Bi-allelic LETM1 variants perturb mitochondrial ion homeostasis leading to a clinical spectrum with predominant nervous system involvement.

Leucine zipper-EF-hand containing transmembrane protein 1 (LETM1) encodes an inner mitochondrial membrane protein with an osmoregulatory function controlling mitochondrial volume and ion homeostasis. The putative association of LETM1 with a human disease was initially suggested in Wolf-Hirschhorn syndrome, a disorder that results from de novo monoallelic deletion of chromosome 4p16.3, a region encompassing LETM1. Utilizing exome sequencing and international gene-matching efforts, we have identified 18 affected individuals from 11 unrelated families harboring ultra-rare bi-allelic missense and loss-of-function LETM1 variants and clinical presentations highly suggestive of mitochondrial disease. These manifested as a spectrum of predominantly infantile-onset (14/18, 78%) and variably progressive neurological, metabolic, and dysmorphic symptoms, plus multiple organ dysfunction associated with neurodegeneration. The common features included respiratory chain complex deficiencies (100%), global developmental delay (94%), optic atrophy (83%), sensorineural hearing loss (78%), and cerebellar ataxia (78%) followed by epilepsy (67%), spasticity (53%), and myopathy (50%). Other features included bilateral cataracts (42%), cardiomyopathy (36%), and diabetes (27%). To better understand the pathogenic mechanism of the identified LETM1 variants, we performed biochemical and morphological studies on mitochondrial K+/H+ exchange activity, proteins, and shape in proband-derived fibroblasts and muscles and in Saccharomyces cerevisiae, which is an important model organism for mitochondrial osmotic regulation. Our results demonstrate that bi-allelic LETM1 variants are associated with defective mitochondrial K+ efflux, swollen mitochondrial matrix structures, and loss of important mitochondrial oxidative phosphorylation protein components, thus highlighting the implication of perturbed mitochondrial osmoregulation caused by LETM1 variants in neurological and mitochondrial pathologies.

AFG3L2 Biallelic Mutation: Clinical Heterogeneity in Two Italian Patients.

AFG3-like matrix AAA peptidase subunit 2 gene (AFG3L2, OMIM * 604,581) biallelic mutations lead to autosomal recessive spastic ataxia-5 SPAX5, OMIM # 614,487), a rare hereditary form of ataxia. The clinical spectrum includes early-onset cerebellar ataxia, spasticity, and progressive myoclonic epilepsy (PME). In Italy, the epidemiology of the disease is probably underestimated. The advent of next generation sequencing (NGS) technologies has speeded up the diagnosis of hereditary diseases and increased the percentage of diagnosis of rare disorders, such as the rare hereditary ataxia groups. Here, we describe two patients from two different villages in the province of Ferrara, who manifested a different clinical ataxia-plus history, although carrying the same biallelic mutation in AFG3L2 (p.Met625Ile) identified through NGS analysis.

A novel homozygous variant in COX5A causes an attenuated phenotype with failure to thrive, lactic acidosis, hypoglycemia, and short stature.

Genetic defect in the nuclear encoded subunits of cytochrome c oxidase are very rare. To date, most deleterious variants affect the mitochondrially encoded subunits of complex IV and the nuclear genes encoded for assembly factors. A biallelic pathogenic variant in the mitochondrial complex IV subunit COX5A was previously reported in a couple of sibs with failure to thrive, lactic acidosis and pulmonary hypertension and a lethal phenotype. Here, we describe a second family with a 11-year-old girl presenting with failure to thrive, lactic acidosis, hypoglycemia and short stature. Clinical exome revealed the homozygous missense variant c.266 T > G in COX5A, which produces a drop of the corresponding protein and a reduction of the COX activity. Compared to the previous observation, this girl showed an attenuated metabolic derangement without involvement of the cardiovascular system and neurodevelopment. Our observation confirms that COX5A recessive variants may cause mitochondrial disease and expands the associated phenotype to less severe presentations.

