Guanylate Kinase 1 Deficiency: A Novel and Potentially Treatable Mitochondrial DNA Depletion/Deletions Disease.

OBJECTIVE: Mitochondrial DNA (mtDNA) depletion/deletions syndrome (MDDS) comprises a group of diseases caused by primary autosomal defects of mtDNA maintenance. Our objective was to study the etiology of MDDS in 4 patients who lack pathogenic variants in known genetic causes. METHODS: Whole exome sequencing of the probands was performed to identify pathogenic variants. We validated the mitochondrial defect by analyzing mtDNA, mitochondrial dNTP pools, respiratory chain activities, and GUK1 activity. To confirm pathogenicity of GUK1 deficiency, we expressed 2 GUK1 isoforms in patient cells. RESULTS: We identified biallelic GUK1 pathogenic variants in all 4 probands who presented with ptosis, ophthalmoparesis, and myopathic proximal limb weakness, as well as variable hepatopathy and altered T-lymphocyte profiles. Muscle biopsies from all probands showed mtDNA depletion, deletions, or both, as well as reduced activities of mitochondrial respiratory chain enzymes. GUK1 encodes guanylate kinase, originally identified as a cytosolic enzyme. Long and short isoforms of GUK1 exist. We observed that the long isoform is intramitochondrial and the short is cytosolic. In probands' fibroblasts, we noted decreased GUK1 activity causing unbalanced mitochondrial dNTP pools and mtDNA depletion in both replicating and quiescent fibroblasts indicating that GUK1 deficiency impairs de novo and salvage nucleotide pathways. Proband fibroblasts treated with deoxyguanosine and/or forodesine, a purine phosphatase inhibitor, ameliorated mtDNA depletion, indicating potential pharmacological therapies. INTERPRETATION: Primary GUK1 deficiency is a new and potentially treatable cause of MDDS. The cytosolic isoform of GUK1 may contribute to the T-lymphocyte abnormality, which has not been observed in other MDDS disorders. ANN NEUROL 2024.

Association between ZASP/LDB3 Pro26Ser and Inclusion Body Myopathy.

Inclusion body myositis (IBM) is a slowly progressive disorder belonging to the idiopathic inflammatory myopathies, and it represents the most common adult-onset acquired myopathy. The main clinical features include proximal or distal muscular asymmetric weakness, with major involvement of long finger flexors and knee extensors. The main histological findings are the presence of fiber infiltrations, rimmed vacuoles, and amyloid inclusions. The etiopathogenesis is a challenge because both environmental and genetic factors are implicated in muscle degeneration and a distinction has been made previously between sporadic and hereditary forms. Here, we describe an Italian patient affected with a hereditary form of IBM with onset in his mid-forties. Next-generation sequencing analysis disclosed a heterozygous mutation c.76C>T (p.Pro26Ser) in the PDZ motif of the LDB3/ZASP gene, a mutation already described in a family with a late-onset myopathy and highly heterogenous degree of skeletal muscle weakness. In the proband's muscle biopsy, the expression of ZASP, myotilin, and desmin were increased. In our family, in addition to the earlier age of onset, the clinical picture is even more peculiar given the evidence, in one of the affected family members, of complete ophthalmoplegia in the vertical gaze. These findings help extend our knowledge of the clinical and genetic background associated with inclusion body myopathic disorders.

The Profiling of 179 miRNA Expression in Serum from Limb Girdle Muscular Dystrophy Patients and Healthy Controls.

