Management of seizures in patients with primary mitochondrial diseases: consensus statement from the InterERNs Mitochondrial Working Group.

BACKGROUND AND PURPOSE: Primary mitochondrial diseases (PMDs) are common inborn errors of energy metabolism, with an estimated prevalence of one in 4300. These disorders typically affect tissues with high energy requirements, including heart, muscle and brain. Epilepsy may be the presenting feature of PMD, can be difficult to treat and often represents a poor prognostic feature. The aim of this study was to develop guidelines and consensus recommendations on safe medication use and seizure management in mitochondrial epilepsy. METHODS: A panel of 24 experts in mitochondrial medicine, pharmacology and epilepsy management of adults and/or children and two patient representatives from seven countries was established. Experts were members of five different European Reference Networks, known as the Mito InterERN Working Group. A Delphi technique was used to allow the panellists to consider draft recommendations on safe medication use and seizure management in mitochondrial epilepsy, using two rounds with predetermined levels of agreement. RESULTS: A high level of consensus was reached regarding the safety of 14 out of all 25 drugs reviewed, resulting in endorsement of National Institute for Health and Care Excellence guidelines for seizure management, with some modifications. Exceptions including valproic acid in POLG disease, vigabatrin in patients with γ-aminobutyric acid transaminase deficiency and topiramate in patients at risk for renal tubular acidosis were highlighted. CONCLUSIONS: These consensus recommendations describe our intent to improve seizure control and reduce the risk of drug-related adverse events in individuals living with PMD-related epilepsy.

IUPHAR ECR review: Cancer-related anorexia-cachexia in cancer patients: Pathophysiology and treatment.

Cancer-related anorexia-cachexia (CRAC) comprises one of the most common syndromes of advanced cancer patients. The prevalence of CRAC increases from 50% to 80% before death. CRAC is associated not only with impaired quality of life in patients and family members but also with shorter survival. The management of CRAC is a great challenge in clinical practice. There are no definite practice guidelines yet for the prevention and treatment of CRAC. A multimodal strategy is the most effective way to treat anorexia-cachexia. Numerous medications have been suggested and used in clinical trials, while others are still being studied on experimental animals. These medications include branched-chain amino acids, eicosapentaenoic acid, thalidomide, cytokine inhibitors, steroids, antiserotoninergic medications, and appetite stimulants. The benefits of supportive care interventions and the advancement of exciting new pharmacological medicines for anorexia-cachexia are becoming more widely recognized. Health care professionals need to be aware of the psychosocial and biological effects of anorexia-cachexia, even though knowledge of the underlying molecular causes of the disorder has advanced significantly.

Gene therapy for mitochondrial disorders.

In this review, we detail the current state of application of gene therapy to primary mitochondrial disorders (PMDs). Recombinant adeno-associated virus-based (rAAV) gene replacement approaches for nuclear gene disorders have been undertaken successfully in more than ten preclinical mouse models of PMDs which has been made possible by the development of novel rAAV technologies that achieve more efficient organ targeting. So far, however, the greatest progress has been made for Leber Hereditary Optic Neuropathy, for which phase 3 clinical trials of lenadogene nolparvovec demonstrated efficacy and good tolerability. Other methods of treating mitochondrial DNA (mtDNA) disorders have also had traction, including refinements to nucleases that degrade mtDNA molecules with pathogenic variants, including transcription activator-like effector nucleases, zinc-finger nucleases, and meganucleases (mitoARCUS). rAAV-based approaches have been used successfully to deliver these nucleases in vivo in mice. Exciting developments in CRISPR-Cas9 gene editing technology have achieved in vivo gene editing in mouse models of PMDs due to nuclear gene defects and new CRISPR-free gene editing approaches have shown great potential for therapeutic application in mtDNA disorders. We conclude the review by discussing the challenges of translating gene therapy in patients both from the point of view of achieving adequate organ transduction as well as clinical trial design.

Current management of primary mitochondrial disorders in EU countries: the European Reference Networks survey.