Novel NDUFA12 variants are associated with isolated complex I defect and variable clinical manifestation.

Isolated biochemical deficiency of mitochondrial complex I is the most frequent signature among mitochondrial diseases and is associated with a wide variety of clinical symptoms. Leigh syndrome represents the most frequent neuroradiological finding in patients with complex I defect and more than 80 monogenic causes have been involved in the disease. In this report, we describe seven patients from four unrelated families harboring novel NDUFA12 variants, with six of them presenting with Leigh syndrome. Molecular genetic characterization was performed using next-generation sequencing combined with the Sanger method. Biochemical and protein studies were achieved by enzymatic activities, blue native gel electrophoresis, and western blot analysis. All patients displayed novel homozygous mutations in the NDUFA12 gene, leading to the virtual absence of the corresponding protein. Surprisingly, despite the fact that in none of the analyzed patients, NDUFA12 protein was detected, they present a different onset and clinical course of the disease. Our report expands the array of genetic alterations in NDUFA12 and underlines phenotype variability associated with NDUFA12 defect.

Expanded phenotype of AARS1-related white matter disease.

PURPOSE: Recent reports of individuals with cytoplasmic transfer RNA (tRNA) synthetase-related disorders have identified cases with phenotypic variability from the index presentations. We sought to assess phenotypic variability in individuals with AARS1-related disease. METHODS: A cross-sectional survey was performed on individuals with biallelic variants in AARS1. Clinical data, neuroimaging, and genetic testing results were reviewed. Alanyl tRNA synthetase (AlaRS) activity was measured in available fibroblasts. RESULTS: We identified 11 affected individuals. Two phenotypic presentations emerged, one with early infantile-onset disease resembling the index cases of AARS1-related epileptic encephalopathy with deficient myelination (n = 7). The second (n = 4) was a later-onset disorder, where disease onset occurred after the first year of life and was characterized on neuroimaging by a progressive posterior predominant leukoencephalopathy evolving to include the frontal white matter. AlaRS activity was significantly reduced in five affected individuals with both early infantile-onset and late-onset phenotypes. CONCLUSION: We suggest that variants in AARS1 result in a broader clinical spectrum than previously appreciated. The predominant form results in early infantile-onset disease with epileptic encephalopathy and deficient myelination. However, a subgroup of affected individuals manifests with late-onset disease and similarly rapid progressive clinical decline. Longitudinal imaging and clinical follow-up will be valuable in understanding factors affecting disease progression and outcome.

A homozygous MRPL24 mutation causes a complex movement disorder and affects the mitoribosome assembly.

Mitochondrial ribosomal protein large 24 (MRPL24) is 1 of the 82 protein components of mitochondrial ribosomes, playing an essential role in the mitochondrial translation process. We report here on a baby girl with cerebellar atrophy, choreoathetosis of limbs and face, intellectual disability and a combined defect of complexes I and IV in muscle biopsy, caused by a homozygous missense mutation identified in MRPL24. The variant predicts a Leu91Pro substitution at an evolutionarily conserved site. Using human mutant cells and the zebrafish model, we demonstrated the pathological role of the identified variant. In fact, in fibroblasts we observed a significant reduction of MRPL24 protein and of mitochondrial respiratory chain complex I and IV subunits, as well a markedly reduced synthesis of the mtDNA-encoded peptides. In zebrafish we demonstrated that the orthologue gene is expressed in metabolically active tissues, and that gene knockdown induced locomotion impairment, structural defects and low ATP production. The motor phenotype was complemented by human WT but not mutant cRNA. Moreover, sucrose density gradient fractionation showed perturbed assembly of large subunit mitoribosomal proteins, suggesting that the mutation leads to a conformational change in MRPL24, which is expected to cause an aberrant interaction of the protein with other components of the 39S mitoribosomal subunit.

ISCA1 mutation in a patient with infantile-onset leukodystrophy causes defects in mitochondrial [4Fe-4S] proteins.