Limb girdle muscular dystrophies (LGMDs) are a group of genetically inherited neuromuscular diseases with a very variable clinical presentation and overlapping traits. Over the last few years there has been an increasing interest in the use of non-invasive circulating biomarkers to monitor disease progression and to evaluate the efficacy of therapeutic approaches. Our aim was to identify the miRNA signature with potential value for LGMD patient screening and stratification. Using miRCURY LNA miRNA qPCR Serum/Plasma Panel, we analyzed 179 miRNAs from 16 patients, divided in four pools based on their genetic diagnosis, and from healthy controls. The miRNAs analysis showed a total of 107 dysregulated miRNAs in LGMD patients when compared to the healthy controls. After filtering via skeletal tissue expression and gene/pathways target analysis, the number of dysregulated miRNAs drastically reduced. Six selected miRNAs-let-7f-5p (in LGMDR1), miR-20a-5p (in LGMDR2), miR-130b-5p, miR-378a-5p (both in LGMDR3), miR-376c-3p and miR-382-5p (both in LGMDR4)-whose expression was significantly lower compared to controls in the different LGMD pools, were further investigated. The bioinformatic analysis of the target genes in each selected miRNA revealed ECM-receptor interaction and TGF-beta signaling as the most involved pathways. The correlation analysis showed a good correlation of let-7f-5p with fibrosis and with the cross sectional area of type I and type II fibers, while miR-130b-5p showed a good correlation with the age of onset of the disease. The receiver operating characteristic curves showed how single miRNAs were able to discriminate a specific group of LGMD patients and how the combination of six miRNAs was able to discriminate LGMD patients from controls.

Extracellular Matrix Disorganization and Sarcolemmal Alterations in COL6-Related Myopathy Patients with New Variants of COL6 Genes.

Collagen VI is a heterotrimeric protein expressed in several tissues and involved in the maintenance of cell integrity. It localizes at the cell surface, creating a microfilamentous network that links the cytoskeleton to the extracellular matrix. The heterotrimer consists of three chains encoded by COL6A1, COL6A2 and COL6A3 genes. Recessive and dominant molecular defects cause two main disorders, the severe Ullrich congenital muscular dystrophy and the relatively mild and slowly progressive Bethlem myopathy. We analyzed the clinical aspects, pathological features and mutational spectrum of 15 COL6-mutated patients belonging to our cohort of muscular dystrophy probands. Patients presented a heterogeneous phenotype ranging from severe forms to mild adult-onset presentations. Molecular analysis by NGS detected 14 different pathogenic variants, three of them so far unreported. Two changes, localized in the triple-helical domain of COL6A1, were associated with a more severe phenotype. Histological, immunological and ultrastructural techniques were employed for the validation of the genetic variants; they documented the high variability in COL6 distribution and the extracellular matrix disorganization, highlighting the clinical heterogeneity of our cohort. The combined use of these different technologies is pivotal in the diagnosis of COL6 patients.

Antisense Morpholino-Based In Vitro Correction of a Pseudoexon-Generating Variant in the SGCB Gene.

Limb-girdle muscular dystrophies (LGMD) are clinically and genetically heterogenous presentations displaying predominantly proximal muscle weakness due to the loss of skeletal muscle fibers. Beta-sarcoglycanopathy (LGMDR4) results from biallelic molecular defects in SGCB and features pediatric onset with limb-girdle involvement, often complicated by respiratory and heart dysfunction. Here we describe a patient who presented at the age of 12 years reporting high creatine kinase levels and onset of cramps after strenuous exercise. Instrumental investigations, including a muscle biopsy, pointed towards a diagnosis of beta-sarcoglycanopathy. NGS panel sequencing identified two variants in the SGCB gene, one of which (c.243+1548T>C) was found to promote the inclusion of a pseudoexon between exons 2 and 3 in the SGCB transcript. Interestingly, we detected the same genotype in a previously reported LGMDR4 patient, deceased more than twenty years ago, who had escaped molecular diagnosis so far. After the delivery of morpholino oligomers targeting the pseudoexon in patient-specific induced pluripotent stem cells, we observed the correction of the physiological splicing and partial restoration of protein levels. Our findings prompt the analysis of the c.243+1548T>C variant in suspected LGMDR4 patients, especially those harbouring monoallelic SGCB variants, and provide a further example of the efficacy of antisense technology for the correction of molecular defects resulting in splicing abnormalities.

