Human aging is heterogeneous and can be explored through development, physiological aging, and premature accelerated aging. We established human induced pluripotent stem cell (iPSC) models derived from neonatal fibroblasts, peripheral blood mononuclear cells from a healthy 60-year-old donor, and cells from a Hutchinson-Gilford progeria syndrome patient. All lines were generated using Sendai virus reprogramming, validated for pluripotency, tri-lineage differentiation, and genomic stability. This collection provides a unique comparative platform to dissect normal and pathological aging, enabling analyses of youthful resilience, progressive age-related alterations, and premature progeroid hallmarks. Beyond technical validation, these models offer a conceptual framework to identify longevity biomarkers

Chromoanasynthesis is a distinct entity within the complex rearrangement phenomena grouped under the name chromoanagenesis. The formation of chromoanasynthesis is linked to defective or interrupted DNA replication, due to various replicative stress factors. Two alternative replication-based mechanisms (FoSTeS and MMBIR) can then act to induce a series of microhomology-dependent templates and switching events before the completion of DNA synthesis on the original template. These mechanisms lead to the formation of clusters of complex chromosome rearrangements, generally characterized by duplications and triplications. Chromoanasynthesis operates in the germline and during early embryonic development through the formation of micronuclei or chromatid bridges during mitotic divisions. The phenomenon has mainly been described in congenital diseases, but more recently cases of chromoanagenesis have been described in cancers.

Chromoplexy is a phenomenon of complex genome rearrangement, occurring during a single cell event and characterized by the formation of chain rearrangements affecting multiple chromosomes. Unlike other genomic rearrangements such as chromothripsis, which involves a single chromosome, chromoplexy affects several chromosomes at once, creating patterns of complex, balanced translocations, and leading to the formation of fusion genes and the simultaneous disruption of several genes. Chromoplexy was first identified in prostate cancers, but it is now observed in various cancers where gene fusions take place. The precise mechanisms behind chromoplexy remain under investigation. The occurrence of these rearrangements follows multiple double-stranded breaks that appear to occur in certain regions or during particular genome configurations (open chromatin, active transcription area), and which lead to an intricate series of inter- and intra-chromosomal translocations and deletions without significant alterations in the number of copies. Although chromoplexy is considered a very early event in oncogenesis, the phenomenon can be repeated and can constitute a mechanism of clonal tumor progression. The occurrence of chromoplexy supports the equilibrium model punctuated by tumor evolution, characterized by periods of relative stability punctuated by sudden and rapid periods of radical genomic changes.

The chromothripsis phenomenon is the first type of chaotic and complex rearrangements discovered since 2011 and now grouped together under the name of chromoanagenesis.Its occurrence has been documented in cancers, congenital diseases as well as in healthy individuals. The phenomenon has also been observed in many animal and plant species, suggesting that it is a mechanism of rapid and deep genome reorganization widely used in response to various cellular stresses.The determination of specific molecular characteristics has allowed chromothripsis to be better characterized and to be distinguished from other types of complex rearrangements. Various non-exclusive exogenous or cellular mechanisms capable of generating chromothripsis have been evoked. Recent experimental models have highlighted three major processes that can generate a cascade of cellular events leading to chromothripsis. These mechanisms are the formation of micronuclei integrating isolated chromosomal material, the occurrence of chromatin bridges around chromosomal material resulting from telomeric fusions, and the abortive apoptosis. In all cases, the cellular and molecular mechanisms of fragmentation, repair, and transmission of damaged chromosomal material are consistent with the characteristics of complex chromosomal rearrangements associated with chromothripsis.Undoubtedly, chromothripsis is one of the most unexpected biological discoveries to emerge from high-resolution genome analysis. As a mechanism for rapid genome modifications in germ lines and early development, chromothripsis supports the concept of macroevolution and can be regarded as a credible mechanism for speciation and organismal evolution.

