Les PNA (peptide nucleic acids) : Des sondes high-tech pour l’analyse génétique et cytogénétique moléculaire

Les PNA (peptide nucleic acids) sont des molécules de synthèse, analogues des acides nucléiques, dans lesquelles le squelette phosphodiester a été remplacé par une chaîne pseudo-peptidique sur laquelle viennent se fixer les bases puriques et pyrimidiques. Du fait de cette structure, les molécules de PNA ne sont pas chargées et s’apparient aux séquences d’acides nucléiques avec une remarquable affinité et une très forte spécificité. De plus, les PNA présentent une remarquable stabilité et une excellente résistance aux protéases et aux nucléases. Depuis leur mise au point en 1991, les PNA ont été incorporées dans de nombreux protocoles d’études et de diagnostics génétiques pour la recherche de mutations, la coupure dirigée de l’ADN ou la thérapie génique. L’élaboration récente de sondes PNA pour l’identification in situ des chromosomes humains a permis d’adapter avec succès cette nouvelle technologie à l’analyse cytogénétique humaine.The peptide nucleic acids (PNAs) constitute a remarkable new class of synthetic nucleic acids analogs, in which the sugar phosphate backbone is replaced by repeating N-(2-aminoethyl) glycine units linked by amine bonds and to which the nucleobases are fixed. This structure gives to PNAs the capacity to hybridize with high affinity and specificity to complementary RNA and DNA sequences, and a great resistance to nucleases and proteinases. Originally conceived as ligands for the study of double stranded DNA, the unique physico-chemical properties of PNAs have led to the development of a large variety of research and diagnostic assays, including antigene and antisense therapy and genome mapping. Several sensitive and robust PNA-dependent methods have been designed for modulating polymerase chain reactions, detecting genomic polymorphisms and mutations or capturing nucleic acids. Over the last few years, the use of PNAs has proven its powerful usefulness in cytogenetics for the rapid in situ identification of human chromosomes and the detection of aneuploidies. Recent studies have reported the successful use of chromosome-specific PNA probes on human lymphocytes, amniocytes, spermatozoa as well as on isolated oocytes and blastomeres. Muticolor PNA protocols have been described for the identification of several human chromosomes, indicating that PNAs could become a powerful tool for in situ chromosomal investigation

Impact of DNA Damage on the Frequency of Sperm Chromosomal Aneuploidy in Normal and Subfertile Men

ABSTRACT Background: Various frequencies of sperm aneuploidy are reported in sperms of subfertile patients compared to normal individuals. Moreover, sperm DNA damage is shown to be associated with male infertility. In this study, the rate of DNA damage and frequencies of aneuploidy in sperms of subfertile patients was investigated. Methods: Semen samples were obtained from healthy normal and subfertile (oligozoospermia, asthenozoospermia, and oligoasthenozoospermia) men. The frequency of aneuploidy was assessed using primed in situ labeling (PRINS) analysis with specific primers for chromosomes 18, 21, X, and Y. Sperm DNA damage was assessed using alkaline comet assay. Results: The mean frequencies of disomy for the patients were significantly higher than normal for all chromosomes (P<0.01). The extent of DNA damage in sperms of subfertiles was significantly higher than in normal individuals (P<0.001). The obtained results indicated that higher rate of DNA damages led to higher frequency of chromosomal disomy except for asthenozoospermia samples which exhibited higher rate of DNA damage and lower frequency of chromosomal disomy. Conclusions: These results demonstrate that men with oligozoospermia and oligoasthenozoospermia have an elevated risk for chromosome abnormalities in their sperm, particularly sex chromosomes. DNA damage might be involved in the process of malsegregation of chromosomes

Position of chromosomes 18, 19, 21 and 22 in 3D-preserved interphase nuclei of human and gorilla and white hand gibbon

<p>Abstract</p> <p>Background</p> <p>Even though comparative nuclear architecture studies in hominoids are sparse, nuclear chromosome architecture was shown to be conserved during hominoid evolution. Thus, it is suspected that yet unknown biological mechanisms must underlie this observation.</p> <p>Results</p> <p>Here for the first time a combination of multicolor banding (MCB) and three-dimensional analysis of interphase cells was used to characterize the position and orientation of human chromosomes #18, #19, #21 and #22 and their homologues in primate B-lymphocytic cells. In general, our data is in concordance with previous studies. The position of the four studied human chromosomes and their homologues were conserved during primate evolution. However, comparison of interphase architecture in human B-lymphocytic cells and sperm revealed differences of localization of acrocentric chromosomes. The latter might be related to the fact that the nucleolus organizing region is not active in sperm.</p> <p>Conclusion</p> <p>Studies in different tissue types may characterize more – potentially biologically relevant differences in nuclear architecture.</p

