Across a variety of Mendelian disorders, ∼50–75% of patients do not receive a genetic diagnosis by exome sequencing indicating disease-causing variants in non-coding regions. Although genome sequencing in principle reveals all genetic variants, their sizeable number and poorer annotation make prioritization challenging. Here, we demonstrate the power of transcriptome sequencing to molecularly diagnose 10% (5 of 48) of mitochondriopathy patients and identify candidate genes for the remainder. We find a median of one aberrantly expressed gene, five aberrant splicing events and six mono-allelically expressed rare variants in patient-derived fibroblasts and establish disease-causing roles for each kind. Private exons often arise from cryptic splice sites providing an important clue for variant prioritization. One such event is found in the complex I assembly factor TIMMDC1 establishing a novel disease-associated gene. In conclusion, our study expands the diagnostic tools for detecting non-exonic variants and provides examples of intronic loss-of-function variants with pathological relevance.

44Across a large variety of Mendelian disorders, ~50-75% of patients do not receive a 45 genetic diagnosis by whole exome sequencing indicative of underlying disease-causing 46 variants in non-coding regions. In contrast, whole genome sequencing facilitates the 47 discovery of all genetic variants, but their sizeable number, coupled with a poor 48 understanding of the non-coding genome, makes their prioritization challenging. Here, we 49 demonstrate the power of transcriptome sequencing to provide a confirmed genetic 50 diagnosis for 10% (5 of 48) of undiagnosed mitochondrial disease patients and identify 51 strong candidate genes for patients remaining without diagnosis. We found a median of 1 52 aberrantly expressed gene, 5 aberrant splicing events, and 6 mono-allelically expressed 53 rare variants in patient-derived fibroblasts and established disease-causing roles for each 54 kind. Private exons often arose from sites that are weakly spliced in other individuals, 55providing an important clue for future variant prioritization. One such intronic exon-56 creating variant was found in three unrelated families in the complex I assembly factor 57 TIMMDC1, which we consequently established as a novel disease-associated gene. In 58 conclusion, our study expands the diagnostic tools for detecting non-exonic variants of 59Mendelian disorders and provides examples of intronic loss-of-function variants with 60 pathological relevance. 61Despite the revolutionizing impact of whole exome sequencing (WES) on the molecular 62 genetics of Mendelian disorders, ~50-75% of the patients do not receive a genetic diagnosis after 63 WES [1][2][3][4][5][6] . The disease-causing variants might be detected by WES but remain as variants of 64 unknown significance (VUS, Methods) or they are missed due to the inability to prioritize them. 65Many of these VUS are synonymous or non-coding variants that may affect RNA abundance or 66 isoform but cannot be prioritized due to the poor understanding of regulatory sequence to date 67 compared to coding sequence. Furthermore, WES covers only the 2% exonic regions of the 68 genome. Accordingly, it is mostly blind to regulatory variants in non-coding regions that could 69 affect RNA sequence and abundance. While the limitation of genome coverage is overcome by 70 whole genome sequencing (WGS), prioritization and interpretation of variants identified by 71 WGS is in turn limited by their amount [7][8][9] . 72With RNA sequencing (RNA-seq), limitations of the sole genetic information can be 73 complemented by directly probing variations in RNA abundance and in RNA sequence, 74 including allele-specific expression and splice isoforms. At least three extreme situations can be 75 directly interpreted to prioritize candidate disease-causing genes for a rare disorder. First, the 76 expression level of a gene can lie outside its physiological range. Genes with expression outside 77 their physical range can be identified as expression outliers, often using a stringent cutoff on 78 expression variat...

Mitochondria are essential for cellular bioenergetics by way of energy production in the form of ATP through the process of oxidative phosphorylation. This crucial task is executed by five multi-protein complexes of which mitochondrial NADH:ubiquinone oxidoreductase or complex I is the largest and most complicated one. During recent years, mutations in nuclear genes encoding structural subunits of complex I have been identified as a cause of devastating neurodegenerative disorders with onset in early childhood. Here, we present a comprehensive overview of clinical, biochemical and cell physiological information of 15 children with isolated, nuclear-encoded complex I deficiency, which was generated in a joint effort of clinical and fundamental research. Our findings point to a rather homogeneous clinical picture in these children and drastically illustrate the severity of the disease. In extensive live cell studies with patient-derived skin fibroblasts we uncovered important cell physiological aspects of complex I deficiency, which point to a central regulatory role of cellular reactive oxygen species production and altered mitochondrial membrane potential in the pathogenesis of the disorder. Moreover, we critically discuss possible interconnections between clinical signs and cellular pathology. Finally, our results indicate apparent differences to drug therapy on the cellular level, depending on the severity of the catalytic defect and identify modulators of cellular Ca(2+) homeostasis as new candidates in the therapy of complex I deficiency.

Mitochondrial oxidative phosphorylation (OXPHOS) sustains organelle function and plays a central role in cellular energy metabolism. The OXPHOS system consists of 5 multisubunit complexes (CI-CV) that are built up of 92 different structural proteins encoded by the nuclear (nDNA) and mitochondrial DNA (mtDNA). Biogenesis of a functional OXPHOS system further requires the assistance of nDNAencoded OXPHOS assembly factors, of which 35 are currently identified. In humans, mutations in both structural and assembly genes and in genes involved in mtDNA maintenance, replication, transcription, and translation induce 'primary' OXPHOS disorders that are associated with neurodegenerative diseases including Leigh syndrome (LS), which is probably the most classical OXPHOS disease during early childhood. Here, we present the current insights regarding function, biogenesis, regulation, and supramolecular architecture of the OXPHOS system, as well as its genetic origin. Next, we provide an inventory of OXPHOS structural and assembly genes which, when mutated, induce human neurodegenerative disorders. Finally, we discuss the consequences of mutations in OXPHOS structural and assembly genes at the single cell level and how this information has advanced our understanding of the role of OXPHOS dysfunction in neurodegeneration.