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Introduction/Aims HyperCKemia is considered a hallmark of neuromuscular diseases. It can be either isolated or associated with cramps, myalgia, weakness, myoglobinuria, or rhabdomyolysis, suggesting a metabolic myopathy. The aim of this work was to investigate possible genetic causes in order to help diagnose patients with recurrent hyperCKemia or clinical suspicion of inherited metabolic myopathy. Methods A cohort of 139 patients (90 adults and 49 children) was analyzed using a custom panel containing 54 genes associated with hyperCKemia. Results A definite genetic diagnosis was obtained in 15.1% of cases, while candidate variants or variants of uncertain significance were found in a further 39.5%. Similar percentages were obtained in patients with infantile or adult onset, with some different causative genes. RYR1 was the gene most frequently identified, either with single or compound heterozygous variants, while ETFDH variants were the most common cause for recessive cases. In one patient, mRNA analysis allowed identifying a large LPIN1 deletion missed by DNA sequencing, leading to a certain diagnosis. Conclusion These data confirm the high genetic heterogeneity of hyperCKemia and metabolic myopathies. The reduced diagnostic yield suggests the existence of additional genes associated with this condition but also allows speculation that a significant number of cases presenting with hyperCKemia or muscle symptoms are due to extrinsic, not genetic, factors.

Objective To characterize ocular motor function in patients with Niemann‐Pick disease type C (NPC). Methods In a multicontinental, cross‐sectional study we characterized ocular‐motor function in 72 patients from 12 countries by video‐oculography. Interlinking with disease severity, we also searched for ocular motor biomarkers. Our study protocol comprised reflexive and self‐paced saccades, smooth pursuit, and gaze‐holding in horizontal and vertical planes. Data were compared with those of 158 healthy controls (HC). Results Some 98.2% of patients generated vertical saccades below the 95% CI of the controls’ peak velocity. Only 46.9% of patients had smooth pursuit gain lower than that of 95% CI of HC. The involvement in both downward and upward directions was similar (51°/s (68.9, [32.7–69.3]) downward versus 78.8°/s (65.9, [60.8–96.8]) upward). Horizontal saccadic peak velocity and latency, vertical saccadic duration and amplitude, and horizontal position smooth pursuit correlated best to disease severity. Compensating strategies such as blinks to elicit saccades, and head and upper body movements to overcome the gaze palsy, were observed. Vertical reflexive saccades were more impaired and slower than self‐paced ones. Gaze‐holding was normal. Ocular‐motor performance depended on the age of onset and disease duration. Conclusions This is the largest cohort of NPC patients investigated for ocular‐motor function. Vertical supranuclear saccade palsy is the hallmark of NPC. Vertical upward and downward saccades are equally impaired. Horizontal saccadic peak velocity and latency, vertical saccadic duration and amplitude, and horizontal position smooth pursuit can be used as surrogate parameters for clinical trials. Compensating strategies can contribute to establishing a diagnosis., In this largest cohort of patients with Niemann‐Pick type C disease examined so far, we found that vertical supranuclear saccade (VSSP), and not gaze palsy (VSGP), is the hallmark of this disease. We also identify the surrogate biomarkers that can be used in clinical trials to track the disease progress and treatment effect.

Objective Dominant optic atrophy (DOA) is the most common inherited optic neuropathy, with a prevalence of 1:12,000 to 1:25,000. OPA1 mutations are found in 70% of DOA patients, with a significant number remaining undiagnosed. Methods We screened 286 index cases presenting optic atrophy, negative for OPA1 mutations, by targeted next generation sequencing or whole exome sequencing. Pathogenicity and molecular mechanisms of the identified variants were studied in yeast and patient-derived fibroblasts. Results Twelve cases (4%) were found to carry novel variants in AFG3L2, a gene that has been associated with autosomal dominant spinocerebellar ataxia 28 (SCA28). Half of cases were familial with a dominant inheritance, whereas the others were sporadic, including de novo mutations. Biallelic mutations were found in 3 probands with severe syndromic optic neuropathy, acting as recessive or phenotype-modifier variants. All the DOA-associated AFG3L2 mutations were clustered in the ATPase domain, whereas SCA28-associated mutations mostly affect the proteolytic domain. The pathogenic role of DOA-associated AFG3L2 mutations was confirmed in yeast, unraveling a mechanism distinct from that of SCA28-associated AFG3L2 mutations. Patients' fibroblasts showed abnormal OPA1 processing, with accumulation of the fission-inducing short forms leading to mitochondrial network fragmentation, not observed in SCA28 patients' cells. Interpretation This study demonstrates that mutations in AFG3L2 are a relevant cause of optic neuropathy, broadening the spectrum of clinical manifestations and genetic mechanisms associated with AFG3L2 mutations, and underscores the pivotal role of OPA1 and its processing in the pathogenesis of DOA. ANN NEUROL 2020 ANN NEUROL 2020;88:18-32.

