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 in silico 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

Dominant ACO2 mutations are a frequent cause of isolated optic atrophy.

Biallelic mutations in ACO2, encoding the mitochondrial aconitase 2, have been identified in individuals with neurodegenerative syndromes, including infantile cerebellar retinal degeneration and recessive optic neuropathies (locus OPA9). By screening European cohorts of individuals with genetically unsolved inherited optic neuropathies, we identified 61 cases harbouring variants in ACO2, among whom 50 carried dominant mutations, emphasizing for the first time the important contribution of ACO2 monoallelic pathogenic variants to dominant optic atrophy. Analysis of the ophthalmological and clinical data revealed that recessive cases are affected more severely than dominant cases, while not significantly earlier. In addition, 27% of the recessive cases and 11% of the dominant cases manifested with extraocular features in addition to optic atrophy. In silico analyses of ACO2 variants predicted their deleterious impacts on ACO2 biophysical properties. Skin derived fibroblasts from patients harbouring dominant and recessive ACO2 mutations revealed a reduction of ACO2 abundance and enzymatic activity, and the impairment of the mitochondrial respiration using citrate and pyruvate as substrates, while the addition of other Krebs cycle intermediates restored a normal respiration, suggesting a possible short-cut adaptation of the tricarboxylic citric acid cycle. Analysis of the mitochondrial genome abundance disclosed a significant reduction of the mitochondrial DNA amount in all ACO2 fibroblasts. Overall, our data position ACO2 as the third most frequently mutated gene in autosomal inherited optic neuropathies, after OPA1 and WFS1, and emphasize the crucial involvement of the first steps of the Krebs cycle in the maintenance and survival of retinal ganglion cells

Mutations in the m-AAA proteases AFG3L2 and SPG7 are causing isolated dominant optic atrophy.

OBJECTIVE: To improve the genetic diagnosis of dominant optic atrophy (DOA), the most frequently inherited optic nerve disease, and infer genotype-phenotype correlations. METHODS: Exonic sequences of 22 genes were screened by new-generation sequencing in patients with DOA who were investigated for ophthalmology, neurology, and brain MRI. RESULTS: We identified 7 and 8 new heterozygous pathogenic variants in SPG7 and AFG3L2. Both genes encode for mitochondrial matricial AAA (m-AAA) proteases, initially involved in recessive hereditary spastic paraplegia type 7 (HSP7) and dominant spinocerebellar ataxia 28 (SCA28), respectively. Notably, variants in AFG3L2 that result in DOA are located in different domains to those reported in SCA28, which likely explains the lack of clinical overlap between these 2 phenotypic manifestations. In comparison, the SPG7 variants identified in DOA are interspersed among those responsible for HSP7 in which optic neuropathy has previously been reported. CONCLUSIONS: Our results position SPG7 and AFG3L2 as candidate genes to be screened in DOA and indicate that regulation of mitochondrial protein homeostasis and maturation by m-AAA proteases are crucial for the maintenance of optic nerve physiology

Genetic landscape of congenital insensitivity to pain and hereditary sensory and autonomic neuropathies

Congenital insensitivity to pain (CIP) and hereditary sensory and autonomic neuropathies (HSAN) are clinically and genetically heterogeneous disorders exclusively or predominantly affecting the sensory and autonomic neurons. Due to the rarity of the diseases and findings based mainly on single case reports or small case series, knowledge about these disorders is limited. Here, we describe the molecular workup of a large international cohort of CIP/HSAN patients including patients from normally under-represented countries. We identify 80 previously unreported pathogenic or likely pathogenic variants in a total of 73 families in the >20 known CIP/HSAN-associated genes. The data expand the spectrum of disease-relevant alterations in CIP/HSAN, including novel variants in previously rarely recognized entities such as ATL3-, FLVCR1- and NGF-associated neuropathies and previously under-recognized mutation types such as larger deletions. In silico predictions, heterologous expression studies, segregation analyses and metabolic tests helped to overcome limitations of current variant classification schemes that often fail to categorize a variant as disease-related or benign. The study sheds light on the genetic causes and disease-relevant changes within individual genes in CIP/HSAN. This is becoming increasingly important with emerging clinical trials investigating subtype or gene-specific treatment strategies

OxPhos Defects Cause Hypermetabolism and Reduce Lifespan in Cells and in Patients With Mitochondrial Diseases

