Bi-allelic variants in SNF8 cause a disease spectrum ranging from severe developmental and epileptic encephalopathy to syndromic optic atrophy.

The endosomal sorting complex required for transport (ESCRT) machinery is essential for membrane remodeling and autophagy and it comprises three multi-subunit complexes (ESCRT I-III). We report nine individuals from six families presenting with a spectrum of neurodevelopmental/neurodegenerative features caused by bi-allelic variants in SNF8 (GenBank: NM_007241.4), encoding the ESCRT-II subunit SNF8. The phenotypic spectrum included four individuals with severe developmental and epileptic encephalopathy, massive reduction of white matter, hypo-/aplasia of the corpus callosum, neurodevelopmental arrest, and early death. A second cohort shows a milder phenotype with intellectual disability, childhood-onset optic atrophy, or ataxia. All mildly affected individuals shared the same hypomorphic variant, c.304G>A (p.Val102Ile). In patient-derived fibroblasts, bi-allelic SNF8 variants cause loss of ESCRT-II subunits. Snf8 loss of function in zebrafish results in global developmental delay and altered embryo morphology, impaired optic nerve development, and reduced forebrain size. In vivo experiments corroborated the pathogenicity of the tested SNF8 variants and their variable impact on embryo development, validating the observed clinical heterogeneity. Taken together, we conclude that loss of ESCRT-II due to bi-allelic SNF8 variants is associated with a spectrum of neurodevelopmental/neurodegenerative phenotypes mediated likely via impairment of the autophagic flux.

Bi-allelic LETM1 variants perturb mitochondrial ion homeostasis leading to a clinical spectrum with predominant nervous system involvement.

Leucine zipper-EF-hand containing transmembrane protein 1 (LETM1) encodes an inner mitochondrial membrane protein with an osmoregulatory function controlling mitochondrial volume and ion homeostasis. The putative association of LETM1 with a human disease was initially suggested in Wolf-Hirschhorn syndrome, a disorder that results from de novo monoallelic deletion of chromosome 4p16.3, a region encompassing LETM1. Utilizing exome sequencing and international gene-matching efforts, we have identified 18 affected individuals from 11 unrelated families harboring ultra-rare bi-allelic missense and loss-of-function LETM1 variants and clinical presentations highly suggestive of mitochondrial disease. These manifested as a spectrum of predominantly infantile-onset (14/18, 78%) and variably progressive neurological, metabolic, and dysmorphic symptoms, plus multiple organ dysfunction associated with neurodegeneration. The common features included respiratory chain complex deficiencies (100%), global developmental delay (94%), optic atrophy (83%), sensorineural hearing loss (78%), and cerebellar ataxia (78%) followed by epilepsy (67%), spasticity (53%), and myopathy (50%). Other features included bilateral cataracts (42%), cardiomyopathy (36%), and diabetes (27%). To better understand the pathogenic mechanism of the identified LETM1 variants, we performed biochemical and morphological studies on mitochondrial K+/H+ exchange activity, proteins, and shape in proband-derived fibroblasts and muscles and in Saccharomyces cerevisiae, which is an important model organism for mitochondrial osmotic regulation. Our results demonstrate that bi-allelic LETM1 variants are associated with defective mitochondrial K+ efflux, swollen mitochondrial matrix structures, and loss of important mitochondrial oxidative phosphorylation protein components, thus highlighting the implication of perturbed mitochondrial osmoregulation caused by LETM1 variants in neurological and mitochondrial pathologies.

Loss-of-Function Variants in CUL3 Cause a Syndromic Neurodevelopmental Disorder.