Impact of COVID-19 on the quality of life of patients with neuromuscular disorders in the Lombardy area, Italy.

INTRODUCTION: /AIMS: Patients with neuromuscular disorders (NMDs), including many elderly, immunosuppressed, and disabled individuals, may have been particularly affected during the coronavirus disease 2019 (COVID-19) pandemic in Lombardy, a COVID-19 high-incidence area between February and May 2020. We aimed to evaluate the effects of the COVID-19 pandemic on the quality of life (QoL) and perceived disease burden of this group of patients. METHODS: We conducted a cross-sectional phone-based survey study between June 1 and June 14, 2020, on a sample of 240 NMD patients followed at our clinic in Milan, Italy. We asked about perceived NMD burden and QoL before and during the COVID-19 pandemic. We collected responses on access to outpatient care and ancillary services. We investigated the presence of symptoms suggestive of COVID-19 infection and confirmed cases. RESULTS: We collected 205 responses: 53 patients (25.9%) reported a subjective worsening of the underlying NMD. QoL measures showed a significant worsening between pre and pandemic time frames (odds ratio, 2.14 95%; confidence interval, 1.82-2.51). Outpatient visits were postponed in more than half of cases (57.1%), with 104 patients (50.7%) experiencing a cancellation of scheduled diagnostic tests. 79 patients (38.5%) reported at least one symptom attributable to COVID-19 infection. Among the 10 patients tested with nasopharyngeal swabs, 6 tested positive and 3 died from respiratory failure, including 2 patients on corticosteroid/ immunosuppressive therapy. DISCUSSION: The COVID-19 pandemic affected QoL and limited access to outpatient care and ancillary services of NMD patients in Lombardy between February and May 2020.

Non-alcoholic beriberi, Wernicke encephalopathy and long-term eating disorder: case report and a mini-review.

INTRODUCTION: Nowadays, reports of beriberi are rare in developed countries. Wernicke encephalopathy may be present in about 25% of patients with beriberi. CASE REPORT: We report the case of a woman with history of depression and chronic eating disorder, who complained Wernicke encephalopathy and beriberi. Sural nerve and muscular biopsy were performed, showing severe axonal neuropathy. Thiamine supplementation was started with rapid improvement of the pulmonary and cardiac affections; improvement of peripheral neuropathy was incomplete. CONCLUSIONS: Thiamine deficiency can be misdiagnosed. Beriberi is an important cause of acute flaccid paralysis; hence, clinicians should consider this diagnosis and prompt start thiamine treatment to avoid permanent neurological sequelae.

Biallelic C1QBP Mutations Cause Severe Neonatal-, Childhood-, or Later-Onset Cardiomyopathy Associated with Combined Respiratory-Chain Deficiencies.

Complement component 1 Q subcomponent-binding protein (C1QBP; also known as p32) is a multi-compartmental protein whose precise function remains unknown. It is an evolutionary conserved multifunctional protein localized primarily in the mitochondrial matrix and has roles in inflammation and infection processes, mitochondrial ribosome biogenesis, and regulation of apoptosis and nuclear transcription. It has an N-terminal mitochondrial targeting peptide that is proteolytically processed after import into the mitochondrial matrix, where it forms a homotrimeric complex organized in a doughnut-shaped structure. Although C1QBP has been reported to exert pleiotropic effects on many cellular processes, we report here four individuals from unrelated families where biallelic mutations in C1QBP cause a defect in mitochondrial energy metabolism. Infants presented with cardiomyopathy accompanied by multisystemic involvement (liver, kidney, and brain), and children and adults presented with myopathy and progressive external ophthalmoplegia. Multiple mitochondrial respiratory-chain defects, associated with the accumulation of multiple deletions of mitochondrial DNA in the later-onset myopathic cases, were identified in all affected individuals. Steady-state C1QBP levels were decreased in all individuals' samples, leading to combined respiratory-chain enzyme deficiency of complexes I, III, and IV. C1qbp-/- mouse embryonic fibroblasts (MEFs) resembled the human disease phenotype by showing multiple defects in oxidative phosphorylation (OXPHOS). Complementation with wild-type, but not mutagenized, C1qbp restored OXPHOS protein levels and mitochondrial enzyme activities in C1qbp-/- MEFs. C1QBP deficiency represents an important mitochondrial disorder associated with a clinical spectrum ranging from infantile lactic acidosis to childhood (cardio)myopathy and late-onset progressive external ophthalmoplegia.