Patients with primary mitochondrial oxidative phosphorylation (OxPhos) defects present with fatigue and multi-system disorders, are often lean, and die prematurely, but the mechanistic basis for this clinical picture remains unclear. By integrating data from 17 cohorts of patients with mitochondrial diseases (n = 690) we find evidence that these disorders increase resting energy expenditure, a state termed hypermetabolism. We examine this phenomenon longitudinally in patient-derived fibroblasts from multiple donors. Genetically or pharmacologically disrupting OxPhos approximately doubles cellular energy expenditure. This cell-autonomous state of hypermetabolism occurs despite near-normal OxPhos coupling efficiency, excluding uncoupling as a general mechanism. Instead, hypermetabolism is associated with mitochondrial DNA instability, activation of the integrated stress response (ISR), and increased extracellular secretion of age-related cytokines and metabokines including GDF15. In parallel, OxPhos defects accelerate telomere erosion and epigenetic aging per cell division, consistent with evidence that excess energy expenditure accelerates biological aging. To explore potential mechanisms for these effects, we generate a longitudinal RNASeq and DNA methylation resource dataset, which reveals conserved, energetically demanding, genome-wide recalibrations. Taken together, these findings highlight the need to understand how OxPhos defects influence the energetic cost of living, and the link between hypermetabolism and aging in cells and patients with mitochondrial diseases

Vaccine breakthrough hypoxemic COVID-19 pneumonia in patients with auto-Abs neutralizing type I IFNs

Life-threatening `breakthrough' cases of critical COVID-19 are attributed to poor or waning antibody response to the SARS- CoV-2 vaccine in individuals already at risk. Pre-existing autoantibodies (auto-Abs) neutralizing type I IFNs underlie at least 15% of critical COVID-19 pneumonia cases in unvaccinated individuals; however, their contribution to hypoxemic breakthrough cases in vaccinated people remains unknown. Here, we studied a cohort of 48 individuals ( age 20-86 years) who received 2 doses of an mRNA vaccine and developed a breakthrough infection with hypoxemic COVID-19 pneumonia 2 weeks to 4 months later. Antibody levels to the vaccine, neutralization of the virus, and auto- Abs to type I IFNs were measured in the plasma. Forty-two individuals had no known deficiency of B cell immunity and a normal antibody response to the vaccine. Among them, ten (24%) had auto-Abs neutralizing type I IFNs (aged 43-86 years). Eight of these ten patients had auto-Abs neutralizing both IFN-a2 and IFN-., while two neutralized IFN-omega only. No patient neutralized IFN-ss. Seven neutralized 10 ng/mL of type I IFNs, and three 100 pg/mL only. Seven patients neutralized SARS-CoV-2 D614G and the Delta variant (B.1.617.2) efficiently, while one patient neutralized Delta slightly less efficiently. Two of the three patients neutralizing only 100 pg/mL of type I IFNs neutralized both D61G and Delta less efficiently. Despite two mRNA vaccine inoculations and the presence of circulating antibodies capable of neutralizing SARS-CoV-2, auto-Abs neutralizing type I IFNs may underlie a significant proportion of hypoxemic COVID-19 pneumonia cases, highlighting the importance of this particularly vulnerable population

COVID-19 symptoms at hospital admission vary with age and sex: results from the ISARIC prospective multinational observational study

Background: The ISARIC prospective multinational observational study is the largest cohort of hospitalized patients with COVID-19. We present relationships of age, sex, and nationality to presenting symptoms. Methods: International, prospective observational study of 60 109 hospitalized symptomatic patients with laboratory-confirmed COVID-19 recruited from 43 countries between 30 January and 3 August 2020. Logistic regression was performed to evaluate relationships of age and sex to published COVID-19 case definitions and the most commonly reported symptoms. Results: ‘Typical’ symptoms of fever (69%), cough (68%) and shortness of breath (66%) were the most commonly reported. 92% of patients experienced at least one of these. Prevalence of typical symptoms was greatest in 30- to 60-year-olds (respectively 80, 79, 69%; at least one 95%). They were reported less frequently in children (≤ 18 years: 69, 48, 23; 85%), older adults (≥ 70 years: 61, 62, 65; 90%), and women (66, 66, 64; 90%; vs. men 71, 70, 67; 93%, each P < 0.001). The most common atypical presentations under 60 years of age were nausea and vomiting and abdominal pain, and over 60 years was confusion. Regression models showed significant differences in symptoms with sex, age and country. Interpretation: This international collaboration has allowed us to report reliable symptom data from the largest cohort of patients admitted to hospital with COVID-19. Adults over 60 and children admitted to hospital with COVID-19 are less likely to present with typical symptoms. Nausea and vomiting are common atypical presentations under 30 years. Confusion is a frequent atypical presentation of COVID-19 in adults over 60 years. Women are less likely to experience typical symptoms than men

Influence de l'instabilité microsatellite et de l'expression d'HSP100deltaE9 sur la survie et la réponse à la chimiothérapie à base de 5-FU dans les cancers colorectaux