BACKGROUND AND PURPOSE: Primary mitochondrial diseases (PMDs) are rare diseases for which diagnosis is challenging, and management and training programs are not well defined in Europe. To capture and assess care needs, five different European Reference Networks have conducted an exploratory survey. METHODS: The survey covering multiple topics relating to PMDs was sent to all ERNs healthcare providers (HCPs) in Europe. RESULTS: We have collected answers from 220 members based in 24/27 European member states and seven non-European member states. Even though most of the responders are aware of neurogenetic diseases, difficulties arise in the ability to deliver comprehensive genetic testing. While single gene analysis is widely available in Europe, whole exome and genome sequencing are not easily accessible, with considerable variation between countries and average waiting time for results frequently above 6 months. Only 12.7% of responders were happy with the ICD-10 codes for classifying patients with PMDs discharged from the hospital, and more than 70% of them consider that PMDs deserve specific ICD codes to improve clinical management, including tailored healthcare, and for reimbursement reasons. Finally, 90% of responders declared that there is a need for further education and training in these diseases. CONCLUSIONS: This survey provides information on the current difficulties in the care of PMDs in Europe. We believe that the results of this survey are important to help rare disease stakeholders in European countries identify key care and research priorities.

IgG4-related retroperitoneal fibrosis: A case report of a challenging disease.

Immunoglobulin G4 (IgG4)-related disease (IgG4-RD) is a multi-organ immune-mediated fibroinflammatory disorder that may imitate malignancy, infectious or any other inflammatory disorder. IgG4-related retroperitoneal fibrosis (IgG4-RPF) is a rare form of IgG4-RD, diagnosis of which is often relied on radiological technology. Herein, we describe a case of 60 year old male, presenting with low back pain and weight loss for a period of 2 months and 15 days. Imaging studies showed a retroperitoneal tumorous mass along with bilateral hydroureteronephrosis, which was later confirmed to be IgG4-related retroperitoneal fibrosis on the basis of extensive histopathological analysis. Immunosuppressive therapy resulted in a decrease in fibrosis and restoration of renal function.

Optimization of a Luteolin-Loaded TPGS/Poloxamer 407 Nanomicelle: The Effects of Copolymers, Hydration Temperature and Duration, and Freezing Temperature on Encapsulation Efficiency, Particle Size, and Solubility.

BACKGROUND: Luteolin is a flavonoid compound that has been widely studied for its various anti-cancer properties and sensitization to multidrug-resistant cells. However, the limited solubility and bioavailability of Lut hindered its potential clinical use. Theoretically, the combination of this compound with vitamin E TPGS and poloxamer 407 can produce a synergistic effect to enhance tumor apoptosis and P-glycoprotein inhibition. This study aimed to develop and optimize vitamin E TPGS/Poloxamer 407 micelles loaded with luteolin through investigating certain factors that can affect the encapsulation efficiency and particle size of the micelle. METHODS: A micelle was prepared using the film hydration method, and the micellar solution was lyophilized. The cake formed was analyzed. The factors investigated include the concentrations of the surfactants, ratio of vitamin E TPGS/Poloxamer 407, temperature of the hydrating solution, duration of hydration, and freezing temperature before lyophilization. The effects of these factors on the encapsulation efficiency and particle size of the micelle were also studied. The encapsulation efficiency was measured using a UV-Vis spectrophotometer, while particle size was measured using dynamic light scattering. RESULTS: The optimized micelle was found to have 90% encapsulation efficiency with a particle size of less than 40 nm, which was achieved using a 10% concentration of surfactants at a vitamin E TPGS/Poloxamer 407 ratio of 3:1. The optimized temperature for hydrating the micellar film was 40 °C, the optimized mixing time was 1 h, and the optimized freezing temperature was -80 °C. The solubility of the luteolin-loaded micelles increased 459-fold compared to pure Lut in water. The critical micelle concentration of the vitamin E TPGS/Poloxamer 407 micelle was 0.001 mg/mL, and the release study showed that luteolin-loaded micelles exhibited sustained release behavior. The release of luteolin from a micelle was found to be higher in pH 6.8 compared to pH 7.4, which signified that luteolin could be accumulated more in a tumor microenvironment compared to blood. CONCLUSION: This study demonstrated that several factors need to be considered when developing such nanoparticles in order to obtain a well-optimized micelle.

Expert Panel Curation of 113 Primary Mitochondrial Disease Genes for the Leigh Syndrome Spectrum.

