Oxidative stress and mitochondrial dynamics malfunction are linked in Pelizaeus-Merzbacher disease

Pelizaeus-Merzbacher disease (PMD) is a fatal hypomyelinating disorder characterized by early impairment of motor development, nystagmus, choreoathetotic movements, ataxia and progressive spasticity. PMD is caused by variations in the proteolipid protein gene PLP1, which encodes the two major myelin proteins of the central nervous system, PLP and its spliced isoform DM20, in oligodendrocytes. Large duplications including the entire PLP1 gene are the most frequent causative mutation leading to the classical form of PMD. The Plp1 overexpressing mouse model (PLP-tg66/66 ) develops a phenotype very similar to human PMD, with early and severe motor dysfunction and a dramatic decrease in lifespan. The sequence of cellular events that cause neurodegeneration and ultimately death is poorly understood. In this work, we analyzed patient-derived fibroblasts and spinal cords of the PLP-tg66/66 mouse model, and identified redox imbalance, with altered antioxidant defense and oxidative damage to several enzymes involved in ATP production, such as glycolytic enzymes, creatine kinase and mitochondrial proteins from the Krebs cycle and oxidative phosphorylation. We also evidenced malfunction of the mitochondria compartment with increased ROS production and depolarization in PMD patient's fibroblasts, which was prevented by the antioxidant N-acetyl-cysteine. Finally, we uncovered an impairment of mitochondrial dynamics in patient's fibroblasts which may help explain the ultrastructural abnormalities of mitochondria morphology detected in spinal cords from PLP-tg66/66 mice. Altogether, these results underscore the link between redox and metabolic homeostasis in myelin diseases, provide insight into the pathophysiology of PMD, and may bear implications for tailored pharmacological intervention

A Solve-RD ClinVar-based reanalysis of 1522 index cases from ERN-ITHACA reveals common pitfalls and misinterpretations in exome sequencing

Purpose Within the Solve-RD project (https://solve-rd.eu/), the European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies aimed to investigate whether a reanalysis of exomes from unsolved cases based on ClinVar annotations could establish additional diagnoses. We present the results of the “ClinVar low-hanging fruit” reanalysis, reasons for the failure of previous analyses, and lessons learned. Methods Data from the first 3576 exomes (1522 probands and 2054 relatives) collected from European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies was reanalyzed by the Solve-RD consortium by evaluating for the presence of single-nucleotide variant, and small insertions and deletions already reported as (likely) pathogenic in ClinVar. Variants were filtered according to frequency, genotype, and mode of inheritance and reinterpreted. Results We identified causal variants in 59 cases (3.9%), 50 of them also raised by other approaches and 9 leading to new diagnoses, highlighting interpretation challenges: variants in genes not known to be involved in human disease at the time of the first analysis, misleading genotypes, or variants undetected by local pipelines (variants in off-target regions, low quality filters, low allelic balance, or high frequency). Conclusion The “ClinVar low-hanging fruit” analysis represents an effective, fast, and easy approach to recover causal variants from exome sequencing data, herewith contributing to the reduction of the diagnostic deadlock

Recherche de nouvelles anomalies en cause dans les leucodystrophies d'origine indéterminée

