Low mitochondrial DNA copy number in buffy coat DNA of primary open-angle glaucoma patients.

Primary open-angle glaucoma (POAG) is characterized by optic nerve degeneration and irreversible loss of retinal ganglion cells (RGCs). The pathophysiology is not fully understood. Since RGCs have a high energy demand, suboptimal mitochondrial function may put the survival of these neurons at risk. In the present study, we explored whether mtDNA copy number or mtDNA deletions could reveal a mitochondrial component in POAG pathophysiology. Buffy coat DNA was isolated from EDTA blood of age- and sex-matched study groups, namely POAG patients with high intraocular pressure (IOP) at diagnosis (high tension glaucoma: HTG; n = 97), normal tension glaucoma patients (NTG, n = 37), ocular hypertensive controls (n = 9), and cataract controls (without glaucoma; n = 32), all without remarkable comorbidities. The number of mtDNA copies was assessed through qPCR quantification of the mitochondrial D-loop and nuclear B2M gene. Presence of the common 4977 base pair mtDNA deletion was assessed by a highly sensitive breakpoint PCR. Analysis showed that HTG patients had a lower number of mtDNA copies per nuclear DNA than NTG patients (p-value <0.01, Dunn test) and controls (p-value <0.001, Dunn test). The common 4977 base pair mtDNA deletion was not detected in any of the participants. A lower mtDNA copy number in blood of HTG patients suggests a role for a genetically defined, deficient mtDNA replication in the pathology of HTG. This may cause a low number of mtDNA copies in RGCs, which together with aging and high IOP, may lead to mitochondrial dysfunction, and contribute to glaucoma pathology.

Consensus recommendations on Epilepsy in Phelan-McDermid syndrome.

Phelan-McDermid syndrome (PMS) is a 22q13.3 deletion syndrome that presents with a disturbed development, neurological and psychiatric characteristics, and sometimes other comorbidities like seizures. The epilepsy manifests itself in a variety of seizure semiologies. Further diagnostics using electroencephalogram (EEG) and brain magnetic resonance imaging (MRI) are important in conjunction with the clinical picture of the seizures to decide whether anticonvulsant therapy is necessary. As part of the development of European consensus guidelines we focussed on the prevalence and semiology of epileptic seizures in PMS associated with a pathogenic variant in the SHANK3 gene or the 22q13 deletion involving SHANK3, in order to then be able to make recommendations regarding diagnosis and therapy.

Phenotypic spectrum and clinical course of single large-scale mitochondrial DNA deletion disease in the paediatric population: a multicentre study.

BACKGROUND: Large-scale mitochondrial DNA deletions (LMD) are a common genetic cause of mitochondrial disease and give rise to a wide range of clinical features. Lack of longitudinal data means the natural history remains unclear. This study was undertaken to describe the clinical spectrum in a large cohort of patients with paediatric disease onset. METHODS: A retrospective multicentre study was performed in patients with clinical onset <16 years of age, diagnosed and followed in seven European mitochondrial disease centres. RESULTS: A total of 80 patients were included. The average age at disease onset and at last examination was 10 and 31 years, respectively. The median time from disease onset to death was 11.5 years. Pearson syndrome was present in 21%, Kearns-Sayre syndrome spectrum disorder in 50% and progressive external ophthalmoplegia in 29% of patients. Haematological abnormalities were the hallmark of the disease in preschool children, while the most common presentations in older patients were ptosis and external ophthalmoplegia. Skeletal muscle involvement was found in 65% and exercise intolerance in 25% of the patients. Central nervous system involvement was frequent, with variable presence of ataxia (40%), cognitive involvement (36%) and stroke-like episodes (9%). Other common features were pigmentary retinopathy (46%), short stature (42%), hearing impairment (39%), cardiac disease (39%), diabetes mellitus (25%) and renal disease (19%). CONCLUSION: Our study provides new insights into the phenotypic spectrum of childhood-onset, LMD-associated syndromes. We found a wider spectrum of more prevalent multisystem involvement compared with previous studies, most likely related to a longer time of follow-up.

Mitochondrial DNA D-loop variants correlate with a primary open-angle glaucoma subgroup.

