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Peroxisomal biogenesis disorders (PBDs) are a heterogeneous group of genetic diseases. Multiple peroxisomal pathways are impaired, and very long chain fatty acids (VLCFA) are the first line biomarkers for the diagnosis. The clinical presentation of PBDs may range from severe, lethal multisystemic disorders to milder, late-onset disease. The vast majority of PBDs belong to Zellweger Spectrum Disordes (ZSDs) and represents a continuum of overlapping clinical symptoms, with Zellweger syndrome being the most severe and Heimler syndrome the less severe disease. Mild clinical conditions frequently present normal or slight biochemical alterations, making the diagnosis of these patients challenging. In the present study we used a combined WES and RNA-seq strategy to diagnose a patient presenting with retinal dystrophy as the main clinical symptom. Results showed the patient was compound heterozygous for mutations in PEX1. VLCFA were normal, but retrospective analysis of lysosphosphatidylcholines (LPC) containing C22:0–C26:0 species was altered. This simple test could avoid the diagnostic odyssey of patients with mild phenotype, such as the individual described here, who was diagnosed very late in adult life. We provide functional data in cell line models that may explain the mild phenotype of the patient by demonstrating the hypomorphic nature of a deep intronic variant altering PEX1 mRNA processing.

Mitochondrial translation defects are a continuously growing group of disorders showing a large variety of clinical symptoms including a wide range of neurological abnormalities. To date, mutations in PTCD3, encoding a component of the mitochondrial ribosome, have only been reported in a single individual with clinical evidence of Leigh syndrome. Here, we describe three additional PTCD3 individuals from two unrelated families, broadening the genetic and phenotypic spectrum of this disorder, and provide definitive evidence that PTCD3 deficiency is associated with Leigh syndrome. The patients presented in the first months of life with psychomotor delay, respiratory insufficiency and feeding difficulties. The neurologic phenotype included dystonia, optic atrophy, nystagmus and tonic-clonic seizures. Brain MRI showed optic nerve atrophy and thalamic changes, consistent with Leigh syndrome. WES and RNA-seq identified compound heterozygous variants in PTCD3 in both families: c.[1453-1GC];[1918CG] and c.[710del];[902CT]. The functional consequences of the identified variants were determined by a comprehensive characterization of the mitochondrial function. PTCD3 protein levels were significantly reduced in patient fibroblasts and, consistent with a mitochondrial translation defect, a severe reduction in the steady state levels of complexes I and IV subunits was detected. Accordingly, the activity of these complexes was also low, and high-resolution respirometry showed a significant decrease in the mitochondrial respiratory capacity. Functional complementation studies demonstrated the pathogenic effect of the identified variants since the expression of wild-type PTCD3 in immortalized fibroblasts restored the steady-state levels of complexes I and IV subunits as well as the mitochondrial respiratory capacity. Additionally, minigene assays demonstrated that three of the identified variants were pathogenic by altering PTCD3 mRNA processing. The fourth variant was a frameshift leading to a truncated protein. In summary, we provide evidence of PTCD3 involvement in human disease confirming that PTCD3 deficiency is definitively associated with Leigh syndrome.

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. This research was supported using resources of the Core Facility Cell Imaging and Ultrastructure Research, University of Vienna, a member of the Vienna Life-Science Instruments (VLSI) and the VetCore Facility (Imaging) of the University of Veterinary Medicine Vienna. We acknowledge International Centre for Genomic Medicine in Neuromuscular Diseases. This research was funded in part, by the Wellcome Trust (WT093205MA, WT104033AIA, and the Synaptopathies Strategic Award, 165908). This study was funded by the Medical Research Council (MR/S01165X/1, MR/S005021/1, G0601943), The National Institute for Health Research University College London Hospitals Biomedical Research Centre, Rosetrees Trust, Ataxia UK, Multiple System Atrophy Trust, Brain Research United Kingdom, Sparks Great Ormond Street Hospital Charity, Muscular Dystrophy United Kingdom (MDUK), Muscular Dystrophy Association (MDA USA) and Senior Non-Clinical Fellow ship to A. Spinazzola, (MC_PC_13029). K.N. and S.E.M.M. were supported by the Austrian Science Funds FWF-P29077 and P31471. A. Spinazzola receives support also from The Lily Foun dation and Brain Research UK. R.K. was supported by European Academy of Neurology Research Training Fellowship and Rosetrees Trust PhD Plus award (PhD2022\100042). info:eu-repo/semantics/publishedVersion