Background Leigh syndrome (LS) is a progressive neurodegenerative disorder associated with primary or secondary dysfunction of mitochondrial oxidative phosphorylation and is the most common mitochondrial disease in childhood. Numerous reports on the biochemical and molecular profiles of LS have been published, but there are limited studies on genetically confirmed large series. We reviewed the clinical, imaging, biochemical and molecular data of 122 patients with a diagnosis of LS collected in the Italian Collaborative Network of Mitochondrial Diseases database. Results Clinical picture was characterized by early onset of several neurological signs dominated by central nervous system involvement associated with both supra- and sub-tentorial grey matter at MRI in the majority of cases. Extraneurological organ involvement is less frequent in LS than expected for a mitochondrial disorder. Complex I and IV deficiencies were the most common biochemical diagnoses, mostly associated with mutations in SURF1 or mitochondrial-DNA genes encoding complex I subunits. Our data showed SURF1 as the genotype with the most unfavorable prognosis, differently from other cohorts reported to date. Conclusion We report on a large genetically defined LS cohort, adding new data on phenotype-genotype correlation, prognostic factors and possible suggestions to diagnostic workup.

Alexander disease (AxD) is a leukodystrophy that primarily affects astrocytes and is caused by dominant variants in the Glial Fibrillary Acidic Protein gene. Three main classifications are currently used, the traditional one defined by the age of onset, and two more recent ones based on both clinical features at onset and brain MRI findings. In this study, we retrospectively included patients with genetically confirmed pediatric-onset AxD. Twenty-one Italian patients were enrolled, and we revised all their clinical and radiological data. Participants were divided according to the current classification systems. We qualitatively analyzed data on neurodevelopment and neurologic decline in order to identify the possible trajectories of the evolution of the disease over time. One patient suffered from a Neonatal presentation and showed a rapidly evolving course which led to death within the second year of life (Type Ia). 16 patients suffered from the Infantile presentation: 5 of them (here defined Type Ib) presented developmental delay and began to deteriorate by the age of 5. A second group (Type Ic) included patients who presented a delay in neuromotor development and started deteriorating after 6 years of age. A third group (Type Id) included patients who presented developmental delay and remained clinically stable beyond adolescence. In 4 patients, the age at last evaluation made it not possible to ascertain whether they belonged to Type Ic or Id, as they were too young to evaluate their neurologic decline. 4 patients suffered from the Juvenile presentation: they had normal neuromotor development with no or only mild cognitive impairment; the subsequent clinical evolution was similar to Type Ic AxD in 2 patients, to Id group in the other 2. In conclusion, our results confirm previously described findings about clinical features at onset; based on follow-up data we might classify patients with Type I AxD into four subgroups (Ia, Ib, Ic, Id). Further studies will be needed to confirm our results and to better highlight the existence of clinical and neuroradiological prognostic factors able to predict disease progression.

International audience; Histone Gene Cluster 1 Member E, HIST1H1E, encodes Histone H1.4, is one of a family of epigenetic regulator genes, acts as a linker histone protein, and is responsible for higher order chromatin structure. HIST1H1E syndrome (also known as Rahman syndrome, OMIM #617537) is a recently described intellectual disability (ID) syndrome. Since the initial description of five unrelated individuals with three different heterozygous protein-truncating variants (PTVs) in the HIST1H1E gene in 2017, we have recruited 30 patients, all with HIST1H1E PTVs that result in the same shift in frame and that cluster to a 94-base pair region in the HIST1H1E carboxy terminal domain. The identification of 30 patients with HIST1H1E variants has allowed the clarification of the HIST1H1E syndrome phenotype. Major findings include an ID and a recognizable facial appearance. ID was reported in all patients and is most frequently of moderate severity. The facial gestalt consists of a high frontal hairline and full lower cheeks in early childhood and, in later childhood and adulthood, affected individuals have a strikingly high frontal hairline, frontal bossing, and deep-set eyes. Other associated clinical features include hypothyroidism, abnormal dentition, behavioral issues, cryptorchidism, skeletal anomalies, and cardiac anomalies. Brain magnetic resonance imaging (MRI) is frequently abnormal with a slender corpus callosum a frequent finding.

LAMA2 mutations cause the most frequent congenital muscular dystrophy subtype MDC1A and a variety of milder phenotypes, characterized by total or partial laminin-α2 deficiency. In both severe and milder cases brain MRI invariably shows abnormal white matter signal intensity. We report clinical, histopathological, imaging and genetic data on two siblings with very subtle, and at first undetected, reduction in laminin-α2 expression, and brain MRI showing minor non-specific abnormalities. Clinical features in the female proband were characterized by muscle weakness involving neck and axial muscles, and pelvic girdle and distal lower limb muscles, reduced tendon reflexes and pes cavus. Clinical features in a younger brother were similar, and remained stable in both siblings during the follow up. Whole exome sequencing (WES) detected two heterozygous truncating LAMA2 mutations. Brain MRI in combination with laminin-α2 immunohistochemistry might not be sufficient and WES might be the only means to reach a diagnosis.