Introduction: Primary open-angle glaucoma (POAG) is a characteristic optic neuropathy, caused by degeneration of the optic nerve-forming neurons, the retinal ganglion cells (RGCs). High intraocular pressure (IOP) and aging have been identified as major risk factors; yet the POAG pathophysiology is not fully understood. Since RGCs have high energy requirements, mitochondrial dysfunction may put the survivability of RGCs at risk. We explored in buffy coat DNA whether mtDNA variants and their distribution throughout the mtDNA could be risk factors for POAG. Methods: The mtDNA was sequenced from age- and sex-matched study groups, being high tension glaucoma (HTG, n=71), normal tension glaucoma patients (NTG, n=33), ocular hypertensive subjects (OH, n=7), and cataract controls (without glaucoma; n=30), all without remarkable comorbidities. Results: No association was found between the number of mtDNA variants in genes encoding proteins, tRNAs, rRNAs, and in non-coding regions in the different study groups. Next, variants that controls shared with the other groups were discarded. A significantly higher number of exclusive variants was observed in the D-loop region for the HTG group (~1.23 variants/subject), in contrast to controls (~0.35 variants/subject). In the D-loop, specifically in the 7S DNA sub-region within the Hypervariable region 1 (HV1), we found that 42% of the HTG and 27% of the NTG subjects presented variants, while this was only 14% for the controls and OH subjects. As we have previously reported a reduction in mtDNA copy number in HTG, we analysed if specific D-loop variants could explain this. While the majority of glaucoma patients with the exclusive D-loop variants m.72T>C, m.16163 A>G, m.16186C>T, m.16298T>C, and m.16390G>A presented a mtDNA copy number below controls median, no significant association between these variants and low copy number was found and their possible negative role in mtDNA replication remains uncertain. Approximately 38% of the HTG patients with reduced copy number did not carry any exclusive D-loop or other mtDNA variants, which indicates that variants in nuclear-encoded mitochondrial genes, environmental factors, or aging might be involved in those cases. Conclusion: In conclusion, we found that variants in the D-loop region may be a risk factor in a subgroup of POAG, possibly by affecting mtDNA replication.

Neurodegenerative and functional signatures of the cerebellar cortex in m.3243A > G patients.

Mutations of the mitochondrial DNA are an important cause of inherited diseases that can severely affect the tissue's homeostasis and integrity. The m.3243A > G mutation is the most commonly observed across mitochondrial disorders and is linked to multisystemic complications, including cognitive deficits. In line with in vitro experiments demonstrating the m.3243A > G's negative impact on neuronal energy production and integrity, m.3243A > G patients show cerebral grey matter tissue changes. However, its impact on the most neuron dense, and therefore energy-consuming brain structure-the cerebellum-remains elusive. In this work, we used high-resolution structural and functional data acquired using 7 T MRI to characterize the neurodegenerative and functional signatures of the cerebellar cortex in m.3243A > G patients. Our results reveal altered tissue integrity within distinct clusters across the cerebellar cortex, apparent by their significantly reduced volume and longitudinal relaxation rate compared with healthy controls, indicating macroscopic atrophy and microstructural pathology. Spatial characterization reveals that these changes occur especially in regions related to the frontoparietal brain network that is involved in information processing and selective attention. In addition, based on resting-state functional MRI data, these clusters exhibit reduced functional connectivity to frontal and parietal cortical regions, especially in patients characterized by (i) a severe disease phenotype and (ii) reduced information-processing speed and attention control. Combined with our previous work, these results provide insight into the neuropathological changes and a solid base to guide longitudinal studies aimed to track disease progression.

Whole exome sequencing reveals a homozygous C1QBP deletion as the cause of progressive external ophthalmoplegia and multiple mtDNA deletions.

Whole exome sequencing (WES), analyzed with GENESIS and WeGET, revealed a homozygous deletion in the C1QBP gene in a patient with progressive external ophthalmoplegia (PEO) and multiple mtDNA deletions. The gene encodes the mitochondria-located complementary 1 Q subcomponent-binding protein, involved in mitochondrial homeostasis. Biallelic mutations in C1QBP cause mitochondrial cardiomyopathy and/or PEO with variable age of onset. Our patient showed only late-onset PEO-plus syndrome without overt cardiac involvement. Available data suggest that early-onset cardiomyopathy variants localize in important structural domains and PEO-plus variants in the coiled-coil region. Our patient demonstrates that C1QBP mutations should be considered in individuals with PEO with or without cardiomyopathy.

Pathogenic SLIRP variants as a novel cause of autosomal recessive mitochondrial encephalomyopathy with complex I and IV deficiency.

