CoQ deficiency causes disruption of mitochondrial sulfide oxidation, a new pathomechanism associated with this syndrome

Coenzyme Q (CoQ) is a key component of the mitochondrial respiratory chain, but it also has several other functions in the cellular metabolism. One of them is to function as an electron carrier in the reaction catalyzed by sulfide:quinone oxidoreductase (SQR), which catalyzes the first reaction in the hydrogen sulfide oxidation pathway. Therefore, SQR may be affected by CoQ deficiency. Using human skin fibroblasts and two mouse models with primary CoQ deficiency, we demonstrate that severe CoQ deficiency causes a reduction in SQR levels and activity, which leads to an alteration of mitochondrial sulfide metabolism. In cerebrum of Coq9R239X mice, the deficit in SQR induces an increase in thiosulfate sulfurtransferase and sulfite oxidase, as well as modifications in the levels of thiols. As a result, biosynthetic pathways of glutamate, serotonin, and catecholamines were altered in the cerebrum, and the blood pressure was reduced. Therefore, this study reveals the reduction in SQR activity as one of the pathomechanisms associated with CoQ deficiency syndrome.Ministerio de Economía y CompetitividadERDF/SAF2013-47761-RERDF/SAF2014-55523-RERDF/RD12/0042/0011ERDF/SAF2015-65786-RJunta de AndalucíaNIH/P01HD08064

The antioxidant Trolox restores mitochondrial membrane potential and Ca2+-stimulated ATP production in human complex I deficiency

Malfunction of mitochondrial complex I caused by nuclear gene mutations causes early-onset neurodegenerative diseases. Previous work using cultured fibroblasts of complex-I-deficient patients revealed elevated levels of reactive oxygen species (ROS) and reductions in both total Ca2+ content of the endoplasmic reticulum (ERCa) and bradykinin(Bk)-induced increases in cytosolic and mitochondrial free Ca2+ ([Ca2+]C; [Ca2+]M) and ATP ([ATP]C; [ATP]M) concentration. Here, we determined the mitochondrial membrane potential (Δψ) in patient skin fibroblasts and show significant correlations with cellular ROS levels and ERCa, i.e., the less negative Δψ, the higher these levels and the lower ERCa. Treatment with 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox) normalized Δψ and Bk-induced increases in [Ca2+]M and [ATP]M. These effects were accompanied by an increase in ERCa and Bk-induced increase in [Ca2+]C. Together, these results provide evidence for an integral role of increased ROS levels in complex I deficiency and point to the potential therapeutic value of antioxidant treatment

Natural History of Patients With Mitochondrial ATPase Deficiency Due to Pathogenic Variants of MT-ATP6 and MT-ATP8

Background and Objectives: The mitochondrial DNA (mtDNA) genes MT-ATP6 and MT-ATP8 encode for subunits α and 8 (A6L) of the adenosine triphosphate synthase complex. Pathogenetic variants in MT-ATP6/8 cause incurable mitochondrial syndromes encompassing a wide spectrum of clinical features including ataxia, motor and language developmental delay, deafness, retinitis pigmentosa, and Leigh pattern in brain MRI. Typically, higher levels of mtDNA variants lead to more severe symptomatology although even individuals with similar mtDNA mutational loads exhibit high clinical variability. Hence, the establishment of potential therapeutics is currently challenging. In this article, we present an international multicenter study designed to provide a retrospective natural history of patients with MT-ATP6/8 deficiency and to identify primary and secondary end points for future clinical trials. Methods: Clinical, biochemical, and molecular genetics data of patients with genetically confirmed MT-ATP6/8 defects were collected and analyzed from Italian, German, US, and Spain national reference centers through ethical committee–approved mitochondrial patients' national registries or local programs. Results: A cohort of 111 patients, 98 unreported, were analyzed (55 male, 56 female). Patients had infantile-onset disease (12 years) in 20%. Kaplan-Meier analysis showed a significant difference (p value = 0.0349) in the survival of infantile and pediatric patients compared with adult patients, although only 8% of patients were not alive at the last follow-up. The CNS was the most frequently affected tissue (93%), followed by the muscle (75%), eye (46%), and heart (18%). Brain MRI showed isolated Leigh-like lesions (58%), Leigh-like lesions and cortical and/or cerebellar atrophy (15%), isolated cerebellar atrophy (10%), and other lesions (21%). At the last follow-up, 11% of patients were wheelchair-bound. Metabolic acidosis or acute deterioration complicated the clinical course in ≅55% of early-onset patients. Molecular genetics studies identified 26 pathogenic variants (6 of them novel). Reduced citrulline levels and increased alanine and lactate levels were reported in 56%, 49%, and 71% of patients, respectively, suggesting their role as potential biomarkers. Discussion: Our results define a more accurate classification based on the age at onset for MT-ATPase deficiency and provide fundamental clinical and biochemical data for disease management

