Supernumerary proteins of the human mitochondrial ribosomal small subunit are integral for assembly and translation.

Mitochondrial ribosomes (mitoribosomes) have undergone substantial evolutionary structural remodeling accompanied by loss of ribosomal RNA, while acquiring unique protein subunits located on the periphery. We generated CRISPR-mediated knockouts of all 14 unique (mitochondria-specific/supernumerary) human mitoribosomal proteins (snMRPs) in the small subunit to study the effect on mitoribosome assembly and protein synthesis, each leading to a unique mitoribosome assembly defect with variable impact on mitochondrial protein synthesis. Surprisingly, the stability of mS37 was reduced in all our snMRP knockouts of the small and large ribosomal subunits and patient-derived lines with mitoribosome assembly defects. A redox-regulated CX9C motif in mS37 was essential for protein stability, suggesting a potential mechanism to regulate mitochondrial protein synthesis. Together, our findings support a modular assembly of the human mitochondrial small ribosomal subunit mediated by essential supernumerary subunits and identify a redox regulatory role involving mS37 in mitochondrial protein synthesis in health and disease.

Leigh syndrome with developmental regression and ataxia due to a novel splicing variant in the PMPCB gene.

Only five children with pathogenic PMPCB gene variants have been described and all carried missense variants. Clinical features included a Leigh-like syndrome of developmental regression, basal ganglia lesions and ataxia with or without dystonia and epilepsy. Three of the five died in childhood and none was older than age six when described. We report the first splice site variant in the PMPCB gene in a 39-year old individual who experienced developmental regression and ataxia following otitis media in childhood. A minigene assay confirms this variant results in aberrant splicing and skipping of exon 12.

Recessive TMOD1 mutation causes childhood cardiomyopathy.

Familial cardiomyopathy in pediatric stages is a poorly understood presentation of heart disease in children that is attributed to pathogenic mutations. Through exome sequencing, we report a homozygous variant in tropomodulin 1 (TMOD1; c.565C>T, p.R189W) in three individuals from two unrelated families with childhood-onset dilated and restrictive cardiomyopathy. To decipher the mechanism of pathogenicity of the R189W mutation in TMOD1, we utilized a wide array of methods, including protein analyses, biochemistry and cultured cardiomyocytes. Structural modeling revealed potential defects in the local folding of TMOD1R189W and its affinity for actin. Cardiomyocytes expressing GFP-TMOD1R189W demonstrated longer thin filaments than GFP-TMOD1wt-expressing cells, resulting in compromised filament length regulation. Furthermore, TMOD1R189W showed weakened activity in capping actin filament pointed ends, providing direct evidence for the variant's effect on actin filament length regulation. Our data indicate that the p.R189W variant in TMOD1 has altered biochemical properties and reveals a unique mechanism for childhood-onset cardiomyopathy.

Inhibition of human cytomegalovirus replication by interferon alpha can involve multiple anti-viral factors.

The shortcomings of current direct-acting anti-viral therapy against human cytomegalovirus (HCMV) has led to interest in host-directed therapy. Here we re-examine the use of interferon proteins to inhibit HCMV replication utilizing both high and low passage strains of HCMV. Pre-treatment of cells with interferon alpha (IFNα) was required for robust and prolonged inhibition of both low and high passage HCMV strains, with no obvious toxicity, and was associated with an increased anti-viral state in HCMV-infected cells. Pre-treatment of cells with IFNα led to poor expression of HCMV immediate-early proteins from both high and low passage strains, which was associated with the presence of the anti-viral factor SUMO-PML. Inhibition of HCMV replication in the presence of IFNα involving ZAP proteins was HCMV strain-dependent, wherein a high passage HCMV strain was obviously restricted by ZAP and a low passage strain was not. This suggested that strain-specific combinations of anti-viral factors were involved in inhibition of HCMV replication in the presence of IFNα. Overall, this work further supports the development of strategies involving IFNα that may be useful to inhibit HCMV replication and highlights the complexity of the anti-viral response to HCMV in the presence of IFNα.

Brain monoamine vesicular transport disease caused by homozygous SLC18A2 variants: A study in 42 affected individuals.