In a Dutch non-consanguineous patient having mitochondrial encephalomyopathy with complex I and complex IV deficiency, whole exome sequencing revealed two compound heterozygous variants in SLIRP. SLIRP gene encodes a stem-loop RNA-binding protein that regulates mitochondrial RNA expression and oxidative phosphorylation (OXPHOS). A frameshift and a deep-intronic splicing variant reduced the amount of functional wild-type SLIRP RNA to 5%. Consequently, in patient fibroblasts, MT-ND1, MT-ND6, and MT-CO1 expression was reduced. Lentiviral transduction of wild-type SLIRP cDNA in patient fibroblasts increased MT-ND1, MT-ND6, and MT-CO1 expression (2.5-7.2-fold), whereas mutant cDNAs did not. A fourfold decrease of citrate synthase versus total protein ratio in patient fibroblasts indicated that the resulting reduced mitochondrial mass caused the OXPHOS deficiency. Transduction with wild-type SLIRP cDNA led to a 2.4-fold increase of this ratio and partly restored OXPHOS activity. This confirmed causality of the SLIRP variants. In conclusion, we report SLIRP variants as a novel cause of mitochondrial encephalomyopathy with OXPHOS deficiency.

Plasma GDF-15 concentration is not elevated in open-angle glaucoma.

AIM: Recently, the level of growth differentiation factor 15 (GDF-15) in blood, was proposed as biomarker to detect mitochondrial dysfunction. In the current study, we evaluate this biomarker in open-angle glaucoma (OAG), as there is increasing evidence that mitochondrial dysfunction plays a role in the pathophysiology of this disease. METHODS: Plasma GDF-15 concentrations were measured with ELISA in 200 OAG patients and 61 age-matched controls (cataract without glaucoma). The OAG patient group consisted of high tension glaucoma (HTG; n = 162) and normal tension glaucoma (NTG; n = 38). Groups were compared using the Kruskal-Wallis nonparametric test with Dunn's multiple comparison post-hoc correction. GDF-15 concentration was corrected for confounders identified with forward linear regression models. RESULTS: Before correcting for confounders, median plasma GDF-15 levels was significantly lower in the combined OAG group (p = 0.04), but not when analysing HTG and NTG patients separately. Forward linear regression analysis showed that age, gender, smoking and systemic hypertension were significant confounders affecting GDF-15 levels. After correction for these confounders, GDF-15 levels in OAG patients were no longer significantly different from controls. Subgroup analysis of the glaucoma patients did not show a correlation between disease severity and plasma GDF-15, but did reveal that for NTG patients, intake of dietary supplements, which potentially improve mitochondrial function, correlated with lower plasma GDF-15. CONCLUSION: The present study suggests that plasma GDF-15 is not suited as biomarker of mitochondrial dysfunction in OAG patients.

Disease characteristics of MCT8 deficiency: an international, retrospective, multicentre cohort study.

BACKGROUND: Disordered thyroid hormone transport, due to mutations in the SLC16A2 gene encoding monocarboxylate transporter 8 (MCT8), is characterised by intellectual and motor disability resulting from cerebral hypothyroidism and chronic peripheral thyrotoxicosis. We sought to systematically assess the phenotypic characteristics and natural history of patients with MCT8 deficiency. METHODS: We did an international, multicentre, cohort study, analysing retrospective data from Jan 1, 2003, to Dec 31, 2019, from patients with MCT8 deficiency followed up in 47 hospitals in 22 countries globally. The key inclusion criterion was genetically confirmed MCT8 deficiency. There were no exclusion criteria. Our primary objective was to analyse the overall survival of patients with MCT8 deficiency and document causes of death. We also compared survival between patients who did or did not attain full head control by age 1·5 years and between patients who were or were not underweight by age 1-3 years (defined as a bodyweight-for-age Z score <-2 SDs or <5th percentile according to WHO definition). Other objectives were to assess neurocognitive function and outcomes, and clinical parameters including anthropometric characteristics, biochemical markers, and neuroimaging findings. FINDINGS: Between Oct 14, 2014, and Jan 17, 2020, we enrolled 151 patients with 73 different MCT8 (SLC16A2) mutations. Median age at diagnosis was 24·0 months (IQR 12·0-60·0, range 0·0-744·0). 32 (21%) of 151 patients died; the main causes of mortality in these patients were pulmonary infection (six [19%]) and sudden death (six [19%]). Median overall survival was 35·0 years (95% CI 8·3-61·7). Individuals who did not attain head control by age 1·5 years had an increased risk of death compared with patients who did attain head control (hazard ratio [HR] 3·46, 95% CI 1·76-8·34; log-rank test p=0·0041). Patients who were underweight during age 1-3 years had an increased risk for death compared with patients who were of normal bodyweight at this age (HR 4·71, 95% CI 1·26-17·58, p=0·021). The few motor and cognitive abilities of patients did not improve with age, as evidenced by the absence of significant correlations between biological age and scores on the Gross Motor Function Measure-88 and Bayley Scales of Infant Development III. Tri-iodothyronine concentrations were above the age-specific upper limit in 96 (95%) of 101 patients and free thyroxine concentrations were below the age-specific lower limit in 94 (89%) of 106 patients. 59 (71%) of 83 patients were underweight. 25 (53%) of 47 patients had elevated systolic blood pressure above the 90th percentile, 34 (76%) of 45 patients had premature atrial contractions, and 20 (31%) of 64 had resting tachycardia. The most consistent MRI finding was a global delay in myelination, which occurred in 13 (100%) of 13 patients. INTERPRETATION: Our description of characteristics of MCT8 deficiency in a large patient cohort reveals poor survival with a high prevalence of treatable underlying risk factors, and provides knowledge that might inform clinical management and future evaluation of therapies. FUNDING: Netherlands Organisation for Health Research and Development, and the Sherman Foundation.