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.

Patients with mitochondrial diseases, who usually manifest a multisystem disease, are considered potentially at-risk for a severe coronavirus disease 2019 (COVID-19). The objective of this study is to analyze the clinical features, prognosis and outcomes of COVID-19 in patients with primary mitochondrial diseases in a cohort of patients followed in Italy. We searched for patients with primary mitochondrial diseases and COVID-19 followed by the Italian Collaborative Network of Mitochondrial Diseases. In a total of 1843 patients followed by the National Network, we have identified from March 1st to January 30th, 2021, 27 SARS-CoV-2 infection. Most of the patients were pauci or asymptomatic (85%) and treated at home. The most common signs of COVID-19 were fever (78,9%), fatigue (47,4%), myalgia (42,1%), cough and headache (36,8%), and dyspnea (31,6%). Those who required COVID-19 therapy were treated with low-molecular-weight heparin, glucocorticoids, and antibiotics (mainly azithromycin) without serious side effects related to the therapy. Five patients (18,5%) clinically deteriorated during the infection, and one of them died for pneumonia. Primary mitochondrial diseases infected individuals seemed to be similarly affected by SARS-CoV-2 compared with the general Italian population in terms of clinical presentation and outcome.

Movement disorders are increasingly being recognized as a manifestation of childhood-onset mitochondrial diseases (MDs). However, the spectrum and characteristics of these conditions have not been studied in detail in the context of a well-defined cohort of patients. We retrospectively explored a cohort of individuals with childhood-onset MDs querying the Nationwide Italian Collaborative Network of Mitochondrial Diseases database. Using a customized online questionnaire, we attempted to collect data from the subgroup of patients with movement disorders. Complete information was available for 102 patients. Movement disorder was the presenting feature of MD in 45 individuals, with a mean age at onset of 11 years. Ataxia was the most common movement disorder at onset, followed by dystonia, tremor, hypokinetic disorders, chorea, and myoclonus. During the disease course, most patients (67.7%) encountered a worsening of their movement disorder. Basal ganglia involvement, cerebral white matter changes, and cerebellar atrophy were the most commonly associated neuroradiological patterns. Forty-one patients harbored point mutations in the mitochondrial DNA, 10 carried mitochondrial DNA rearrangements, and 41 cases presented mutations in nuclear-DNA-encoded genes, the latter being associated with an earlier onset and a higher impairment in activities of daily living. Among our patients, 32 individuals received pharmacological treatment; clonazepam and oral baclofen were the most commonly used drugs, whereas levodopa and intrathecal baclofen administration were the most effective. A better delineation of the movement disorders phenotypes starting in childhood may improve our diagnostic workup in MDs, fine tuning management, and treatment of affected patients.

Leigh syndrome is a progressive neurodegenerative disorder, most commonly observed in paediatric mitochondrial disease, and is often associated with pathogenic variants in complex I structural subunits or assembly factors resulting in isolated respiratory chain complex I deficiency. Clinical heterogeneity has been reported, but key diagnostic findings are developmental regression, elevated lactate and characteristic neuroimaging abnormalities. Here, we describe three affected children from two unrelated families who presented with Leigh syndrome due to homozygous variants (c.346_*7del and c.173A>T p.His58Leu) in NDUFC2, encoding a complex I subunit. Biochemical and functional investigation of subjects’ fibroblasts confirmed a severe defect in complex I activity, subunit expression and assembly. Lentiviral transduction of subjects’ fibroblasts with wild‐type NDUFC2 cDNA increased complex I assembly supporting the association of the identified NDUFC2 variants with mitochondrial pathology. Complexome profiling confirmed a loss of NDUFC2 and defective complex I assembly, revealing aberrant assembly intermediates suggestive of stalled biogenesis of the complex I holoenzyme and indicating a crucial role for NDUFC2 in the assembly of the membrane arm of complex I, particularly the ND2 module., This work describes the first confirmed pathogenic variants in NDUFC2 in a case of mitochondrial disease presenting with symptoms of Leigh syndrome and a severe deficiency in OXPHOS complex I. NDUFC2 was shown to be important for the assembly of the membrane arm of complex I.

