Treatment of Mitochondrial Phenylalanyl-tRNa-Synthetase Deficiency (FARS2) with Oral Phenylalanine.

OBJECTIVE: By loading transfer RNAs with their cognate amino acids, aminoacyl-tRNA synthetases (ARS) are essential for protein translation. Both cytosolic ARS1-deficiencies and mitochondrial ARS2 deficiencies can cause severe diseases. Amino acid supplementation has shown to positively influence the clinical course of four individuals with cytosolic ARS1 deficiencies. We hypothesize that this intervention could also benefit individuals with mitochondrial ARS2 deficiencies. METHODS: This study was designed as a N-of-1 trial. Daily oral L-phenylalanine supplementation was used in a 3-year-old girl with FARS2 deficiency. A period without supplementation was implemented to discriminate the effects of treatment from age-related developments and continuing physiotherapy. Treatment effects were measured through a physiotherapeutic testing battery, including movement assessment battery for children, dynamic gait index, gross motor function measure 66, and quality of life questionnaires. RESULTS: The individual showed clear improvement in all areas tested, especially in gross motor skills, movement abilities, and postural stability. In the period without supplementation, she lost newly acquired motor skills but regained these upon restarting supplementation. No adverse effects and good tolerance of treatment were observed. INTERPRETATION AND CONCLUSION: Our positive results encourage further studies both on L-phenylalanine for other individuals with FARS2 deficiency and the exploration of this treatment rationale for other ARS2 deficiencies. Additionally, treatment costs were relatively low at 1.10 €/day.

Genotypic and phenotypic spectrum of infantile liver failure due to pathogenic TRMU variants.

PURPOSE: This study aimed to define the genotypic and phenotypic spectrum of reversible acute liver failure (ALF) of infancy resulting from biallelic pathogenic TRMU variants and determine the role of cysteine supplementation in its treatment. METHODS: Individuals with biallelic (likely) pathogenic variants in TRMU were studied within an international retrospective collection of de-identified patient data. RESULTS: In 62 individuals, including 30 previously unreported cases, we described 47 (likely) pathogenic TRMU variants, of which 17 were novel, and 1 intragenic deletion. Of these 62 individuals, 42 were alive at a median age of 6.8 (0.6-22) years after a median follow-up of 3.6 (0.1-22) years. The most frequent finding, occurring in all but 2 individuals, was liver involvement. ALF occurred only in the first year of life and was reported in 43 of 62 individuals; 11 of whom received liver transplantation. Loss-of-function TRMU variants were associated with poor survival. Supplementation with at least 1 cysteine source, typically N-acetylcysteine, improved survival significantly. Neurodevelopmental delay was observed in 11 individuals and persisted in 4 of the survivors, but we were unable to determine whether this was a primary or a secondary consequence of TRMU deficiency. CONCLUSION: In most patients, TRMU-associated ALF was a transient, reversible disease and cysteine supplementation improved survival.

Thiamine Treatment and Favorable Outcome in an Infant with Biallelic TPK1 Variants.

Episodic encephalopathy due to mutations in the thiamine pyrophosphokinase 1 (TPK1) gene is a rare autosomal recessive metabolic disorder. Patients reported so far have onset in early childhood of acute encephalopathic episodes, which result in a progressive neurologic dysfunction including ataxia, dystonia, and spasticity. Here, we report the case of an infant with TPK1 deficiency (compound heterozygosity for two previously described pathogenic variants) presenting with two encephalopathic episodes and clinical stabilization under oral thiamine and biotin supplementation. In contrast to other reported cases, our patient showed an almost normal psychomotor development, which might be due to an early diagnosis and subsequent therapy.

Expanding the clinical and genetic spectrum of CAD deficiency: an epileptic encephalopathy treatable with uridine supplementation.