OBJECTIVE: De novo variants in cullin-3 ubiquitin ligase (CUL3) have been strongly associated with neurodevelopmental disorders (NDDs), but no large case series have been reported so far. Here, we aimed to collect sporadic cases carrying rare variants in CUL3, describe the genotype-phenotype correlation, and investigate the underlying pathogenic mechanism. METHODS: Genetic data and detailed clinical records were collected via multicenter collaboration. Dysmorphic facial features were analyzed using GestaltMatcher. Variant effects on CUL3 protein stability were assessed using patient-derived T-cells. RESULTS: We assembled a cohort of 37 individuals with heterozygous CUL3 variants presenting a syndromic NDD characterized by intellectual disability with or without autistic features. Of these, 35 have loss-of-function (LoF) and 2 have missense variants. CUL3 LoF variants in patients may affect protein stability leading to perturbations in protein homeostasis, as evidenced by decreased ubiquitin-protein conjugates in vitro. Notably, we show that 4E-BP1 (EIF4EBP1), a prominent substrate of CUL3, fails to be targeted for proteasomal degradation in patient-derived cells. INTERPRETATION: Our study further refines the clinical and mutational spectrum of CUL3-associated NDDs, expands the spectrum of cullin RING E3 ligase-associated neuropsychiatric disorders, and suggests haploinsufficiency via LoF variants is the predominant pathogenic mechanism. ANN NEUROL 2024.

Digenic Leigh syndrome on the background of the m.11778G>A Leber hereditary optic neuropathy variant.

Leigh syndrome spectrum (LSS) is a primary mitochondrial disorder defined neuropathologically by a subacute necrotizing encephalomyelopathy and characterized by bilateral basal ganglia and/or brainstem lesions. LSS is associated with variants in several mitochondrial DNA genes and more than 100 nuclear genes, most often related to mitochondrial complex I (CI) dysfunction. Rarely, LSS has been reported in association with primary Leber hereditary optic neuropathy (LHON) variants of the mitochondrial DNA, coding for CI subunits (m.3460G>A in MT-ND1, m.11778G>A in MT-ND4 and m.14484T>C in MT-ND6). The underlying mechanism by which these variants manifest as LSS, a severe neurodegenerative disease, as opposed to the LHON phenotype of isolated optic neuropathy, remains an open question. Here, we analyse the exome sequencing of six probands with LSS carrying primary LHON variants, and report digenic co-occurrence of the m.11778G > A variant with damaging heterozygous variants in nuclear disease genes encoding CI subunits as a plausible explanation. Our findings suggest a digenic mechanism of disease for m.11778G>A-associated LSS, consistent with recent reports of digenic disease in individuals manifesting with LSS due to biallelic variants in the recessive LHON-associated disease gene DNAJC30 in combination with heterozygous variants in CI subunits.

Associations of carotid intima media thickness with gene expression in whole blood and genetically predicted gene expression across 48 tissues.

Carotid intima media thickness (cIMT) is a biomarker of subclinical atherosclerosis and a predictor of future cardiovascular events. Identifying associations between gene expression levels and cIMT may provide insight to atherosclerosis etiology. Here, we use two approaches to identify associations between mRNA levels and cIMT: differential gene expression analysis in whole blood and S-PrediXcan. We used microarrays to measure genome-wide whole blood mRNA levels of 5647 European individuals from four studies. We examined the association of mRNA levels with cIMT adjusted for various potential confounders. Significant associations were tested for replication in three studies totaling 3943 participants. Next, we applied S-PrediXcan to summary statistics from a cIMT genome-wide association study (GWAS) of 71 128 individuals to estimate the association between genetically determined mRNA levels and cIMT and replicated these analyses using S-PrediXcan on an independent GWAS on cIMT that included 22 179 individuals from the UK Biobank. mRNA levels of TNFAIP3, CEBPD and METRNL were inversely associated with cIMT, but these associations were not significant in the replication analysis. S-PrediXcan identified associations between cIMT and genetically determined mRNA levels for 36 genes, of which six were significant in the replication analysis, including TLN2, which had not been previously reported for cIMT. There was weak correlation between our results using differential gene expression analysis and S-PrediXcan. Differential expression analysis and S-PrediXcan represent complementary approaches for the discovery of associations between phenotypes and gene expression. Using these approaches, we prioritize TNFAIP3, CEBPD, METRNL and TLN2 as new candidate genes whose differential expression might modulate cIMT.