Niemann-Pick type C (NPC) is a rare lysosomal storage disorder with ocular motor involvement. In a multicontinental, cross-sectional study we characterized ocular motor function in 72 genetically proven patients from twelve countries by means of video-oculography. Interlinking with disease severity, we also searched for ocular motor biomarkers. Our study protocol comprised reflexive and self-paced saccades, smooth pursuit, and gaze-holding in horizontal and vertical planes. Data were compared with those of 158 healthy controls. The Modified Disability Rating Scale, Scale for Assessment and Rating of Ataxia, Spinocerebellar Ataxia Functional Index for neurological status, and Montreal Cognitive Assessment for cognition were also performed.In contrast to previous publications and the common belief that the “downward saccadic system degenerates to greater extent than the upward one”, our measurements of vertical saccades demonstrated that the involvement in both directions was similar. Mean saccadic peak velocity to 20° stimulus was 63.5°/s (SD, 95% CIs of the mean: 59.5, [47.9-79.2]) in NPC patients and 403.1°/s (69.0, [392.0-414.2°/s]) in healthy subjects (pThe number of patient-specific saccadic patterns, incl. slow-pursuit like, hypometric and staircase-pattern saccades suggest varying involvement of the saccadic system with fragmentation of the velocity profile as a sign of omnipause neuron dysfunction. Observed compensating strategies, such as blinks to elicit saccades, head and upper body movements to overcome the gaze palsy, should be used clinically to establish a diagnosis.Vertical reflexive saccades were more impaired and slower than self-paced ones. Ocular motor performance depended on age of onset and disease duration.We found that peak velocity and latency of horizontal saccades, vertical saccadic duration and amplitude, and horizontal position smooth pursuit can be used as surrogate parameters for clinical trials, as they showed the strongest correlation to disease severity. By comparing saccadic with pursuit movements, we showed that 98.2% of patients generated vertical saccades (both up and down) that were below the 95% confidence intervals of the controls’ peak velocity. Only 46.9% of patients had smooth pursuit gain lower than that of 95% of healthy controls. Vertical supranuclear saccade palsy and not vertical supranuclear gaze palsy is the hallmark of NPC disease. The distinction between saccadic and gaze palsy is also important in other neurodegenerative diseases and inborn errors of metabolism with ocular motor involvement, such as progressive supranuclear palsy or Gaucher disease type 3.

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.

Many aspects of epilepsy in mitochondrial disorders (MDs) need to be further clarified. To this aim, we explored retrospectively a cohort of individuals with MDs querying the “Nationwide Italian Collaborative Network of Mitochondrial Diseases” (NICNMD) database (1467 patients included since 2010 to December 2016). We collected information on age at epilepsy onset, seizure type and frequency, genetic findings, and antiepileptic drugs (AEDs). At the time of our survey, 147/1467 (10%) patients in the NICNMD database had epilepsy. Complete information was available only for 98 patients, 52 males and 46 females, aged 5–92years (mean age 40.4 ± 18.4; 14/98 children/teenagers and 84 adults). Epilepsy was the presenting feature of MD in 46/98 (47%) individuals, with onset at a median age of 19years (range, 0.2–68; < 3years in 14/97 (14%), 3–19years in 36/97 (37%), > 19years in 47/97 (49%)). Moreover, 91/98 patients (93%) displayed multiple seizures, with daily or weekly frequency in 25/91 (28%). Interictal EEG was abnormal in 70/78 (90%) patients, displaying abnormal background (47/70; 67%) and/or interictal paroxysms (53/70; 76%). Eighty of 90 patients (89%) displayed a 50–100% reduction of seizures on AEDs; levetiracetam was the most commonly used. Forty-one patients (42%) carried the m.3243A>G mutation, 16 (16%) the m.8344A>G, and 9 (9%) nuclear DNA (nDNA) mutations. Individuals with early-onset seizures mainly carried nDNA mutations and had a more severe epilepsy phenotype, higher seizure frequency, and disorganized background EEG activity. A better definition of epilepsy in MDs may foster the diagnostic workup, management, and treatment of affected patients, and allow more homogeneous patient stratification.

Background Biallelic mutations in TBC1-domain containing kinase (TBCK) lead to hypotonia, global developmental delay with severe cognitive and motor deficits, and variable presentation of dysmorphic facial features and brain malformations. It remains unclear whether hypotonia in these individuals is purely neurogenic, or also caused by progressive muscle disease. Methods Whole exome sequencing was performed on a family diagnosed with nonspecific myopathic changes by means of histological analysis and immunohistochemistry of muscle biopsy samples. Results A novel homozygous truncation in TBCK was found in two sisters diagnosed with muscle disease and severe psychomotor delay. TBCK was completely absent in these patients. Conclusions Our findings identify a novel early truncating variant in TBCK associated with a severe presentation and add muscle disease to the variability of phenotypes associated with TBCK mutations. Inconsistent genotype/phenotype correlation could be ascribed to the multiple roles of TBCK in intracellular signaling and endolysosomal function in different tissues.

Background and Methods Mutations in WDR45 cause β-propeller protein-associated neurodegeneration (BPAN), a type of neurodegeneration with brain iron accumulation (NBIA). We reviewed clinical and MRI findings in 4 patients with de novo WDR45 mutations. Results Psychomotor delay and movement disorders were present in all cases; early-onset epileptic encephalopathy was present in 3. In all cases, first MRI showed: prominent bilateral SN enlargement, bilateral dentate nuclei T2-hyperintensity, and corpus callosum thinning. Iron deposition in the SN and globus pallidus (GP) only became evident later. Diffuse cerebral atrophy was present in 3 cases. Conclusions In this series, SN swelling and dentate nucleus T2 hyperintensity were early signs of BPAN, later followed by progressive iron deposition in the SN and GP. When clinical suspicion is raised, MRI is crucial for identifying early features suggesting this type of NBIA.