PURPOSE: Biallelic CAD variants underlie CAD deficiency (or early infantile epileptic encephalopathy-50, [EIEE-50]), an error of pyrimidine de novo biosynthesis amenable to treatment via the uridine salvage pathway. We further define the genotype and phenotype with a focus on treatment. METHODS: Retrospective case series of 20 patients. RESULTS: Our study confirms CAD deficiency as a progressive EIEE with recurrent status epilepticus, loss of skills, and dyserythropoietic anemia. We further refine the phenotype by reporting a movement disorder as a frequent feature, and add that milder courses with isolated developmental delay/intellectual disability can occur as well as onset with neonatal seizures. With no biomarker available, the diagnosis relies on genetic testing and functional validation in patient-derived fibroblasts. Underlying pathogenic variants are often rated as variants of unknown significance, which could lead to underrecognition of this treatable disorder. Supplementation with uridine, uridine monophosphate, or uridine triacetate in ten patients was safe and led to significant clinical improvement in most patients. CONCLUSION: We advise a trial with uridine (monophosphate) in all patients with developmental delay/intellectual disability, epilepsy, and anemia; all patients with status epilepticus; and all patients with neonatal seizures until (genetically) proven otherwise or proven unsuccessful after 6 months. CAD deficiency might represent a condition for genetic newborn screening.

Choline-related-inherited metabolic diseases-A mini review.

In humans, the important water soluble, vitamin-like nutrient choline, is taken up with the diet or recycled in the liver. Deficiencies of choline have only been reported in experimental situations or total parenteral nutrition. Currently, no recommended dietary allowances are published; only an adequate daily intake is defined. Choline is involved in three main physiological processes: structural integrity and lipid-derived signaling for cell membranes, cholinergic neurotransmission, and methylation. Choline is gaining increasing public attention due to studies reporting a relation of low choline levels to subclinical organ dysfunction (nonalcoholic fatty liver or muscle damage), stunting, and neural tube defects. Furthermore, positive effects on memory and a lowering of cardiovascular risks and inflammatory markers have been proposed. On the other hand, dietary choline has been associated with increased atherosclerosis in mice. This mini review will provide a summary of the biochemical pathways, in which choline is involved and their respective inborn errors of metabolism (caused by mutations in SLC5A7, CHAT, SLC44A1, CHKB, PCYT1A, CEPT1, CAD; DHODH, UMPS, FMO3, DMGDH, and GNMT). The broad phenotypic spectrum ranging from malodor, intellectual disability, to epilepsy, anemia, or congenital myasthenic syndrome is presented, highlighting the central role of choline within human metabolism.

Clinical, biochemical and genetic spectrum of 70 patients with ACAD9 deficiency: is riboflavin supplementation effective?

BACKGROUND: Mitochondrial acyl-CoA dehydrogenase family member 9 (ACAD9) is essential for the assembly of mitochondrial respiratory chain complex I. Disease causing biallelic variants in ACAD9 have been reported in individuals presenting with lactic acidosis and cardiomyopathy. RESULTS: We describe the genetic, clinical and biochemical findings in a cohort of 70 patients, of whom 29 previously unpublished. We found 34 known and 18 previously unreported variants in ACAD9. No patients harbored biallelic loss of function mutations, indicating that this combination is unlikely to be compatible with life. Causal pathogenic variants were distributed throughout the entire gene, and there was no obvious genotype-phenotype correlation. Most of the patients presented in the first year of life. For this subgroup the survival was poor (50% not surviving the first 2 years) comparing to patients with a later presentation (more than 90% surviving 10 years). The most common clinical findings were cardiomyopathy (85%), muscular weakness (75%) and exercise intolerance (72%). Interestingly, severe intellectual deficits were only reported in one patient and severe developmental delays in four patients. More than 70% of the patients were able to perform the same activities of daily living when compared to peers. CONCLUSIONS: Our data show that riboflavin treatment improves complex I activity in the majority of patient-derived fibroblasts tested. This effect was also reported for most of the treated patients and is mirrored in the survival data. In the patient group with disease-onset below 1 year of age, we observed a statistically-significant better survival for patients treated with riboflavin.

Biallelic Mutations in LIPT2 Cause a Mitochondrial Lipoylation Defect Associated with Severe Neonatal Encephalopathy.