Chromosome distribution in human sperm – a 3D multicolor banding-study

<p>Abstract</p> <p>Background</p> <p>Nuclear architecture studies in human sperm are sparse. By now performed ones were practically all done on flattened nuclei. Thus, studies close at the <it>in vivo </it>state of sperm, i.e. on three-dimensionally conserved interphase cells, are lacking by now. Only the position of 14 chromosomes in human sperm was studied.</p> <p>Results</p> <p>Here for the first time a combination of multicolor banding (MCB) and three-dimensional analysis of interphase cells was used to characterize the position and orientation of all human chromosomes in sperm cells of a healthy donor. The interphase nuclei of human sperm are organized in a non-random way, driven by the gene density and chromosome size.</p> <p>Conclusion</p> <p>Here we present the first comprehensive results on the nuclear architecture of normal human sperm. Future studies in this tissue type, e.g. also in male patients with unexplained fertility problems, may characterize yet unknown mechanisms of infertility.</p

Aneuploidy and Confined Chromosomal Mosaicism in the Developing Human Brain

BACKGROUND: Understanding the mechanisms underlying generation of neuronal variability and complexity remains the central challenge for neuroscience. Structural variation in the neuronal genome is likely to be one important mechanism for neuronal diversity and brain diseases. Large-scale genomic variations due to loss or gain of whole chromosomes (aneuploidy) have been described in cells of the normal and diseased human brain, which are generated from neural stem cells during intrauterine period of life. However, the incidence of aneuploidy in the developing human brain and its impact on the brain development and function are obscure. METHODOLOGY/PRINCIPAL FINDINGS: To address genomic variation during development we surveyed aneuploidy/polyploidy in the human fetal tissues by advanced molecular-cytogenetic techniques at the single-cell level. Here we show that the human developing brain has mosaic nature, being composed of euploid and aneuploid neural cells. Studying over 600,000 neural cells, we have determined the average aneuploidy frequency as 1.25-1.45% per chromosome, with the overall percentage of aneuploidy tending to approach 30-35%. Furthermore, we found that mosaic aneuploidy can be exclusively confined to the brain. CONCLUSIONS/SIGNIFICANCE: Our data indicates aneuploidization to be an additional pathological mechanism for neuronal genome diversification. These findings highlight the involvement of aneuploidy in the human brain development and suggest an unexpected link between developmental chromosomal instability, intercellural/intertissular genome diversity and human brain diseases

Chromothripsis: how does such a catastrophic event impact human reproduction?

International audienceThe recent discovery of a new kind of massive chromosomal rearrangement, baptized chromothripsis (chromo for chromosomes, thripsis for shattering into pieces), greatly modifies our understanding of molecular mechanisms implicated in the repair of DNA damage and the genesis of complex chromosomal rearrangements. Initially described in cancers, and then in constitutional rearrangements, chromothripsis is characterized by the shattering of one (or a few) chromosome(s) segments followed by a chaotic reassembly of the chromosomal fragments, occurring during one unique cellular event. The diversity and the high complexity of chromothripsis events raise questions about their origin, their ties to chromosome instability and their impact in pathology. Several causative mechanisms, involving abortive apoptosis, telomere erosion, mitotic errors, micronuclei formation and p53 inactivation, have been proposed. The remarkable point is that all these mechanisms have been identified in the field of human reproduction as causal factors for reproductive failures and chromosomal abnormalities. Consequently, it seems important to consider this unexpected catastrophic phenomenon in the context of fertilization and early embryonic development in order to discuss its potential impact on human reproduction

Âge maternel et anomalies chromosomiques dans les ovocytes humains

L’étude chromosomique des ovocytes humains issus d’échecs de fécondation in vitro, constitue une approche directe des mécanismes de non-disjonction chromosomique, malgré les difficultés techniques inhérentes à l’analyse de ces cellules. Une étude réalisée sur un vaste échantillon de 1 397 caryotypes ovocytaires a permis de réaliser une analyse fiable et précise de la corrélation existant entre la formation méiotique de ces anomalies et l’âge maternel. Deux modes de non-disjonction méiotique ont été identifiés : la non-disjonction classique de chromosomes entiers et un type particulier de séparation prématurée des chromatides aboutissant à la transmission méiotique de chromatides isolées. Ces deux formes de non-disjonction sont corrélées à l’âge maternel, mais il apparaît que la corrélation est beaucoup plus significative pour le mode de séparation prématurée des chromatides. Ce phénomène constitue donc un mécanisme essentiel de non-disjonction dans le sexe féminin, et pourrait être lié à une perte progressive de la cohésion moléculaire assurée par des protéines spécifiques (les cohésines) entre les chromatides homologues

