Dries Dobbelaere

Diagnosis, Symptoms, Frequency and Mortality of 260 Patients with Urea Cycle Disorders from a 21-Year, Multicentre Study of Acute Hyperammonaemic Episodes

Aim: A large longitudinal interventional study of patients with a urea cycle disorder (UCD) in hyperammonaemic crisis was undertaken to amass a significant body of data on their presenting symptoms and survival.

Creatine biosynthesis and transport in health and disease

Creatine is physiologically provided equally by diet and by endogenous synthesis from arginine and glycine with successive involvements of arginine glycine amidinotransferase [AGAT] and guanidinoacetate methyl transferase [GAMT]. A specific plasma membrane transporter, creatine transporter [CRTR] (SLC6A8), further enables cells to incorporate creatine and through uptake of its precursor, guanidinoacetate, also directly contributes to creatine biosynthesis. Breakthrough in the role of creatine has arisen from studies on creatine deficiency disorders. Primary creatine disorders are inherited as autosomal recessive (mutations affecting GATM [for glycine-amidinotransferase, mitochondrial]) and GAMT genes) or X-linked (SLC6A8 gene) traits. They have highlighted the role of creatine in brain functions altered in patients (global developmental delay, intellectual disability, behavioral disorders). Creatine modulates GABAergic and glutamatergic cerebral pathways, presynaptic CRTR (SLC6A8) ensuring re-uptake of synaptic creatine. Secondary creatine disorders, addressing other genes, have stressed the extraordinary imbrication of creatine metabolism with many other cellular pathways. This high dependence on multiple pathways supports creatine as a cellular sensor, to cell methylation and energy status. Creatine biosynthesis consumes 40% of methyl groups produced as S-adenosylmethionine, and creatine uptake is controlled by AMP activated protein kinase, a ubiquitous sensor of energy depletion. Today, creatine is considered as a potential sensor of cell methylation and energy status, a neurotransmitter influencing key (GABAergic and glutamatergic) CNS neurotransmission, therapeutic agent with anaplerotic properties (towards creatine kinases [creatine-creatine phosphate cycle] and creatine neurotransmission), energetic and antioxidant compound (benefits in degenerative diseases through protection against energy depletion and oxidant species) with osmolyte behavior (retention of water by muscle). This review encompasses all these aspects by providing an illustrated metabolic account for brain and body creatine in health and disease, an algorithm to diagnose metabolic and gene bases of primary and secondary creatine deficiencies, and a metabolic exploration by (1)H-MRS assessment of cerebral creatine levels and response to therapeutic measures.

Comprehensive cDNA study and quantitative analysis of mutant HADHA and HADHB transcripts in a French cohort of 52 patients with mitochondrial trifunctional protein deficiency

BACKGROUND Deficiency of mitochondrial trifunctional protein (MTP) is caused by mutations in the HADHA and HADHB genes, which have been mostly delineated at the genomic DNA level and have not been always elucidated. AIM To identify mutations in a French cohort of 52 MTP deficient patients and the susceptibility of mutations generating premature termination codons (PTCs) to the nonsense mRNA mediated decay (NMD). METHODS Mutation screening in fibroblasts was performed at the cDNA level and real-time RT-PCR was used to compare the levels of the different PTC-bearing mRNAs before and after a treatment of fibroblasts by emetine, a translation inhibitor. RESULTS A mutation detection rate of 100% was achieved. A total of 22 novel mutations were identified, including a large-sized genomic deletion in HADHB gene. A high proportion of all identified mutations were non-sense, frameshift and splicing mutations, generating (PTCs), distributed essentially on HADHA coding regions. We could demonstrate that the majority of mutations resulting in PTCs conform to the established rules governing the susceptibility to NMD. CONCLUSION Our results emphasize the value of cDNA analysis in the characterization of HADHA and HADHB mutations and further strengthen the model of haploinsufficiency as a major pathomechanism in MTP defects.