The presence of redundant copy number variants (CNVs) in groups of patients with neurological diseases suggests that these variants could have pathogenic effect. We have collected array comparative genomic hybridization (CGH) data of about 2,500 patients affected by neurocognitive disorders and we observed that CNVs in 2p16.3 locus were as frequent as those in 15q11.2, being both the most frequent unbalances in our cohort of patients. Focusing to 2p16.3 region, unbalances involving NRXN1 coding region have been already associated with neuropsychiatric disorders, although with incomplete penetrance, but little is known about CNVs located proximal to the gene, in the long noncoding RNA AK127244. We found that, in our cohort of patients with neuropsychiatric disorders, the frequency of CNVs involving AK127244 was comparable to that of NRXN1 gene. Patients carrying 2p16.3 unbalances shared some common clinical characteristics regardless NRXN1 and AK127244 CNVs localization, suggesting that the AK127244 long noncoding RNA could be involved in neurocognitive disease with the same effect of NRXN1 unbalances. AK127244 as well as NRXN1 unbalances seem to have a particular influence on language development, behavior or mood, according with the topographic correlation between NRXN1 expression and prefrontal cortex functions.

A 4‐year‐old boy born full term after an uneventful pregnancy and with normal early developmental milestones achievement presented hypotonia, motor coordination disorder, and childhood apraxia of speech. Brain MRI revealed globi pallidi T2 symmetrical signal abnormalities (figure). An extensive diagnostic workup panel was performed to exclude metabolic and acquired conditions.1,2 Urine organic acid profile showed marked γ-hydroxybutyrate aciduria (222 µg/mg creatinine, normal

GM1 ganglioside, a monosialic glycosphingolipid and a crucial component of plasma membranes, accumulates in lysosomal storage disorders, primarily in GM1 gangliosidosis. The development of biomarkers for simplifying diagnosis, monitoring disease progression and evaluating drug therapies is an important objective in research into neurodegenerative lysosomal disorders. With this in mind, we established fluorescent imaging and flow-cytometric methods to track changes in GM1 ganglioside levels in patients with GM1 gangliosidosis and in control cells. We also evaluated GM1 ganglioside content in patients’ cells treated with the commercially available Miglustat, a substrate inhibitor potentially suitable for the treatment of late-onset GM1 gangliosidosis. The flow-cytometric method proved to be sensitive, unbiased, and rapid in determining variations in GM1 ganglioside content in human lymphocytes derived from small amounts of fresh blood. We detected a strong correlation between GM1 ganglioside content and the clinical severity of GM1 gangliosidosis. We confirm the ability of Miglustat to act as a substrate reduction agent in the patients’ treated cells. As well as being suitable for diagnosing and managing patients with GM1 gangliosidosis this method could be useful in the diagnosis and management of other lysosomal diseases, such as galactosialidosis, Type C Niemann-Pick, and any other disease with pathologic variations of GM1 ganglioside.

Next Generation Sequencing (NGS) technologies are revolutionizing the diagnostic screening for rare disease entities, including primary mitochondrial disorders, particularly those caused by nuclear gene defects. NGS approaches are able to identify the causative gene defects in small families and even single individuals, unsuitable for investigation by traditional linkage analysis. These technologies are contributing to fill the gap between mitochondrial disease cases defined on the basis of clinical, neuroimaging and biochemical readouts, which still outnumber by approximately 50% the cases for which a molecular-genetic diagnosis is attained. We have been using a combined, two-step strategy, based on targeted genes panel as a first NGS screening, followed by whole exome sequencing (WES) in still unsolved cases, to analyze a large cohort of subjects, that failed to show mutations in mtDNA and in ad hoc sets of specific nuclear genes, sequenced by the Sanger's method. Not only this approach has allowed us to reach molecular diagnosis in a significant fraction (20%) of these difficult cases, but it has also revealed unexpected and conceptually new findings. These include the possibility of marked variable penetrance of recessive mutations, the identification of large-scale DNA rearrangements, which explain spuriously heterozygous cases, and the association of mutations in known genes with unusual, previously unreported clinical phenotypes. Importantly, WES on selected cases has unraveled the presence of pathogenic mutations in genes encoding non-mitochondrial proteins (e.g. the transcription factor E4F1), an observation that further expands the intricate genetics of mitochondrial disease and suggests a new area of investigation in mitochondrial medicine. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.

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.