Chromoplexy is a massive and complex rearrangement mechanism, occurring during a single cell event and characterized by the formation of chain rearrangements affecting multiple chromosomes. This can lead to the formation of fusion genes and the simultaneous disruption of several genes. Chromoplexy was first identified in prostate cancers, but it is now being observed in various cancers where gene fusions take place. The mechanisms underlying chromoplexy formation are still unclear. The occurrence of these rearrangements follows multiple double-stranded breaks that appear to occur in particular genome configurations (open chromatin, active transcription area, etc.) and which lead to intricate series of inter- and intra-chromosomal translocations and deletions without significant alterations in the number of copies. Although chromoplexy is considered a very early event in oncogenesis, the phenomenon can be repeated and constitute a mechanism of clonal tumor progression. The occurrence of chromoplexy supports the equilibrium model punctuated by tumor evolution, characterized by periods of relative stability punctuated by sudden and rapid periods of radical genomic changes.

Next-generation sequencing (NGS) has revolutionized clinical genomics, enhancing structural variant (SV) analysis, especially found in chromothripsis. This guide focuses on key steps of SV interpretation using IGV, detailing methods for assessing SV veracity and distinguishing true SVs from artifacts. This resource aims to aid clinicians and researchers in effectively interpreting NGS-derived SV calling data to improve diagnostic accuracy and patient care.

Chromothripsis and chromoanasynthesis have been described as new complex chromosomal rearrangements and are grouped under the term chromoanagenesis.Various mechanisms of formation of these rearrangements have been identified and reproduced experimentally, including the sequestration of chromosomes in micronuclei, the premature condensation of chromosomes, or abortive apoptosis. All these phenomena can occur during human spermatogenesis, particularly abortive apoptosis which leads to DNA fragmentation in spermatozoa.Given the paternal origin of most cases of chromothripsis and chromoanasynthesis, their presence is researched in human sperm samples with high DNA fragmentation rate.Magnetic-activated cell sorting (MACS) is performed on sperm samples to separate spermatozoa according to their fragmentation rate. Sorting efficiency is assessed by using the Annexin V-FITC staining and the fragmentation rate is assessed by using TUNEL (Terminal deoxynucleotidyl transferase-mediated dUTP Nick-End Labeling) assay.Pools of spermatozoa with a high DNA fragmentation rate are thus constituted, on which genomic analysis can be performed to identify complex rearrangements, such as chromothripsis and chromoanasynthesis.

Introduction et objectifs L’usage des technologies de séquençage de nouvelle génération prend une part de plus en plus importante dans le diagnostic prénatal génétique des anomalies du développement fœtal et tend à effacer l’intérêt du caryotype et de la cytogénétique. Bien que ces technologies soient capables d’identifier toutes les variations du génome du fœtus, le regard du cytogénéticien reste indispensable en matière de mécanisme chromosomique. Matériels, patients et méthodes Au décours la première grossesse de Mme H., l’échographie du 2e trimestre montre une association polymalformative du fœtus et un retard de croissance intra-utérin. Il proposé à Mme H. de participer à une étude en aveugle du séquençage du génome entier en parallèle des explorations de cytogénétique en soins courants : analyse chromosomique par puce à ADN (ACPA) et caryotype standard. Mme H. donne son consentement à ce double parcours à visée diagnostique et une amniocentèse est pratiquée. Résultats L’ACPA montre 2 variations du nombre de copie (CNV) aux extrémités des chromosomes 2 et 9, orientant le cytogénéticien vers l’hypothèse la plus probable de la présence d’un dérivé chromosomique issu d’une translocation réciproque. L’étude des CNV identifiés par séquençage du génome, obtenu en quelques jours, aboutit à la même hypothèse. Après culture du liquide amniotique, le caryotype montre un mécanisme plus complexe, infirmant la première hypothèse et orientant vers la présence d’un recombinant chromosomique d’une insertion. Un second examen plus poussé du génome, analysant les variants structuraux (SV) confirme cette dernière hypothèse et précise le parent porteur de l’insertion équilibrée, lui-même confirmé par le caryotype et la FISH des parents. Conclusions Toutes les informations génétiques et cytogénétiques du fœtus étaient présentes dès la fin du séquençage du génome entier. Cependant, il a fallu le regard du cytogénéticien pour reconstituer les remaniements et identifier les points de cassure à partir des données de séquences.