Les maladies génétiques qui touchent la myéline du système nerveux central (SNC), ou leucodystrophies, sont responsables de tableaux cliniques extrêmement variés. Dans ce travail, nous nous sommes intéressées aux formes avec paucité de la myéline, ou hypomyélinisation, impliquant le gène PLP1 (Xq23) codant pour les protéines majeures constitutives de la myéline du SNC : PLP et DM20. Les "PLP-pathies" se traduisent par un déficit moteur variable selon les différentes mutations allant des formes sévères de la maladie de Pelizaeus-Merzbacher (PMD) à des formes modérées et tardives de paraplégie spastique (SPG2). Cependant, pour une grande partie des patients souffrant de PMD et de SPG2, l'implication du gène PLP1 n'a pas pu être montrée suggérant soit une hétérogénéité génétique soit des mécanismes mutationnels alternatifs de PLP1. Cette cohorte de patients, dénommée PMLD(Pelizaeus Merzbacheer like disease), a fait l'objet de cette étude. Nous avons, d'une part testé plusieurs gènes candidats dont le gène MBP, codant pour la 2ème protéine constitutive majeure de la myéline du SNC, le gène GPM6B et le gène OLIG2 codant pour un facteur de transcription, puis d'autre part recherché des méchanismes mutationnels alternatifs de PLP1 en recherchant des remaniements intragéniques et en explorant les régions non codantes et le niveau d'expression de PLP1 par l'étude des transcrits PLP et DM20 à partir des fibroblastes. Si nous avons pu montrer que les gènes candidats testés ainsi que les remaniements intragéniques de PLP1 ne sont pas majoritairement impliqués dans ce groupe de leucodystrophie, l'étude des transcrits a montré, en revanche, l'intérêt de l'utilisation des fibroblastes comme modèle d'étude de l'épissage et du niveau d'expression de PLP1 et a permis de suggérer qu'une anomalie de régulation de l'expression de PLP1 pourrait être impliquée chez certains patients PMLD.Genetic diseases affecting primarily the myelin of the central nervous system (CNS), or leucodystrophies, include a large variety of phenotypes. The aims of this work lead with hypomyelinating forms, displaying a defect in myelin production and implicating the PLP1 gene which encodes the major proteins of CNS myelin, i.e. PLP and DM20. "PLP-pathies" are characterized by motor development impairment and encompass a wide continuum spectrum extending from severe forms of Pelizaeus-Merzbacher disease (PMD) to relatively mild late onset spastic paraplegia (SPG2). However, a large proportion of patients presenting with a PMD or a SPG2 phenotype remains without identified PLP1 mutations suggesting either genetic heterogeneity or PLP1 gene alternative mutational mechanisms. This cohort of patients, called PMD like (PMLD), has been studied in this work to test both hypotheses. First, different candidate genes have been tested, including MBP which encodes the second major proteins of CNS myelin; GPM6B, which present close similarities with DM20 and OLIG2 which encodes a transcription factor oligodendrocyte specific. Secondly, the existence of PLP1 alternative mutational mechanisms has been evaluated by looking for small intragenic rearrangements, and for qualitative and quantitative abnormalities analyzing PLP and DM20 transcripts from patients' fibroblasts. Obtained results have excluded the implication of the tested candidate genes as well as PLP1 small intragenic rearrangements in the aetiology of PMLD. On the other hand, qualitative and quantitative analysis of PLP/DM20 transcripts from fibroblasts have demonstrated their usefulness and suggested that a PLP1 gene expression dysregulation could be involved in a subset of PMLD patients.CLERMONT FD-BCIU-Santé (631132104) / SudocSudocFranceF

Cohorte française de 41 patients porteurs d’une délétion 2q37

International audienceLe locus 2q37 est l’une des régions subtélomériques les plus fréquemment délétées, pouvant être à l’origine du syndrome microdélétionnel 2q37, aussi appelé syndrome d’Ostéodystrophie Héréditaire d’Albright-like (AHO-like) ou syndrome retard mental-brachydactylie (BDMR) (MIM 60043), d’expression clinique variable. Suite à un appel à collaboration nationale, 41 patients porteurs d’une délétion 2q37 isolée ont été recensés. Tous les diagnostics ont été posés par l’analyse chromosomique sur puces à ADN, et confirmés par FISH avec une sonde locus-spécifique 2q37. Les délétions sont de taille variable, de 14kb intragénique DIS3L2 à 9.6 Mb. La majorité des cas est non héritée, de probable survenue de novo. Cette cohorte, pédiatrique et adulte, permet de confirmer la variabilité phénotypique et d’affiner le phénotype post-natal (1 seul cas prénatal). Les deux signes principaux mais inconstants sont les difficultés légères à modérées des apprentissages associées à des troubles comportementaux notamment des difficultés attentionnelles, et la brachydactylie. La morphologie faciale typique précédemment rapportée est fréquente également dans notre cohorte. L’obésité (6/26), le surpoids (3/26), la petite taille (2/29) sont absents dans plus de 70% des cas. L’épilepsie est décrite dans 15% des cas. Les malformations sont le plus souvent cardiaques et rénales, de bon pronostic. D’autres particularités cliniques ont été soulignées (notamment malformations cérébrales non spécifiques, troubles du transit, trouble du sommeil, troubles squelettiques et hyperlaxité). Il s’agit de la plus grosse cohorte de patients non publiés (28/41) décrite à ce jour

OXIDATIVE STRESS AND MITOCHONDRIAL DYNAMICS MALFUNCTION ARE LINKED IN PELIZAEUS-MERZBACHER DISEASE

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Familial transmission of chromoanagenesis leads to unpredictable unbalanced rearrangements through meiotic recombination