Alexander disease is an hereditary leukodystrophy related to mutations of GFAP. Classically AxD was divided in infantile, juvenile, and adult subgroups. Recent data suggested considering only two subtypes: type I (infantile onset with lesions extending to the cerebral hemispheres); type II (adult onset with primary involvement of subtentorial structures). We report two related and one unrelated patients presenting with a peculiar association of clinical and neuroradiological features. GFAP analysis disclosed the presence of one novel and two previously reported mutations. Our patients underline the importance of considering AxD in patients with bulbar symptoms and autonomic dysfunction even if MRI shows only posterior fossa abnormalities, supporting the hypothesis of a third type of AxD sharing features of both type I and type II. The evidence of an intrafamilial phenotypic variability suggests the possible role of still unknown factors influencing the effect of GFAP mutation and determining the phenotype.

OBJECTIVES To determine the prevalence of epilepsy in children with early-onset mitochondrial diseases (MDs) and to evaluate the epileptic phenotypes and associated features. MATERIALS AND METHODS Children affected by MD with onset during the first year of life were enrolled. Patients were classified according to their mitochondrial phenotype, and all findings in patients with epilepsy versus patients without were compared. The epileptic features were analyzed. RESULTS The series includes 129 patients (70 females) with median age at disease onset of 3 months. The median time of follow-up was 5 years. Non-syndromic mitochondrial encephalopathy and pyruvate dehydrogenase complex deficiency were the main mitochondrial diseases associated with epilepsy (P

Cristina Dallabona, Truus E. M. Abbink, Rosalba Carrozzo, Alessandra Torraco, Andrea Legati, Carola G. M. van Berkel, Marcello Niceta, Tiziana Langella, Daniela Verrigni, Teresa Rizza, Daria Diodato, Fiorella Piemonte, Eleonora Lamantea, Mingyan Fang, Jianguo Zhang, Diego Martinelli, Elsa Bevivino, Carlo Dionisi-Vici, Adeline Vanderver, Sunny G. Philip, Manju A. Kurian, Ishwar C. Verma, Sunita Bijarnia-Mahay, Sandra Jacinto, Fatima Furtado, Patrizia Accorsi, Anna Ardissone, Isabella Moroni, Ileana Ferrero, Marco Tartaglia, Paola Goffrini, Daniele Ghezzi, Marjo S. van der Knaap and Enrico Bertini. LYRM7 mutations cause a multifocal cavitating leukoencephalopathy with distinct MRI appearance. Brain 2016; 139: 782-794. doi:10.1093/brain/awv392. The authors apologize for mixing the mutations of Patients 5 and 7 on pages 788 and 789 in Figure 2 and its legend, in the main text and the online Supplementary Tables. This has now been corrected online. On page 788, the text should read: Sanger sequencing of the entire coding sequence of LYRM7 and intron-exon boundaries was performed in three additional patients. Patient 5 was found homozygous for the c.37delA (p.Thr13Hisfs∗17) mutation, which is predicted to dramatically affect recognition of the splice donor motif of exon 4 and likely impair proper RNA splicing (Fig. 2M and N), while Patients 6 and 7 were found to be homozygous for the c.214C4T (p.Q72∗) (Fig. 2L), and c.243-244 + 2delGAGT (p.?) (Fig. 2H) mutations, respectively. The Supplementary Tables have been corrected online. On page 789, Figure 2 and legend should be as follows.

Background KARS encodes lysyl- transfer ribonucleic acid (tRNA) synthetase, which catalyzes the aminoacylation of tRNA-Lys in the cytoplasm and mitochondria. Eleven families/sporadic patients and 16 different mutations in KARS have been reported to date. The associated clinical phenotype is heterogeneous ranging from early onset encephalopathy to isolated peripheral neuropathy or nonsyndromic hearing impairment. Recently additional presentations including leukoencephalopathy as predominant cerebral involvement or cardiomyopathy, isolated or associated with muscular and cerebral involvement, have been reported. A progressive Leukoencephalopathy with brainstem and spinal cord calcifications was previously described in a singleton patient and in two siblings, without the identification of the genetic cause. We reported here about a new severe phenotype associated with biallelic KARS mutations and sharing some common points with the other already reported phenotypes, but with a distinct clinical and neuroimaging picture. Review of KARS mutant patients published to date will be also discussed. Results Herein, we report the clinical, biochemical and molecular findings of 2 unreported Italian patients affected by developmental delay, acquired microcephaly, spastic tetraparesis, epilepsy, sensory-neural hypoacusia, visual impairment, microcytic hypochromic anaemia and signs of hepatic dysfunction. MRI pattern in our patients was characterized by progressive diffuse leukoencephalopathy and calcifications extending in cerebral, brainstem and cerebellar white matter, with spinal cord involvement. Genetic analysis performed on these 2 patients and in one subject previously described with similar MRI pattern revealed the presence of biallelic mutations in KARS in all 3 subjects. Conclusions With our report we define the molecular basis of the previously described Leukoencephalopathy with Brainstem and Spinal cord Calcification widening the spectrum of KARS related disorders, particularly in childhood onset disease suggestive for mitochondrial impairment. The review of previous cases does not suggest a strict and univocal genotype/phenotype correlation for this highly heterogeneous entity. Moreover, our cases confirm the usefulness of search for common brain and spine MR imaging pattern and of broad genetic screening, in syndromes clinically resembling mitochondrial disorders in spite of normal biochemical assay. Electronic supplementary material The online version of this article (10.1186/s13023-018-0788-4) contains supplementary material, which is available to authorized users.

