Fifty years of research on mitochondrial fatty acid oxidation disorders: The remaining challenges.

Since the identification of the first disorder of mitochondrial fatty acid oxidation defects (FAOD) in 1973, more than 20 defects have been identified. Although there are some differences, most FAOD have similar clinical signs, which are mainly due to energy depletion and toxicity of accumulated metabolites. However, some of them have an unusual clinical phenotype or specific clinical signs. This manuscript focuses on what we have learnt so far on the pathophysiology of these disorders, which present with clinical signs that are not typical of categorical FAOD. It also highlights that some disorders have not yet been identified and tries to make assumptions to explain why. It also deals with new treatments under consideration in FAOD, including triheptanoin and similar anaplerotic substrates, ketone body treatments, RNA and gene therapy approaches. Finally, it suggests challenges for the diagnosis of FAOD in the coming years, both for symptomatic patients and for those diagnosed through newborn screening. The ultimate goal would be to identify all the patients born with FAOD and ensure for them the best possible quality of life.

The role of ERNDIM diagnostic proficiency schemes in improving the quality of diagnostic testing for inherited metabolic diseases.

External quality assurance (EQA) is crucial to monitor and improve the quality of biochemical genetic testing. ERNDIM (www.erndim.org), established in 1994, aims at reliable and standardized procedures for diagnosis, treatment and monitoring of inherited metabolic disease (IMD) by providing EQA schemes and educational activities. Currently, ERNDIM provides 16 different EQA schemes including quantitative schemes for various metabolite groups, and interpretive schemes such as diagnostic proficiency testing (DPT). DPT schemes focus on the ability of laboratories to correctly identify and interpret abnormalities in authentic urine samples across a wide range of IMDs. In the DPT schemes, six samples each year are distributed together with clinical information. Laboratories choose and perform the tests needed to reach a diagnosis. Data were collected on 345 samples, distributed to up to 105 laboratories worldwide. Diagnostic proficiency (the % of total points possible for all participating laboratories within a scheme for analysis and interpretation) ranged widely: amino acid disorders (n = 20), range 33%-100%, mean 84%; organic acid disorders (n = 35), range 14%-100%, mean 84%; lysosomal storage disorders (n = 13), range 20%-97%, mean 73%; purine/pyrimidine disorders (n = 9), range 37%-100%, mean 70%; miscellaneous disorders (n = 8), range 17%-100%, mean 65%; no IMD, range 65%-95%, mean 85%. When a sample with the same disorder was distributed in a subsequent survey, performance improved in 75 cases with no improvement seen in 32, suggesting overall improvement of performance. ERNDIM diagnostic proficiency testing is a valuable activity which can help to assess laboratory performance, identify methodological/technical challenges, be informative during quality audits and contribute to a better clinical appreciation of diagnostic uncertainty.

Three cases of adult-onset Brown-Vialetto-Van Laere syndrome: Novel variants in SLC52A3 gene and MRI abnormalities.

Brown-Vialetto-Van Laere syndrome is a rare, autosomal, recessive neurological condition caused by variants in the riboflavin transporter genes SLC52A2 and SLC52A3. Here, we report on three cases. Case 1 was a 35-year-old woman from a consanguineous family who presented with progressive deafness, subacute multiple cranial nerve impairments (III, VII, IX, XII), and MRI abnormalities (including as hypersignal from the cranial nerves). The patient was homozygous for a novel SLC52A3variant. Case 2 was the woman's brother, who presented similar symptoms. Case 3 was an 18-year-old woman experiencing progressive hearing loss, bilateral steppage gait and a cranial nerves impairment (VII and XII). MRI revealed hypersignal in the root nerves and cauda equina. A novel heterozygous variant in SLC52A3 was identified. A subacute history of polyradiculoneuropathy along with progressive deafness, cranial nerve impairment, and MRI abnormalities should raise suspicion for Brown-Vialetto-Van Laere syndrome.

Clinical outcomes in a series of 18 patients with long chain fatty acids oxidation disorders treated with triheptanoin for a median duration of 22 months.

