A snapshot of some pLI score pitfalls

The pLI score reflects the tolerance of a given gene to the loss of function on the basis of the number of protein truncating variants, that is, the frameshift, splice donor, splice acceptor, and stop-gain variants referenced for this gene in control databases weighted by the size of the gene and the sequencing coverage. It is frequently used to prioritize candidate genes when analyzing whole exome or whole genome data. We list here the main pitfalls to consider before using this score. Concrete illustrations are given for each of these pitfalls

Warburg-like effect is a hallmark of complex I assembly defects

Due to its pivotal role in NADH oxidation and ATP synthesis, mitochondrial complex I (CI) emerged as a crucial regulator of cellular metabolism. A functional CI relies on the sequential assembly of nuclear- and mtDNA-encoded subunits; however, whether CI assembly status is involved in the metabolic adaptations in CI deficiency still remains largely unknown. Here, we investigated the relationship between CI functions, its structure and the cellular metabolism in 29 patient fibroblasts representative of most CI mitochondrial diseases. Our results show that, contrary to the generally accepted view, a complex I deficiency does not necessarily lead to a glycolytic switch, i.e. the so-called Warburg effect, but that this particular metabolic adaptation is a feature of CI assembly defect. By contrast, a CI functional defect without disassembly induces a higher catabolism to sustain the oxidative metabolism. Mechanistically, we demonstrate that reactive oxygen species overproduction by CI assembly intermediates and subsequent AMPK-dependent Pyruvate Dehydrogenase inactivation are key players of this metabolic reprogramming. Thus, this study provides a two-way-model of metabolic responses to CI deficiencies that are central not only in defining therapeutic strategies for mitochondrial diseases, but also in all pathophysiological conditions involving a CI deficiency

Bioinformatics Tools and Databases to Assess the Pathogenicity of Mitochondrial DNA Variants in the Field of Next Generation Sequencing

The development of next generation sequencing (NGS) has greatly enhanced the diagnosis of mitochondrial disorders, with a systematic analysis of the whole mitochondrial DNA (mtDNA) sequence and better detection sensitivity. However, the exponential growth of sequencing data renders complex the interpretation of the identified variants, thereby posing new challenges for the molecular diagnosis of mitochondrial diseases. Indeed, mtDNA sequencing by NGS requires specific bioinformatics tools and the adaptation of those developed for nuclear DNA, for the detection and quantification of mtDNA variants from sequence alignment to the calling steps, in order to manage the specific features of the mitochondrial genome including heteroplasmy, i.e., coexistence of mutant and wildtype mtDNA copies. The prioritization of mtDNA variants remains difficult, relying on a limited number of specific resources: population and clinical databases, and tools providing a prediction of the variant pathogenicity. An evaluation of the most prominent bioinformatics tools showed that their ability to predict the pathogenicity was highly variable indicating that special efforts should be directed at developing new bioinformatics tools dedicated to the mitochondrial genome. In addition, massive parallel sequencing raised several issues related to the interpretation of very low mtDNA mutational loads, discovery of variants of unknown significance, and mutations unrelated to patient phenotype or the co-occurrence of mtDNA variants. This review provides an overview of the current strategies and bioinformatics tools for accurate annotation, prioritization and reporting of mtDNA variations from NGS data, in order to carry out accurate genetic counseling in individuals with primary mitochondrial diseases

Novel gene mutation in an atypical late-onset mitochondrial form of multifocal dystonia

Mitochondrial complex I, the largest component of the mitochondrial respiratory chain, comprises 44 subunits of which 7 are encoded by the mitochondrial genome and the remainder by the nuclear genome. Isolated complex I deficiencies represent a major contribution within the group of respiratory chain defects. We report an atypical case carrying a homozygous NDUFS4 missense mutation, with late-onset multifocal dystonia, in contrast to expected clinical phenotypes due to other NDUFS4 mutations, which have been constantly reported to be responsible for Leigh syndrome of early onset and death

Next generation sequencing in family with MNGIE syndrome associated to optic atrophy: Novel homozygous POLG mutation in the C-terminal sub-domain leading to mtDNA depletion