Biallelic mutations in NDUFAF6 have been identified as responsible for cases of autosomal recessive Leigh syndrome associated with mitochondrial complex I deficiency. Here we report two siblings and two unrelated subjects with Leigh syndrome, in which we found the same compound heterozygous missense (c.532G>C:p.A178P) and deep intronic (c.420+784C>T) variants in NDUFAF6. We demonstrated that the identified intronic variant creates an alternative splice site, leading to the production of an aberrant transcript. A detailed analysis of whole-exome sequencing data together with the functional validation based on mRNA analysis may reveal pathogenic variants even in non-exonic regions.

Congenital myasthenic syndromes (CMS) are rare and heterogeneous genetic diseases characterized by compromised neuromuscular transmission and clinical features of fatigable weakness; age at onset, presenting symptoms, distribution of weakness, and response to treatment differ depending on the underlying molecular defect. Mutations in one of the multiple genes, encoding proteins expressed at the neuromuscular junction, are currently known to be associated with subtypes of CMS. The most common CMS syndrome identified is associated with mutation in the CHRNE gene, causing principally muscle nicotinic acetylcholine receptor deficiency, that results in reduced receptor density on the postsynaptic membrane. We describe the clinical, neurophysiological and molecular features of two unrelated CMS Italian families with marked phenotypic variability, carrying the already reported p.T159P mutation in the CHRNE gene. Our report highlights clinical heterogeneity, intrafamily variability in spite of the same genotype and a possible gender effect; it confirms the efficacy and safety of salbutamol in patients who harbor mutations in the epsilon subunit of acetylcholine receptor.

Ocular myopathy, typically manifesting as progressive external ophthalmoplegia (PEO), is among the most common mitochondrial phenotypes. The purpose of this study is to better define the clinical phenotypes associated with ocular myopathy. This is a retrospective study on a large cohort from the database of the “Nation-wide Italian Collaborative Network of Mitochondrial Diseases”. We distinguished patients with ocular myopathy as part of a multisystem mitochondrial encephalomyopathy (PEO-encephalomyopathy), and then PEO with isolated ocular myopathy from PEO-plus when PEO was associated with additional features of multisystemic involvement. Ocular myopathy was the most common feature in our cohort of mitochondrial patients. Among the 722 patients with a definite genetic diagnosis, ocular myopathy was observed in 399 subjects (55.3%) and was positively associated with mtDNA single deletions and POLG mutations. Ocular myopathy as manifestation of a multisystem mitochondrial encephalomyopathy (PEO-encephalomyopathy, n=131) was linked to the m.3243A>G mutation, whereas the other “PEO” patients (n=268) were associated with mtDNA single deletion and Twinkle mutations. Increased lactate was associated with central neurological involvement. We then defined, among the PEO group, as “pure PEO” the patients with isolated ocular myopathy and “PEO-plus” those with ocular myopathy and other features of neuromuscular and multisystem involvement, excluding central nervous system. The male proportion was significantly lower in pure PEO than PEO-plus. This study reinforces the need for research on the role of gender in mitochondrial diseases. The phenotype definitions here revisited may contribute to a more homogeneous patient categorization, useful in future studies and clinical trials

Background Heterozygous mutations in OPA1 are a common cause of autosomal dominant optic atrophy, sometimes associated with extra-ocular manifestations. Few cases harboring compound heterozygous OPA1 mutations have been described manifesting complex neurodegenerative disorders in addition to optic atrophy. Results We report here three patients: one boy showing an early-onset mitochondrial disorder with hypotonia, ataxia and neuropathy that was severely progressive, leading to early death because of multiorgan failure; two unrelated sporadic girls manifesting a spastic ataxic syndrome associated with peripheral neuropathy and, only in one, optic atrophy. Using a targeted resequencing of 132 genes associated with mitochondrial disorders, in two probands we found compound heterozygous mutations in OPA1: in the first a 5 nucleotide deletion, causing a frameshift and insertion of a premature stop codon (p.Ser64Asnfs*7), and a missense change (p.Ile437Met), which has recently been reported to have clinical impact; in the second, a novel missense change (p.Val988Phe) co-occurred with the p.Ile437Met substitution. In the third patient a homozygous mutation, c.1180G > A (p.Ala394Thr) in OPA1 was detected by a trio-based whole exome sequencing approach. One of the patients presented also variants in mitochondrial DNA that may have contributed to the peculiar phenotype. The deleterious effect of the identified missense changes was experimentally validated in yeast model. OPA1 level was reduced in available patients’ biological samples, and a clearly fragmented mitochondrial network was observed in patients’ fibroblasts. Conclusions This report provides evidence that bi-allelic OPA1 mutations may lead to complex and severe multi-system recessive mitochondrial disorders, where optic atrophy might not represent the main feature. Electronic supplementary material The online version of this article (doi:10.1186/s13023-017-0641-1) contains supplementary material, which is available to authorized users.