Multiple mitochondrial dysfunction syndromes (MMDS) comprise a group of severe autosomal recessive diseases characterized by impaired respiration and lipoic acid metabolism, resulting in infantile-onset mitochondrial encephalopathy, non-ketotic hyperglycinemia, myopathy, lactic acidosis and early death. Four different MMDS have been analyzed in detail according to the genes involved in the disease, MMDS1 (NFU1), MMDS2 (BOLA3), MMDS3 (IBA57) and MMDS4 (ISCA2). MMDS5 has recently been described in a clinical case report of patients carrying a mutation in ISCA1, but with no further functional analysis. ISCA1 encodes a mitochondrial protein essential for the assembly of [4Fe-4S] clusters in key metabolic and respiratory enzymes. Here, we describe a patient with a severe early onset leukodystrophy, multiple defects of respiratory complexes and a severe impairment of lipoic acid synthesis. A homozygous missense mutation in ISCA1 (c.29T>G; p.V10G) identified by targeted MitoExome sequencing resulted in dramatic reduction of ISCA1 protein level. The mutation located in the uncleaved presequence severely affected both mitochondrial import and stability of ISCA1. Down-regulation of ISCA1 in HeLa cells by RNAi impaired the biogenesis of mitochondrial [4Fe-4S] proteins, yet could be complemented by expression of wild-type ISCA1. In contrast, the ISCA1 p.V10G mutant protein only partially complemented the defects, closely resembling the biochemical phenotypes observed for ISCA1 patient fibroblasts. Collectively, our comprehensive clinical and biochemical investigations show that the ISCA1 p.V10G mutation functionally impaired mitochondrial [4Fe-4S] protein assembly and hence was causative for the observed clinical defects.

Clinical-genetic features and peculiar muscle histopathology in infantile DNM1L-related mitochondrial epileptic encephalopathy.

Mitochondria are highly dynamic organelles, undergoing continuous fission and fusion. The DNM1L (dynamin-1 like) gene encodes for the DRP1 protein, an evolutionary conserved member of the dynamin family, responsible for fission of mitochondria, and having a role in the division of peroxisomes, as well. DRP1 impairment is implicated in several neurological disorders and associated with either de novo dominant or compound heterozygous mutations. In five patients presenting with severe epileptic encephalopathy, we identified five de novo dominant DNM1L variants, the pathogenicity of which was validated in a yeast model. Fluorescence microscopy revealed abnormally elongated mitochondria and aberrant peroxisomes in mutant fibroblasts, indicating impaired fission of these organelles. Moreover, a very peculiar finding in our cohort of patients was the presence, in muscle biopsy, of core like areas with oxidative enzyme alterations, suggesting an abnormal distribution of mitochondria in the muscle tissue.

Clinical, radiological, and genetic characteristics of 16 patients with ACO2 gene defects: Delineation of an emerging neurometabolic syndrome.

Mitochondrial aconitase is the second enzyme in the tricarboxylic acid (TCA) cycle catalyzing the interconversion of citrate into isocitrate and encoded by the nuclear gene ACO2. A homozygous pathogenic variant in the ACO2 gene was initially described in 2012 resulting in a novel disorder termed "infantile cerebellar retinal degeneration" (ICRD, OMIM#614559). Subsequently, additional studies reported patients with pathogenic ACO2 variants, further expanding the genetic and clinical spectrum of this disorder to include milder and later onset manifestations. Here, we report an international multicenter cohort of 16 patients (of whom 7 are newly diagnosed) with biallelic pathogenic variants in ACO2 gene. Most patients present in early infancy with severe truncal hypotonia, truncal ataxia, variable seizures, evolving microcephaly, and ophthalmological abnormalities of which the most dominant are esotropia and optic atrophy with later development of retinal dystrophy. Most patients remain nonambulatory and do no acquire any language, but a subgroup of patients share a more favorable course. Brain magnetic resonance imaging (MRI) is typically normal within the first months but global atrophy gradually develops affecting predominantly the cerebellum. Ten of our patients were homozygous to the previously reported c.336C>G founder mutation while the other six patients were all compound heterozygotes displaying 10 novel mutations of whom 2 were nonsense predicting a deleterious effect on enzyme function. Structural protein modeling predicted significant impairment in aconitase substrate binding in the additional missense mutations. This study provides the most extensive cohort of patients and further delineates the clinical, radiological, biochemical, and molecular features of ACO2 deficiency.