Multi-omics in MECP2 duplication syndrome patients and carriers.

MECP2 duplication syndrome (MDS) is an X-linked neurodevelopmental disorder caused by the gain of dose of at least the genes MECP2 and IRAK1 and is characterised by intellectual disability (ID), developmental delay, hypotonia, epilepsy and recurrent infections. It mainly affects males, and females can be affected or asymptomatic carriers. Rett syndrome (RTT) is mainly triggered by loss of function mutations in MECP2 and is a well described syndrome that presents ID, epilepsy, lack of purposeful hand use and impaired speech, among others. As a result of implementing omics technology, altered biological pathways in human RTT samples have been reported, but such molecular characterisation has not been performed in patients with MDS. We gathered human skin fibroblasts from 17 patients with MDS, 10 MECP2 duplication carrier mothers and 21 patients with RTT, and performed multi-omics (RNAseq and proteomics) analysis. Here, we provide a thorough description and compare the shared and specific dysregulated biological processes between the cohorts. We also highlight the genes TMOD2, SRGAP1, COPS2, CNPY2, IGF2BP1, MOB2, VASP, FZD7, ECSIT and KIF3B as biomarker and therapeutic target candidates due to their implication in neuronal functions. Defining the RNA and protein profiles has shown that our four cohorts are less alike than expected by their shared phenotypes.

Biallelic USP14 variants cause a syndromic neurodevelopmental disorder.

PURPOSE: Imbalances in protein homeostasis affect human brain development, with the ubiquitin-proteasome system (UPS) and autophagy playing crucial roles in neurodevelopmental disorders (NDD). This study explores the impact of biallelic USP14 variants on neurodevelopment, focusing on its role as a key hub connecting UPS and autophagy. METHODS: Here, we identified biallelic USP14 variants in 4 individuals from 3 unrelated families: 1 fetus, a newborn with a syndromic NDD and 2 siblings affected by a progressive neurological disease. Specifically, the 2 siblings from the latter family carried 2 compound heterozygous variants c.8T>C p.(Leu3Pro) and c.988C>T p.(Arg330∗), whereas the fetus had a homozygous frameshift c.899_902del p.(Lys300Serfs∗24) variant, and the newborn patient harbored a homozygous frameshift c.233_236del p.(Leu78Glnfs∗11) variant. Functional studies were conducted using sodium dodecyl-sulfate polyacrylamide gel electrophoresis, western blotting, and mass spectrometry analyses in both patient-derived and CRISPR-Cas9-generated cells. RESULTS: Our investigations indicated that the USP14 variants correlated with reduced N-terminal methionine excision, along with profound alterations in proteasome, autophagy, and mitophagy activities. CONCLUSION: Biallelic USP14 variants in NDD patients perturbed protein degradation pathways, potentially contributing to disorder etiology. Altered UPS, autophagy, and mitophagy activities underscore the intricate interplay, elucidating their significance in maintaining proper protein homeostasis during brain development.

De novo variants in RNF213 are associated with a clinical spectrum ranging from Leigh syndrome to early-onset stroke.

PURPOSE: RNF213, encoding a giant E3 ubiquitin ligase, has been recognized for its role as a key susceptibility gene for moyamoya disease. Case reports have also implicated specific variants in RNF213 with an early-onset form of moyamoya disease with full penetrance. We aimed to expand the phenotypic spectrum of monogenic RNF213-related disease and to evaluate genotype-phenotype correlations. METHODS: Patients were identified through reanalysis of exome sequencing data of an unselected cohort of unsolved pediatric cases and through GeneMatcher or ClinVar. Functional characterization was done by proteomics analysis and oxidative phosphorylation enzyme activities using patient-derived fibroblasts. RESULTS: We identified 14 individuals from 13 unrelated families with (de novo) missense variants in RNF213 clustering within or around the Really Interesting New Gene (RING) domain. Individuals presented either with early-onset stroke (n = 11) or with Leigh syndrome (n = 3). No genotype-phenotype correlation could be established. Proteomics using patient-derived fibroblasts revealed no significant differences between clinical subgroups. 3D modeling revealed a clustering of missense variants in the tertiary structure of RNF213 potentially affecting zinc-binding suggesting a gain-of-function or dominant negative effect. CONCLUSION: De novo missense variants in RNF213 clustering in the E3 RING or other regions affecting zinc-binding lead to an early-onset syndrome characterized by stroke or Leigh syndrome.