Âge maternel et anomalies chromosomiques dans les ovocytes humains

L’étude chromosomique des ovocytes humains issus d’échecs de fécondation in vitro, constitue une approche directe des mécanismes de non-disjonction chromosomique, malgré les difficultés techniques inhérentes à l’analyse de ces cellules. Une étude réalisée sur un vaste échantillon de 1 397 caryotypes ovocytaires a permis de réaliser une analyse fiable et précise de la corrélation existant entre la formation méiotique de ces anomalies et l’âge maternel. Deux modes de non-disjonction méiotique ont été identifiés : la non-disjonction classique de chromosomes entiers et un type particulier de séparation prématurée des chromatides aboutissant à la transmission méiotique de chromatides isolées. Ces deux formes de non-disjonction sont corrélées à l’âge maternel, mais il apparaît que la corrélation est beaucoup plus significative pour le mode de séparation prématurée des chromatides. Ce phénomène constitue donc un mécanisme essentiel de non-disjonction dans le sexe féminin, et pourrait être lié à une perte progressive de la cohésion moléculaire assurée par des protéines spécifiques (les cohésines) entre les chromatides homologues.Maternal ageing is the only etiological factor unequivocally associated with the occurrence of aneuploid conceptuses. Molecular studies of trisomies have demonstrated that the pattern of recombinaison was an important predisposing factor to meiotic nondisjunction. To complete this data, a large chromosomal study has been undertaken on 1,397 unfertilised human oocytes recovered from women participating in in vitro fertilization programmes. Conventional whole chromosome nondisjunction and premature chromatid separation were the major types of numerical abnormalities observed. A positive relationship was found between maternal age and these two types of nondisjunction, but the most significant correlation was observed with chromatid separation resulting in the presence of free chromatid in metaphase II oocyte. These data revealed that chromatid separation was an essential factor in the age-dependent occurrence of aneuploidy. This finding provided new insights into the mechanism of nondisjunction in female meiosis since disturbance in molecular chromatid cohesion by cohesins might be a causal mechanism predisposing to nondisjunction and involved in the maternal age effect

Chromoanagenesis: cataclysms behind complex chromosomal rearrangements

Abstract Background During the last decade, genome sequencing projects in cancer genomes as well as in patients with congenital diseases and healthy individuals have led to the identification of new types of massive chromosomal rearrangements arising during single chaotic cellular events. These unanticipated catastrophic phenomenon are termed chromothripsis, chromoanasynthesis and chromoplexis., and are grouped under the name of “chromoanagenesis”. Results For each process, several specific features have been described, allowing each phenomenon to be distinguished from each other and to understand its mechanism of formation and to better understand its aetiology. Thus, chromothripsis derives from chromosome shattering followed by the random restitching of chromosomal fragments with low copy-number change whereas chromoanasynthesis results from erroneous DNA replication of a chromosome through serial fork stalling and template switching with variable copy-number gains, and chromoplexy refers to the occurrence of multiple inter-and intra-chromosomal translocations and deletions with little or no copy-number alterations in prostate cancer. Cumulating data and experimental models have shown that chromothripsis and chromoanasynthesis may essentially result from lagging chromosome encapsulated in micronuclei or telomere attrition and end-to-end telomere fusion. Conclusion The concept of chromanagenesis has provided new insight into the aetiology of complex structural rearrangements, the connection between defective cell cycle progression and genomic instability, and the complexity of cancer evolution. Increasing reported chromoanagenesis events suggest that these chaotic mechanisms are probably much more frequent than anticipated

Chromothripsis and the Macroevolution Theory.

International audienceThe recent discovery of a new class of massive chromosomal rearrangements, occurring during one unique cellular event and baptized "chromothripsis," deeply modifies our perception on the genesis of complex genomic rearrangements, but also it raises the question of the potential driving role of chromothripsis in species evolution. The occurrence of chromothripsis appears to be in good agreement with macroevolution models proposed as a complement to phyletic gradualism. The emergence of this unexpected phenomenon may help to demonstrate the contribution of chromosome rearrangements to speciation process