PURPOSE: Brain monoamine vesicular transport disease is an infantile-onset movement disorder that mimics cerebral palsy. In 2013, the homozygous SLC18A2 variant, p.Pro387Leu, was first reported as a cause of this rare disorder, and dopamine agonists were efficient for treating affected individuals from a single large family. To date, only 6 variants have been reported. In this study, we evaluated genotype-phenotype correlations in individuals with biallelic SLC18A2 variants. METHODS: A total of 42 affected individuals with homozygous SLC18A2 variant alleles were identified. We evaluated genotype-phenotype correlations and the missense variants in the affected individuals based on the structural modeling of rat VMAT2 encoded by Slc18a2, with cytoplasm- and lumen-facing conformations. A Caenorhabditis elegans model was created for functional studies. RESULTS: A total of 19 homozygous SLC18A2 variants, including 3 recurrent variants, were identified using exome sequencing. The affected individuals typically showed global developmental delay, hypotonia, dystonia, oculogyric crisis, and autonomic nervous system involvement (temperature dysregulation/sweating, hypersalivation, and gastrointestinal dysmotility). Among the 58 affected individuals described to date, 16 (28%) died before the age of 13 years. Of the 17 patients with p.Pro237His, 9 died, whereas all 14 patients with p.Pro387Leu survived. Although a dopamine agonist mildly improved the disease symptoms in 18 of 21 patients (86%), some affected individuals with p.Ile43Phe and p.Pro387Leu showed milder phenotypes and presented prolonged survival even without treatment. The C. elegans model showed behavioral abnormalities. CONCLUSION: These data expand the phenotypic and genotypic spectra of SLC18A2-related disorders.

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.

Phenotypic continuum of NFU1-related disorders.

Bi-allelic variants in Iron-Sulfur Cluster Scaffold (NFU1) have previously been associated with multiple mitochondrial dysfunctions syndrome 1 (MMDS1) characterized by early-onset rapidly fatal leukoencephalopathy. We report 19 affected individuals from 10 independent families with ultra-rare bi-allelic NFU1 missense variants associated with a spectrum of early-onset pure to complex hereditary spastic paraplegia (HSP) phenotype with a longer survival (16/19) on one end and neurodevelopmental delay with severe hypotonia (3/19) on the other. Reversible or irreversible neurological decompensation after a febrile illness was common in the cohort, and there were invariable white matter abnormalities on neuroimaging. The study suggests that MMDS1 and HSP could be the two ends of the NFU1-related phenotypic continuum.

HIDEA syndrome is caused by biallelic, pathogenic, rare or founder P4HTM variants impacting the active site or the overall stability of the P4H-TM protein.

HIDEA syndrome is caused by biallelic pathogenic variants in P4HTM. The phenotype is characterized by muscular and central hypotonia, hypoventilation including obstructive and central sleep apneas, intellectual disability, dysautonomia, epilepsy, eye abnormalities, and an increased tendency to develop respiratory distress during pneumonia. Here, we report six new patients with HIDEA syndrome caused by five different biallelic P4HTM variants, including three novel variants. We describe two Finnish enriched pathogenic P4HTM variants and demonstrate that these variants are embedded within founder haplotypes. We review the clinical data from all previously published patients with HIDEA and characterize all reported P4HTM pathogenic variants associated with HIDEA in silico. All known pathogenic variants in P4HTM result in either premature stop codons, an intragenic deletion, or amino acid changes that impact the active site or the overall stability of P4H-TM protein. In all cases, normal P4H-TM enzyme function is expected to be lost or severely decreased. This report expands knowledge of the genotypic and phenotypic spectrum of the disease.

An overview of international genetic evaluations of show jumping in sport horses.