Genome and RNA sequencing were essential to reveal cryptic intronic variants associated to defective ATP6AP1 mRNA processing.

The diagnosis of Mendelian disorders has notably advanced with integration of whole exome and genome sequencing (WES and WGS) in clinical practice. However, challenges in variant interpretation and uncovered variants by WES still leave a substantial percentage of patients undiagnosed. In this context, integrating RNA sequencing (RNA-seq) improves diagnostic workflows, particularly for WES inconclusive cases. Additionally, functional studies are often necessary to elucidate the impact of prioritized variants on gene expression and protein function. Our study focused on three unrelated male patients (P1-P3) with ATP6AP1-CDG (congenital disorder of glycosylation), presenting with intellectual disability and varying degrees of hepatopathy, glycosylation defects, and an initially inconclusive diagnosis through WES. Subsequent RNA-seq was pivotal in identifying the underlying genetic causes in P1 and P2, detecting ATP6AP1 underexpression and aberrant splicing. Molecular studies in fibroblasts confirmed these findings and identified the rare intronic variants c.289-233C > T and c.289-289G > A in P1 and P2, respectively. Trio-WGS also revealed the variant c.289-289G > A in P3, which was a de novo change in both patients. Functional assays expressing the mutant alleles in HAP1 cells demonstrated the pathogenic impact of these variants by reproducing the splicing alterations observed in patients. Our study underscores the role of RNA-seq and WGS in enhancing diagnostic rates for genetic diseases such as CDG, providing new insights into ATP6AP1-CDG molecular bases by identifying the first two deep intronic variants in this X-linked gene. Additionally, our study highlights the need to integrate RNA-seq and WGS, followed by functional validation, in routine diagnostics for a comprehensive evaluation of patients with an unidentified molecular etiology.

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.

Recessive NUP54 Variants Underlie Early-Onset Dystonia with Striatal Lesions.

Infantile striatonigral degeneration is caused by a homozygous variant of the nuclear-pore complex (NPC) gene NUP62, involved in nucleo-cytoplasmic trafficking. By querying sequencing-datasets of patients with dystonia and/or Leigh(-like) syndromes, we identified 3 unrelated individuals with biallelic variants in NUP54. All variants clustered in the C-terminal protein region that interacts with NUP62. Associated phenotypes were similar to those of NUP62-related disease, including early-onset dystonia with dysphagia, choreoathetosis, and T2-hyperintense lesions in striatum. In silico and protein-biochemical studies gave further evidence for the argument that the variants were pathogenic. We expand the spectrum of NPC component-associated dystonic conditions with localized basal-ganglia abnormalities. ANN NEUROL 2023;93:330-335.

Identification of molecular signatures and pathways involved in Rett syndrome using a multi-omics approach.