Purkinje cell COX deficiency and mtDNA depletion in an animal model of spinocerebellar ataxia type 1.

Spinocerebellar ataxias (SCAs) are a genetically heterogeneous group of cerebellar degenerative disorders, characterized by progressive gait unsteadiness, hand incoordination, and dysarthria. Ataxia type 1 (SCA1) is caused by the expansion of a CAG trinucleotide repeat in the SCA1 gene resulting in the atypical extension of a polyglutamine (polyQ) tract within the ataxin-1 protein. Our main objective was to investigate the mitochondrial oxidative metabolism in the cerebellum of transgenic SCA1 mice. SCA1 transgenic mice develop clinical features in the early life stages (around 5 weeks of age) presenting pathological cerebellar signs with concomitant progressive Purkinje neuron atrophy and relatively little cell loss; this evidence suggests that the SCA1 phenotype is not the result of cell death per se, but a possible effect of cellular dysfunction that occurs before neuronal demise. We studied the mitochondrial oxidative metabolism in cerebellar cells from both homozygous and heterozygous transgenic SCA1 mice, aged 2 and 6 months. Histochemical examination showed a cytochrome-c-oxidase (COX) deficiency in the Purkinje cells (PCs) of both heterozygous and homozygous mice, the oxidative defect being more prominent in older mice, in which the percentage of COX-deficient PC was up to 30%. Using a laser-microdissector, we evaluated the mitochondrial DNA (mtDNA) content on selectively isolated COX-competent and COX-deficient PC by quantitative Polymerase Chain Reaction and we found mtDNA depletion in those with oxidative dysfunction. In conclusion, the selective oxidative metabolism defect observed in neuronal PC expressing mutant ataxin occurs as early as 8 weeks of age thus representing an early step in the PC degeneration process in SCA1 disease.

The italian limb girdle muscular dystrophy registry: Relative frequency, clinical features, and differential diagnosis.

INTRODUCTION: Limb girdle muscular dystrophies (LGMDs) are characterized by high molecular heterogeneity, clinical overlap, and a paucity of specific biomarkers. Their molecular definition is fundamental for prognostic and therapeutic purposes. METHODS: We created an Italian LGMD registry that included 370 molecularly defined patients. We reviewed detailed retrospective and prospective data and compared each LGMD subtype for differential diagnosis purposes. RESULTS: LGMD types 2A and 2B are the most frequent forms in Italy. The ages at disease onset, clinical progression, and cardiac and respiratory involvement can vary greatly between each LGMD subtype. In a set of extensively studied patients, targeted next-generation sequencing (NGS) identified mutations in 36.5% of cases. CONCLUSION: Detailed clinical characterization combined with muscle tissue analysis is fundamental to guide differential diagnosis and to address molecular tests. NGS is useful for diagnosing forms without specific biomarkers, although, at least in our study cohort, several LGMD disease mechanisms remain to be identified. Muscle Nerve 55: 55-68, 2017.

Linear mtDNA fragments and unusual mtDNA rearrangements associated with pathological deficiency of MGME1 exonuclease.