The breeding of sport horses to compete in the Olympic disciplines of show jumping, eventing, and dressage is fast becoming a global industry with the increased use of reproductive technologies, including artificial insemination and embryo transfer. Reproductive technologies have facilitated the dissemination of genetics from elite horses across multiple countries and breeds as breeders are no longer limited by location. Due to this increased level of crossbreeding, there is an increased need for estimated breeding values (EBVs) for sport horse performance that can be compared across breeds and countries. However, the implementation of across-breed or across-country genetic evaluations has been limited by the differences in each studbook's individual breeding programs and genetic evaluations. Consequently, the aim of this review was to compare the genetic evaluations for show jumping of sport horse studbooks worldwide. The top sport horse studbooks in the world according to the World Breeding Federation for Sport Horses Studbook Rankings 2019 were contacted by email to request information on their current breeding programs and genetic evaluations. Twenty-six of the 51 studbooks contacted replied to this request but only 18 of these studbooks conducted their own genetic evaluations or were part of a larger genetic evaluation in their country of origin. The other eight studbooks were not involved in genetic evaluations at present but expressed an interest in the implementation of such in the future. Overall, many differences were identified among the genetic evaluations of each studbook or each country. The definition of show jumping performance differed within each evaluation and the methods and models utilized also differed. Despite some stallions and mares being registered in multiple studbooks or having progeny in multiple studbooks, these differences make comparison of EBVs across studbooks difficult. Further transparency and collaboration of sport horse studbooks with organizations such as Interstallion, will be essential to facilitate any future implementation of international genetic evaluations for show jumping performance.

Duplication/triplication mosaicism of EBF3 and expansion of the EBF3 neurodevelopmental disorder phenotype.

Deleterious variants in the transcription factor early B-cell factor 3 (EBF3) are known to cause a neurodevelopmental disorder (EBF3-NDD). We report eleven individuals with EBF3 variants, including an individual with a duplication/triplication mosaicism of a region encompassing EBF3 and a phenotype consistent with EBF3-NDD, which may reflect the importance of EBF3 gene-dosage for neurodevelopment. The phenotype of individuals in this cohort was quite mild compared to the core phenotype of previously described individuals. Although ataxia tended to wane with age, we show that cognitive difficulties may increase, and we recommend that individuals with EBF3-NDD have systematic neuropsychological follow-up.

MED27 Variants Cause Developmental Delay, Dystonia, and Cerebellar Hypoplasia.

The Mediator multiprotein complex functions as a regulator of RNA polymerase II-catalyzed gene transcription. In this study, exome sequencing detected biallelic putative disease-causing variants in MED27, encoding Mediator complex subunit 27, in 16 patients from 11 families with a novel neurodevelopmental syndrome. Patient phenotypes are highly homogeneous, including global developmental delay, intellectual disability, axial hypotonia with distal spasticity, dystonic movements, and cerebellar hypoplasia. Seizures and cataracts were noted in severely affected individuals. Identification of multiple patients with biallelic MED27 variants supports the critical role of MED27 in normal human neural development, particularly for the cerebellum. ANN NEUROL 2021;89:828-833.

SUCLA2 mutations cause global protein succinylation contributing to the pathomechanism of a hereditary mitochondrial disease.

Mitochondrial acyl-coenzyme A species are emerging as important sources of protein modification and damage. Succinyl-CoA ligase (SCL) deficiency causes a mitochondrial encephalomyopathy of unknown pathomechanism. Here, we show that succinyl-CoA accumulates in cells derived from patients with recessive mutations in the tricarboxylic acid cycle (TCA) gene succinyl-CoA ligase subunit-ß (SUCLA2), causing global protein hyper-succinylation. Using mass spectrometry, we quantify nearly 1,000 protein succinylation sites on 366 proteins from patient-derived fibroblasts and myotubes. Interestingly, hyper-succinylated proteins are distributed across cellular compartments, and many are known targets of the (NAD+)-dependent desuccinylase SIRT5. To test the contribution of hyper-succinylation to disease progression, we develop a zebrafish model of the SCL deficiency and find that SIRT5 gain-of-function reduces global protein succinylation and improves survival. Thus, increased succinyl-CoA levels contribute to the pathology of SCL deficiency through post-translational modifications.

Clinical and molecular description of 19 patients with GATAD2B-Associated Neurodevelopmental Disorder (GAND).

De novo pathogenic variants in the GATAD2B gene have been associated with a syndromic neurodevelopmental disorder (GAND) characterized by severe intellectual disability (ID), impaired speech, childhood hypotonia, and dysmorphic features. Since its first description in 2013, nine patients have been reported in case reports and a series of 50 patients was recently published, which is consistent with the relative frequency of GATAD2B pathogenic variants in public databases. We report the detailed phenotype of 19 patients from various ethnic backgrounds with confirmed pathogenic GATAD2B variants including intragenic deletions. All individuals presented developmental delay with a median age of 2.5 years for independent walking and of 3 years for first spoken words. GATAD2B variant carriers showed very little subsequent speech progress, two patients over 30 years of age remaining non-verbal. ID was mostly moderate to severe, with one profound and one mild case, which shows a wider spectrum of disease severity than previously reported. We confirm macrocephaly as a major feature in GAND (53%). Most common dysmorphic features included broad forehead, deeply set eyes, hypertelorism, wide nasal base, and pointed chin. Conversely, prenatal abnormalities, non-cerebral malformations, epilepsy, and autistic behavior were uncommon. Other features included feeding difficulties, behavioral abnormalities, and unspecific abnormalities on brain MRI. Improving our knowledge of the clinical phenotype is essential for correct interpretation of the molecular results and accurate patient management.

