Expanding the spectrum of neonatal-onset AIFM1-associated disorders.

OBJECTIVES: Pathogenic variants in AIFM1 have been associated with a wide spectrum of disorders, spanning from CMT4X to mitochondrial encephalopathy. Here we present a novel phenotype and review the existing literature on AIFM1-related disorders. METHODS: We performed EEG recordings, brain MRI and MR Spectroscopy, metabolic screening, echocardiogram, clinical exome sequencing (CES) and family study. Effects of the variant were established on cultured fibroblasts from skin punch biopsy. RESULTS: The patient presented with drug-resistant, electro-clinical, multifocal seizures 6 h after birth. Brain MRI revealed prominent brain swelling of both hemispheres and widespread signal alteration in large part of the cortex and of the thalami, with sparing of the basal nuclei. CES analysis revealed the likely pathogenic variant c.5T>C; p.(Phe2Ser) in the AIFM1 gene. The affected amino acid residue is located in the mitochondrial targeting sequence. Functional studies on cultured fibroblast showed a clear reduction in AIFM1 protein amount and defective activities of respiratory chain complexes I, III and IV. No evidence of protein mislocalization or accumulation of precursor protein was observed. Riboflavin, Coenzyme Q10 and thiamine supplementation was therefore given. At 6 months of age, the patient exhibited microcephaly but did not experience any further deterioration. He is still fed orally and there is no evidence of muscle weakness or atrophy. INTERPRETATION: This is the first AIFM1 case associated with neonatal seizures and diffuse white matter involvement with relative sparing of basal ganglia, in the absence of clinical signs suggestive of myopathy or motor neuron disease.

Diagnostic Challenges in Late Onset Multiple Acyl-CoA Dehydrogenase Deficiency: Clinical, Morphological, and Genetic Aspects.

Background: Multiple acyl-CoA dehydrogenase deficiency (MADD) is an autosomal recessive disorder of fatty acid oxidation due to deficiency of the mitochondrial electron transfer chain. The late-onset form is characterized by exercise intolerance, muscle weakness, and lipid storage in myofibers. Most MADD patients greatly benefit from riboflavin supplementation. Patients and methods: A retrospective study was conducted on patients with a diagnosis of vacuolar myopathy with lipid storage followed in our neuromuscular unit in the last 20 years. We selected 10 unrelated patients with the diagnosis of MADD according to clinical, morphological, and biochemical aspects. Clinical features, blood tests including serum acylcarnitines, EMG, and ENG were revised. Muscle biopsy was performed in all, and one individual underwent also a sural nerve biopsy. Gene sequencing of ETFA, ETFB, and ETFDH was performed as a first-tier genetic analysis followed by next-generation sequencing of an hyperCKemia gene panel in patients with undefined genotypes. Results: Clinical evaluation at onset in all our patients showed fatigue and muscle weakness; four patients showed difficulties in chewing, three patients complained of dysphagia, two patients had a dropped head, and a patient had an unexpected ataxia with numbness and dysesthesia. Laboratory blood tests revealed a variable increase in serum CK (266-6,500) and LDH levels (500-2,000). Plasma acylcarnitine profile evidenced increased levels of different chains intermediates. EMG was either normal or showed myogenic or neurogenic patterns. NCS demonstrated sensory neuropathy in two patients. Muscle biopsies showed a vacuolar myopathy with a variable increase in lipid content. Nerve biopsy evidenced an axonal degeneration with the loss of myelinated fibers. ETFDH genetic analysis identifies 14 pathogenic variants. Patients were treated with high doses of riboflavin (400 mg/die). All of them showed a rapid muscle strength improvement and normalization of abnormal values in laboratory tests. Neuropathic symptoms did not improve. Conclusion: Our data confirmed that clinical features in MADD patients are extremely variable in terms of disease onset and symptoms making diagnosis difficult. Laboratory investigations, such as serum acylcarnitine profile and muscle biopsy evaluation, may strongly address to a correct diagnosis. The favorable response to riboflavin supplementation strengthens the importance of an early diagnosis of these disorders among the spectrum of metabolic myopathies.

The zebrafish orthologue of the human hepatocerebral disease gene MPV17 plays pleiotropic roles in mitochondria.

