Double administration of self-complementary AAV9NDUFS4 prevents Leigh disease in Ndufs4-/- mice.

Leigh disease, or subacute necrotizing encephalomyelopathy, a genetically heterogeneous condition consistently characterized by defective mitochondrial bioenergetics, is the most common oxidative-phosphorylation related disease in infancy. Both neurological signs and pathological lesions of Leigh disease are mimicked by the ablation of the mouse mitochondrial respiratory chain subunit Ndufs4-/-, which is part of, and crucial for, normal Complex I activity and assembly, particularly in the brains of both children and mice. We previously conveyed the human NDUFS4 gene to the mouse brain using either single-stranded adeno-associated viral 9 recombinant vectors or the PHP.B adeno-associated viral vector. Both these approaches significantly prolonged the lifespan of the Ndufs4-/- mouse model but the extension of the survival was limited to a few weeks by the former approach, whereas the latter was applicable to a limited number of mouse strains, but not to primates. Here, we exploited the recent development of new, self-complementary adeno-associated viral 9 vectors, in which the transcription rate of the recombinant gene is markedly increased compared with the single-stranded adeno-associated viral 9 and can be applied to all mammals, including humans. Either single intra-vascular or double intra-vascular and intra-cerebro-ventricular injections were performed at post-natal Day 1. The first strategy ubiquitously conveyed the human NDUFS4 gene product in Ndufs4-/- mice, doubling the lifespan from 45 to ≈100 days after birth, when the mice developed rapidly progressive neurological failure. However, the double, contemporary intra-vascular and intra-cerebroventricular administration of self-complementary-adeno-associated viral NDUFS4 prolonged healthy lifespan up to 9 months of age. These mice were well and active at euthanization, at 6, 7, 8 and 9 months of age, to investigate the brain and other organs post-mortem. Robust expression of hNDUFS4 was detected in different cerebral areas preserving normal morphology and restoring Complex I activity and assembly. Our results warrant further investigation on the translatability of self-complementary-adeno-associated viral 9 NDUFS4-based therapy in the prodromal phase of the disease in mice and eventually humans.

Next-generation sequencing of Tunisian Leigh syndrome patients reveals novel variations: impact for diagnosis and treatment.

Mitochondrial cytopathies, among which the Leigh syndrome (LS), are caused by variants either in the mitochondrial or the nuclear genome, affecting the oxidative phosphorylation process. The aim of the present study consisted in defining the molecular diagnosis of a group of Tunisian patients with LS. Six children, belonging to five Tunisian families, with clinical and imaging presentations suggestive of LS were recruited. Whole mitochondrial DNA and targeted next-generation sequencing of a panel of 281 nuclear genes involved in mitochondrial physiology were performed. Bioinformatic analyses were achieved in order to identify deleterious variations. A single m.10197G>A (p.Ala47Thr) variant was found in the mitochondrial MT-ND3 gene in one patient, while the others were related to autosomal homozygous variants: two c.1412delA (p.Gln471ArgfsTer42) and c.1264A>G (p.Thr422Ala) in SLC19A3, one c.454C>G (p.Pro152Ala) in SLC25A19 and one c.122G>A (p.Gly41Asp) in ETHE1. Our findings demonstrate the usefulness of genomic investigations to improve LS diagnosis in consanguineous populations and further allow for treating the patients harboring variants in SLC19A3 and SLC25A19 that contribute to thiamine transport, by thiamine and biotin supplementation. Considering the Tunisian genetic background, the newly identified variants could be screened in patients with similar clinical presentation in related populations.

Hypoxia ameliorates brain hyperoxia and NAD+ deficiency in a murine model of Leigh syndrome.