Genetic background of ataxia in children younger than 5 years in Finland.

OBJECTIVE: To characterize the genetic background of molecularly undefined childhood-onset ataxias in Finland. METHODS: This study examined a cohort of patients from 50 families with onset of an ataxia syndrome before the age of 5 years collected from a single tertiary center, drawing on the advantages offered by next generation sequencing. A genome-wide genotyping array (Illumina Infinium Global Screening Array MD-24 v.2.0) was used to search for copy number variation undetectable by exome sequencing. RESULTS: Exome sequencing led to a molecular diagnosis for 20 probands (40%). In the 23 patients examined with a genome-wide genotyping array, 2 additional diagnoses were made. A considerable proportion of probands with a molecular diagnosis had de novo pathogenic variants (45%). In addition, the study identified a de novo variant in a gene not previously linked to ataxia: MED23. Patients in the cohort had medically actionable findings. CONCLUSIONS: There is a high heterogeneity of causative mutations in this cohort despite the defined age at onset, phenotypical overlap between patients, the founder effect, and genetic isolation in the Finnish population. The findings reflect the heterogeneous genetic background of ataxia seen worldwide and the substantial contribution of de novo variants underlying childhood ataxia.

Fibroblast Growth Factor 21 Drives Dynamics of Local and Systemic Stress Responses in Mitochondrial Myopathy with mtDNA Deletions.

Mitochondrial dysfunction elicits stress responses that safeguard cellular homeostasis against metabolic insults. Mitochondrial integrated stress response (ISRmt) is a major response to mitochondrial (mt)DNA expression stress (mtDNA maintenance, translation defects), but the knowledge of dynamics or interdependence of components is lacking. We report that in mitochondrial myopathy, ISRmt progresses in temporal stages and development from early to chronic and is regulated by autocrine and endocrine effects of FGF21, a metabolic hormone with pleiotropic effects. Initial disease signs induce transcriptional ISRmt (ATF5, mitochondrial one-carbon cycle, FGF21, and GDF15). The local progression to 2nd metabolic ISRmt stage (ATF3, ATF4, glucose uptake, serine biosynthesis, and transsulfuration) is FGF21 dependent. Mitochondrial unfolded protein response marks the 3rd ISRmt stage of failing tissue. Systemically, FGF21 drives weight loss and glucose preference, and modifies metabolism and respiratory chain deficiency in a specific hippocampal brain region. Our evidence indicates that FGF21 is a local and systemic messenger of mtDNA stress in mice and humans with mitochondrial disease.

A urinary biosignature for mitochondrial myopathy, encephalopathy, lactic acidosis and stroke like episodes (MELAS).

We used a comprehensive metabolomics approach to study the altered urinary metabolome of two mitochondrial myopathy, encephalopathy lactic acidosis and stroke like episodes (MELAS) cohorts carrying the m.3243A>G mutation. The first cohort were used in an exploratory phase, identifying 36 metabolites that were significantly perturbed by the disease. During the second phase, the 36 selected metabolites were able to separate a validation cohort of MELAS patients completely from their respective control group, suggesting usefulness of these 36 markers as a diagnostic set. Many of the 36 perturbed metabolites could be linked to an altered redox state, fatty acid catabolism and one-carbon metabolism. However, our evidence indicates that, of all the metabolic perturbations caused by MELAS, stalled fatty acid oxidation prevailed as being particularly disturbed. The strength of our study was the utilization of five different analytical platforms to generate the robust metabolomics data reported here. We show that urine may be a useful source for disease-specific metabolomics data, linking, amongst others, altered one-carbon metabolism to MELAS. The results reported here are important in our understanding of MELAS and might lead to better treatment options for the disease.