OBJECTIVE: Primary mitochondrial diseases (PMDs) are heterogeneous disorders caused by inherited mitochondrial dysfunction. Classically defined neuropathologically as subacute necrotizing encephalomyelopathy, Leigh syndrome spectrum (LSS) is the most frequent manifestation of PMD in children, but may also present in adults. A major challenge for accurate diagnosis of LSS in the genomic medicine era is establishing gene-disease relationships (GDRs) for this syndrome with >100 monogenic causes across both nuclear and mitochondrial genomes. METHODS: The Clinical Genome Resource (ClinGen) Mitochondrial Disease Gene Curation Expert Panel (GCEP), comprising 40 international PMD experts, met monthly for 4 years to review GDRs for LSS. The GCEP standardized gene curation for LSS by refining the phenotypic definition, modifying the ClinGen Gene-Disease Clinical Validity Curation Framework to improve interpretation for LSS, and establishing a scoring rubric for LSS. RESULTS: The GDR with LSS across the nuclear and mitochondrial genomes was classified as definitive for 31 of 114 GDRs curated (27%), moderate for 38 (33%), limited for 43 (38%), and disputed for 2 (2%). Ninety genes were associated with autosomal recessive inheritance, 16 were maternally inherited, 5 were autosomal dominant, and 3 were X-linked. INTERPRETATION: GDRs for LSS were established for genes across both nuclear and mitochondrial genomes. Establishing these GDRs will allow accurate variant interpretation, expedite genetic diagnosis of LSS, and facilitate precision medicine, multisystem organ surveillance, recurrence risk counseling, reproductive choice, natural history studies, and determination of eligibility for interventional clinical trials. ANN NEUROL 2023;94:696-712.

Leigh syndrome.

Leigh syndrome, or subacute necrotizing encephalomyelopathy, was initially recognized as a neuropathological entity in 1951. Bilateral symmetrical lesions, typically extending from the basal ganglia and thalamus through brainstem structures to the posterior columns of the spinal cord, are characterized microscopically by capillary proliferation, gliosis, severe neuronal loss, and relative preservation of astrocytes. Leigh syndrome is a pan-ethnic disorder usually with onset in infancy or early childhood, but late-onset forms occur, including in adult life. Over the last six decades it has emerged that this complex neurodegenerative disorder encompasses more than 100 separate monogenic disorders associated with enormous clinical and biochemical heterogeneity. This chapter discusses clinical, biochemical and neuropathological aspects of the disorder, and postulated pathomechanisms. Known genetic causes, including defects of 16 mitochondrial DNA (mtDNA) genes and approaching 100 nuclear genes, are categorized into disorders of subunits and assembly factors of the five oxidative phosphorylation enzymes, disorders of pyruvate metabolism and vitamin and cofactor transport and metabolism, disorders of mtDNA maintenance, and defects of mitochondrial gene expression, protein quality control, lipid remodeling, dynamics, and toxicity. An approach to diagnosis is presented, together with known treatable causes and an overview of current supportive management options and emerging therapies on the horizon.

OxPhos defects cause hypermetabolism and reduce lifespan in cells and in patients with mitochondrial diseases.

Patients with primary mitochondrial oxidative phosphorylation (OxPhos) defects present with fatigue and multi-system disorders, are often lean, and die prematurely, but the mechanistic basis for this clinical picture remains unclear. By integrating data from 17 cohorts of patients with mitochondrial diseases (n = 690) we find evidence that these disorders increase resting energy expenditure, a state termed hypermetabolism. We examine this phenomenon longitudinally in patient-derived fibroblasts from multiple donors. Genetically or pharmacologically disrupting OxPhos approximately doubles cellular energy expenditure. This cell-autonomous state of hypermetabolism occurs despite near-normal OxPhos coupling efficiency, excluding uncoupling as a general mechanism. Instead, hypermetabolism is associated with mitochondrial DNA instability, activation of the integrated stress response (ISR), and increased extracellular secretion of age-related cytokines and metabokines including GDF15. In parallel, OxPhos defects accelerate telomere erosion and epigenetic aging per cell division, consistent with evidence that excess energy expenditure accelerates biological aging. To explore potential mechanisms for these effects, we generate a longitudinal RNASeq and DNA methylation resource dataset, which reveals conserved, energetically demanding, genome-wide recalibrations. Taken together, these findings highlight the need to understand how OxPhos defects influence the energetic cost of living, and the link between hypermetabolism and aging in cells and patients with mitochondrial diseases.

Expanding the neurodevelopmental phenotype associated with HK1 de novo heterozygous missense variants.