A novel mitochondrial m.4414T>C MT-TM gene variant causing progressive external ophthalmoplegia and myopathy.

We report a novel mitochondrial m.4414T>C variant in the mt-tRNAMet (MT-TM) gene in an adult patient with chronic progressive external ophthalmoplegia and myopathy whose muscle biopsy revealed focal cytochrome c oxidase (COX)-deficient and ragged red fibres. The m.4414T>C variant occurs at a strongly evolutionary conserved sequence position, disturbing a canonical base pair and disrupting the secondary and tertiary structure of the mt-tRNAMet. Definitive evidence of pathogenicity is provided by clear segregation of m.4414T>C mutant levels with COX deficiency in single muscle fibres. Interestingly, the variant is present in skeletal muscle at relatively low levels (30%) and undetectable in accessible, non-muscle tissues from the patient and her asymptomatic brother, emphasizing the continuing requirement for a diagnostic muscle biopsy as the preferred tissue for mtDNA genetic investigations of mt-tRNA variants leading to mitochondrial myopathy.

Effectiveness and safety of the tri-iodothyronine analogue Triac in children and adults with MCT8 deficiency: an international, single-arm, open-label, phase 2 trial.

BACKGROUND: Deficiency of the thyroid hormone transporter monocarboxylate transporter 8 (MCT8) causes severe intellectual and motor disability and high serum tri-iodothyronine (T3) concentrations (Allan-Herndon-Dudley syndrome). This chronic thyrotoxicosis leads to progressive deterioration in bodyweight, tachycardia, and muscle wasting, predisposing affected individuals to substantial morbidity and mortality. Treatment that safely alleviates peripheral thyrotoxicosis and reverses cerebral hypothyroidism is not yet available. We aimed to investigate the effects of treatment with the T3 analogue Triac (3,3',5-tri-iodothyroacetic acid, or tiratricol), in patients with MCT8 deficiency. METHODS: In this investigator-initiated, multicentre, open-label, single-arm, phase 2, pragmatic trial, we investigated the effectiveness and safety of oral Triac in male paediatric and adult patients with MCT8 deficiency in eight countries in Europe and one site in South Africa. Triac was administered in a predefined escalating dose schedule-after the initial dose of once-daily 350 µg Triac, the daily dose was increased progressively in 350 µg increments, with the goal of attaining serum total T3 concentrations within the target range of 1·4-2·5 nmol/L. We assessed changes in several clinical and biochemical signs of hyperthyroidism between baseline and 12 months of treatment. The prespecified primary endpoint was the change in serum T3 concentrations from baseline to month 12. The co-primary endpoints were changes in concentrations of serum thyroid-stimulating hormone (TSH), free and total thyroxine (T4), and total reverse T3 from baseline to month 12. These analyses were done in patients who received at least one dose of Triac and had at least one post-baseline evaluation of serum throid function. This trial is registered with ClinicalTrials.gov, number NCT02060474. FINDINGS: Between Oct 15, 2014, and June 1, 2017, we screened 50 patients, all of whom were eligible. Of these patients, four (8%) patients decided not to participate because of travel commitments. 46 (92%) patients were therefore enrolled in the trial to receive Triac (median age 7·1 years [range 0·8-66·8]). 45 (98%) participants received Triac and had at least one follow-up measurement of thyroid function and thus were included in the analyses of the primary endpoints. Of these 45 patients, five did not complete the trial (two patients withdrew [travel burden, severe pre-existing comorbidity], one was lost to follow-up, one developed of Graves disease, and one died of sepsis). Patients required a mean dose of 38.3 µg/kg of bodyweight (range 6·4-84·3) to attain T3 concentrations within the target range. Serum T3 concentration decreased from 4·97 nmol/L (SD 1·55) at baseline to 1·82 nmol/L (0·69) at month 12 (mean decrease 3·15 nmol/L, 95% CI 2·68-3·62; p<0·0001), while serum TSH concentrations decreased from 2·91 mU/L (SD 1·68) to 1·02 mU/L (1·14; mean decrease 1·89 mU/L, 1·39-2·39; p<0·0001) and serum free T4 concentrations decreased from 9·5 pmol/L (SD 2·5) to 3·4 (1·6; mean decrease 6·1 pmol/L (5·4-6·8; p<0·0001). Additionally, serum total T4 concentrations decreased by 31·6 nmol/L (28·0-35·2; p<0·0001) and reverse T3 by 0·08 nmol/L (0·05-0·10; p<0·0001). Seven treatment-related adverse events (transiently increased perspiration or irritability) occurred in six (13%) patients. 26 serious adverse events that were considered unrelated to treatment occurred in 18 (39%) patients (mostly hospital admissions because of infections). One patient died from pulmonary sepsis leading to multi-organ failure, which was unrelated to Triac treatment. INTERPRETATION: Key features of peripheral thyrotoxicosis were alleviated in paediatric and adult patients with MCT8 deficiency who were treated with Triac. Triac seems a reasonable treatment strategy to ameliorate the consequences of untreated peripheral thyrotoxicosis in patients with MCT8 deficiency. FUNDING: Dutch Scientific Organization, Sherman Foundation, NeMO Foundation, Wellcome Trust, UK National Institute for Health Research Cambridge Biomedical Centre, Toulouse University Hospital, and Una Vita Rara ONLUS.