Combined genomics and proteomics unveils elusive variants and vast aetiologic heterogeneity in dystonia

Dystonia is a rare-disease trait for which large-scale genomic investigations are still underrepresented. Genetic heterogeneity among patients with unexplained dystonia warrants interrogation of entire genome sequences, but this has not yet been systematically evaluated. To significantly enhance our understanding of the genetic contribution to dystonia, we (re)analyzed 2,874 whole-exome sequencing (WES), 564 whole-genome sequencing (WGS), as well as 80 fibroblast-derived proteomics datasets, representing the output of high-throughput analyses in 1,990 patients and 973 unaffected relatives from 1,877 families. Recruitment and precision-phenotyping procedures were driven by long-term collaborations of international experts with access to overlooked populations. By exploring WES data, we found that continuous scaling of sample sizes resulted in steady gains in the number of associated disease genes without plateauing. On average, every second diagnosis involved a gene not previously implicated in our cohort. Second-line WGS focused on a subcohort of undiagnosed individuals with high likelihood of having monogenic forms of dystonia, comprising large proportions of patients with early onset (81.3%), generalized symptom distribution (50.8%) and/or coexisting features (68.9%). We undertook extensive searches for variants in nuclear and mitochondrial genomes to uncover 38 (ultra)rare diagnostic-grade findings in 37 of 305 index patients (12.1%), many of which had remained undetected due to methodological inferiority of WES or pipeline limitations. WGS-identified elusive variations included alterations in exons poorly covered by WES, RNA-gene variants, mitochondrial-DNA mutations, small copy-number variants, complex rearranged genome structure, and short tandem repeats. For improved variant interpretation in WGS-inconclusive cases, we employed systematic integration of quantitative proteomics. This aided in verifying diagnoses related to technically challenging variants and in upgrading a variant of uncertain significance (3 of 70 WGS-inconclusive index patients, 4.3%). Further, unsupervised proteomic outlier-analysis supplemented with transcriptome sequencing revealed pathological gene underexpression induced by transcript disruptions in three more index patients with underlying (deep) intronic variants (3/70, 4.3%), highlighting the potential for targeted antisense-oligonucleotide therapy development. Finally, trio-WGS prioritized a de-novo missense change in the candidate PRMT1, encoding a histone-methyltransferase. Data-sharing strategies supported the discovery of three distinct PRMT1 de-novo variants in four phenotypically similar patients, associated with loss-of-function effects in in-vitro assays. This work underscores the importance of continually expanding sequencing cohorts to characterize the extensive spectrum of gene aberrations in dystonia. We show that a pool of unresolved cases is amenable to WGS and complementary multi-omic studies, directing advanced etiopathological concepts and future diagnostic-practice workflows for dystonia

Impact of genetic and non-genetic factors on phenotypic diversity in NBAS-associated disease

Biallelic pathogenic variants in neuroblastoma-amplified sequence (NBAS) cause a pleiotropic multisystem disorder. Three clinical subgroups have been defined correlating with the localisation of pathogenic variants in the NBAS gene; variants affecting the C-terminal region of NBAS result in SOPH syndrome (short stature, optic atrophy, Pelger-Huët anomaly), variants affecting the Sec 39 domain are associated with infantile liver failure syndrome type 2 (ILFS2) and variants affecting the ß-propeller domain give rise to a combined phenotype. However, there is still unexplained phenotypic diversity across the three subgroups, challenging the current concept of genotype-phenotype correlations in NBAS-associated disease. Therefore, besides examining the genetic influence, we aim to elucidate the potential impact of pre-symptomatic diagnosis, emergency management and other modifying variables on the clinical phenotype. We investigated genotype-phenotype correlations in individuals sharing the same genotypes (n = 30 individuals), and in those sharing the same missense variants with a loss-of-function variant in trans (n = 38 individuals). Effects of a pre-symptomatic diagnosis and emergency management on the severity of acute liver failure (ALF) episodes also were analysed, comparing liver function tests (ALAT, ASAT, INR) and mortality. A strong genotype-phenotype correlation was demonstrated in individuals sharing the same genotype; this was especially true for the ILFS2 subgroup. Genotype-phenotype correlation in patients sharing only one missense variant was still high, though at a lower level. Pre-symptomatic diagnosis in combination with an emergency management protocol leads to a trend of reduced severity of ALF. High genetic impact on clinical phenotype in NBAS-associated disease facilitates monitoring and management of affected patients sharing the same genotype. Pre-symptomatic diagnosis and an emergency management protocol do not prevent ALF but may reduce its clinical severity