MGME1, also known as Ddk1 or C20orf72, is a mitochondrial exonuclease found to be involved in the processing of mitochondrial DNA (mtDNA) during replication. Here, we present detailed insights on the role of MGME1 in mtDNA maintenance. Upon loss of MGME1, elongated 7S DNA species accumulate owing to incomplete processing of 5' ends. Moreover, an 11-kb linear mtDNA fragment spanning the entire major arc of the mitochondrial genome is generated. In contrast to control cells, where linear mtDNA molecules are detectable only after nuclease S1 treatment, the 11-kb fragment persists in MGME1-deficient cells. In parallel, we observed characteristic mtDNA duplications in the absence of MGME1. The fact that the breakpoints of these mtDNA rearrangements do not correspond to either classical deletions or the ends of the linear 11-kb fragment points to a role of MGME1 in processing mtDNA ends, possibly enabling their repair by homologous recombination. In agreement with its functional involvement in mtDNA maintenance, we show that MGME1 interacts with the mitochondrial replicase PolgA, suggesting that it is a constituent of the mitochondrial replisome, to which it provides an additional exonuclease activity. Thus, our results support the viewpoint that MGME1-mediated mtDNA processing is essential for faithful mitochondrial genome replication and might be required for intramolecular recombination of mtDNA.

Mutations in DNA2 link progressive myopathy to mitochondrial DNA instability.

Syndromes associated with multiple mtDNA deletions are due to different molecular defects that can result in a wide spectrum of predominantly adult-onset clinical presentations, ranging from progressive external ophthalmoplegia (PEO) to multisystemic disorders of variable severity. The autosomal-dominant form of PEO is genetically heterogeneous. Recently, causative mutations have been reported in several nuclear genes that encode proteins of the mtDNA replisome machinery (POLG, POLG2, and C10orf2) or that are involved in pathways for the synthesis of deoxyribonuclotides (ANT1 and RRM2B). Despite these findings, putative mutations remain unknown in half of the subjects with PEO. We report the identification, by exome sequencing, of mutations in DNA2 in adult-onset individuals with a form of mitochondrial myopathy featuring instability of muscle mtDNA. DNA2 encodes a helicase/nuclease family member that is most likely involved in mtDNA replication, as well as in the long-patch base-excision repair (LP-BER) pathway. In vitro biochemical analysis of purified mutant proteins revealed a severe impairment of nuclease, helicase, and ATPase activities. These results implicate human DNA2 and the LP-BER pathway in the pathogenesis of adult-onset disorders of mtDNA maintenance.

Transcriptomic profiling of TK2 deficient human skeletal muscle suggests a role for the p53 signalling pathway and identifies growth and differentiation factor-15 as a potential novel biomarker for mitochondrial myopathies.

BACKGROUND: Mutations in the gene encoding thymidine kinase 2 (TK2) result in the myopathic form of mitochondrial DNA depletion syndrome which is a mitochondrial encephalomyopathy presenting in children. In order to unveil some of the mechanisms involved in this pathology and to identify potential biomarkers and therapeutic targets we have investigated the gene expression profile of human skeletal muscle deficient for TK2 using cDNA microarrays. RESULTS: We have analysed the whole transcriptome of skeletal muscle from patients with TK2 mutations and compared it to normal muscle and to muscle from patients with other mitochondrial myopathies. We have identified a set of over 700 genes which are differentially expressed in TK2 deficient muscle. Bioinformatics analysis reveals important changes in muscle metabolism, in particular, in glucose and glycogen utilisation, and activation of the starvation response which affects aminoacid and lipid metabolism. We have identified those transcriptional regulators which are likely to be responsible for the observed changes in gene expression. CONCLUSION: Our data point towards the tumor suppressor p53 as the regulator at the centre of a network of genes which are responsible for a coordinated response to TK2 mutations which involves inflammation, activation of muscle cell death by apoptosis and induction of growth and differentiation factor 15 (GDF-15) in muscle and serum. We propose that GDF-15 may represent a potential novel biomarker for mitochondrial dysfunction although further studies are required.

Mitochondrial disease heterogeneity: a prognostic challenge.