Mutations in APOPT1, encoding a mitochondrial protein, cause cavitating leukoencephalopathy with cytochrome c oxidase deficiency.

Cytochrome c oxidase (COX) deficiency is a frequent biochemical abnormality in mitochondrial disorders, but a large fraction of cases remains genetically undetermined. Whole-exome sequencing led to the identification of APOPT1 mutations in two Italian sisters and in a third Turkish individual presenting severe COX deficiency. All three subjects presented a distinctive brain MRI pattern characterized by cavitating leukodystrophy, predominantly in the posterior region of the cerebral hemispheres. We then found APOPT1 mutations in three additional unrelated children, selected on the basis of these particular MRI features. All identified mutations predicted the synthesis of severely damaged protein variants. The clinical features of the six subjects varied widely from acute neurometabolic decompensation in late infancy to subtle neurological signs, which appeared in adolescence; all presented a chronic, long-surviving clinical course. We showed that APOPT1 is targeted to and localized within mitochondria by an N-terminal mitochondrial targeting sequence that is eventually cleaved off from the mature protein. We then showed that APOPT1 is virtually absent in fibroblasts cultured in standard conditions, but its levels increase by inhibiting the proteasome or after oxidative challenge. Mutant fibroblasts showed reduced amount of COX holocomplex and higher levels of reactive oxygen species, which both shifted toward control values by expressing a recombinant, wild-type APOPT1 cDNA. The shRNA-mediated knockdown of APOPT1 in myoblasts and fibroblasts caused dramatic decrease in cell viability. APOPT1 mutations are responsible for infantile or childhood-onset mitochondrial disease, hallmarked by the combination of profound COX deficiency with a distinctive neuroimaging presentation.

Novel mutation in mitochondrial Elongation Factor EF-Tu associated to dysplastic leukoencephalopathy and defective mitochondrial DNA translation.

The mitochondrial Elongation Factor Tu (EF-Tu), encoded by the TUFM gene, is a highly conserved GTPase, which is part of the mitochondrial protein translation machinery. In its activated form it delivers the aminoacyl-tRNAs to the A site of the mitochondrial ribosome. We report here on a baby girl with severe infantile macrocystic leukodystrophy with micropolygyria and a combined defect of complexes I and IV in muscle biopsy, caused by a novel mutation identified in TUFM. Using human mutant cells and the yeast model, we demonstrate the pathological role of the novel variant. Moreover, results of a molecular modeling study suggest that the mutant is inactive in mitochondrial polypeptide chain elongation, probably as a consequence of its reduced ability to bind mitochondrial aa-tRNAs. Four patients have so far been described with mutations in TUFM, and, following the first description of the disease in a single patient, we describe similar clinical and neuroradiological features in an additional patient.

LYRM7 mutations cause a multifocal cavitating leukoencephalopathy with distinct MRI appearance.