Multi-omics-based phenotyping of AFG3L2-mutant lymphoblasts determines key factors of a pathophysiological interplay between mitochondrial vulnerability and neurodegeneration in spastic ataxia type 5

Mitochondrial integrity is fundamental to cellular function, upheld by a network of proteases that regulate proteostasis and mitochondrial dynamics. Among these proteases, AFG3L2 is critical due to its roles in maintaining mitochondrial homeostasis, regulating mitochondrial protein quality, and facilitating mitochondrial biogenesis. Mutations in AFG3L2 are implicated in a spectrum of diseases, including spinocerebellar ataxia type 28 (SCA28) and spastic ataxia 5 (SPAX5), as well as other systemic conditions. This study employs a multi-omics approach to investigate the biochemical impact of AFG3L2 mutations in immortalized lymphoblastoid cell lines derived from a patient with biallelic variants leading to spastic ataxia (SPAX5). Our proteomic analysis revealed AFG3L2 impairment, with significant dysregulation of proteins critical for mitochondrial function, cytoskeletal integrity, and cellular metabolism. Specifically, disruptions were observed in mitochondrial dynamics and calcium homeostasis, alongside downregulation of key proteins like COX11, a copper chaperone for complex IV assembly, and NFU1, an iron-sulfur cluster protein linked to spastic paraparesis and infection-related worsening. Lipidomic analysis highlighted substantial alterations in lipid composition, with significant decreases in sphingomyelins, phosphatidylethanolamine, and phosphatidylcholine, reflecting disruptions in lipid metabolism and membrane integrity. Metabolomic profiling did not reveal any significant findings. Our comprehensive investigation into loss of functional AFG3L2 elucidates a pathophysiology extending beyond mitochondrial proteostasis, implicating a wide array of cellular processes. The findings reveal substantial cellular disturbances at multiple levels, contributing to neurodegeneration through disrupted mitochondrial respiratory chain, calcium homeostasis, cytoskeletal integrity, and altered lipid homeostasis. This study underscores the complexity of SPAX5 pathophysiology and the importance of multi-omics approaches in developing effective strategies to address the impact of loss of functional AFG3L2. Our data also highlight the value of immortalized lymphoblastoid cells as a tool for pre-clinical testing and research, offering a detailed biochemical fingerprint that enhances our understanding of SPAX5 and identifies potential areas for further investigation

ZSCAN10 deficiency causes a neurodevelopmental disorder with characteristic oto-facial malformations

Neurodevelopmental disorders are major indications for genetic referral and have been linked to more than 1500 loci including genes encoding transcriptional regulators. The dysfunction of transcription factors often results in characteristic syndromic presentations; however, at least half of these patients lack a genetic diagnosis. The implementation of machine learning approaches has the potential to aid in the identification of new disease genes and delineate associated phenotypes. Next generation sequencing was performed in seven affected individuals with neurodevelopmental delay and dysmorphic features. Clinical characterization included reanalysis of available neuroimaging datasets and 2D portrait image analysis with GestaltMatcher. The functional consequences of ZSCAN10 loss were modelled in mouse embryonic stem cells (mESCs), including a knockout and a representative ZSCAN10 protein truncating variant. These models were characterized by gene expression and western blot analyses, chromatin immunoprecipitation and quantitative PCR (ChIP-qPCR) and immunofluorescence staining. Zscan10 knockout mouse embryos were generated and phenotyped. We prioritized bi-allelic ZSCAN10 loss-of-function variants in seven affected individuals from five unrelated families as the underlying molecular cause. RNA-sequencing analyses in Zscan10−/− mESCs indicated dysregulation of genes related to stem cell pluripotency. In addition, we established in mESCs the loss-of-function mechanism for a representative human ZSCAN10 protein truncating variant by showing alteration of its expression levels and subcellular localization, interfering with its binding to DNA enhancer targets. Deep phenotyping revealed global developmental delay, facial asymmetry and malformations of the outer ear as consistent clinical features. Cerebral MRI showed dysplasia of the semicircular canals as an anatomical correlate of sensorineural hearing loss. Facial asymmetry was confirmed as a clinical feature by GestaltMatcher and was recapitulated in the Zscan10 mouse model along with inner and outer ear malformations. Our findings provide evidence of a novel syndromic neurodevelopmental disorder caused by bi-allelic loss-of-function variants in ZSCAN10

Clinico-Genetic, Imaging and Molecular Delineation of COQ8A-Ataxia: A Multicenter Study of 59 Patients.