Lipoate serves as a cofactor for the glycine cleavage system (GCS) and four 2-oxoacid dehydrogenases functioning in energy metabolism (α-oxoglutarate dehydrogenase [α-KGDHc] and pyruvate dehydrogenase [PDHc]), or amino acid metabolism (branched-chain oxoacid dehydrogenase, 2-oxoadipate dehydrogenase). Mitochondrial lipoate synthesis involves three enzymatic steps catalyzed sequentially by lipoyl(octanoyl) transferase 2 (LIPT2), lipoic acid synthetase (LIAS), and lipoyltransferase 1 (LIPT1). Mutations in LIAS have been associated with nonketotic hyperglycinemia-like early-onset convulsions and encephalopathy combined with a defect in mitochondrial energy metabolism. LIPT1 deficiency spares GCS deficiency and has been associated with a biochemical signature of combined 2-oxoacid dehydrogenase deficiency leading to early death or Leigh-like encephalopathy. We report on the identification of biallelic LIPT2 mutations in three affected individuals from two families with severe neonatal encephalopathy. Brain MRI showed major cortical atrophy with white matter abnormalities and cysts. Plasma glycine was mildly increased. Affected individuals' fibroblasts showed reduced oxygen consumption rates, PDHc, α-KGDHc activities, leucine catabolic flux, and decreased protein lipoylation. A normalization of lipoylation was observed after expression of wild-type LIPT2, arguing for LIPT2 requirement in intramitochondrial lipoate synthesis. Lipoic acid supplementation did not improve clinical condition nor activities of PDHc, α-KGDHc, or leucine metabolism in fibroblasts and was ineffective in yeast deleted for the orthologous LIP2.

Effects of alpha-melanocyte-stimulating hormone on mitochondrial energy metabolism in rats of different age-groups.

Hypothalamic alpha-melanocyte-stimulating hormone (α-MSH) is a key catabolic mediator of energy homeostasis. Its anorexigenic and hypermetabolic effects show characteristic age-related alterations that may be part of the mechanism of middle-aged obesity and geriatric anorexia/cachexia seen in humans and other mammals. We aimed to investigate the role of α-MSH in mitochondrial energy metabolism during the course of aging in a rodent model. To determine the role of α-MSH in mitochondrial energy metabolism in muscle, we administered intracerebroventricular (ICV) infusions of α-MSH for 7-days to different age-groups of male Wistar rats. The activities of oxidative phosphorylation complexes I to V and citrate synthase were determined and compared to those of age-matched controls. We also quantified mitochondrial DNA (mtDNA) copy number and measured the expression of the master regulators of mitochondrial biogenesis, peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and peroxisome proliferator-activated receptor gamma (PPARγ). The peptide reduced weight gain in juvenile rats to one fifth of that of controls and increased the weight loss in older animals by about five fold. Mitochondrial DNA copy number inversely correlated with changes in body weight in controls, but not in α-MSH-treated animals. The strong increase in body weight in young rats was associated with a low mtDNA copy number and high PPARγ mRNA levels in controls. Expression of PGC-1α and PPARγ declined with age, whereas OXPHOS and citrate synthase enzyme activities were unchanged. In contrast, α-MSH treatment suppressed OXPHOS enzyme and citrate synthase activity. In conclusion, our results showed age-related differences in the metabolic effects of α-MSH. In addition, administration of α-MSH suppressed citrate synthase and OXPHOS activities independent of age. These findings suggest that α-MSH exposure may inhibit mitochondrial biogenesis.

NAXE Mutations Disrupt the Cellular NAD(P)HX Repair System and Cause a Lethal Neurometabolic Disorder of Early Childhood.