L instabilité microsatellite (MSI) dans les cancers colorectaux (CCR), est considérée comme un facteur de bon pronostic et un marqueur de mauvaise réponse au 5-FU. Dans les tumeurs MSI, la présence d une délétion au niveau du microsatellite T17 de l intron 8 d HSP110 induit l expression d une protéine tronquée HSP110 E9. Son expression au détriment d HSP110 est présentée comme un marqueur prédictif de bonne réponse à la chimiothérapie et de bon pronostic. Nous avons évalué la taille du T17 dans le tissu sain et la tumeur, l expression d HSP110 E9, la réponse à la chimiothérapie et la survie dans deux populations de patients : 86 présentant un CCR métastatique (CCRm) traités par FOLFIRI-CETUX ; puis 120 présentant une tumeur MSI. Le T17 est peu polymorphique dans le tissu sain. Nous confirmons que la délétion du T7 est spécifique des tumeurs MSI, et que sa taille dépend du type de déficit du MMR. On observe que le niveau d expression d HSP110 E9 est fonction de la taille de la délétion. L analyse de la réponse et de la survie ne montre pas de différence en fonction du statut MSI dans les CCRm, ni en fonction du taux d HSP110 E9 dans les CCR MSI. Le phénotype MSI n apparaît pas comme étant un marqueur de bon pronostic, ni de bonne réponse à la chimiothérapie dans les CCRm. L expression d HSP110 E9 est spécifique des tumeurs MSI, même à un stade tumoral avancé. Son niveau d expression semble dépendre du type de déficit du MMR, mais ne semble pas améliorer le pronostic des patients. Des études complémentaires fondamentales et cliniques sont donc nécessaires pour préciser l action d HSP110 E9 et son influence sur la survie et la réponse à la chimiothérapie.Microsatellite instability (MSI) in colorectal cancer (CRC) is considered a good prognosis factor, it is however a bad predictive marker to 5-FU-based chemotherapy. Deletions in the microsatellite T17 located in intron 8 of HSP110 lead to a truncated isoform of the protein. The expression of the truncated protein constitutes a major determinant for both prognosis and treatment response in CRC. For two series of subjects, 86 with metastatic CRC (mCRC) and 120 with MSI tumors, we studied the length of the T17 in healthy tissue and tumor, expression rate of HSP110 E9, response to chemotherapy and survival rate. T17 is weakly polymorphic in healthy tissue. We confirmed that deletion is specific to MSI tumors, and that the deletion length depends on MMR system defect. We noticed that the HSP110 E9 expression rate is conditioned by T17 length deletion. Finally, we observed that response to chemotherapy and survival rate analysis did not show any differences neither in terms of MSI status in mCRC nor in terms of HSP110 E9 expression rate in MSI tumors. MSI does not appear to be a good prognosis factor or a bad response marker to 5-FU-based chemotherapy in mCRC. HSP110 truncated isoform is only expressed in MSI tumors, even in advanced CRC. HSP110 E9 expression rate seems to be influenced by the MMR defect, but does not seem to improved patient s prognosis. Several fundamental and clinical studies have to be conducted to determine the cellular actions of HSP110 E9 and its influence on response to chemotherapy and survival rate.ANGERS-BU Médecine-Pharmacie (490072105) / SudocSudocFranceF

Aggressive, early resistant and relapsed mantle cell lymphoma distinct extrinsic microenvironment highlighted by transcriptome analysis

Abstract Immunotherapy strategies relying on innate or adaptive immune components are increasingly used in onco‐haematology. However, little is known about the infiltrated lymph nodes (LN) or bone marrow (BM) landscape of mantle cell lymphoma (MCL). The original transcriptomic approach of reverse transcriptase multiplex ligation‐dependent probe amplification (RT‐MLPA) was applied here to explore the expression of 24 genes of interest in MCL at diagnosis (21 LN and 15 BM) or relapse (18 LN). This allowed us to identify that at baseline, samples from MCL patients with an aggressive morphology (i.e. blastoid or pleomorphic) or a high proliferative profile, displayed significantly higher monocyte/macrophage‐associated transcripts (CD14 and CD163) in LN and BM. Regarding T‐cells, aggressive MCL forms had significantly lower amounts of LN CD3E transcripts, yet an increased expression of cytotoxic markers in LN (CD8) and BM (CD94). A very high‐risk group with early treatment resistance displayed, at diagnosis, high proliferation (KI67) and high macrophages and cytotoxic transcript levels. Post‐immunochemotherapy relapsed samples revealed lower levels of T‐ and natural killer‐cells markers, while monocyte/macrophage markers remained similar to diagnosis. This study suggests that rapid analysis of MCL microenvironment transcriptome signatures by RT‐MLPA could allow for an early distinction of patient subgroups candidates for adapted treatment strategies