INTRODUCTION: Triheptanoin provides long-chain fatty acid oxidation disorder (LC-FAOD) patients with an alternative to medium-even-chain triglycerides therapy. MATERIAL-METHODS: Retrospective analysis of 18 French LC-FAOD patients benefiting from early access to triheptanoin treatment. RESULTS: Eight female and 10 male patients with LC-FAOD (VLCAD, LCHAD, CACT, CPTII and MTP) were treated with triheptanoin for a median duration of 22 months (range: 9-228 months). At last consultation, triheptanoin accounted for 15-35% of their daily caloric intake. In the year following the introduction of triheptanoin, patients reported a reduction of intermittent snacking and nocturnal meals. Three patients, including 1 adult, became free of severe hypoglycaemic events. Ten of 12 paediatric patients and 4 of 6 adult patients reported reduced fatigue with reductions in the number and severity of episodes of myalgia. Of 6 patients, including 1 adult, that had required the use of a wheelchair in the year prior to triheptanoin, all but one no longer required its use. The number of emergency hospitalizations decreased, and none were recorded for paediatric patients during these 12 months. Cumulative annual days of emergency care in the home were reduced from 286 to 51 days in the year before and after initiation, respectively, and 13 patients required no such interventions. Adverse events were limited to digestive issues that dissipated over time. CONCLUSIONS: Our case-series suggests that long-term treatment of LC-FAOD paediatric and adult patients with triheptanoin is safe and leads to marked improvement of symptoms and an improved quality of life.

Adherence to cysteamine in nephropathic cystinosis: A unique electronic monitoring experience for a better understanding. A prospective cohort study: CrYSTobs.

INTRODUCTION: In nephropathic cystinosis (NC), adherence to cysteamine remains challenging; poor adherence is worsening the disease progression with a decline of kidney function and increase of extrarenal morbidities. Our objective was to describe adherence to cysteamine in NC patients, using electronic monitoring systems. METHODS: Patients with confirmed NC, aged > 4 years and receiving oral cysteamine (short acting or delayed release formulation as standard of care) from 3 French reference centers, were included. Adherence to treatment was primarily assessed as the percentage of days with a good adherence score, adherence score rating from 0 (poor) to 2 (good). A descriptive analysis was performed after 1-year follow-up. RESULTS: Seventeen patients (10 girls, median age: 13.9 (5.4-33.0) years) were included. Median age at diagnosis was 17.0 (3.0-76.9) months and age at start of cysteamine was 21.0 (15.5-116.3) months. Median daily dose of cysteamine was 1.05 (0.55-1.63) g/m2/day. Over the year, the median percentage of days with a good adherence score was 80 (1-99)% decreasing to 68 (1-99)% in patients > 11 years old. The median of average number of hours covered by treatment in a day was 22.5 (6.1-23.9) versus 14.9 (9.2-20.5) hours for delayed release versus short acting cysteamine. CONCLUSION: Our data are the first describing a rather good adherence to cysteamine, decreasing in adolescents and adults. We described a potential interest of the delayed release formulation. Our data highlight the need for a multidisciplinary approach including therapeutic education and individualized approaches in NC patients transitioning to adulthood. Graphical abstract.

Collaboration between academics, small pharmaceutical company and patient organizations in the development of a new formulation of cysteamine in nephropathic cystinosis: A successful story.

Rare diseases usually concern small and disseminated population. Implementing clinical research with the right design, outcomes measures and the recruitment of patients are challenges. Collaborations, training and multidisciplinary approach are often required. In this article, we provide an overview of a successful collaboration in nephropathic cystinosis (NC), focusing on what was the key of success, the interactions between academics, the pharmaceutical company and patients organizations. NC is considered as a very rare disease. In 2010, a new formulation of cysteamine, the only available treatment to improve renal outcome of the disease, was proposed by a small American company. Studies were implemented in France under the coordination of an expert of the disease and the clinical investigation center of Lyon. The collaboration resulted in a good recruitment and retention of the patients in the study and most of all in the availability of the new formulation in France. Patients could have facilitated the research by being involved in the early stages of the studies. Involving patients and public early in the process is particularly important in rare diseases as the patient is a great source of knowledge and has his own expectations. Priorities of research, design, conduct and reporting of clinical trials can be defined in collaboration with adults but also with young patients or public, the first concerned in rare diseases. This concept is still to be developed and improved especially with paediatric patients.

Neuropsychological and neuroanatomical phenotype in 17 patients with cystinosis.