This study focused on the molecular characterization of patients with leukoencephalopathy associated with a specific biochemical defect of mitochondrial respiratory chain complex III, and explores the impact of a distinct magnetic resonance imaging pattern of leukoencephalopathy to detect biallelic mutations in LYRM7 in patients with biochemically unclassified leukoencephalopathy. 'Targeted resequencing' of a custom panel including genes coding for mitochondrial proteins was performed in patients with complex III deficiency without a molecular genetic diagnosis. Based on brain magnetic resonance imaging findings in these patients, we selected additional patients from a database of unclassified leukoencephalopathies who were scanned for mutations in LYRM7 by Sanger sequencing. Targeted sequencing revealed homozygous mutations in LYRM7, encoding mitochondrial LYR motif-containing protein 7, in four patients from three unrelated families who had a leukoencephalopathy and complex III deficiency. Two subjects harboured previously unreported variants predicted to be damaging, while two siblings carried an already reported pathogenic homozygous missense change. Sanger sequencing performed in the second cohort of patients revealed LYRM7 mutations in three additional patients, who were selected on the basis of the magnetic resonance imaging pattern. All patients had a consistent magnetic resonance imaging pattern of progressive signal abnormalities with multifocal small cavitations in the periventricular and deep cerebral white matter. Early motor development was delayed in half of the patients. All patients but one presented with subacute neurological deterioration in infancy or childhood, preceded by a febrile infection, and most patients had repeated episodes of subacute encephalopathy with motor regression, irritability and stupor or coma resulting in major handicap or death. LYRM7 protein was strongly reduced in available samples from patients; decreased complex III holocomplex was observed in fibroblasts from a patient carrying a splice site variant; functional studies in yeast confirmed the pathogenicity of two novel mutations. Mutations in LYRM7 were previously found in a single patient with a severe form of infantile onset encephalopathy. We provide new molecular, clinical, and neuroimaging data allowing us to characterize more accurately the molecular spectrum of LYRM7 mutations highlighting that a distinct and recognizable magnetic resonance imaging pattern is related to mutations in this gene. Inter- and intrafamilial variability exists and we observed one patient who was asymptomatic by the age of 6 years.

Clinical and molecular study in a long-surviving patient with MLASA syndrome due to novel PUS1 mutations.

Myopathy-lactic acidosis-sideroblastic anemia (MLASA) syndrome is a rare autosomal recessive disease. We studied a 43-year-old female presenting since childhood with mild cognitive impairment and sideroblastic anemia. She later developed hepatopathy, cardiomyopathy, and insulin-dependent diabetes. Muscle weakness appeared in adolescence and, at age 43, she was unable to walk. Two novel different mutations in the PUS1 gene were identified: c.487delA (p.I163Lfs*4) and c.884 G>A (p.R295Q). Quantitative analysis of DNA from skeletal muscle biopsies showed a significant increase in mitochondrial DNA (mtDNA) content in the patient compared to controls. Clinical and molecular findings of this patient widen the genotype-phenotype spectrum in MLASA syndrome.

Partial complex I deficiency due to the CNS conditional ablation of Ndufa5 results in a mild chronic encephalopathy but no increase in oxidative damage.

Deficiencies in the complex I (CI; NADH-ubiquinone oxidoreductase) of the respiratory chain are frequent causes of mitochondrial diseases and have been associated with other neurodegenerative disorders, such as Parkinson's disease. The NADH-ubiquinone oxidoreductase 1 alpha subcomplex subunit 5 (NDUFA5) is a nuclear-encoded structural subunit of CI, located in the peripheral arm. We inactivated Ndufa5 in mice by the gene-trap methodology and found that this protein is required for embryonic survival. Therefore, we have created a conditional Ndufa5 knockout (KO) allele by introducing a rescuing Ndufa5 cDNA transgene flanked by loxP sites, which was selectively ablated in neurons by the CaMKIIα-Cre. At the age of 11 months, mice with a central nervous system knockout of Ndufa5 (Ndufa5 CNS-KO) showed lethargy and loss of motor skills. In these mice cortices, the levels of NDUFA5 protein were reduced to 25% of controls. Fully assembled CI levels were also greatly reduced in cortex and CI activity in homogenates was reduced to 60% of controls. Despite the biochemical phenotype, no oxidative damage, neuronal death or gliosis were detected in the Ndufa5 CNS-KO brain at this age. These results showed that a partial defect in CI in neurons can lead to late-onset motor phenotypes without neuronal loss or oxidative damage.