Leigh syndrome is a severe mitochondrial neurodegenerative disease with no effective treatment. In the Ndufs4-/- mouse model of Leigh syndrome, continuously breathing 11% O2 (hypoxia) prevents neurodegeneration and leads to a dramatic extension (~5-fold) in lifespan. We investigated the effect of hypoxia on the brain metabolism of Ndufs4-/- mice by studying blood gas tensions and metabolite levels in simultaneously sampled arterial and cerebral internal jugular venous (IJV) blood. Relatively healthy Ndufs4-/- and wildtype (WT) mice breathing air until postnatal age ~38 d were compared to Ndufs4-/- and WT mice breathing air until ~38 days old followed by 4-weeks of breathing 11% O2. Compared to WT control mice, Ndufs4-/- mice breathing air have reduced brain O2 consumption as evidenced by an elevated partial pressure of O2 in IJV blood (PijvO2) despite a normal PO2 in arterial blood, and higher lactate/pyruvate (L/P) ratios in IJV plasma revealed by metabolic profiling. In Ndufs4-/- mice, hypoxia treatment normalized the cerebral venous PijvO2 and L/P ratios, and decreased levels of nicotinate in IJV plasma. Brain concentrations of nicotinamide adenine dinucleotide (NAD+) were lower in Ndufs4-/- mice breathing air than in WT mice, but preserved at WT levels with hypoxia treatment. Although mild hypoxia (17% O2) has been shown to be an ineffective therapy for Ndufs4-/- mice, we find that when combined with nicotinic acid supplementation it provides a modest improvement in neurodegeneration and lifespan. Therapies targeting both brain hyperoxia and NAD+ deficiency may hold promise for treating Leigh syndrome.

TRMU deficiency: A broad clinical spectrum responsive to cysteine supplementation.

TRMU is a nuclear gene crucial for mitochondrial DNA translation by encoding tRNA 5-methylaminomethyl-2-thiouridylate methyltransferase, which thiolates mitochondrial tRNA. Biallelic pathogenic variants in TRMU are associated with transient infantile liver failure. Other less common presentations such as Leigh syndrome, myopathy, and cardiomyopathy have been reported. Recent studies suggested that provision of exogenous L-cysteine or N-acetylcysteine may ameliorate the effects of disease-causing variants and improve the natural history of the disease. Here, we report six infants with biallelic TRMU variants, including four previously unpublished patients, all treated with exogenous cysteine. We highlight the first report of an affected patient undergoing orthotopic liver transplantation, the long-term effects of cysteine supplementation, and the ability of the initial presentation to mimic multiple inborn errors of metabolism. We propose that TRMU deficiency should be suspected in all children presenting with persistent lactic acidosis and hypoglycemia, and that combined N-acetylcysteine and L-cysteine supplementation should be considered prior to molecular diagnosis, as this is a low-risk approach that may increase survival and mitigate the severity of the disease course.

Thiamine transporter-2 deficiency: outcome and treatment monitoring.

BACKGROUND: The clinical characteristics distinguishing treatable thiamine transporter-2 deficiency (ThTR2) due to SLC19A3 genetic defects from the other devastating causes of Leigh syndrome are sparse. METHODS: We report the clinical follow-up after thiamine and biotin supplementation in four children with ThTR2 deficiency presenting with Leigh and biotin-thiamine-responsive basal ganglia disease phenotypes. We established whole-blood thiamine reference values in 106 non-neurological affected children and monitored thiamine levels in SLC19A3 patients after the initiation of treatment. We compared our results with those of 69 patients with ThTR2 deficiency after a review of the literature. RESULTS: At diagnosis, the patients were aged 1 month to 17 years, and all of them showed signs of acute encephalopathy, generalized dystonia, and brain lesions affecting the dorsal striatum and medial thalami. One patient died of septicemia, while the remaining patients evidenced clinical and radiological improvements shortly after the initiation of thiamine. Upon follow-up, the patients received a combination of thiamine (10-40 mg/kg/day) and biotin (1-2 mg/kg/day) and remained stable with residual dystonia and speech difficulties. After establishing reference values for the different age groups, whole-blood thiamine quantification was a useful method for treatment monitoring. CONCLUSIONS: ThTR2 deficiency is a reversible cause of acute dystonia and Leigh encephalopathy in the pediatric years. Brain lesions affecting the dorsal striatum and medial thalami may be useful in the differential diagnosis of other causes of Leigh syndrome. Further studies are needed to validate the therapeutic doses of thiamine and how to monitor them in these patients.