Age at disease onset and peak ammonium level rather than interventional variables predict the neurological outcome in urea cycle disorders

BackgroundPatients with urea cycle disorders (UCDs) have an increased risk of neurological disease manifestation.AimsDetermining the effect of diagnostic and therapeutic interventions on the neurological outcome.MethodsEvaluation of baseline, regular follow-up and emergency visits of 456 UCD patients prospectively followed between 2011 and 2015 by the E-IMD patient registry.ResultsAbout two-thirds of UCD patients remained asymptomatic until age 12 days [i.e. the median age at diagnosis of patients identified by newborn screening (NBS)] suggesting a potential benefit of NBS. In fact, NBS lowered the age at diagnosis in patients with late onset of symptoms (>28 days), and a trend towards improved long-term neurological outcome was found for patients with argininosuccinate synthetase and lyase deficiency as well as argininemia identified by NBS. Three to 17 different drug combinations were used for maintenance therapy, but superiority of any single drug or specific drug combination above other combinations was not demonstrated. Importantly, non-interventional variables of disease severity, such as age at disease onset and peak ammonium level of the initial hyperammonemic crisis (cut-off level: 500 μmol/L) best predicted the neurological outcome.ConclusionsPromising results of NBS for late onset UCD patients are reported and should be re-evaluated in a larger and more advanced age group. However, non-interventional variables affect the neurological outcome of UCD patients. Available evidence-based guideline recommendations are currently heterogeneously implemented into practice, leading to a high variability of drug combinations that hamper our understanding of optimised long-term and emergency treatment.

Spinocerebellar Ataxia: A Rational Approach to Aetiological Diagnosis

The objective of this study was to determine the main causal diagnosis for spinocerebellar ataxia (SCA) in a geographically defined population of ataxia patients and to suggest a rational basis for choosing appropriate clinical and paraclinical assessments. Given the many aetiologies responsible for SCA, the diagnosis requires the performance of a wide range of paraclinical analyses. At present, there is no consensus on the diagnostic value of these examinations. Furthermore, most of the currently available data gathered by reference centres suffer from selection bias. We performed a prospective study of consecutive cerebellar ataxia patients referred by their family doctors to a university hospital in northern France. Multiple system atrophy and obvious secondary causes (e.g. alcoholism) were excluded by our screening process. The patient’s family members were also assessed. Of the 204 patients examined, 47% presented autosomal dominant ataxia and 33% presented sporadic ataxia. Autosomal recessive ataxia was rare (8%) and age at onset was significantly earlier for this condition than for other forms. An aetiological diagnosis was established in 44% of patients, a plausible hypothesis could be formed in 13% of cases, and no diagnosis was made in the remaining 44%. Established diagnoses included SCA1, SCA2, SCA3 and SCA6 mutations, Friedreich’s ataxia, and one rare case of ataxia associated with anti-glutamic acid decarboxylase antibodies. Two families presented ataxia associated with autosomal, dominant, optic atrophy with an OPA1 mutation. Mitochondrial diseases were suspected in about 10% of patients. In SCA, reliable determination of the transmission mode always requires the assessment of family members. Mitochondrial disease may be an emerging cause of ataxia. Metabolite assays appeared to be of little value when systematically performed and so should be prescribed only by metabolic disorder specialists in selected cases of sporadic and recessive ataxia. Ophthalmological examination was the most helpful physiological assessment.

Impact of miglustat on evolution of atypical presentation of late-infantile-onset Niemann–Pick disease type C with early cognitive impairment, behavioral dysfunction, epilepsy, ophthalmoplegia, and cerebellar involvement: a case report