BackgroundThe spectrum of mitochondrial disease is genetically and phenotypically diverse, resulting from pathogenic variants in over 400 genes, with aerobic energy metabolism defects as a common denominator. Such heterogeneity poses a significant challenge in making an accurate diagnosis, critical for precision medicine.MethodsIn an international collaboration initiated by the European Network for Mitochondrial Diseases (GENOMIT) we recruited 2,023 pediatric patients at 11 specialist referral centers between October 2010 and January 2021, accumulating exome sequencing and HPO-encoded phenotype data. An exome-wide search for variants in known and potential novel disease genes, complemented by functional studies, followed ACMG guidelines.Results1,109 cases (55%) received a molecular diagnosis, of which one fifth have potential disease-modifying treatments (236/1,109, 21%). Functional studies enabled diagnostic uplift from 36% to 55% and discovery of 62 novel disease genes. Pathogenic variants were identified within genes encoding mitochondrial proteins or RNAs in 801 cases (72%), while, given extensive phenotype overlap, the remainder involved proteins targeted to other cellular compartments. To delineate genotype-phenotype associations, our data was complemented with registry and literature data to develop “GENOMITexplorer”, an open access resource detailing patient- (n=3,940), gene- (n=427), and variant-level (n=1,492) associations (prokischlab.github.io/GENOMITexplorer/).ConclusionsReaching a molecular diagnosis was essential for implementation of precision medicine and clinical trial eligibility, underlining the need for genome-wide screening given inability to accurately define mitochondrial diseases clinically. Key to diagnostic success were functional studies, encouraging early acquisition of patient- derived tissues and routine integration of high-throughput functional data to improve patient care by uplifting diagnostic rate.

BackgroundLack of functional evidence hampers variant interpretation, leaving a large proportion of cases with a suspected Mendelian disorder without genetic diagnosis after genome or whole exome sequencing (WES). Research studies advocate to further sequence transcriptomes to directly and systematically probe gene expression defects. However, collection of additional biopsies, and establishment of lab workflows, analytical pipelines, and defined concepts in clinical interpretation of aberrant gene expression are still needed for adopting RNA-sequencing (RNA-seq) in routine diagnostics.MethodsWe implemented an automated RNA-seq protocol and a computational workflow with which we analyzed skin fibroblasts of 303 individuals with a suspected mitochondrial disease which previously underwent WES.ResultsWe detected on average 12,500 genes per sample including around 60% disease genes - a coverage substantially higher than with whole blood, supporting the use of skin biopsies. We prioritized genes demonstrating aberrant expression, aberrant splicing, or mono-allelic expression. The pipeline required less than one week from sample preparation to result reporting and provided a median of eight disease-associated genes per patient for inspection. A genetic diagnosis was established for 16% of the 205 WES-inconclusive cases. Detection of aberrant expression was a major contributor to diagnosis including instances of 50% reduction, which, together with mono-allelic expression, allowed for the diagnosis of dominant disorders caused by haploinsufficiency. Moreover, calling aberrant splicing and variants from RNA-seq data enabled detecting and validating splice-disrupting variants, of which the majority fell outside WES-covered regions.ConclusionTogether, these results show that streamlined experimental and computational processes can accelerate the implementation of RNA-seq in routine diagnostics.One sentence summaryImplementation of RNA-seq as a complementary tool in standard diagnostics achieves a 16% in diagnosis rate over whole exome sequencing.

By lack of functional evidence, genome-based diagnostic rates cap at approximately 50% across diverse Mendelian diseases. Here, we demonstrate the effectiveness of combining genomics, transcriptomics, and, for the first time, proteomics and phenotypic descriptors, in a systematic diagnostic approach to discover the genetic cause of mitochondrial diseases. On fibroblast cell lines from 145 individuals, tandem mass tag labelled proteomics detected approximately 8,000 proteins per sample and covered over 50% of all Mendelian disease-associated genes. Aberrant protein expression analysis allowed the validation of candidate protein-destabilising variants, in addition to providing independent complementary functional evidence to variants leading to aberrant RNA expression. Overall, our integrative computational workflow led to genetic resolution for 22% of 121 genetically unsolved whole exome or whole genome negative cases and to the discovery of two novel disease genes. With increasing democratization of high-throughput omics assays, our approach and code provide a blueprint for implementing multi-omics based Mendelian disease diagnostics in routine clinical practice.