Mitochondrial DNA depletion syndromes (MDS) are a group of rare autosomal recessive disorders with early onset and no cure available. MDS are caused by mutations in nuclear genes involved in mitochondrial DNA (mtDNA) maintenance, and characterized by both a strong reduction in mtDNA content and severe mitochondrial defects in affected tissues. Mutations in MPV17, a nuclear gene encoding a mitochondrial inner membrane protein, have been associated with hepatocerebral forms of MDS. The zebrafish mpv17 null mutant lacks the guanine-based reflective skin cells named iridophores and represents a promising model to clarify the role of Mpv17. In this study, we characterized the mitochondrial phenotype of mpv17-/- larvae and found early and severe ultrastructural alterations in liver mitochondria, as well as significant impairment of the respiratory chain, leading to activation of the mitochondrial quality control. Our results provide evidence for zebrafish Mpv17 being essential for maintaining mitochondrial structure and functionality, while its effects on mtDNA copy number seem to be subordinate. Considering that a role in nucleotide availability had already been postulated for MPV17, that embryos blocked in pyrimidine synthesis do phenocopy mpv17-/- knockouts (KOs) and that mpv17-/- KOs have impaired Dihydroorotate dehydrogenase activity, we provided mpv17 mutants with the pyrimidine precursor orotic acid (OA). Treatment with OA, an easily available food supplement, significantly increased both iridophore number and mtDNA content in mpv17-/- mutants, thus linking the loss of Mpv17 to pyrimidine de novo synthesis and opening a new simple therapeutic approach for MPV17-related MDS.

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.

Leukoencephalopathy with thalamus and brainstem involvement and high lactate 'LTBL' caused by EARS2 mutations.

In the large group of genetically undetermined infantile-onset mitochondrial encephalopathies, multiple defects of mitochondrial DNA-related respiratory-chain complexes constitute a frequent biochemical signature. In order to identify responsible genes, we used exome-next-generation sequencing in a selected cohort of patients with this biochemical signature. In an isolated patient, we found two mutant alleles for EARS2, the gene encoding mitochondrial glutamyl-tRNA synthetase. The brain magnetic resonance imaging of this patient was hallmarked by extensive symmetrical cerebral white matter abnormalities sparing the periventricular rim and symmetrical signal abnormalities of the thalami, midbrain, pons, medulla oblongata and cerebellar white matter. Proton magnetic resonance spectroscopy showed increased lactate. We matched this magnetic resonance imaging pattern with that of a cohort of 11 previously selected unrelated cases. We found mutations in the EARS2 gene in all. Subsequent detailed clinical and magnetic resonance imaging based phenotyping revealed two distinct groups: mild and severe. All 12 patients shared an infantile onset and rapidly progressive disease with severe magnetic resonance imaging abnormalities and increased lactate in body fluids and proton magnetic resonance spectroscopy. Patients in the 'mild' group partially recovered and regained milestones in the following years with striking magnetic resonance imaging improvement and declining lactate levels, whereas those of the 'severe' group were characterized by clinical stagnation, brain atrophy on magnetic resonance imaging and persistent lactate increases. This new neurological disease, early-onset leukoencephalopathy with thalamus and brainstem involvement and high lactate, is hallmarked by unique magnetic resonance imaging features, defined by a peculiar biphasic clinical course and caused by mutations in a single gene, EARS2, expanding the list of medically relevant defects of mitochondrial DNA translation.

Severe X-linked mitochondrial encephalomyopathy associated with a mutation in apoptosis-inducing factor.

We investigated two male infant patients who were given a diagnosis of progressive mitochondrial encephalomyopathy on the basis of clinical, biochemical, and morphological features. These patients were born from monozygotic twin sisters and unrelated fathers, suggesting an X-linked trait. Fibroblasts from both showed reduction of respiratory chain (RC) cIII and cIV, but not of cI activities. We found a disease-segregating mutation in the X-linked AIFM1 gene, encoding the Apoptosis-Inducing Factor (AIF) mitochondrion-associated 1 precursor that deletes arginine 201 (R201 del). Under normal conditions, mature AIF is a FAD-dependent NADH oxidase of unknown function and is targeted to the mitochondrial intermembrane space (this form is called AIF(mit)). Upon apoptogenic stimuli, a soluble form (AIF(sol)) is released by proteolytic cleavage and migrates to the nucleus, where it induces "parthanatos," i.e., caspase-independent fragmentation of chromosomal DNA. In vitro, the AIF(R201 del) mutation decreases stability of both AIF(mit) and AIF(sol) and increases the AIF(sol) DNA binding affinity, a prerequisite for nuclear apoptosis. In AIF(R201 del) fibroblasts, staurosporine-induced parthanatos was markedly increased, whereas re-expression of AIF(wt) induced recovery of RC activities. Numerous TUNEL-positive, caspase 3-negative nuclei were visualized in patient #1's muscle, again indicating markedly increased parthanatos in the AIF(R201 del) critical tissues. We conclude that AIF(R201 del) is an unstable mutant variant associated with increased parthanatos-linked cell death. Our data suggest a role for AIF in RC integrity and mtDNA maintenance, at least in some tissues. Interestingly, riboflavin supplementation was associated with prolonged improvement of patient #1's neurological conditions, as well as correction of RC defects in mutant fibroblasts, suggesting that stabilization of the FAD binding in AIF(mit) is beneficial.