BACKGROUND: Rett syndrome (RTT) is a neurodevelopmental disorder mainly caused by mutations in the methyl-CpG-binding protein 2 gene (MECP2). MeCP2 is a multi-functional protein involved in many cellular processes, but the mechanisms by which its dysfunction causes disease are not fully understood. The duplication of the MECP2 gene causes a distinct disorder called MECP2 duplication syndrome (MDS), highlighting the importance of tightly regulating its dosage for proper cellular function. Additionally, some patients with mutations in genes other than MECP2 exhibit phenotypic similarities with RTT, indicating that these genes may also play a role in similar cellular functions. The purpose of this study was to characterise the molecular alterations in patients with RTT in order to identify potential biomarkers or therapeutic targets for this disorder. METHODS: We used a combination of transcriptomics (RNAseq) and proteomics (TMT mass spectrometry) to characterise the expression patterns in fibroblast cell lines from 22 patients with RTT and detected mutation in MECP2, 15 patients with MDS, 12 patients with RTT-like phenotypes and 13 healthy controls. Transcriptomics and proteomics data were used to identify differentially expressed genes at both RNA and protein levels, which were further inspected via enrichment and upstream regulator analyses and compared to find shared features in patients with RTT. RESULTS: We identified molecular alterations in cellular functions and pathways that may contribute to the disease phenotype in patients with RTT, such as deregulated cytoskeletal components, vesicular transport elements, ribosomal subunits and mRNA processing machinery. We also compared RTT expression profiles with those of MDS seeking changes in opposite directions that could lead to the identification of MeCP2 direct targets. Some of the deregulated transcripts and proteins were consistently affected in patients with RTT-like phenotypes, revealing potentially relevant molecular processes in patients with overlapping traits and different genetic aetiology. CONCLUSIONS: The integration of data in a multi-omics analysis has helped to interpret the molecular consequences of MECP2 dysfunction, contributing to the characterisation of the molecular landscape in patients with RTT. The comparison with MDS provides knowledge of MeCP2 direct targets, whilst the correlation with RTT-like phenotypes highlights processes potentially contributing to the pathomechanism leading these disorders.

Variants in ATP5F1B are associated with dominantly inherited dystonia.

ATP5F1B is a subunit of the mitochondrial ATP synthase or complex V of the mitochondrial respiratory chain. Pathogenic variants in nuclear genes encoding assembly factors or structural subunits are associated with complex V deficiency, typically characterized by autosomal recessive inheritance and multisystem phenotypes. Movement disorders have been described in a subset of cases carrying autosomal dominant variants in structural subunits genes ATP5F1A and ATP5MC3. Here, we report the identification of two different ATP5F1B missense variants (c.1000A>C; p.Thr334Pro and c.1445T>C; p.Val482Ala) segregating with early-onset isolated dystonia in two families, both with autosomal dominant mode of inheritance and incomplete penetrance. Functional studies in mutant fibroblasts revealed no decrease of ATP5F1B protein amount but severe reduction of complex V activity and impaired mitochondrial membrane potential, suggesting a dominant-negative effect. In conclusion, our study describes a new candidate gene associated with isolated dystonia and confirms that heterozygous variants in genes encoding subunits of the mitochondrial ATP synthase may cause autosomal dominant isolated dystonia with incomplete penetrance, likely through a dominant-negative mechanism.

ACOX1 Gain-of-Function Variant in Two German Pediatric Patients, in One Case Mimicking Autoimmune Inflammatory Disease.

Mitchell syndrome is a very rare genetic disorder due to a specific de novo gain-of-function variant in acyl-CoA oxidase 1 (ACOX1). So far, only five patients with this disease have been described worldwide. We present here two additional unrelated German patients found to carry the same heterozygous ACOX1 N237S variant through exome sequencing (ES). Both patients showed neurodegenerative clinical features starting from ∼4 to 5 years of age including progressive hearing loss, ataxia, ichthyosis, as well as progressive visual impairment leading to amaurosis, and died at the ages of 16 and 8 years, respectively. The first patient was clinically suspected to have anti-myelin oligodendrocyte glycoprotein-antibody-associated myelitis, but the disease course overall deteriorated despite extensive immunomodulatory therapy. The second patient was originally suspected to have a mitochondrial disorder due to intermittent elevated blood lactate. Since Mitchell syndrome has only been identified in 2020, the diagnosis in this second patient was only established through re-evaluation of ES data years after the original analysis. Comparison of all seven reported patients suggests that Mitchell syndrome often (but not always) clinically mimics autoimmune-inflammatory disease. Therefore, in patients with autoimmune central nervous system disease who do not respond adequately to standard therapies, re-evaluation of this diagnosis is needed and genetic analyses such as trio ES should be considered.

Mitochondrial Protein Interaction Mapping Identifies Regulators of Respiratory Chain Function.