Leigh syndrome caused by mutations in MTFMT is associated with a better prognosis.

Objectives: Mitochondrial methionyl-tRNA formyltransferase (MTFMT) is required for the initiation of translation and elongation of mitochondrial protein synthesis. Pathogenic variants in MTFMT have been associated with Leigh syndrome (LS) and mitochondrial multiple respiratory chain deficiencies. We sought to elucidate the spectrum of clinical, neuroradiological and molecular genetic findings of patients with bi-allelic pathogenic variants in MTFMT. Methods: Retrospective cohort study combining new cases and previously published cases. Results: Thirty-eight patients with pathogenic variants in MTFMT were identified, including eight new cases. The median age of presentation was 14 months (range: birth to 17 years, interquartile range [IQR] 4.5 years), with developmental delay and motor symptoms being the most frequent initial manifestation. Twenty-nine percent of the patients survived into adulthood. MRI headings in MTFMT pathogenic variants included symmetrical basal ganglia changes (62%), periventricular and subcortical white matter abnormalities (55%), and brainstem lesions (48%). Isolated complex I and combined respiratory chain deficiencies were identified in 31% and 59% of the cases, respectively. Reduction of the mitochondrial complex I and complex IV subunits was identified in the fibroblasts (13/13). Sixteen pathogenic variants were identified, of which c.626C>T was the most common. Seventy-four percent of the patients were alive at their last clinical review (median 6.8 years, range: 14 months to 31 years, IQR 14.5 years). Interpretation: Patients that harbour pathogenic variants in MTFMT have a milder clinical phenotype and disease progression compared to LS caused by other nuclear defects. Fibroblasts may preclude the need for muscle biopsy, to prove causality of any novel variant.

Whole Exome Sequencing Is the Preferred Strategy to Identify the Genetic Defect in Patients With a Probable or Possible Mitochondrial Cause.