Mitochondrial diseases are a heterogeneous group of progressive, genetically transmitted, multisystem disorders caused by impaired mitochondrial function. The disease course for individuals with mitochondrial myopathies varies greatly from patient to patient because disease progression largely depends on the type of disease and on the degree of involvement of various organs which makes the prognosis unpredictable both within the same family and among families with the same mutation. This is particularly, but not exclusively, true for mitochondrial disorders caused by mtDNA point mutations, which are maternally inherited and subject to the randomness of the heteroplasmy. For this reason, the prognosis cannot be given by single mitochondrial disease, but should be formulated by any single mitochondrial disease-related event or complication keeping in mind that early recognition and treatment of symptoms are crucial for the prognosis. The following approach can help prevent severe organ dysfunctions or at least allow early diagnosis and treatment of disease-related complications.

POLG1 mutations and stroke like episodes: a distinct clinical entity rather than an atypical MELAS syndrome.

BACKGROUND: POLG1 mutations have been associated with MELAS-like phenotypes. However given several clinical differences it is unknown whether POLG1 mutations are possible causes of MELAS or give raise to a distinct clinical and genetic entity, named POLG1-associated encephalopathy. CASE PRESENTATION: We describe a 74 years old man carrying POLG1 mutations presenting with strokes, myopathy and ragged red fibers with some atypical aspects for MELAS such as late onset, lack of cerebral calcification and presence of frontal and occipital MRI lesions better consistent with the POLG associated-encephalopathy spectrum. CONCLUSION: The lack of available data hampers a definite diagnosis in our patient as well as makes it difficult to compare MELAS, which is a clearly defined clinical syndrome, with POLG1-associated encephalopathy, which is so far a purely molecularly defined syndrome with a quite heterogeneous clinical picture. However, the present report contributes to expand the phenotypic spectrum of POLG1 mutations underlining the importance of searching POLG1 mutations in patients with mitochondrial signs and MELAS like phenotypes but negative for common mtDNA mutations.

Next-generation sequencing reveals DGUOK mutations in adult patients with mitochondrial DNA multiple deletions.

The molecular diagnosis of mitochondrial disorders still remains elusive in a large proportion of patients, but advances in next generation sequencing are significantly improving our chances to detect mutations even in sporadic patients. Syndromes associated with mitochondrial DNA multiple deletions are caused by different molecular defects resulting in a wide spectrum of predominantly adult-onset clinical presentations, ranging from progressive external ophthalmoplegia to multi-systemic disorders of variable severity. The mutations underlying these conditions remain undisclosed in half of the affected subjects. We applied next-generation sequencing of known mitochondrial targets (MitoExome) to probands presenting with adult-onset mitochondrial myopathy and harbouring mitochondrial DNA multiple deletions in skeletal muscle. We identified autosomal recessive mutations in the DGUOK gene (encoding mitochondrial deoxyguanosine kinase), which has previously been associated with an infantile hepatocerebral form of mitochondrial DNA depletion. Mutations in DGUOK occurred in five independent subjects, representing 5.6% of our cohort of patients with mitochondrial DNA multiple deletions, and impaired both muscle DGUOK activity and protein stability. Clinical presentations were variable, including mitochondrial myopathy with or without progressive external ophthalmoplegia, recurrent rhabdomyolysis in a young female who had received a liver transplant at 9 months of age and adult-onset lower motor neuron syndrome with mild cognitive impairment. These findings reinforce the concept that mutations in genes involved in deoxyribonucleotide metabolism can cause diverse clinical phenotypes and suggest that DGUOK should be screened in patients harbouring mitochondrial DNA deletions in skeletal muscle.

MERRF Mutation A8344G in a Four-Generation Family without Central Nervous System Involvement: Clinical and Molecular Characterization.