BACKGROUND: Cystinosis is a rare autosomal recessive disorder caused by intracellular cystine accumulation. Proximal tubulopathy (Fanconi syndrome) is one of the first signs, leading to end-stage renal disease between the age of 12 and 16. Other symptoms occur later and encompass endocrinopathies, distal myopathy and deterioration of the central nervous system. Treatment with cysteamine if started early can delay the progression of the disease. Little is known about the neurological impairment which occurs later. The goal of the present study was to find a possible neuroanatomical dysmorphic pattern that could help to explain the cognitive profile of cystinosis patients. We also performed a detailed review of the literature on neurocognitive complications associated with cystinosis. METHODS: 17 patients (mean age = 17.6 years, [5.4-33.3]) with cystinosis were included in the study. Neuropsychological assessment was performed including intelligence (Intelligence Quotient (IQ) with Wechsler's scale), memory (Children Memory Scale and Wechsler Memory Scale), visuo-spatial (Rey's figure test) and visuo-perceptual skills assessments. Structural brain MRI (3 T) was also performed in 16 out of 17 patients, with high resolution 3D T1-weighted, 3D FLAIR and spectroscopy sequences. RESULTS: Intellectual efficiency was normal in patients with cystinosis (mean Total IQ = 93). However the Perceptual Reasoning Index (mean = 87, [63-109]) was significantly lower than the Verbal Comprehension Index (mean = 100, [59-138], p = 0.003). Memory assessment showed no difference between visual and verbal memory. But the working memory was significantly impaired in comparison with the general memory skills (p = 0.003). Visuospatial skills assessment revealed copy and reproduction scores below the 50th percentile rank in more than 70% of the patients. Brain MRI showed cortical and sub-cortical cerebral atrophy, especially in the parieto-occipital region and FLAIR hypersignals in parietal, occipital and brain stem/cerebellum. Patients with atrophic brain had lower Total IQ scores compared to non-atrophic cystinosis patients. CONCLUSIONS: Patients with cystinosis have a specific neuropsychological and neuroanatomical profile. We suggest performing a systematic neuropsychological assessment in such children aiming at considering adequate management.

Asparagine synthetase deficiency: A novel case with an unusual molecular mechanism.

We report the case of a girl with Asparagine synthetase deficiency, an autosomal recessive metabolic disorder characterized by severe microcephaly and epileptic encephalopathy secondary to pathogenic variants in the ASNS gene. Genetic explorations found a deletion of ASNS and a missense variant on the other allele detected respectively by array comparative genomic hybridization (CGH) and Sanger sequencing. Amino acid analysis provided a biochemical confirmation. Previous cases of Asparagine synthetase deficiency were diagnosed though exome Sequencing. The combination of several techniques (array CGH, sequencing, and biochemical analysis) improves the opportunity to provide accurate diagnosis.

Contribution of tandem mass spectrometry to the diagnosis of lysosomal storage disorders.

Tandem mass spectrometry (MS/MS) is a highly sensitive and specific technique. Thanks to the development of triple quadrupole analyzers, it is becoming more widely used in laboratories working in the field of inborn errors of metabolism. We review here the state of the art of this technique applied to the diagnosis of lysosomal storage disorders (LSDs) and how MS/MS has changed the diagnostic rationale in recent years. This fine technology brings more sensitive, specific, and reliable methods than the previous biochemical ones for the analysis of urinary glycosaminoglycans, oligosaccharides, and sialic acid. In sphingolipidoses, the quantification of urinary sphingolipids (globotriaosylceramide, sulfatides) is possible. The measurement of new plasmatic biomarkers such as oxysterols, bile acids, and lysosphingolipids allows the screening of many sphingolipidoses and related disorders (Niemann-Pick type C), replacing tedious biochemical techniques. Applied to amniotic fluid, a more reliable prenatal diagnosis or screening of LSDs is now available for fetuses presenting with antenatal manifestations. Applied to enzyme measurements, it allows high throughput assays for the screening of large populations, even newborn screening. The advent of this new method can modify the diagnostic rationale behind LSDs.

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 ß-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.

Correction: The genetic basis of classic nonketotic hyperglycinemia due to mutations in GLDC and AMT.

The original supplementary information included with this article contained several minor errors. Corrected Supplementary Information accompanies this corrigendum.