Mitochondrial disorders are monogenic disorders characterized by a defect in oxidative phosphorylation and caused by pathogenic variants in one of over 340 different genes. The implementation of whole-exome sequencing has led to a revolution in their diagnosis, duplicated the number of associated disease genes, and significantly increased the diagnosed fraction. However, the genetic etiology of a substantial fraction of patients exhibiting mitochondrial disorders remains unknown, highlighting limitations in variant detection and interpretation, which calls for improved computational and DNA sequencing methods, as well as the addition of OMICS tools. More intriguingly, this also suggests that some pathogenic variants lie outside of the protein-coding genes and that the mechanisms beyond the Mendelian inheritance and the mtDNA are of relevance. This review covers the current status of the genetic basis of mitochondrial diseases, discusses current challenges and perspectives, and explores the contribution of factors beyond the protein-coding regions and monogenic inheritance in the expansion of the genetic spectrum of disease.

RNA sequencing (RNA-seq) has emerged as a powerful approach to discover disease-causing gene regulatory defects in individuals affected by genetically undiagnosed rare disorders. Pioneering studies have shown that RNA-seq could increase the diagnosis rates over DNA sequencing alone by 8–36%, depending on the disease entity and tissue probed. To accelerate adoption of RNA-seq by human genetics centers, detailed analysis protocols are now needed. We present a step-by-step protocol that details how to robustly detect aberrant expression levels, aberrant splicing and mono-allelic expression in RNA-seq data using dedicated statistical methods. We describe how to generate and assess quality control plots and interpret the analysis results. The protocol is based on the detection of RNA outliers pipeline (DROP), a modular computational workflow that integrates all the analysis steps, can leverage parallel computing infrastructures and generates browsable web page reports. The authors describe a modular pipeline to detect aberrant gene expression events (expression level, splicing and mono-allelic expression) from patient RNA sequencing data, which can complement DNA-based diagnosis by enhancing the functional interpretation of variants.

ADP-ribosylation is a reversible posttranslational modification used to regulate protein function. ADP-ribosyltransferases transfer ADP-ribose from NAD(+) to the target protein, and ADP-ribosylhydrolases, such as ADPRHL2, reverse the reaction. We used exome sequencing to identify five different bi-allelic pathogenic ADPRHL2 variants in 12 individuals from 8 families affected by a neurodegenerative disorder manifesting in childhood or adolescence with key clinical features including developmental delay or regression, seizures, ataxia, and axonal (sensori-)motor neuropathy. ADPRHL2 was virtually absent in available affected individuals’ fibroblasts, and cell viability was reduced upon hydrogen peroxide exposure, although it was rescued by expression of wild-type ADPRHL2 mRNA as well as treatment with a PARP1 inhibitor. Our findings suggest impaired protein ribosylation as another pathway that, if disturbed, causes neurodegenerative diseases.

Recently, heterozygous de novo mutations in SCL1A2 have been reported to underlie severe early-onset epileptic encephalopathy. In one male presenting with epileptic seizures and visual impairment, we identified a novel homozygous splicing variant in SCL1A2 (c.1421 + 1G > C) by using exome sequencing. Functional studies on cDNA level confirmed a consecutive loss of function. Our findings suggest that not only de novo mutations but also biallelic variants in SLC1A2 can cause epilepsy and that there is an additional autosomal recessive mode of inheritance. These findings also contribute to the understanding of the genetic mechanism of autosomal dominant SLC1A2-related epileptic encephalopathy as they exclude haploinsufficiency as exclusive genetic mechanism.