Mitochondrial disorders, characterized by clinical symptoms and/or OXPHOS deficiencies, are caused by pathogenic variants in mitochondrial genes. However, pathogenic variants in some of these genes can lead to clinical manifestations which overlap with other neuromuscular diseases, which can be caused by pathogenic variants in non-mitochondrial genes as well. Mitochondrial pathogenic variants can be found in the mitochondrial DNA (mtDNA) or in any of the 1,500 nuclear genes with a mitochondrial function. We have performed a two-step next-generation sequencing approach in a cohort of 117 patients, mostly children, in whom a mitochondrial disease-cause could likely or possibly explain the phenotype. A total of 86 patients had a mitochondrial disorder, according to established clinical and biochemical criteria. The other 31 patients had neuromuscular symptoms, where in a minority a mitochondrial genetic cause is present, but a non-mitochondrial genetic cause is more likely. All patients were screened for pathogenic variants in the mtDNA and, if excluded, analyzed by whole exome sequencing (WES). Variants were filtered for being pathogenic and compatible with an autosomal or X-linked recessive mode of inheritance in families with multiple affected siblings and/or consanguineous parents. Non-consanguineous families with a single patient were additionally screened for autosomal and X-linked dominant mutations in a predefined gene-set. We identified causative pathogenic variants in the mtDNA in 20% of the patient-cohort, and in nuclear genes in 49%, implying an overall yield of 68%. We identified pathogenic variants in mitochondrial and non-mitochondrial genes in both groups with, obviously, a higher number of mitochondrial genes affected in mitochondrial disease patients. Furthermore, we show that 31% of the disease-causing genes in the mitochondrial patient group were not included in the MitoCarta database, and therefore would have been missed with MitoCarta based gene-panels. We conclude that WES is preferable to panel-based approaches for both groups of patients, as the mitochondrial gene-list is not complete and mitochondrial symptoms can be secondary. Also, clinically and genetically heterogeneous disorders would require sequential use of multiple different gene panels. We conclude that WES is a comprehensive and unbiased approach to establish a genetic diagnosis in these patients, able to resolve multi-genic disease-causes.

Clinical, biochemical and genetic spectrum of 70 patients with ACAD9 deficiency: is riboflavin supplementation effective?

BACKGROUND: Mitochondrial acyl-CoA dehydrogenase family member 9 (ACAD9) is essential for the assembly of mitochondrial respiratory chain complex I. Disease causing biallelic variants in ACAD9 have been reported in individuals presenting with lactic acidosis and cardiomyopathy. RESULTS: We describe the genetic, clinical and biochemical findings in a cohort of 70 patients, of whom 29 previously unpublished. We found 34 known and 18 previously unreported variants in ACAD9. No patients harbored biallelic loss of function mutations, indicating that this combination is unlikely to be compatible with life. Causal pathogenic variants were distributed throughout the entire gene, and there was no obvious genotype-phenotype correlation. Most of the patients presented in the first year of life. For this subgroup the survival was poor (50% not surviving the first 2 years) comparing to patients with a later presentation (more than 90% surviving 10 years). The most common clinical findings were cardiomyopathy (85%), muscular weakness (75%) and exercise intolerance (72%). Interestingly, severe intellectual deficits were only reported in one patient and severe developmental delays in four patients. More than 70% of the patients were able to perform the same activities of daily living when compared to peers. CONCLUSIONS: Our data show that riboflavin treatment improves complex I activity in the majority of patient-derived fibroblasts tested. This effect was also reported for most of the treated patients and is mirrored in the survival data. In the patient group with disease-onset below 1 year of age, we observed a statistically-significant better survival for patients treated with riboflavin.

Anatomic & metabolic brain markers of the m.3243A>G mutation: A multi-parametric 7T MRI study.

One of the most common mitochondrial DNA (mtDNA) mutations, the A to G transition at base pair 3243, has been linked to changes in the brain, in addition to commonly observed hearing problems, diabetes and myopathy. However, a detailed quantitative description of m.3243A>G patients' brains has not been provided so far. In this study, ultra-high field MRI at 7T and volume- and surface-based data analyses approaches were used to highlight morphology (i.e. atrophy)-, microstructure (i.e. myelin and iron concentration)- and metabolism (i.e. cerebral blood flow)-related differences between patients (N = 22) and healthy controls (N = 15). The use of quantitative MRI at 7T allowed us to detect subtle changes of biophysical processes in the brain with high accuracy and sensitivity, in addition to typically assessed lesions and atrophy. Furthermore, the effect of m.3243A>G mutation load in blood and urine epithelial cells on these MRI measures was assessed within the patient population and revealed that blood levels were most indicative of the brain's state and disease severity, based on MRI as well as on neuropsychological data. Morphometry MRI data showed a wide-spread reduction of cortical, subcortical and cerebellar gray matter volume, in addition to significantly enlarged ventricles. Moreover, surface-based analyses revealed brain area-specific changes in cortical thickness (e.g. of the auditory cortex), and in T1, T2* and cerebral blood flow as a function of mutation load, which can be linked to typically m.3243A>G-related clinical symptoms (e.g. hearing impairment). In addition, several regions linked to attentional control (e.g. middle frontal gyrus), the sensorimotor network (e.g. banks of central sulcus) and the default mode network (e.g. precuneus) were characterized by alterations in cortical thickness, T1, T2* and/or cerebral blood flow, which has not been described in previous MRI studies. Finally, several hypotheses, based either on vascular, metabolic or astroglial implications of the m.3243A>G mutation, are discussed that potentially explain the underlying pathobiology. To conclude, this is the first 7T and also the largest MRI study on this patient population that provides macroscopic brain correlates of the m.3243A>G mutation indicating potential MRI biomarkers of mitochondrial diseases and might guide future (longitudinal) studies to extensively track neuropathological and clinical changes.