Persistent pulmonary arterial hypertension in the newborn (PPHN): a frequent manifestation of TMEM70 defective patients.

INTRODUCTION: Mutations in the TMEM70 are the most common cause of nuclear ATP synthase deficiency resulting in a distinctive phenotype characterized by severe neonatal hypotonia, hypertrophic cardiomyopathy (HCMP), facial dysmorphism, severe lactic acidosis, hyperammonemia and 3-methylglutaconic aciduria (3-MGA). METHODS AND RESULTS: We collected 9 patients with genetically confirmed TMEM70 defect from 8 different families. Six were homozygous for the c.317-2A>G mutation, 2 were compound heterozygous for mutations c.317-2A>G and c.628A>C and 1 was homozygous for the novel c.701A>C mutation. Generalized hypotonia, lactic acidosis, hyperammonemia and 3-MGA were present in all since birth. Five patients presented acute respiratory distress at birth requiring intubation and ventilatory support. HCMP was detected in 5 newborns and appeared a few months later in 3 additional children. Five patients showed a severe and persistent neonatal pulmonary hypertension (PPHN) requiring Nitric Oxide (NO) and/or sildenafil administration combined in 2 cases with high-frequency oscillatory (HFO) ventilation. In 3 of these patients, echocardiography detected signs of HCMP at birth. CONCLUSIONS: PPHN is a life-threatening poorly understood condition with bad prognosis if untreated. Pulmonary hypertension has rarely been reported in mitochondrial disorders and, so far, it has been described in association with TMEM70 deficiency only in one patient. This report further expands the clinical and genetic spectrum of the syndrome indicating PPHN as a frequent and life-threatening complication regardless of the type of mutation. Moreover, in these children PPHN appears even in the absence of an overt cardiomyopathy, thus representing an early sign and a clue for diagnosis.

Frataxin silencing inactivates mitochondrial Complex I in NSC34 motoneuronal cells and alters glutathione homeostasis.

Friedreich's ataxia (FRDA) is a hereditary neurodegenerative disease characterized by a reduced synthesis of the mitochondrial iron chaperon protein frataxin as a result of a large GAA triplet-repeat expansion within the first intron of the frataxin gene. Despite neurodegeneration being the prominent feature of this pathology involving both the central and the peripheral nervous system, information on the impact of frataxin deficiency in neurons is scant. Here, we describe a neuronal model displaying some major biochemical and morphological features of FRDA. By silencing the mouse NSC34 motor neurons for the frataxin gene with shRNA lentiviral vectors, we generated two cell lines with 40% and 70% residual amounts of frataxin, respectively. Frataxin-deficient cells showed a specific inhibition of mitochondrial Complex I (CI) activity already at 70% residual frataxin levels, whereas the glutathione imbalance progressively increased after silencing. These biochemical defects were associated with the inhibition of cell proliferation and morphological changes at the axonal compartment, both depending on the frataxin amount. Interestingly, at 70% residual frataxin levels, the in vivo treatment with the reduced glutathione revealed a partial rescue of cell proliferation. Thus, NSC34 frataxin silenced cells could be a suitable model to study the effect of frataxin deficiency in neurons and highlight glutathione as a potential beneficial therapeutic target for FRDA.

Biallelic variants in GTPBP3: New patients, phenotypic spectrum, and outcome.

INTRODUCTION: COXPD23 is a rare mitochondrial disease caused by biallelic pathogenic variants in GTPBP3. We report on two siblings with a mild phenotype. CASE REPORTS: The young boy presented with global developmental delay, ataxic gait and upper limbs tremor, and the older sister with absence seizures and hypertrophic cardiomyopathy. Respiratory chain impairment was confirmed in muscle. DISCUSSION: Reviewed cases point toward clustering around two prevalent phenotypes: an early-onset presentation with severe fatal encephalopathy and a late milder presentation with global developmental delay/ID and cardiopathy, with the latter as, is the main feature. Our patients showed an intermediate phenotype with intrafamilial variability.