Abstract Chromoanagenesis is a cellular mechanism that leads to complex chromosomal rearrangements (CCR) during a single catastrophic event. It may result in loss and/or gain of genetic material and may be responsible for various phenotypes. These rearrangements are usually sporadic. However, some familial cases have been reported. Here, we studied six families in whom an asymptomatic or paucisymptomatic parent transmitted a CCR to its offspring in an unbalanced manner. The rearrangements were characterized by karyotyping, fluorescent in situ hybridization, chromosomal microarray (CMA) and/or whole genome sequencing (WGS) in the carrier parents and offspring. We then hypothesized meiosis‐pairing figures between normal and abnormal parental chromosomes that may have led to the formation of new unbalanced rearrangements through meiotic recombination. Our work indicates that chromoanagenesis might be associated with a normal phenotype and normal fertility, even in males, and that WGS may be the only way to identify these events when there is no imbalance. Subsequently, the CCR can be transmitted to the next generation in an unbalanced and unpredictable manner following meiotic recombination. Thereby, prenatal diagnosis using CMA should be proposed to these families to detect any pathogenic imbalances in the offspring

10q26 deletion syndrome: a French cohort study

International audience10q26 deletion syndrome (OMIM #609625) is a rare autosomal dominant genetic disorder with about 100 patients reported. Most cases are sporadic. Global development delay, short stature, microcephaly and typical facial appearance with triangular face, large forehead, low-set malformed ears, hypertelorism, prominent nose and a thin vermilion of the upper lip constitute the main clinical features. The clinical spectrum is very heterogeneous and neurobehavioral manifestations, deafness, limb malformations, cardiac and urogenital abnormalities can be associated. Thus, patients with 10q26 chromosomal deletion need multidisciplinary management strategies from birth. One of the main reasons for this heterogeneity is the variety of 10qter region chromosomal deletions summarized into the “10q26 deletion syndrome”. Various studies proposed critical regions to explain the main phenotype (Yatzenko et al., 2009; Choucair et al., 2015; Lin S et al., 2016) or more specific features (Vera-Carbonell et al., 2015; Choucair et al., 2015). In addition, these studies proposed about 20 genes of interest such as DOCK1 and FGFR2 to explain the different clinical features observed. We report a French ACLF cohort of 35 patients from 9 centers presenting 10q26 complete or partial deletions (size: 64kb to 12.5Mb), complex chromosomal rearrangement and derivative chromosomes diagnosed using DNA-array, to bring a further insight of the genotype/phenotype correlation

Episignatures in practice: independent evaluation of published episignatures for the molecular diagnostics of ten neurodevelopmental disorders

Variants of uncertain significance (VUS) are a significant issue for the molecular diagnosis of rare diseases. The publication of episignatures as effective biomarkers of certain Mendelian neurodevelopmental disorders has raised hopes to help classify VUS. However, prediction abilities of most published episignatures have not been independently investigated yet, which is a prerequisite for an informed and rigorous use in a diagnostic setting. We generated DNA methylation data from 101 carriers of (likely) pathogenic variants in ten different genes, 57 VUS carriers, and 25 healthy controls. Combining published episignature information and new validation data with a k-nearest-neighbour classifier within a leave-one-out scheme, we provide unbiased specificity and sensitivity estimates for each of the signatures. Our procedure reached 100% specificity, but the sensitivities unexpectedly spanned a very large spectrum. While ATRX, DNMT3A, KMT2D , and NSD1 signatures displayed a 100% sensitivity, CREBBP-RSTS and one of the CHD8 signatures reached <40% sensitivity on our dataset. Remaining Cornelia de Lange syndrome, KMT2A , KDM5C and CHD7 signatures reached 70–100% sensitivity at best with unstable performances, suffering from heterogeneous methylation profiles among cases and rare discordant samples. Our results call for cautiousness and demonstrate that episignatures do not perform equally well. Some signatures are ready for confident use in a diagnostic setting. Yet, it is imperative to characterise the actual validity perimeter and interpretation of each episignature with the help of larger validation sample sizes and in a broader set of episignatures

Classification of 101 BRCA1 and BRCA2 variants of uncertain significance by cosegregation study: A powerful approach

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Classification of 101 BRCA1 and BRCA2 variants of uncertain significance by cosegregation study: A powerful approach

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