Mitochondria are essential for numerous cellular processes, yet hundreds of their proteins lack robust functional annotation. To reveal functions for these proteins (termed MXPs), we assessed condition-specific protein-protein interactions for 50 select MXPs using affinity enrichment mass spectrometry. Our data connect MXPs to diverse mitochondrial processes, including multiple aspects of respiratory chain function. Building upon these observations, we validated C17orf89 as a complex I (CI) assembly factor. Disruption of C17orf89 markedly reduced CI activity, and its depletion is found in an unresolved case of CI deficiency. We likewise discovered that LYRM5 interacts with and deflavinates the electron-transferring flavoprotein that shuttles electrons to coenzyme Q (CoQ). Finally, we identified a dynamic human CoQ biosynthetic complex involving multiple MXPs whose topology we map using purified components. Collectively, our data lend mechanistic insight into respiratory chain-related activities and prioritize hundreds of additional interactions for further exploration of mitochondrial protein function.

Digenic Inheritance in Rare Disorders and Mitochondrial Disease-Crossing the Frontier to a More Comprehensive Understanding of Etiology.

Our understanding of rare disease genetics has been shaped by a monogenic disease model. While the traditional monogenic disease model has been successful in identifying numerous disease-associated genes and significantly enlarged our knowledge in the field of human genetics, it has limitations in explaining phenomena like phenotypic variability and reduced penetrance. Widening the perspective beyond Mendelian inheritance has the potential to enable a better understanding of disease complexity in rare disorders. Digenic inheritance is the simplest instance of a non-Mendelian disorder, characterized by the functional interplay of variants in two disease-contributing genes. Known digenic disease causes show a range of pathomechanisms underlying digenic interplay, including direct and indirect gene product interactions as well as epigenetic modifications. This review aims to systematically explore the background of digenic inheritance in rare disorders, the approaches and challenges when investigating digenic inheritance, and the current evidence for digenic inheritance in mitochondrial disorders.

Bi-Allelic UQCRFS1 Variants Are Associated with Mitochondrial Complex III Deficiency, Cardiomyopathy, and Alopecia Totalis.

Isolated complex III (CIII) deficiencies are among the least frequently diagnosed mitochondrial disorders. Clinical symptoms range from isolated myopathy to severe multi-systemic disorders with early death and disability. To date, we know of pathogenic variants in genes encoding five out of 10 subunits and five out of 13 assembly factors of CIII. Here we describe rare bi-allelic variants in the gene of a catalytic subunit of CIII, UQCRFS1, which encodes the Rieske iron-sulfur protein, in two unrelated individuals. Affected children presented with low CIII activity in fibroblasts, lactic acidosis, fetal bradycardia, hypertrophic cardiomyopathy, and alopecia totalis. Studies in proband-derived fibroblasts showed a deleterious effect of the variants on UQCRFS1 protein abundance, mitochondrial import, CIII assembly, and cellular respiration. Complementation studies via lentiviral transduction and overexpression of wild-type UQCRFS1 restored mitochondrial function and rescued the cellular phenotype, confirming UQCRFS1 variants as causative for CIII deficiency. We demonstrate that mutations in UQCRFS1 can cause mitochondrial disease, and our results thereby expand the clinical and mutational spectrum of CIII deficiencies.

Pathogenic Bi-allelic Mutations in NDUFAF8 Cause Leigh Syndrome with an Isolated Complex I Deficiency.