Genetic Basis of Severe Childhood-Onset Cardiomyopathies.

BACKGROUND: Childhood cardiomyopathies are progressive and often lethal disorders, forming the most common cause of heart failure in children. Despite severe outcomes, their genetic background is still poorly characterized. OBJECTIVES: The purpose of this study was to characterize the genetics of severe childhood cardiomyopathies in a countrywide cohort. METHODS: The authors collected a countrywide cohort, KidCMP, of 66 severe childhood cardiomyopathies from the sole center in Finland performing cardiac transplantation. For genetic diagnosis, next-generation sequencing and subsequent validation using genetic, cell biology, and computational approaches were used. RESULTS: The KidCMP cohort presents remarkable early-onset and severe disorders: the median age of diagnosis was 0.33 years, and 17 patients underwent cardiac transplantation. The authors identified the pathogenic variants in 39% of patients: 46% de novo, 34% recessive, and 20% dominantly-inherited. The authors report NRAP underlying childhood dilated cardiomyopathy, as well as novel phenotypes for known heart disease genes. Some genetic diagnoses have immediate implications for treatment: CALM1 with life-threatening arrhythmias, and TAZ with good cardiac prognosis. The disease genes converge on metabolic causes (PRKAG2, MRPL44, AARS2, HADHB, DNAJC19, PPA2, TAZ, BAG3), MAPK pathways (HRAS, PTPN11, RAF1, TAB2), development (NEK8 and TBX20), calcium signaling (JPH2, CALM1, CACNA1C), and the sarcomeric contraction cycle (TNNC1, TNNI3, ACTC1, MYH7, NRAP). CONCLUSIONS: Childhood cardiomyopathies are typically caused by rare, family-specific mutations, most commonly de novo, indicating that next-generation sequencing of trios is the approach of choice in their diagnosis. Genetic diagnoses may suggest intervention strategies and predict prognosis, offering valuable tools for prioritization of patients for transplantation versus conservative treatment.

Metabolomes of mitochondrial diseases and inclusion body myositis patients: treatment targets and biomarkers.

Mitochondrial disorders (MDs) are inherited multi-organ diseases with variable phenotypes. Inclusion body myositis (IBM), a sporadic inflammatory muscle disease, also shows mitochondrial dysfunction. We investigated whether primary and secondary MDs modify metabolism to reveal pathogenic pathways and biomarkers. We investigated metabolomes of 25 mitochondrial myopathy or ataxias patients, 16 unaffected carriers, six IBM and 15 non-mitochondrial neuromuscular disease (NMD) patients and 30 matched controls. MD and IBM metabolomes clustered separately from controls and NMDs. MDs and IBM showed transsulfuration pathway changes; creatine and niacinamide depletion marked NMDs, IBM and infantile-onset spinocerebellar ataxia (IOSCA). Low blood and muscle arginine was specific for patients with m.3243A>G mutation. A four-metabolite blood multi-biomarker (sorbitol, alanine, myoinositol, cystathionine) distinguished primary MDs from others (76% sensitivity, 95% specificity). Our omics approach identified pathways currently used to treat NMDs and mitochondrial stroke-like episodes and proposes nicotinamide riboside in MDs and IBM, and creatine in IOSCA and IBM as novel treatment targets. The disease-specific metabolic fingerprints are valuable "multi-biomarkers" for diagnosis and promising tools for follow-up of disease progression and treatment effect.

Defective mitochondrial ATPase due to rare mtDNA m.8969G>A mutation-causing lactic acidosis, intellectual disability, and poor growth.

Mutations in mitochondrial ATP synthase 6 (MT-ATP6) are a frequent cause of NARP (neurogenic muscle weakness, ataxia, and retinitis pigmentosa) or Leigh syndromes, especially a point mutation at nucleotide position 8993. M.8969G>A is a rare MT-ATP6 mutation, previously reported only in three individuals, causing multisystem disorders with mitochondrial myopathy, lactic acidosis, and sideroblastic anemia or IgA nephropathy. We present two siblings with the m.8969G>A mutation and a novel, substantially milder phenotype with lactic acidosis, poor growth, and intellectual disability. Our findings expand the phenotypic spectrum and show that mtDNA mutations should be taken account also with milder, stable phenotypes.