Leber's hereditary optic neuropathy (LHON) is an inherited mitochondrial disease that usually leads to acute or subacute bilateral central vision loss. In 95% of cases, LHON is caused by one of three primary mutations of the mitochondrial DNA (mtDNA), m.11778G>A in the MT-ND4 gene, m.14484T>C in the MT-ND6 gene, or m.3460G>A in the MT-ND1 gene. Here we characterize clinically, genetically, and biochemically a LHON family with multiple patients harboring two of these primary LHON mutations, m.11778G>A homoplasmic and m.14484T>C heteroplasmic. The unusually low male-to-female ratio of affected family members is also seen among the other patients previously reported with two primary LHON mutations m.11778G>A and m.14484T>C. While the index patient had very late onset of symptoms at 75years and severe visual loss, her two daughters had both onset in childhood (6 and 9years), with moderate to mild visual loss. A higher degree of heteroplasmy of the m.14484T>C mutation was found to correlate with an earlier age at onset in this family. Ours is the first LHON family harboring two primary LHON mutations where functional studies were performed in several affected family members. A more pronounced bioenergetic defect was found to correlate with an earlier age at onset. The patient with the earliest age at onset had a more significant complex I dysfunction than all controls, including the LHON patient with only the m.11778G>A mutation, suggesting a synergistic effect of the two primary LHON mutations in this patient.

RNA sequencing (RNA-seq) has emerged as a powerful approach to discover disease-causing gene regulatory defects for individuals affected with a genetically undiagnosed rare disorder. Pioneer studies have shown that RNA-seq could increase diagnostic rates over DNA sequencing alone by 8% to 36 % depending on disease entities and probed tissues. To accelerate the adoption of RNA-seq among human genetic centers, detailed analysis protocols are now needed. Here, we present a step-by-step protocol that instructs how to robustly detect aberrant expression, aberrant splicing, and mono-allelic expression of a rare allele in RNA-seq data using dedicated statistical methods. We describe how to generate and assess quality control plots and interpret the analysis results. The protocol is based on DROP (Detection of RNA Outliers Pipeline), a modular computational workflow that integrates all analysis steps, can leverage parallel computing infrastructures, and generates browsable web page reports.

Aberrant splicing is a major cause of rare diseases, yet its prediction from genome sequence remains in most cases inconclusive. Recently, RNA sequencing has proven to be an effective complementary avenue to detect aberrant splicing. Here, we developed FRASER, an algorithm to detect aberrant splicing from RNA sequencing data. Unlike existing methods, FRASER captures not only alternative splicing but also intron retention events. This typically doubles the number of detected aberrant events and identified a pathogenic intron retention in MCOLN1. FRASER automatically controls for latent confounders, which are widespread and substantially affect sensitivity. Moreover, FRASER is based on a count distribution and multiple testing correction, reducing the number of calls by two orders of magnitude over commonly applied z score cutoffs, with a minor sensitivity loss. The application to rare disease diagnostics is demonstrated by reprioritizing a pathogenic aberrant exon truncation in TAZ from a published dataset. FRASER is easy to use and freely available.

Inherited optic neuropathies include complex phenotypes, mostly driven by mitochondrial dysfunction. We report an optic atrophy spectrum disorder, including retinal macular dystrophy and kidney insufficiency leading to transplantation, associated with mitochondrial DNA (mtDNA) depletion without accumulation of multiple deletions. By whole-exome sequencing, we identified mutations affecting the mitochondrial single-strand binding protein (SSBP1) in 4 families with dominant and 1 with recessive inheritance. We show that SSBP1 mutations in patient-derived fibroblasts variably affect the amount of SSBP1 protein and alter multimer formation, but not the binding to ssDNA. SSBP1 mutations impaired mtDNA, nucleoids, and 7S-DNA amounts as well as mtDNA replication, affecting replisome machinery. The variable mtDNA depletion in cells was reflected in severity of mitochondrial dysfunction, including respiratory efficiency, OXPHOS subunits, and complex amount and assembly. mtDNA depletion and cytochrome c oxidase-negative cells were found ex vivo in biopsies of affected tissues, such as kidney and skeletal muscle. Reduced efficiency of mtDNA replication was also reproduced in vitro, confirming the pathogenic mechanism. Furthermore, ssbp1 suppression in zebrafish induced signs of nephropathy and reduced optic nerve size, the latter phenotype complemented by WT mRNA but not by SSBP1 mutant transcripts. This previously unrecognized disease of mtDNA maintenance implicates SSBP1 mutations as a cause of human pathology.