Preclinical Efficacy and Safety Evaluation of Hematopoietic Stem Cell Gene Therapy in a Mouse Model of MNGIE.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder caused by thymidine phosphorylase (TP) deficiency resulting in systemic accumulation of thymidine (d-Thd) and deoxyuridine (d-Urd) and characterized by early-onset neurological and gastrointestinal symptoms. Long-term effective and safe treatment is not available. Allogeneic bone marrow transplantation may improve clinical manifestations but carries disease and transplant-related risks. In this study, lentiviral vector-based hematopoietic stem cell gene therapy (HSCGT) was performed in Tymp-/-Upp1-/- mice with the human phosphoglycerate kinase (PGK) promoter driving TYMP. Supranormal blood TP activity reduced intestinal nucleoside levels significantly at low vector copy number (median, 1.3; range, 0.2-3.6). Furthermore, we covered two major issues not addressed before. First, we demonstrate aberrant morphology of brain astrocytes in areas of spongy degeneration, which was reversed by HSCGT. Second, long-term follow-up and vector integration site analysis were performed to assess safety of the therapeutic LV vectors in depth. This report confirms and supplements previous work on the efficacy of HSCGT in reducing the toxic metabolites in Tymp-/-Upp1-/- mice, using a clinically applicable gene transfer vector and a highly efficient gene transfer method, and importantly demonstrates phenotypic correction with a favorable risk profile, warranting further development toward clinical implementation.

Genetic defects in mtDNA-encoded protein translation cause pediatric, mitochondrial cardiomyopathy with early-onset brain disease.

This study aims to identify gene defects in pediatric cardiomyopathy and early-onset brain disease with oxidative phosphorylation (OXPHOS) deficiencies. We applied whole-exome sequencing in three patients with pediatric cardiomyopathy and early-onset brain disease with OXPHOS deficiencies. The brain pathology was studied by MRI analysis. In consanguineous patient 1, we identified a homozygous intronic variant (c.850-3A > G) in the QRSL1 gene, which was predicted to cause abnormal splicing. The variant segregated with the disease and affected the protein function, which was confirmed by complementation studies, restoring OXPHOS function only with wild-type QRSL1. Patient 2 was compound heterozygous for two novel affected and disease-causing variants (c.[253G > A];[938G > A]) in the MTO1 gene. In patient 3, we detected one unknown affected and disease-causing variants (c.2872C > T) and one known disease-causing variant (c.1774C > T) in the AARS2 gene. The c.1774C > T variant was present in the paternal copy of the AARS2 gene, the c.2872C > T in the maternal copy. All genes were involved in translation of mtDNA-encoded proteins. Defects in mtDNA-encoded protein translation lead to severe pediatric cardiomyopathy and brain disease with OXPHOS abnormalities. This suggests that the heart and brain are particularly sensitive to defects in mitochondrial protein synthesis during late embryonic or early postnatal development, probably due to the massive mitochondrial biogenesis occurring at that stage. If both the heart and brain are involved, the prognosis is poor with a likely fatal outcome at young age.

Peripheral Neuropathy, Episodic Rhabdomyolysis, and Hypoparathyroidism in a Patient with Mitochondrial Trifunctional Protein Deficiency.