Contains fulltext : 137503.pdf (Publisher’s version ) (Open Access) 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.

To explore the genetic architecture of human overgrowth syndromes and human growth control, we performed experimental and bioinformatic analyses of 710 individuals with overgrowth (height and/or head circumference ≥+2 SD) and intellectual disability (OGID). We identified a causal mutation in 1 of 14 genes in 50% (353/710). This includes HIST1H1E, encoding histone H1.4, which has not been associated with a developmental disorder previously. The pathogenic HIST1H1E mutations are predicted to result in a product that is less effective in neutralizing negatively charged linker DNA because it has a reduced net charge, and in DNA binding and protein-protein interactions because key residues are truncated. Functional network analyses demonstrated that epigenetic regulation is a prominent biological process dysregulated in individuals with OGID. Mutations in six epigenetic regulation genes—NSD1, EZH2, DNMT3A, CHD8, HIST1H1E, and EED—accounted for 44% of individuals (311/710). There was significant overlap between the 14 genes involved in OGID and 611 genes in regions identified in GWASs to be associated with height (p = 6.84 × 10−8), suggesting that a common variation impacting function of genes involved in OGID influences height at a population level. Increased cellular growth is a hallmark of cancer and there was striking overlap between the genes involved in OGID and 260 somatically mutated cancer driver genes (p = 1.75 × 10−14). However, the mutation spectra of genes involved in OGID and cancer differ, suggesting complex genotype-phenotype relationships. These data reveal insights into the genetic control of human growth and demonstrate that exome sequencing in OGID has a high diagnostic yield.

Defects of the Fe/S cluster biosynthesis represent a subgroup of diseases affecting the mitochondrial energy metabolism. In the last years, mutations in four genes (NFU1, BOLA3, ISCA2 and IBA57) have been related to a new group of multiple mitochondrial dysfunction syndromes characterized by lactic acidosis, hyperglycinemia, multiple defects of the respiratory chain complexes, and impairment of four lipoic acid-dependent enzymes: α-ketoglutarate dehydrogenase complex, pyruvic dehydrogenase, branched-chain α-keto acid dehydrogenase complex and the H protein of the glycine cleavage system. Few patients have been reported with mutations in IBA57 and with variable clinical phenotype. Herein, we describe four unrelated patients carrying novel mutations in IBA57. All patients presented with combined or isolated defect of complex I and II. Clinical features varied widely, ranging from fatal infantile onset of the disease to acute and severe psychomotor regression after the first year of life. Brain MRI was characterized by cavitating leukodystrophy. The identified mutations were never reported previously and all had a dramatic effect on IBA57 stability. Our study contributes to expand the array of the genotypic variation of IBA57 and delineates the leukodystrophic pattern of IBA57 deficient patients.

Background Assembly of cytochrome c oxidase (COX, complex IV, cIV), the terminal component of the mitochondrial respiratory chain, is assisted by several factors, most of which are conserved from yeast to humans. However, some of them, including COA7, are found in humans but not in yeast. COA7 is a 231aa-long mitochondrial protein present in animals, containing five Sel1-like tetratricopeptide repeat sequences, which are likely to interact with partner proteins. Methods Whole exome sequencing was carried out on a 19 year old woman, affected by early onset, progressive severe ataxia and peripheral neuropathy, mild cognitive impairment and a cavitating leukodystrophy of the brain with spinal cord hypotrophy. Biochemical analysis of the mitochondrial respiratory chain revealed the presence of isolated deficiency of cytochrome c oxidase (COX) activity in skin fibroblasts and skeletal muscle. Mitochondrial localization studies were carried out in isolated mitochondria and mitoplasts from immortalized control human fibroblasts. Results We found compound heterozygous mutations in COA7 : a paternal c.410A>G, p.Y137C, and a maternal c.287+1G>T variants. Lentiviral-mediated expression of recombinant wild-type COA7 cDNA in the patient fibroblasts led to the recovery of the defect in COX activity and restoration of normal COX amount. In mitochondrial localization experiments, COA7 behaved as the soluble matrix protein Citrate Synthase. Conclusions We report here the first patient carrying pathogenic mutations of COA7 , causative of isolated COX deficiency and progressive neurological impairment. We also show that COA7 is a soluble protein localized to the matrix, rather than in the intermembrane space as previously suggested.