The phenomena of chromoanagenesis have profoundly changed our conceptions regarding the genesis and etiology of complex rearrangements. These chaotic genomic events have also led to a better understanding of the mechanisms underlying the maintenance and fluctuations of genomic stability. Chromoanagenesis phenomena are now recognized as important drivers of karyotypic evolution, both in cancer and in developmental disorders. They question the conventional view of gradual evolution, supporting the role of genomic chaos in rapid evolutionary transitions.The advent of new genome sequencing technologies and their use in combination with powerful bioinformatics tools or single-cell analysis techniques have led to a better understanding of the complexity and heterogeneity of chromoanagenesis and to the description of new forms of complex and chaotic genomic rearrangements.

Introduction Le locus 8p23.1 est le siège de nombreux remaniements chromosomiques [1] impliquant les régions REPD et REPP, pouvant associer des duplications, des délétions, des inversions et des zones de disomie uniparentale (DUP) responsables de phénotypes variables. Patient Le patient examiné est un homme de 60ans, présentant une déficience intellectuelle sévère à début précoce, une cataracte, des troubles de la réfraction et quelques particularités morphologiques.L’analyse chromosomique par puce à ADN montre un remaniement du locus 8p23.1 associant une délétion interstitielle distale de 2,6 Mb à une duplication interstitielle proximale de 0,8 Mb faisant suspecter un mécanisme de remaniement chromosomique complexe avec présence d’une DUP de la région terminale [2]. Ces deux déséquilibres ne permettent pas à eux seuls d’expliquer l’intégralité du tableau clinique, indiquant alors un séquençage short-reads du génome. Résultats Le séquençage a précisé la présence d’une duplication en miroir avec insertion du segment dupliqué en lieu et place de la délétion. Les deux fragments dupliqués sont séparés par une portion non dupliquée et non inversée de génome, mais la portion terminale ne présente pas de DUP. Ces résultats font suspecter un mécanisme de recombinaison homologue non allélique faisant suite à une cassure double brin de l’ADN [2], [3]. L’analyse a également montré deux mutations ponctuelles pathogènes dans les gènes SMARCD1 et NR2F1 permettant de compléter le diagnostic. Discussion et conclusion Bien que les techniques actuelles de séquençage génomique short-reads soient utiles pour élucider les remaniements chromosomiques, l’interprétation globale des données de structure du génome peut être longue et fastidieuse. Elles demeurent insuffisantes pour localiser les points de cassure exacts dans les zones répétées du génome, en particulier dans les cas de duplications segmentaires. Les nouvelles technologies de séquençage long-reads et de cartographie optique du génome permettront d’améliorer et de simplifier le diagnostic chromosomique.

Single-gene copy number variants (CNVs) limited to placenta although rarely identified may have clinical implications. We describe a pregnant woman referred for chorionic villus sampling due to increased fetal nuchal translucency. Incident intragenic deletion of Duchenne muscular dystrophy (DMD) gene, affecting exons 56 and 57, was identified in a male fetus in ~23-30% of placental cells by chromosomal microarray and confirmed using multiplex ligation-dependent probe amplification (MLPA). Rapid aneuploidy testing showed normal results and the deletion was not detected in the mother. Subsequent analyses on amniotic cells yielded a normal DMD gene result, corroborating the confined placental nature of the mosaicism. Hence, this report emphasizes the importance of conducting amniocentesis following detection of mosaicism for single gene CNVs on chorionic villi, in order to preclude confined placental mosaicism (CPM). As far as we know, this report marks only the second documented situation of CPM involving an intragenic DMD deletion.

The factors influencing mother-to-child cell trafficking and persistence over children’s lives have yet to be established. The quantification of maternal microchimerism was previously reported through HLA-based approaches, which introduced bias regarding the tolerogenic environment. We aimed to identify cells of maternal origin irrespective of the HLA repertoire and to ascertain the determinants of microchimeric cells. This case–control study enrolled 40 male infants attending pediatric surgery from January 2022 to October 2022. Female cells were quantified in infants’ tonsil tissue by using cytogenetic fluorescent in situ hybridization (FISH) coupled with optimized automated microscopy. Out of the 40 infants, half (47.4%) had been breastfed for more than one month, a quarter for less a month, and 10 children (26.3%) were never breastfed. XX cells were observed in male tonsils in two-thirds of participants at a median density of 5 cells per 100,000 cells. In univariate analyses, child age was negatively associated with a high female cell density. In exploratory multivariate analyses, previous breastfeeding is a likely determinant of the persistence of these cells in the host, as well as the rank among siblings. Part of the benefit of breastmilk for child health may therefore be driven by breastfeeding-related microchimerism.