BackgroundNiemann–Pick disease type C is a rare inherited neurodegenerative disease involving impaired intracellular lipid trafficking and accumulation of glycolipids in various tissues, including the brain. Miglustat, a reversible inhibitor of glucosylceramide synthase, has been shown to be effective in the treatment of progressive neurological manifestations in pediatric and adult patients with Niemann–Pick disease type C, and has been used in that indication in Europe since 2010.Case presentationWe describe the case of a 16-year-old white French boy with late-infantile-onset Niemann–Pick disease type C who had the unusual presentation of early-onset behavioral disturbance and learning difficulties (aged 5) alongside epileptic seizures. Over time he developed characteristic, progressive vertical ophthalmoplegia, ataxic gait, and cerebellar syndrome; at age 10 he was diagnosed as having Niemann–Pick disease type C based on filipin staining and genetic analysis (heterozygous I1061T/R934X NPC1 mutations). He was commenced on miglustat therapy aged 11 and over the course of approximately 3 years he showed a global improvement as well as improved cognitive and ambulatory function. During this period he remained seizure free on antiepileptic therapy, using valproate and lamotrigine.ConclusionsMiglustat improved the neurological status of our patient, including seizure control. Based on our findings in this patient and previous published data, we discuss the importance of effective seizure control in neurological improvement in Niemann–Pick disease type C, and the relevance of cerebellar involvement as a possible link between these clinical phenomena. Thus the therapeutic efficacy of miglustat could be hypothesized as a substrate reduction effect on Purkinje cells.

Fluxomic evidence for impaired contribution of short-chain acyl-CoA dehydrogenase to mitochondrial palmitate β-oxidation in symptomatic patients with ACADS gene susceptibility variants

BACKGROUND Despite ACADS (acyl-CoA dehydrogenase, short-chain) gene susceptibility variants (c.511C>T and c.625G>A) are considered to be non-pathogenic, encoded proteins are known to exhibit altered kinetics. Whether or not, they might affect overall fatty acid β-oxidation still remains, however, unclear. METHODS De novo biosynthesis of acylcarnitines by whole blood samples incubated with deuterated palmitate (16-2H3,15-2H2-palmitate) is suitable as a fluxomic exploration to distinguish between normal and disrupted β-oxidation, abnormal profiles and ratios of acylcarnitines with different chain-lengths being indicative of the site for enzymatic blockade. Determinations in 301 control subjects of ratios between deuterated butyrylcarnitine and sum of deuterated C2 to C14 acylcarnitines served here as reference values to state specifically functional SCAD impairment in patients addressed for clinical and/or biological suspicion of a β-oxidation disorder. RESULTS Functional SCAD impairment was found in 39 patients. The 27 patients accepting subsequent gene studies were all positive for ACADS mutations. Twenty-six of 27 patients were positive for c.625G>A variant. Twenty-three of 27 patients harbored susceptibility variants as sole ACADS alterations (18 homozygous and 3 heterozygous for c.625G>A, 2 compound heterozygous for c.625G>A/c.511C>T). CONCLUSION Our present fluxomic assessment of SCAD suggests a link between ACADS susceptibility variants and abnormal β-oxidation consistent with known altered kinetics of these variants.

Propranolol et lactatémie durant un choc hypovolémique non septique : à propos d’une observation

Lactate production results from anaerobic glycolysis. This pathway is recruited physiologically during intense and sustained muscular contractions. Hyperlactatemia may develop when tissue oxygenation is jeopardized such as in shock, its absence having been, however, sometimes reported in sepsis in which interactions between infectious agents and the organisms cells might blunt or disrupt hyperlactatemia development. During the course of acute rotavirus gastroenteritis, a 9-month-old girl developed severe dehydration (capillary-refill time, 5 s) leading to hypovolemic shock without signs of sepsis and with hypotension at 62/21 mmHg Surprisingly, the child failed to develop hyperlactatemia during shock. An etiologic search to understand why hyperlactatemia did not occur revealed that this patient had been receiving propranolol since the age of four months for the treatment of a Cyrano hemangioma. Via its inhibitory action on β-adrenergic receptors, propranolol antagonizes the stimulation of glycolysis by catecholamines, which may be rationally proposed to have contributed to preventing hyperlactatemia during hypovolemic shock in this patient. Mechanisms by which propranolol can mediate this antihyperlactatemia action are further illustrated and discussed.