LC-MS/MS multiplex analysis of lysosphingolipids in plasma and amniotic fluid: A novel tool for the screening of sphingolipidoses and Niemann-Pick type C disease.

BACKGROUND: The biological diagnosis of sphingolipidoses currently relies on the measurement of specific enzymatic activities and/or genetic studies. Lysosphingolipids have recently emerged as potential biomarkers of sphingolipidoses and Niemann-Pick type C in plasma. METHODOLOGY: We developed a sensitive and specific method enabling the simultaneous quantification of lysosphingolipids by LC-MS/MS: lysoglobotriaosylceramide for Fabry disease, lysohexosylceramide (i.e. lysoglucosylceramide and/or lysogalactosylceramide) for Gaucher and Krabbe diseases, lysosphingomyelin and its carboxylated analogue lysosphingomyelin-509 for Niemann-Pick type A or B, and C diseases, lysoGM1 ganglioside for GM1gangliosidosis and lysoGM2 ganglioside for GM2 gangliosidosis. FINDINGS: The diagnostic performances were validated in plasma samples analysing a large series of patients affected with sphingolipidoses and Niemann-Pick type C disease (n = 98), other inborn errors of metabolism (n = 23), and controls (n = 228). The multiplex measurement of lysosphingolipids allowed the screening of Fabry (including female patients and late-onset variants), Gaucher and infantile Krabbe, Niemann-Pick type A/B and C diseases with high sensitivity and specificity. LysoGM1 and LysoGM2 were elevated in most of the patients affected with GM1 and GM2 gangliosidosis respectively. In amniotic fluid supernatant from pregnancies presenting non-immune hydrops fetalis (n = 77, including previously diagnosed Gaucher (n = 5), GM1 gangliosidosis (n = 4) and galactosialidosis (n = 4) fetuses) and from normal pregnancies (n = 15), a specific and dramatic increase of lysohexosylceramide was observed only in the Gaucher amniotic fluid samples. INTERPRETATION: This multiplex assay which allows the simultaneous measurement of lysosphingolipids in plasma modifies the diagnostic strategy of sphingolipidoses and Niemann-Pick type C. Furthermore, in pregnancies presenting non-immune hydrops fetalis, lysohexosylceramide measurement in amniotic fluid offers a rapid screening of fetal Gaucher disease without waiting for glucocerebrosidase activity measurement in cultured amniocytes.

Fluxomic evidence for impaired contribution of short-chain acyl-CoA dehydrogenase to mitochondrial palmitate ß-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 ß-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 ß-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 ß-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 ß-oxidation consistent with known altered kinetics of these variants.

Development of a new tandem mass spectrometry method for urine and amniotic fluid screening of oligosaccharidoses.

RATIONALE: The first step in the diagnosis of oligosaccharidoses is to evidence abnormal oligosaccharides excreted in urine, usually performed by the poorly sensitive but efficient thin layer chromatography (TLC) method. Developing a tandem mass spectrometry (MS/MS) technique could be of great interest to replace TLC. METHODS: Abnormal underivatized oligosaccharides have been recently studied using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, allowing the unambiguous identification of oligosaccharidoses. Based on this previous work, we developed an advantageous and efficient liquid chromatography (LC)/MS/MS method using a more common triple quadrupole tandem mass spectrometer for oligosaccharides analysis. RESULTS: Oligosaccharidoses (n = 97) and control (n = 240) urine samples were analysed. A specific pattern was obtained for each oligosaccharidosis using this method. In urine, it allows not only the identification of all the oligosaccharidoses previously identified by TLC (fucosidosis, alphamannosidosis, aspartylglucosaminuria, GM1 gangliosidosis, sialidosis, galactosialidosis and Schindler disease), but also extends the field of diagnosis to mucolipidosis type II, Sandhoff disease, and ß-mannosidosis. The same technique was applied to 16 amniotic fluid supernatants from oligosaccharidosis-affected foetuses (n = 16) compared with 37 unaffected. All the affected foetuses could be clearly identified: sialidosis (n = 3), galactosialidosis (n = 4), aspartylglucosaminuria (n = 1), mucolipidosis type II (n = 4) or GM1 gangliosidosis (n = 4). This technique can be applied to early prenatal diagnosis as well as to the oligosaccharidosis screening in the case of non-immune hydrops fetalis. CONCLUSIONS: The method is quick and easy to run, with an LC analysis time of 13 min per sample. The quantitative validation could not be obtained in the absence of a specific standard and of a labelled internal standard for each compound. Even if this LC/MS/MS method is only qualitative, it is very specific and much more sensitive than TLC. It allows the urinary screening of oligosaccharidoses, even mild or late-onset forms, and the screening of antenatal forms in amniotic fluid. Copyright © 2017 John Wiley & Sons, Ltd.