A combination of unexplained peripheral neuropathy, hypoparathyroidism, and the inability to cope with metabolic stress could point to a rare inborn error of metabolism, such as mitochondrial trifunctional protein (MTP) deficiency.Here, we describe a 20-year-old woman who was known since childhood with axonal motor sensory polyneuropathy of unknown origin. She presented with progressive dyspnoea, and increased muscle weakness, preceded by 6 days of fever, vomiting, and diarrhoea. Laboratory testing showed rhabdomyolysis, and hypocalcaemia with low parathyroid levels. The patient was intubated because of respiratory insufficiency and a viral and bacterial pneumonia was diagnosed. She was discharged after 16 days of admission. Metabolic screening, performed at the time of rhabdomyolysis, showed increased concentrations of long-chain 3-hydroxyacyl carnitine species, together with elevated urinary excretion of 3-hydroxy dicarboxylic acids. Decreased activity of long-chain 3-hydroxyacyl-CoA dehydrogenase and long-chain 3-ketoacyl-CoA thiolase in peripheral lymphocytes and fibroblasts confirmed a MTP deficiency. Sequence analysis of the HADHB gene showed two heterozygous variants: c.209+1G>C (splicing defect) and c.980T>C (p.Leu327Leu). When the acylcarnitine profile was repeated after the episode of rhabdomyolysis had resolved it showed no abnormalities.Our case illustrates a cluster of peripheral neuropathy, episodic rhabdomyolysis, and hypoparathyroidism in a patient with MTP deficiency caused by mutations in the HADHB gene. It stresses the importance of performing metabolic screening when patients are most symptomatic, as normal results can be found at times when no metabolic stress is present. Screening is relatively easy and timely diagnosis has important implications for treatment.

Phenotype-genotype correlations in Leigh syndrome: new insights from a multicentre study of 96 patients.

BACKGROUND: Leigh syndrome is a phenotypically and genetically heterogeneous mitochondrial disorder. While some genetic defects are associated with well-described phenotypes, phenotype-genotype correlations in Leigh syndrome are not fully explored. OBJECTIVE: We aimed to identify phenotype-genotype correlations in Leigh syndrome in a large cohort of systematically evaluated patients. METHODS: We studied 96 patients with genetically confirmed Leigh syndrome diagnosed and followed in eight European centres specialising in mitochondrial diseases. RESULTS: We found that ataxia, ophthalmoplegia and cardiomyopathy were more prevalent among patients with mitochondrial DNA defects. Patients with mutations in MT-ND and NDUF genes with complex I deficiency shared common phenotypic features, such as early development of central nervous system disease, followed by high occurrence of cardiac and ocular manifestations. The cerebral cortex was affected in patients with NDUF mutations significantly more often than the rest of the cohort. Patients with the m.8993T>G mutation in MT-ATP6 gene had more severe clinical and radiological manifestations and poorer disease outcome compared with patients with the m.8993T>C mutation. CONCLUSION: Our study provides new insights into phenotype-genotype correlations in Leigh syndrome and particularly in patients with complex I deficiency and with defects in the mitochondrial ATP synthase.

Selection and Characterization of Palmitic Acid Responsive Patients with an OXPHOS Complex I Defect.

Mitochondrial disorders are genetically and clinically heterogeneous, mainly affecting high energy-demanding organs due to impaired oxidative phosphorylation (OXPHOS). Currently, effective treatments for OXPHOS defects, with complex I deficiency being the most prevalent, are not available. Yet, clinical practice has shown that some complex I deficient patients benefit from a high-fat or ketogenic diet, but it is unclear how these therapeutic diets influence mitochondrial function and more importantly, which complex I patients could benefit from such treatment. Dietary studies in a complex I deficient patient with exercise intolerance showed increased muscle endurance on a high-fat diet compared to a high-carbohydrate diet. We performed whole-exome sequencing to characterize the genetic defect. A pathogenic homozygous p.G212V missense mutation was identified in the TMEM126B gene, encoding an early assembly factor of complex I. A complementation study in fibroblasts confirmed that the p.G212V mutation caused the complex I deficiency. The mechanism turned out to be an incomplete assembly of the peripheral arm of complex I, leading to a decrease in the amount of mature complex I. The patient clinically improved on a high-fat diet, which was supported by the 25% increase in maximal OXPHOS capacity in TMEM126B defective fibroblast by the saturated fatty acid palmitic acid, whereas oleic acid did not have any effect in those fibroblasts. Fibroblasts of other patients with a characterized complex I gene defect were tested in the same way. Patient fibroblasts with complex I defects in NDUFS7 and NDUFAF5 responded to palmitic acid, whereas ACAD9, NDUFA12, and NDUFV2 defects were non-responding. Although the data are too limited to draw a definite conclusion on the mechanism, there is a tendency that protein defects involved in early assembly complexes, improve with palmitic acid, whereas proteins defects involved in late assembly, do not. Our data show at a clinical and biochemical level that a high fat diet can be beneficial for complex I patients and that our cell line assay will be an easy tool for the selection of patients, who might potentially benefit from this therapeutic diet.