To safeguard the cell from the accumulation of potentially harmful metabolic intermediates, specific repair mechanisms have evolved. APOA1BP, now renamed NAXE, encodes an epimerase essential in the cellular metabolite repair for NADHX and NADPHX. The enzyme catalyzes the epimerization of NAD(P)HX, thereby avoiding the accumulation of toxic metabolites. The clinical importance of the NAD(P)HX repair system has been unknown. Exome sequencing revealed pathogenic biallelic mutations in NAXE in children from four families with (sub-) acute-onset ataxia, cerebellar edema, spinal myelopathy, and skin lesions. Lactate was elevated in cerebrospinal fluid of all affected individuals. Disease onset was during the second year of life and clinical signs as well as episodes of deterioration were triggered by febrile infections. Disease course was rapidly progressive, leading to coma, global brain atrophy, and finally to death in all affected individuals. NAXE levels were undetectable in fibroblasts from affected individuals of two families. In these fibroblasts we measured highly elevated concentrations of the toxic metabolite cyclic-NADHX, confirming a deficiency of the mitochondrial NAD(P)HX repair system. Finally, NAD or nicotinic acid (vitamin B3) supplementation might have therapeutic implications for this fatal disorder.

Biallelic IARS Mutations Cause Growth Retardation with Prenatal Onset, Intellectual Disability, Muscular Hypotonia, and Infantile Hepatopathy.

tRNA synthetase deficiencies are a growing group of genetic diseases associated with tissue-specific, mostly neurological, phenotypes. In cattle, cytosolic isoleucyl-tRNA synthetase (IARS) missense mutations cause hereditary weak calf syndrome. Exome sequencing in three unrelated individuals with severe prenatal-onset growth retardation, intellectual disability, and muscular hypotonia revealed biallelic mutations in IARS. Studies in yeast confirmed the pathogenicity of identified mutations. Two of the individuals had infantile hepatopathy with fibrosis and steatosis, leading in one to liver failure in the course of infections. Zinc deficiency was present in all affected individuals and supplementation with zinc showed a beneficial effect on growth in one.

Free-thiamine is a potential biomarker of thiamine transporter-2 deficiency: a treatable cause of Leigh syndrome.

Thiamine transporter-2 deficiency is caused by mutations in the SLC19A3 gene. As opposed to other causes of Leigh syndrome, early administration of thiamine and biotin has a dramatic and immediate clinical effect. New biochemical markers are needed to aid in early diagnosis and timely therapeutic intervention. Thiamine derivatives were analysed by high performance liquid chromatography in 106 whole blood and 38 cerebrospinal fluid samples from paediatric controls, 16 cerebrospinal fluid samples from patients with Leigh syndrome, six of whom harboured mutations in the SLC19A3 gene, and 49 patients with other neurological disorders. Free-thiamine was remarkably reduced in the cerebrospinal fluid of five SLC19A3 patients before treatment. In contrast, free-thiamine was slightly decreased in 15.2% of patients with other neurological conditions, and above the reference range in one SLC19A3 patient on thiamine supplementation. We also observed a severe deficiency of free-thiamine and low levels of thiamine diphosphate in fibroblasts from SLC19A3 patients. Surprisingly, pyruvate dehydrogenase activity and mitochondrial substrate oxidation rates were within the control range. Thiamine derivatives normalized after the addition of thiamine to the culture medium. In conclusion, we found a profound deficiency of free-thiamine in the CSF and fibroblasts of patients with thiamine transporter-2 deficiency. Thiamine supplementation led to clinical improvement in patients early treated and restored thiamine values in fibroblasts and cerebrospinal fluid.

The spectrum of pyruvate oxidation defects in the diagnosis of mitochondrial disorders.