Leigh syndrome is one of the most common neurological phenotypes observed in pediatric mitochondrial disease presentations. It is characterized by symmetrical lesions found on neuroimaging in the basal ganglia, thalamus, and brainstem and by a loss of motor skills and delayed developmental milestones. Genetic diagnosis of Leigh syndrome is complicated on account of the vast genetic heterogeneity with >75 candidate disease-associated genes having been reported to date. Candidate genes are still emerging, being identified when "omics" tools (genomics, proteomics, and transcriptomics) are applied to manipulated cell lines and cohorts of clinically characterized individuals who lack a genetic diagnosis. NDUFAF8 is one such protein; it has been found to interact with the well-characterized complex I (CI) assembly factor NDUFAF5 in a large-scale protein-protein interaction screen. Diagnostic next-generation sequencing has identified three unrelated pediatric subjects, each with a clinical diagnosis of Leigh syndrome, who harbor bi-allelic pathogenic variants in NDUFAF8. These variants include a recurrent splicing variant that was initially overlooked due to its deep-intronic location. Subject fibroblasts were found to express a complex I deficiency, and lentiviral transduction with wild-type NDUFAF8-cDNA ameliorated both the assembly defect and the biochemical deficiency. Complexome profiling of subject fibroblasts demonstrated a complex I assembly defect, and the stalled assembly intermediates corroborate the role of NDUFAF8 in early complex I assembly. This report serves to expand the genetic heterogeneity associated with Leigh syndrome and to validate the clinical utility of orphan protein characterization. We also highlight the importance of evaluating intronic sequence when a single, definitively pathogenic variant is identified during diagnostic testing.

ATP2B2 de novo variants as a cause of variable neurodevelopmental disorders that feature dystonia, ataxia, intellectual disability, behavioral symptoms, and seizures.

PURPOSE: ATP2B2 encodes the variant-constrained plasma-membrane calcium-transporting ATPase-2, expressed in sensory ear cells and specialized neurons. ATP2B2/Atp2b2 variants were previously linked to isolated hearing loss in patients and neurodevelopmental deficits with ataxia in mice. We aimed to establish the association between ATP2B2 and human neurological disorders. METHODS: Multinational case recruitment, scrutiny of trio-based genomics data, in silico analyses, and functional variant characterization were performed. RESULTS: We assembled 7 individuals harboring rare, predicted deleterious heterozygous ATP2B2 variants. The alleles comprised 5 missense substitutions that affected evolutionarily conserved sites and 2 frameshift variants in the penultimate exon. For 6 variants, a de novo status was confirmed. Unlike described patients with hearing loss, the individuals displayed a spectrum of neurological abnormalities, ranging from ataxia with dystonic features to complex neurodevelopmental manifestations with intellectual disability, autism, and seizures. Two cases with recurrent amino-acid variation showed distinctive overlap with cerebellar atrophy-associated ataxia and epilepsy. In cell-based studies, all variants caused significant alterations in cytosolic calcium handling with both loss- and gain-of-function effects. CONCLUSION: Presentations in our series recapitulate key phenotypic aspects of Atp2b2-mouse models and underline the importance of precise calcium regulation for neurodevelopment and cerebellar function. Our study documents a role for ATP2B2 variants in causing heterogeneous neurodevelopmental and movement-disorder syndromes.

Genotypic and phenotypic spectrum of infantile liver failure due to pathogenic TRMU variants.

PURPOSE: This study aimed to define the genotypic and phenotypic spectrum of reversible acute liver failure (ALF) of infancy resulting from biallelic pathogenic TRMU variants and determine the role of cysteine supplementation in its treatment. METHODS: Individuals with biallelic (likely) pathogenic variants in TRMU were studied within an international retrospective collection of de-identified patient data. RESULTS: In 62 individuals, including 30 previously unreported cases, we described 47 (likely) pathogenic TRMU variants, of which 17 were novel, and 1 intragenic deletion. Of these 62 individuals, 42 were alive at a median age of 6.8 (0.6-22) years after a median follow-up of 3.6 (0.1-22) years. The most frequent finding, occurring in all but 2 individuals, was liver involvement. ALF occurred only in the first year of life and was reported in 43 of 62 individuals; 11 of whom received liver transplantation. Loss-of-function TRMU variants were associated with poor survival. Supplementation with at least 1 cysteine source, typically N-acetylcysteine, improved survival significantly. Neurodevelopmental delay was observed in 11 individuals and persisted in 4 of the survivors, but we were unable to determine whether this was a primary or a secondary consequence of TRMU deficiency. CONCLUSION: In most patients, TRMU-associated ALF was a transient, reversible disease and cysteine supplementation improved survival.