A 53-year-old man approached our Neuromuscular Unit following an incidental finding of hyperckemia. Similar to his mother who had died at the age of 77 years, he was diabetic and had a few lipomas. The patient's two sisters, aged 60 and 50 years, did not have any neurological symptoms. Proband's skeletal muscle biopsy showed several COX-negative fibers, many of which were "ragged red". Genetic analysis revealed the presence of the A8344G mtDNA mutation, which is most commonly associated with a maternally inherited multisystem mitochondrial disorder known as MERRF (myoclonus epilepsy with ragged-red fibers). The two sisters also carry the mutation. Family members on the maternal side were reported healthy. Although atypical phenotypes have been reported in association with the A8344G mutation, central nervous system (CSN) manifestations other than myoclonic epilepsy are always reported in the family tree. If present, our four-generation family manifestations are late-onset and do not affect CNS. This could be explained by the fact that the mutational load remains low and therefore prevents tissues/organs from reaching the pathologic threshold. The fact that this occurs throughout generations and that CNS, which has the highest energetic demand, is clinically spared, suggests that regulatory genes and/or pathways affect mitochondrial segregation and replication, and protect organs from progressive dysfunction.

Characterization of Skeletal Muscle Biopsy and Derived Myoblasts in a Patient Carrying Arg14del Mutation in Phospholamban Gene.

Phospholamban is involved in the regulation of the activity and storage of calcium in cardiac muscle. Several mutations have been identified in the PLN gene causing cardiac disease associated with arrhythmogenic and dilated cardiomyopathy. The patho-mechanism underlying PLN mutations is not fully understood and a specific therapy is not yet available. PLN mutated patients have been deeply investigated in cardiac muscle, but very little is known about the effect of PLN mutations in skeletal muscle. In this study, we investigated both histological and functional features in skeletal muscle tissue and muscle-derived myoblasts from an Italian patient carrying the Arg14del mutation in PLN. The patient has a cardiac phenotype, but he also reported lower limb fatigability, cramps and fasciculations. The evaluation of a skeletal muscle biopsy showed histological, immunohistochemical and ultrastructural alterations. In particular, we detected an increase in the number of centronucleated fibers and a reduction in the fiber cross sectional area, an alteration in p62, LC3 and VCP proteins and the formation of perinuclear aggresomes. Furthermore, the patient's myoblasts showed a greater propensity to form aggresomes, even more marked after proteasome inhibition compared with control cells. Further genetic and functional studies are necessary to understand whether a definition of PLN myopathy, or cardiomyopathy plus, can be introduced for selected cases with clinical evidence of skeletal muscle involvement. Including skeletal muscle examination in the diagnostic process of PLN-mutated patients can help clarify this issue.

A biallelic variant in COX18 cause isolated Complex IV deficiency associated with neonatal encephalo-cardio-myopathy and axonal sensory neuropathy.

Pathogenic variants impacting upon assembly of mitochondrial respiratory chain Complex IV (Cytochrome c Oxidase or COX) predominantly result in early onset mitochondrial disorders often leading to CNS, skeletal and cardiac muscle manifestations. The aim of this study is to describe a molecular defect in the COX assembly factor gene COX18 as the likely cause of a neonatal form of mitochondrial encephalo-cardio-myopathy and axonal sensory neuropathy. The proband is a 19-months old female displaying hypertrophic cardiomyopathy at birth and myopathy with axonal sensory neuropathy and failure to thrive developing in the first months of life. Serum lactate was consistently increased. Whole exome sequencing allowed the prioritization of the unreported homozygous substitution NM_001297732.2:c.667 G > C p.(Asp223His) in COX18. Patient's muscle biopsy revealed severe and diffuse COX deficiency and striking mitochondrial abnormalities. Biochemical and enzymatic studies in patient's myoblasts and in HEK293 cells after COX18 silencing showed a severe impairment of both COX activity and assembly. The biochemical defect was partially rescued by delivery of wild-type COX18 cDNA into patient's myoblasts. Our study identifies a novel defect of COX assembly and expands the number of nuclear genes involved in a mitochondrial disorder due to isolated COX deficiency.