in man.

Nonketotic hyperglycinemia in a newborn infant.

A neonate with nonketotic hyperglycinemia who experienced apnea, hiccups and tonic-clonic seizures on the first day of life is reported. The physical findings and laboratory tests including arterial blood gases were normal. However, serial blood and CSF amino-acid analyses demonstrated elevated glycine levels. Serum and CSF glycine levels were 1949 mumol/L and 415.5 mumol/L, respectively. (Normal serum level is 104-254 mumol/L and CSF level is 5 +/- 2 mumol/L). The CSF/plasma glycine ratio was 0.11. Oral sodium benzoate and folic acid therapy was initiated. After two weeks of assisted ventilation and clinical improvement, the patient was discharged with a protein-restricted diet.