OBJECTIVE: To foster trial-readiness of coenzyme Q8A (COQ8A)-ataxia, we map the clinicogenetic, molecular, and neuroimaging spectrum of COQ8A-ataxia in a large worldwide cohort, and provide first progression data, including treatment response to coenzyme Q10 (CoQ10). METHODS: Cross-modal analysis of a multicenter cohort of 59 COQ8A patients, including genotype-phenotype correlations, 3D-protein modeling, in vitro mutation analyses, magnetic resonance imaging (MRI) markers, disease progression, and CoQ10 response data. RESULTS: Fifty-nine patients (39 novel) with 44 pathogenic COQ8A variants (18 novel) were identified. Missense variants demonstrated a pleiotropic range of detrimental effects upon protein modeling and in vitro analysis of purified variants. COQ8A-ataxia presented as variable multisystemic, early-onset cerebellar ataxia, with complicating features ranging from epilepsy (32%) and cognitive impairment (49%) to exercise intolerance (25%) and hyperkinetic movement disorders (41%), including dystonia and myoclonus as presenting symptoms. Multisystemic involvement was more prevalent in missense than biallelic loss-of-function variants (82-93% vs 53%; p = 0.029). Cerebellar atrophy was universal on MRI (100%), with cerebral atrophy or dentate and pontine T2 hyperintensities observed in 28%. Cross-sectional (n = 34) and longitudinal (n = 7) assessments consistently indicated mild-to-moderate progression of ataxia (SARA: 0.45/year). CoQ10 treatment led to improvement by clinical report in 14 of 30 patients, and by quantitative longitudinal assessments in 8 of 11 patients (SARA: -0.81/year). Explorative sample size calculations indicate that ≥48 patients per arm may suffice to demonstrate efficacy for interventions that reduce progression by 50%. INTERPRETATION: This study provides a deeper understanding of the disease, and paves the way toward large-scale natural history studies and treatment trials in COQ8A-ataxia. ANN NEUROL 2020;88:251-263

De novo DHDDS variants cause a neurodevelopmental and neurodegenerative disorder with myoclonus

Subcellular membrane systems are highly enriched in dolichol, whose role in organelle homeostasis and endosomal-lysosomal pathway remains largely unclear besides being involved in protein glycosylation. DHDDS encodes for the catalytic subunit (DHDDS) of the enzyme cis-prenyltransferase (cis-PTase), involved in dolichol biosynthesis and dolichol-dependent protein glycosylation in the endoplasmic reticulum. An autosomal recessive form of retinitis pigmentosa (retinitis pigmentosa 59) has been associated with a recurrent DHDDS variant. Moreover, two recurring de novo substitutions were detected in a few cases presenting with neurodevelopmental disorder, epilepsy, and movement disorder. We evaluated a large cohort of patients (n=25) with de novo pathogenic variants in DHDDS and provided the first systematic description of the clinical features and long-term outcome of this new neurodevelopmental and neurodegenerative disorder. The functional impact of the identified variants was explored by yeast complementation system and enzymatic assay. Patients presented during infancy or childhood with a variable association of neurodevelopmental disorder, generalized epilepsy, action myoclonus/cortical tremor, and ataxia. Later in the disease course they experienced a slow neurological decline with the emergence of hyperkinetic and/or hypokinetic movement disorder, cognitive deterioration, and psychiatric disturbances. Storage of lipidic material and altered lysosomes were detected in myelinated fibers and fibroblasts, suggesting a dysfunction of the lysosomal enzymatic scavenger machinery. Serum glycoprotein hypoglycosylation was not detected and, in contrast to retinitis pigmentosa and other congenital disorders of glycosylation involving dolichol metabolism, the urinary dolichol D18/D19 ratio was normal. Mapping the disease-causing variants into the protein structure revealed that most of them clustered around the active site of the DHDDS subunit. Functional studies using yeast complementation assay and in vitro activity measurements confirmed that these changes affected the catalytic activity of the cis-PTase and showed growth defect in yeast complementation system as compared with the wild-type enzyme and retinitis pigmentosa-associated protein. In conclusion, we characterized a distinctive neurodegenerative disorder due to de novo DHDDS variants, which clinically belongs to the spectrum of genetic progressive encephalopathies with myoclonus. Clinical and biochemical data from this cohort depicted a condition at the intersection of congenital disorders of glycosylation and inherited storage diseases with several features akin to of progressive myoclonus epilepsy such as neuronal ceroid lipofuscinosis and other lysosomal disorders