Pyruvate oxidation defects (PODs) are among the most frequent causes of deficiencies in the mitochondrial energy metabolism and represent a substantial subset of classical mitochondrial diseases. PODs are not only caused by deficiency of subunits of the pyruvate dehydrogenase complex (PDHC) but also by various disorders recently described in the whole pyruvate oxidation route including cofactors, regulation of PDHC and the mitochondrial pyruvate carrier. Our own patients from 2000 to July 2014 and patients identified by a systematic survey of the literature from 1970 to July 2014 with a pyruvate oxidation disorder and a genetically proven defect were included in the study (n=628). Of these defects 74.2% (n=466) belong to PDHC subunits, 24.5% (n=154) to cofactors, 0.5% (n=3) to PDHC regulation and 0.8% (n=5) to mitochondrial pyruvate import. PODs are underestimated in the field of mitochondrial diseases because not all diagnostic centres include biochemical investigations of PDHC in their routine analysis. Cofactor and transport defects can be missed, if pyruvate oxidation is not measured in intact mitochondria routinely. Furthermore deficiency of the X-chromosomal PDHA1 can be biochemically missed depending on the X-inactivation pattern. This is reflected by an increasing number of patients diagnosed recently by genetic high throughput screening approaches. PDHC deficiency including regulation and import affect mainly the glucose dependent central and peripheral nervous system and skeletal muscle. PODs with combined enzyme defects affect also other organs like heart, lung and liver. The spectrum of clinical presentation of PODs is still expanding. PODs are a therapeutically interesting group of mitochondrial diseases since some can be bypassed by ketogenic diet or treated by cofactor supplementation. PDHC kinase inhibition, chaperone therapy and PGC1α stimulation is still a matter of further investigations.

Expanding the clinical and molecular spectrum of thiamine pyrophosphokinase deficiency: a treatable neurological disorder caused by TPK1 mutations.

Thiamine pyrophosphokinase (TPK) produces thiamine pyrophosphate, a cofactor for a number of enzymes, including pyruvate dehydrogenase and 2-ketoglutarate dehydrogenase. Episodic encephalopathy type thiamine metabolism dysfunction (OMIM 614458) due to TPK1 mutations is a recently described rare disorder. The mechanism of the disease, its phenotype and treatment are not entirely clear. We present two patients with novel homozygous TPK1 mutations (Patient 1 with p.Ser160Leu and Patient 2 with p.Asp222His). Unlike the previously described phenotype, Patient 2 presented with a Leigh syndrome like non-episodic early-onset global developmental delay, thus extending the phenotypic spectrum of the disorder. We, therefore, propose that TPK deficiency may be a better name for the condition. The two cases help to further refine the neuroradiological features of TPK deficiency and show that MRI changes can be either fleeting or progressive and can affect either white or gray matter. We also show that in some cases lactic acidosis can be absent and 2-ketoglutaric aciduria may be the only biochemical marker. Furthermore, we have established the assays for TPK enzyme activity measurement and thiamine pyrophosphate quantification in frozen muscle and blood. These tests will help to diagnose or confirm the diagnosis of TPK deficiency in a clinical setting. Early thiamine supplementation prevented encephalopathic episodes and improved developmental progression of Patient 1, emphasizing the importance of early diagnosis and treatment of TPK deficiency. We present evidence suggesting that thiamine supplementation may rescue TPK enzyme activity. Lastly, in silico protein structural analysis shows that the p.Ser160Leu mutation is predicted to interfere with TPK dimerization, which may be a novel mechanism for the disease.

Impaired riboflavin transport due to missense mutations in SLC52A2 causes Brown-Vialetto-Van Laere syndrome.

Brown-Vialetto-Van Laere syndrome (BVVLS [MIM 211530]) is a rare neurological disorder characterized by infancy onset sensorineural deafness and ponto-bulbar palsy. Mutations in SLC52A3 (formerly C20orf54), coding for riboflavin transporter 2 (hRFT2), have been identified as the molecular genetic correlate in several individuals with BVVLS. Exome sequencing of just one single case revealed that compound heterozygosity for two pathogenic mutations in the SLC52A2 gene coding for riboflavin transporter 3 (hRFT3), another member of the riboflavin transporter family, is also associated with BVVLS. Overexpression studies confirmed that the gene products of both mutant alleles have reduced riboflavin transport activities. While mutations in SLC52A3 cause decreased plasma riboflavin levels, concordant with a role of SLC52A3 in riboflavin uptake from food, the SLC52A2-mutant individual had normal plasma riboflavin concentrations, a finding in line with a postulated function of SLC52A2 in riboflavin uptake from blood into target cells. Our results contribute to the understanding of human riboflavin metabolism and underscore its role in the pathogenesis of BVVLS, thereby providing a rational basis for a high-dose riboflavin treatment.