La recherche d'évènements cellulaires rares dans un échantillon biologique présente de nombreux intérêts médicaux : dépistage de cellules fœtales dans le sang maternel ou autres tissus, évaluation du chimérisme après allogreffe, recherche de cellules pathologiques, … La rareté de ces cellules est un frein à l'utilisation de méthodes innovantes telles que le séquençage nouvelle génération et nécessite le développement de stratégies alternatives. À l'aide de la plateforme de microscopie automatisée Metafer (MetaSystem®) disponible dans notre laboratoire, nous mettons au point un système de détection d'évènements rares cytologiques ou cytogénétiques sur échantillons humains. Différentes matrices biologiques sont testées : cellules sanguines circulantes, biopsies d'organes lymphoïdes secondaires, frottis cervicovaginaux. Un étalon interne positif est ajouté à chaque type de prélèvement dont la quantité est contrôlée par comptage au microscope optique. Ces étalons internes sont sélectionnés en fonction de leurs propriétés cytologiques (présence d'un antigène de surface) et/ou cytogénétiques (sexe chromosomique). Les paramètres du logiciel de détection (« classifiers ») sont adaptés aux caractéristiques des cellules à identifier sur plusieurs cycles de détection/analyse. Les premiers tests réalisés sur ces 3 matrices révèlent des caractéristiques analytiques satisfaisantes : limite de détection, corrélation entre le nombre d'évènements détectés et le nombre attendu, rapidité d'acquisition/interprétation. À ce jour, ces tests nécessitent d'être validés sur des séries plus importantes. Cette approche automatisée par microscopie est un outil fiable et robuste. Elle permettra de détecter des mosaïcismes ou des chimérismes très faibles qui échappent aux technologies de nouvelle génération.

Titin protein is responsible for muscle elasticity. The TTN gene, composed of 364 exons, is subjected to extensive alternative splicing and leads to different isoforms expressed in skeletal and cardiac muscle. Variants in TTN are responsible for myopathies with a wide phenotypic spectrum and autosomal dominant or recessive transmission. The I-band coding domain, highly subject to alternative splicing, contains a three-zone block of repeated sequences with 99% homology. Sequencing and localization of variants in these areas are complex when using short-reads sequencing, a second-generation sequencing technique. We have implemented a protocol based on the third-generation sequencing technology (long-reads sequencing). This new method allows us to localize variants in these repeated areas to improve the diagnosis of TTN-related myopathies and offer the analysis of relatives in postnatal or in prenatal screening.

Designated under the name of chromoanagenesis, the phenomena of chromothripsis, chromanasynthesis and chromoplexy constitute new types of complex rearrangements, including many genomic alterations localized on a few chromosomal regions, and whose discovery over the last decade has changed our perception about the formation of chromosomal abnormalities and their etiology. Although exhibiting specific features, these new catastrophic mechanisms generally occur within a single cell cycle and their emergence is closely linked to genomic instability. Various non-exclusive exogenous or cellular mechanisms capable of generating chromoanagenesis have been evoked. However, recent experimental data shed light on 2 major processes, which following a defect in the mitotic segregation of chromosomes, can generate a cascade of cellular events leading to chromoanagenesis. These mechanisms are the formation of micronuclei integrating isolated chromosomal material, and the occurrence of chromatin bridges around chromosomal material resulting from telomeric fusions. In both cases, the cellular and molecular mechanisms of fragmentation, repair and transmission of damaged chromosomal material are consistent with the features of chromoanagenesis-related complex chromosomal rearrangements. In this review, we introduce each type of chromoanagenesis, and describe the experimental models that have allowed to validate the existence of chromoanagenesis events and to better understand their cellular mechanisms of formation and transmission, as well as their impact on the stability and the plasticity of the genome.