Neurodevelopmental disorder with visual defects and brain anomalies (NEDVIBA) is a recently described genetic condition caused by de novo missense HK1 variants. Phenotypic data is currently limited; only seven patients have been published to date. This descriptive case series of a further four patients with de novo missense HK1 variants, alongside integration of phenotypic data with the reported cases, aims to improve our understanding of the associated phenotype. We provide further evidence that de novo HK1 variants located within the regulatory-terminal domain and alpha helix are associated with neurological problems and visual problems. We highlight for the first time an association with a raised cerebrospinal fluid lactate and specific abnormalities to the basal ganglia on brain magnetic resonance imaging, as well as associated respiratory issues and swallowing/feeding difficulties. We propose that this distinctive neurodevelopmental phenotype could arise through disruption of the regulatory glucose-6-phosphate binding site and subsequent gain of function of HK1 within the brain.

Biallelic variants in OGDH encoding oxoglutarate dehydrogenase lead to a neurodevelopmental disorder characterized by global developmental delay, movement disorder, and metabolic abnormalities.

PURPOSE: This study aimed to establish the genetic cause of a novel autosomal recessive neurodevelopmental disorder characterized by global developmental delay, movement disorder, and metabolic abnormalities. METHODS: We performed a detailed clinical characterization of 4 unrelated individuals from consanguineous families with a neurodevelopmental disorder. We used exome sequencing or targeted-exome sequencing, cosegregation, in silico protein modeling, and functional analyses of variants in HEK293 cells and Drosophila melanogaster, as well as in proband-derived fibroblast cells. RESULTS: In the 4 individuals, we identified 3 novel homozygous variants in oxoglutarate dehydrogenase (OGDH) (NM_002541.3), which encodes a subunit of the tricarboxylic acid cycle enzyme α-ketoglutarate dehydrogenase. In silico homology modeling predicts that c.566C>T:p.(Pro189Leu) and c.890C>A:p.(Ser297Tyr) variants interfere with the structure and function of OGDH. Fibroblasts from individual 1 showed that the p.(Ser297Tyr) variant led to a higher degradation rate of the OGDH protein. OGDH protein with p.(Pro189Leu) or p.(Ser297Tyr) variants in HEK293 cells showed significantly lower levels than the wild-type protein. Furthermore, we showed that expression of Drosophila Ogdh (dOgdh) carrying variants homologous to p.(Pro189Leu) or p.(Ser297Tyr), failed to rescue developmental lethality caused by loss of dOgdh. SpliceAI, a variant splice predictor, predicted that the c.935G>A:p.(Arg312Lys)/p.(Phe264_Arg312del) variant impacts splicing, which was confirmed through a mini-gene assay in HEK293 cells. CONCLUSION: We established that biallelic variants in OGDH cause a neurodevelopmental disorder with metabolic and movement abnormalities.

Transcriptomic analyses reveal neuronal specificity of Leigh syndrome associated genes.

Leigh syndrome is a rare, inherited, complex neurometabolic disorder with genetic and clinical heterogeneity. Features present in affected patients range from classical stepwise developmental regression to ataxia, seizures, tremor, and occasionally psychiatric manifestations. Currently, more than 100 monogenic causes of Leigh syndrome have been identified, yet the pathophysiology remains unknown. Here, we sought to determine the cellular specificity within the brain of all genes currently associated with Leigh syndrome. Further, we aimed to investigate potential genetic commonalities between Leigh syndrome and other disorders with overlapping clinical features. Enrichment of our target genes within the brain was evaluated with co-expression (CoExp) network analyses constructed using existing UK Brain Expression Consortium data. To determine the cellular specificity of the Leigh associated genes, we employed expression weighted cell type enrichment (EWCE) analysis of single-cell RNA-Seq data. Finally, CoExp network modules demonstrating enrichment of Leigh syndrome associated genes were then utilised for synaptic gene ontology analysis and heritability analysis. CoExp network analyses revealed that Leigh syndrome associated genes exhibit the highest levels of expression in brain regions most affected on MRI in affected patients. EWCE revealed significant enrichment of target genes in hippocampal and somatosensory pyramidal neurons and interneurons of the brain. Analysis of CoExp modules enriched with our target genes revealed preferential association with pre-synaptic structures. Heritability studies suggested some common enrichment between Leigh syndrome and Parkinson disease and epilepsy. Our findings suggest a primary mitochondrial dysfunction as the underlying basis of Leigh syndrome, with associated genes primarily expressed in neuronal cells.