We report four unrelated children with homozygous loss-of-function variants in TASP1 and an overlapping phenotype comprising developmental delay with hypotonia and microcephaly, feeding difficulties with failure-to-thrive, recurrent respiratory infections, cardiovascular malformations, cryptorchidism, happy demeanor, and distinctive facial features. Two children had a homozygous founder deletion encompassing exons 5–11 of TASP1, the third had a homozygous missense variant, c.701 C>T (p.Thr234Met), affecting the active site of the encoded enzyme, and the fourth had a homozygous nonsense variant, c.199 C>T (p.Arg67*). TASP1 encodes taspase 1 (TASP1), which is responsible for cleaving, thus activating, the lysine methyltransferases KMT2A and KMT2D, which are essential for histone methylation and transcription regulation. The consistency of the phenotype, the critical biological function of TASP1, the deleterious nature of the TASP1 variants, and the overlapping features with Wiedemann–Steiner and Kabuki syndromes respectively caused by pathogenic variants in KMT2A and KMT2D all support that TASP1 is a disease-related gene.

Accurate quantification of cellular and mitochondrial bioenergetic activity is of great interest in medicine and biology. Mitochondrial stress tests performed with Seahorse Bioscience XF Analyzers allow estimating different bioenergetic measures by monitoring oxygen consumption rates (OCR) of living cells in multi-well plates. However, studies of statistical best practices for determining OCR measurements and comparisons have been lacking so far. Therefore, we performed mitochondrial stress tests in 126 96-well plates involving 203 fibroblast cell lines to understand how OCR behaves across different biosamples, wells, and plates. We show that the noise of OCR is multiplicative, that outlier data points can concern individual measurements or all measurements of a well, and that the inter-plate variation is greater than intra-plate variation. Based on these insights, we developed a novel statistical method, OCR-Stats, that: i) robustly estimates OCR levels modeling multiplicative noise and automatically identifying outlier data points and outlier wells; and ii) performs statistical testing between samples, taking into account the different magnitudes of the between- and within-plates variations. This led to a significant reduction of the coefficient of variation across plates of basal respiration by 36% and of maximal respiration by 32%. Moreover, using positive and negative controls, we show that our statistical test outperforms existing methods, which either suffer from an excess of false positives (within-plates methods), or of false negatives (between-plates methods). Altogether, the aim of this study is to propose statistical good practices to support experimentalists in designing, analyzing, testing and reporting results of mitochondrial stress tests using this high throughput platform.

Respiratory chain complex I deficiency is the most frequently identified biochemical defect in childhood mitochondrial diseases. Clinical symptoms range from fatal infantile lactic acidosis to Leigh syndrome and other encephalomyopathies or cardiomyopathies. To date, disease-causing variants in genes coding for 27 complex I subunits, including 7 mitochondrial DNA genes, and in 11 genes encoding complex I assembly factors have been reported. Here, we describe rare biallelic variants in NDUFB8 encoding a complex I accessory subunit revealed by whole-exome sequencing in two individuals from two families. Both presented with a progressive course of disease with encephalo(cardio) myopathic features including muscular hypotonia, cardiac hypertrophy, respiratory failure, failure to thrive, and developmental delay. Blood lactate was elevated. Neuroimaging disclosed progressive changes in the basal ganglia and either brain stem or internal capsule. Biochemical analyses showed an isolated decrease in complex I enzymatic activity in muscle and fibroblasts. Complementation studies by expression of wild-type NDUFB8 in cells from affected individuals restored mitochondrial function, confirming NDUFB8 variants as the cause of complex I deficiency. Hereby we establish NDUFB8 as a relevant gene in childhood-onset mitochondrial disease.

Despite their importance in determining protein abundance, a comprehensive catalogue of sequence features controlling protein-to-mRNA (PTR) ratios and a quantification of their effects is still lacking. Here we quantified PTR ratios for 11,575 proteins across 29 human tissues using matched transcriptomes and proteomes. We analyzed the contribution of known sequence determinants of protein synthesis and degradation and 15 novel mRNA and protein sequence motifs that we found by association testing. While the dynamic range of PTR ratios spans more than 2 orders of magnitude, our integrative model predicts PTR ratios at a median precision of 3.2-fold. A reporter assay provided significant functional support for two novel UTR motifs and a proteome-wide competition-binding assay identified motif-specific bound proteins for one motif. Moreover, our direct comparison of protein to RNA levels led to a new metrics of codon optimality. Altogether, this study shows that a large fraction of PTR ratio variance across genes can be predicted from sequence and identified many new candidate post-transcriptional regulatory elements in the human genome.