Les avancées technologiques permises par l'analyse de l'ADN libre circulant ont profondément modifié la prise en charge des patientes enceintes. Les premières grandes évolutions ont concerné le dépistage du sexe fœtal et le génotypage Rhésus du fœtus. Ces dernières années, c'est le dépistage de la trisomie 21 qui a été révolutionnée, avec la possibilité de dépister un très grand nombre de patientes par séquençage nouvelle génération, sur une simple prise de sang. Rapidement, cette technologie s'est appliquée aux autres principales aneuploïdies des autosomes, comme la trisomie 13 et la trisomie 18. Il est maintenant parfaitement possible de quantifier tous les chromosomes fœtaux à partir d'une prise de sang. La vision pangénomique, ou plutôt panchromosomique, qu'offre le DPNI s'accompagne avec la découverte de données non initialement désirées appelées « données incidentes ». Les cytogénéticiens ont bien sûr l'habitude de traiter ce type de données par leur expertise lors de l'analyse chromosomique par puce à ADN (ACPA) ou le caryotype et savent les interpréter dans le contexte pathologique qui mène à un geste invasif anténatal, comme la présence de signes d'appel échographiques. Cependant, le contexte du dépistage de la trisomie 21 est radicalement différent puisqu'il concerne l'ensemble de grossesses. La probabilité bien que faible mais non négligeable d'obtenir ce type de données pour un fœtus sans signe d'appel échographique a poussé les pays où le DPNI panchromosomique est proposé systématiquement à toutes les femmes enceintes et en première intention, à établir des recommandations nationales. C'est le cas de la Belgique qui, au travers de la BeSHG (Belgian Society of Human Genetics), propose de classer ces données incidentes en fonction de leur caractères « actionnables », c'est-à-dire de leur conséquence sur la prise en charge du fœtus et/ou de la patiente. Au travers d'exemples concrets nous balayerons les différentes catégories de données incidentes proposées par les recommandations Belges. Nous discuterons également de la possible application de ces recommandations dans le cadre du dépistage de la trisomie 21 fœtale en France.

Designated under the name of chromoanagenesis, the phenomena of chromothripsis, chromoanasynthesis, and chromoplexy constitute new types of complex rearrangements, including many genomic alterations localized on a few chromosomal regions, and whose discovery over the last decade has changed our perception about the formation of chromosomal abnormalities and their etiology. Their occurrence has been documented both in cancers and in subjects carrying congenital diseases, or even in healthy individuals. Although exhibiting specific features, these new catastrophic mechanisms generally occur within a single cell cycle and their emergence is closely linked to genomic instability. Various nonexclusive exogenous or cellular mechanisms capable of generating chromoanagenesis have been evoked. However, recent experimental data shed light on two major processes, which following a defect in the mitotic segregation of chromosomes can generate a cascade of cellular events leading to chromoanagenesis. These mechanisms are the formation of micronuclei integrating isolated chromosomal material and the occurrence of chromatin bridges around chromosomal material resulting from telomeric fusions. In both cases, the cellular and molecular mechanisms of fragmentation, repair, and transmission of damaged chromosomal material are consistent with the features of chromoanagenesis-related complex chromosomal rearrangements. In this review, we introduce each type of chromoanagenesis and describe the experimental cytogenomic models that have allowed to validate the existence of chromoanagenesis events and to better understand their cellular mechanisms of formation and transmission, as well as their impact on the stability and the plasticity of the genome.

To understand the role of the extensive senescence-associated 3D genome reorganization, we generated genome-wide chromatin interaction maps, epigenome, replication-timing, whole-genome bisulfite sequencing, and gene expression profiles from cells entering replicative senescence (RS) or upon oncogene-induced senescence (OIS). We identify senescence-associated heterochromatin domains (SAHDs). Differential intraversus inter-SAHD interactions lead to the formation of senescence-associated heterochromatin foci (SAHFs) in OIS but not in RS. This OIS-specific configuration brings active genes located in genomic regions adjacent to SAHDs in close spatial proximity and favors their expression, We also identify DNMT1 as a factor that induces SAHFs by promoting HMGA2 expression. Upon DNMT1 depletion, OIS cells transition to a 3D genome conformation akin to that of cells in replicative senescence. These data show how multi-omics and imaging can identify critical features of RS and OIS and discover determinants of acute senescence and SAHF formation.