Introduction Mitochondrial diseases are a group of disorders caused mainly by the impairment of the mitochondrial oxidative phosphorylation process, due to mutations either in the mitochondrial or nuclear genome. Among them, the mitochondrial neuro-gastrointestinal encephalo-myopathy (MNGIE) syndrome affects adolescents or young adults, and is mostly caused by TYMP mutations encoding a cytosolic thymidine phosphorylase (TP). Patients and methods The present study reports the molecular investigation by next-generation re-sequencing of 281 nuclear genes, encoding mitochondrial proteins, of consanguineous family including two individuals with MNGIE syndrome associated to optic atrophy. Bioinformatic analysis was also performed in addition to mtDNA deletion screening and mtDNA copy number quantification in blood of the two patients which were carried out by solf clipping program and qPCR respectively. Results Next-generation re-sequencing revealed a novel homozygous c.2391G > T POLG mutation (p.M797I) co-occurring with the hypomorphic c.1311A > G OPA1 variant (p.I437M). Analysis of the mitochondrial genome in the two patients disclosed mtDNA depletion in blood, but no deletion. Bio-informatics investigations supported the pathogenicity of the novel POLG mutation that is located in the C-terminal subdomain and might change POLG 3D structure, stability and function. Conclusion The novel homozygous p.M797I POLG mutation is responsible for MNGIE combined to optic atrophy and mtDNA depletion in the two patients

Neurologic Phenotypes Associated With Mutations in RTN4IP1 (OPA10) in Children and Young Adults

Importance: Neurologic disorders with isolated symptoms or complex syndromes are relatively frequent among mitochondrial inherited diseases. Recessive RTN4IP1 gene mutations have been shown to cause isolated and syndromic optic neuropathies. Objective: To define the spectrum of clinical phenotypes associated with mutations in RTN4IP1 encoding a mitochondrial quinone oxidoreductase. Design, Setting, and Participants: This study involved 12 individuals from 11 families with severe central nervous system diseases and optic atrophy. Targeted and whole-exome sequencing were performed-at Hospital Angers (France), Institute of Neurology Milan (Italy), Imagine Institute Paris (France), Helmoltz Zentrum of Munich (Germany), and Beijing Genomics Institute (China)-to clarify the molecular diagnosis of patients. Each patient\u27s neurologic, ophthalmologic, magnetic resonance imaging, and biochemical features were investigated. This study was conducted from May 1, 2014, to June 30, 2016. Main Outcomes and Measures: Recessive mutations in RTN4IP1 were identified. Clinical presentations ranged from isolated optic atrophy to severe encephalopathies. Results: Of the 12 individuals in the study, 6 (50%) were male and 6 (50%) were female. They ranged in age from 5 months to 32 years. Of the 11 families, 6 (5 of whom were consanguineous) had a member or members who presented isolated optic atrophy with the already reported p.Arg103His or the novel p.Ile362Phe, p.Met43Ile, and p.Tyr51Cys amino acid changes. The 5 other families had a member or members who presented severe neurologic syndromes with a common core of symptoms, including optic atrophy, seizure, intellectual disability, growth retardation, and elevated lactate levels. Additional clinical features of those affected were deafness, abnormalities on magnetic resonance images of the brain, stridor, and abnormal electroencephalographic patterns, all of which eventually led to death before age 3 years. In these patients, novel and very rare homozygous and compound heterozygous mutations were identified that led to the absence of the protein and complex I disassembly as well as mild mitochondrial network fragmentation. Conclusions and Relevance: A broad clinical spectrum of neurologic features, ranging from isolated optic atrophy to severe early-onset encephalopathies, is associated with RTN4IP1 biallelic mutations and should prompt RTN4IP1 screening in both syndromic neurologic presentations and nonsyndromic recessive optic neuropathies