Pharmacological inhibition of carnitine palmitoyltransferase 1 restores mitochondrial oxidative phosphorylation in human trifunctional protein deficient fibroblasts.

BACKGROUND: Mitochondrial Trifunctional Protein deficiency (TFPD) is a severe genetic disease characterized by altered energy metabolism and accumulation of long-chain (LC) acylcarnitines in blood and tissues. This accumulation could impair the mitochondrial oxidative phosphorylation (OxPhos), contributing to the non-optimal outcome despite conventional diet therapy with medium-chain triglycerides (MCT). METHOD: Acylcarnitine and OxPhos parameters were measured in TFPD-fibroblasts obtained from 8 children and cultured in medium mimicking fasting (LCFA) or conventional treatment (MCT), with or without Etomoxir (ETX) an inhibitor of carnitine palmitoyltransferase 1 (CPT1) activity, and were compared to results obtained with fibroblasts from 5 healthy-control children. The effects of various acylcarnitines were also tested on control fibroblasts. RESULTS: In the LCFA-condition, TFPD-fibroblasts demonstrated a large accumulation of LC-acylcarnitines associated with decreased O2-consumption (63±3% of control, P<0.001) and ATP production (67±5%, P<0.001) without modification of coupling efficiency. A dose-dependent decrease in O2-consumption was reproduced in control fibroblasts by addition of increasing dose of LC-acylcarnitines, while it was almost preserved with MC-acylcarnitines. The MCT-condition reduced LC-acylcarnitine accumulation and partially improved O2-consumption (80±3%, P<0.01) in TFPD-fibroblasts. The addition of ETX in both LCFA- and MCT-conditions normalized acylcarnitine profiles and restored O2-consumption and ATP production at the same levels than control. CONCLUSION: Accumulation of LC-acylcarnitines plays a major role in the pathophysiology of TFPD, reducing OxPhos capacities. These deleterious effects could be partially prevented by MCT-therapy and totally corrected by ETX. Inhibition of CPT1 may be view as a new therapeutic target for patients with a severe form of TFPD.

Mitochondrial acetoacetyl-CoA thiolase deficiency: basal ganglia impairment may occur independently of ketoacidosis.

BACKGROUND: Mitochondrial acetoacetyl-CoA thiolase (T2) deficiency affects ketone body and isoleucine catabolism. Neurological impairment may occur secondary to ketoacidotic episodes. However, we observed neuromotor abnormalities without ketoacidotic events in two T2-deficient families. We hypothesized that the neurological signs were related to the genetic defect and may occur independently of ketoacidotic episodes. We therefore conducted a retrospective review on a French T2-deficient patient series searching for neuromotor impairment. METHODS: In total, 26 cases were retrospectively analysed for clinical, biological and neuroimaging data. RESULTS: Neurological findings were observed for 6/26 (23%) patients. Among these, two had never experienced ketoacidotic episodes, though they developed extrapyramidal signs with putamen involvement. Two of the other four patients developed neurological abnormalities before the first ketoacidotic crisis, with putamen involvement in one case. The third patient developed extrapyramidal symptoms more than 10 years after the initial decompensation with globus pallidus involvement. The last patient developed extrapyramidal signs immediately after a severe ketoacidotic crisis with putaminal lesions. CONCLUSIONS: Most T2-deficient patients achieved normal neurodevelopment. However, on account of the role of T2 in isoleucine catabolism, these patients are potentially exposed to accumulation of toxic isoleucine-derived metabolites, which may contribute to neurological impairment. Our findings confirm previous observations that neurological symptoms in T2 deficiency may occur unrelated to ketoacidosis. The role of protein restriction as a preventive measure against neurological symptoms could not be established in this study and deserves further evaluation. Long-term follow-up data on children diagnosed by newborn screening may clarify the pathogenesis of this neurometabolic association.