Rare disease gene association discovery from burden analysis of the 100,000 Genomes Project data.

To discover rare disease-gene associations, we developed a gene burden analytical framework and applied it to rare, protein-coding variants from whole genome sequencing of 35,008 cases with rare diseases and their family members recruited to the 100,000 Genomes Project (100KGP). Following in silico triaging of the results, 88 novel associations were identified including 38 with existing experimental evidence. We have published the confirmation of one of these associations, hereditary ataxia with UCHL1 , and independent confirmatory evidence has recently been published for four more. We highlight a further seven compelling associations: hypertrophic cardiomyopathy with DYSF and SLC4A3 where both genes show high/specific heart expression and existing associations to skeletal dystrophies or short QT syndrome respectively; monogenic diabetes with UNC13A with a known role in the regulation of ß cells and a mouse model with impaired glucose tolerance; epilepsy with KCNQ1 where a mouse model shows seizures and the existing long QT syndrome association may be linked; early onset Parkinson's disease with RYR1 with existing links to tremor pathophysiology and a mouse model with neurological phenotypes; anterior segment ocular abnormalities associated with POMK showing expression in corneal cells and with a zebrafish model with developmental ocular abnormalities; and cystic kidney disease with COL4A3 showing high renal expression and prior evidence for a digenic or modifying role in renal disease. Confirmation of all 88 associations would lead to potential diagnoses in 456 molecularly undiagnosed cases within the 100KGP, as well as other rare disease patients worldwide, highlighting the clinical impact of a large-scale statistical approach to rare disease gene discovery.

Specialist multidisciplinary input maximises rare disease diagnoses from whole genome sequencing.

Diagnostic whole genome sequencing (WGS) is increasingly used in rare diseases. However, standard, semi-automated WGS analysis may overlook diagnoses in complex disorders. Here, we show that specialist multidisciplinary analysis of WGS, following an initial 'no primary findings' (NPF) report, improves diagnostic rates and alters management. We undertook WGS in 102 adults with diagnostically challenging primary mitochondrial disease phenotypes. NPF cases were reviewed by a genomic medicine team, thus enabling bespoke informatic approaches, co-ordinated phenotypic validation, and functional work. We enhanced the diagnostic rate from 16.7% to 31.4%, with management implications for all new diagnoses, and detected strong candidate disease-causing variants in a further 3.9% of patients. This approach presents a standardised model of care that supports mainstream clinicians and enhances diagnostic equity for complex disorders, thereby facilitating access to the potential benefits of genomic healthcare. This research was made possible through access to the data and findings generated by the 100,000 Genomes Project: http://www.genomicsengland.co.uk .

Premature Ovarian Insufficiency in CLPB Deficiency: Transcriptomic, Proteomic and Phenotypic Insights.

CONTEXT: Premature ovarian insufficiency (POI) is a common form of female infertility that usually presents as an isolated condition but can be part of various genetic syndromes. Early diagnosis and treatment of POI can minimize comorbidity and improve health outcomes. OBJECTIVE: We aimed to determine the genetic cause of syndromic POI, intellectual disability, neutropenia, and cataracts. METHODS: We performed whole-exome sequencing (WES) followed by functional validation via RT-PCR, RNAseq, and quantitative proteomics, as well as clinical update of previously reported patients with variants in the caseinolytic peptidase B (CLPB) gene. RESULTS: We identified causative variants in CLPB, encoding a mitochondrial disaggregase. Variants in this gene are known to cause an autosomal recessive syndrome involving 3-methylglutaconic aciduria, neurological dysfunction, cataracts, and neutropenia that is often fatal in childhood; however, there is likely a reporting bias toward severe cases. Using RNAseq and quantitative proteomics we validated causation and gained insight into genotype:phenotype correlation. Clinical follow-up of patients with CLPB deficiency who survived to adulthood identified POI and infertility as a common postpubertal ailment. CONCLUSION: A novel splicing variant is associated with CLPB deficiency in an individual who survived to adulthood. POI is a common feature of postpubertal female individuals with CLPB deficiency. Patients with CLPB deficiency should be referred to pediatric gynecologists/endocrinologists for prompt POI diagnosis and hormone replacement therapy to minimize associated comorbidities.