CLUH couples mitochondrial distribution to the energetic and metabolic status

Mitochondrial dynamics and distribution are critical for supplying ATP in response to energy demand. CLUH is a protein involved in mitochondrial distribution whose dysfunction leads to mitochondrial clustering, the metabolic consequences of which remain unknown. To gain insight into the role of CLUH on mitochondrial energy production and cellular metabolism, we have generated CLUH-knockout cells using CRISPR/Cas9. Mitochondrial clustering was associated with a smaller cell size and with decreased abundance of respiratory complexes, resulting in oxidative phosphorylation (OXPHOS) defects. This energetic impairment was found to be due to the alteration of mitochondrial translation and to a metabolic shift towards glucose dependency. Metabolomic profiling by mass spectroscopy revealed an increase in the concentration of some amino acids, indicating a dysfunctional Krebs cycle, and increased palmitoylcarnitine concentration, indicating an alteration of fatty acid oxidation, and a dramatic decrease in the concentrations of phosphatidylcholine and sphingomyeline, consistent with the decreased cell size. Taken together, our study establishes a clear function for CLUH in coupling mitochondrial distribution to the control of cell energetic and metabolic status

Mitochondrial DNA content reduction in the most fertile spermatozoa is accompanied by increased mitochondrial DNA rearrangement

Abstract STUDY QUESTION Is there an association between male fertility and spermatozoa mitochondrial DNA (mtDNA) copy number and genome rearrangements? SUMMARY ANSWER Normal spermatozoa not only have a lower mtDNA copy number but also more DNA rearrangements than spermatozoa of men with severe oligoasthenospermia (SOA). WHAT IS KNOWN ALREADY While there is a consensus that mtDNA content is decreased in the most fertile spermatozoa, the role of mtDNA sequence alteration in male infertility is unclear. High-throughput sequencing, which allows an exhaustive analysis of mtDNA rearrangements and mutations, could be helpful in this context, but has yet to be used. STUDY DESIGN, SIZE, DURATION This is an observational study of semen samples obtained from 44 men undergoing ART at an academic infertility centre in France, from October 2018 to November 2020. The men were classified into two groups: 20 men in the SOA group and 24 men with normal semen parameters in the control group. PARTICIPANTS/MATERIALS, SETTING, METHODS For each patient and control, mtDNA was isolated from sperm fractions from the 40% and 90% layers of the density gradient. The average mtDNA content of each sample was assessed using digital PCR. Deep sequencing was performed using next-generation sequencing. Signal processing and base calling were performed via the embedded pre-processing pipeline, the variants were analysed using an in-house workflow and a dedicated tool, based on soft-clipping, was used to study large mtDNA rearrangements. The distribution and the type of rearrangements and variants were compared between patients with SOA and controls on one hand, and between the 40% and 90% gradient layers, on the other hand. MAIN RESULTS AND THE ROLE OF CHANCE The mtDNA content of spermatozoa in the SOA group was significantly higher than in the control group (P &amp;lt; 0.0001). Moreover, mtDNA content was significantly higher in spermatozoa from the 40% layer (the most fertile spermatozoa) compared to the 90% layer, both in the SOA (P = 0.02) and the control group (P &amp;lt; 0.0001). The frequency of large mtDNA deletions and duplications was significantly higher in the control group (P = 0.002). Most of these rearrangements are potentially related to DNA breaks and their number was reduced by the removal of the linear mtDNA from the samples. Heteroplasmic variants were found more frequently in the SOA group (P = 0.05) and in the 40% layer (P = 0.03), but none had any obvious functional consequence. LIMITATIONS, REASONS FOR CAUTION Our findings are novel and significant but should be verified in larger cohorts and other types of male infertility. WIDER IMPLICATIONS OF THE FINDINGS Our findings suggest that sperm mtDNA rearrangements are not necessarily associated with mitochondrial dysfunction and male infertility. Instead, they seem to be concomitant with the process of mtDNA content reduction in the most potentially fertile spermatozoa. Furthermore, they refute the hypothesis that, in the case of mtDNA alteration, a compensatory mechanism allows an increase in mtDNA copy number to rectify the energy deficit. The increased frequency of mtDNA rearrangements in the most fertile spermatozoa is a novel result that offers new insight into the relation between sperm quality and mtDNA. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by Angers University Hospital (grant AOI CHU Angers 2018), Angers University and the French national research centres INSERM and CNRS. There are no competing interests. TRIAL REGISTRATION NUMBER N/A. </jats:sec