Fluxomic assay-assisted diagnosis orientation in a cohort of 11 patients with myopathic form of CPT2 deficiency

Carnitine palmitoyltransferase type 2 (CPT2) deficiency, a mitochondrial fatty acid oxidation disorder (MFAOD), is a cause of myopathy in its late clinical presentation. As for other MFAODs, its diagnosis may be evocated when blood acylcarnitine profile is abnormal. However, a lack of abnormalities or specificity in this profile is not exclusive of CPT2 deficiency. Our retrospective study reports clinical and biological data in a cohort of 11 patients with circulating acylcarnitine profile unconclusive enough for a specific diagnosis orientation. In these patients, CPT2 gene studies was prompted by prior fluxomic explorations of mitochondrial β-oxidation on intact whole blood cells incubated with pentadeuterated ([16-2H3, 15-2H2])-palmitate. Clinical indication for fluxomic explorations was at least one acute rhabdomyolysis episode complicated, in 5 of 11 patients, by acute renal failure. Major trigger of rhabdomyolysis was febrile infection. In all patients, fluxomic data indicated deficient CPT2 function showing normal deuterated palmitoylcarnitine (C16-Cn) formation rates associated with increased ratios between generated C16-Cn and downstream deuterated metabolites (Σ deuterated C2-Cn to C14-Cn). Subsequent gene studies showed in all patients pathogenic gene variants in either homozygous or compound heterozygous forms. Consistent with literature data, allelic frequency of the c.338C > T[p.Ser113Leu] mutation amounted to 68.2% in our cohort. Other missense mutations included c.149C > A[p.Pro50His] (9%), c.200C > G[p.Ala200Gly] (4.5%) and previously unreported c.1171A > G[p.ser391Gly] (4.5%) and c.1420G > C[p.Ala474Pro] (4.5%) mutations. Frameshift c.1666-1667delTT[p.Leu556val*16] mutation (9%) was observed in two patients unknown to be related.

Coupled brain and urine spectroscopy - in vivo metabolomic characterization of HMG-CoA lyase deficiency in 5 patients

BACKGROUND 3-Hydroxy-3-Methylglutaryl-Coenzyme A (HMG-CoA) lyase deficiency is a rare inborn error of leucine metabolism and ketogenesis. Despite recurrent hypoglycemia and metabolic decompensations, most patients have a good clinical and neurological outcome contrasting with abnormal brain magnetic resonance imaging (MRI) signals and consistent abnormal brain proton magnetic resonance spectroscopy (1H-MRS) metabolite peaks. Identifying these metabolites could provide surrogate markers of the disease and improve understanding of MRI-clinical discrepancy and follow-up of affected patients. METHODS Urine samples, brain MRI and 1H-MRS in 5 patients with HMG-CoA lyase deficiency (4 boys and 1 girl aged from 25days to 10years) were, for each patient, obtained on the same day. Brain and urine spectroscopy were performed at the same pH by studying urine at pH 7.4. Due to pH-induced modifications in chemical shifts and because reference 1H NMR spectra are obtained at pH 2.5, spectroscopy of normal urine added with the suspected metabolite was further performed at this pH to validate the correct identification of compounds. RESULTS Mild to extended abnormal white matter MRI signals were observed in all cases. Brain spectroscopy abnormal peaks at 0.8-1.1ppm, 1.2-1.4ppm and 2.4ppm were also detected by urine spectroscopy at pH 7.4. Taking into account pH-induced changes in chemical shifts, brain abnormal peaks in patients were formally identified to be those of 3-hydroxyisovaleric, 3-methylglutaconic, 3-methylglutaric and 3-hydroxy-3-methylglutaric acids. CONCLUSION 3-Methylglutaric, 3-hydroxyisovaleric and 3-hydroxy-3-methylglutaric acids identified on urine 1H-NMR spectra of 5 patients with HMG-CoA lyase deficiency are responsible for the cerebral spectroscopy signature seen in these patients, validating their local involvement in brain and putative contribution to brain neuropathology.