Recombinaison entre chromosomes non-disjoints dans la Trisomie 21 et correlations génotypes/phénotypes dans le syndrome de Down

Le syndrome de Down (SD), dont l'origine génétique est majoritairement une trisomie 21, se caractérise par un grand nombre de traits phénotypiques dont la plupart présente une variabilité élevée de sévérité. Une partie de cette variabilité pourrait être due à certaines combinaisons tri-alléliques de polymorphismes nucléotidiques (SNP). Par ailleurs, un événement unique de recombinaison dans la région télomérique des chromosomes 21 homologues augmenterait de 5 fois le risque de non-disjonction. Néanmoins, la distribution fine de ces événements n'est pas connue. Nous avons entrepris d'aborder ces deux types de questions en typant une centaine de SNP du chromosome 21 sur lame de verre chez des patients SD. Nous avons mis au point le typage de deux groupes de cinquante SNP permettant chacun de répondre à l'une des questions énoncées ci-dessus. Le premier groupe couvre 2 mégabases de la région critique du syndrome de Down (21q22.13-q22.2). Nous avons génotypé ces SNP chez 81 patients et étudié les répartitions des génotypes entre patients avec et sans un trait phénotypique donné (dont le retard mental et les cardiopathies). Nous avons observé une association significative après correction de Bonferroni (p<0,001) et une association plus faible (p<0,05) entre deux variants intragéniques du gène KCNJ6 et la présence de cardiopathies congénitales de type anomalie de cloisonnement chez les patients SD. Le rôle potentiel de variants du gène KCNJ6 dans l'apparition d'une cardiopathie constitue une nouvelle piste de travail pour la compréhension de ce phénotype. Le second groupe de SNP étudié couvre les 7,9 Mb télomérique du chromosome 21. Nous n'avons pas observé de différence statistique entre les distributions des recombinaisons associées à une non-disjonction et les contrôles. Néanmoins, la présence d'une recombinaison unique dans une région d'environ 2 Mb, située à 2 Mb du télomère du chromosome 21, semble particulièrement décisive pour l'apparition d'une non-disjonction.Down syndrome (DS), which is mainly caused by trisomy 21, is characterized by many abnormalities affecting most organ systems. The majority of DS features are highly variable between patients in term of presence and severity. Nucleotidic variations (SNPs) within or around some triplicated genes could be partly responsible for this variability. On another hand, fine scale analysis of recombination in the sub-telomeric region should make it more precise the relationship previously reported between altered recombination and meiotic non-disjunction. We addressed these two questions by typing a hundred of SNPs along chromosome 21 in families with a trisomic child. Two set of 50 SNPs have been designed, one for each project. The first 50 SNPs set covers 2 megabases of the Down Syndrome Critical Region (21q22.13-q22.2). We have genotyped 81 patients and studied, for each SNP, the proportion of genotypes between patients with and without a DS trait (including mental retardation and cardiac malformation). We have observed two associations, one strong (p<0,001) and one weaker (p<0,05), between two intragenic SNPs and the presence of a septal cardiac defect. The concerned gene is KCNJ6 a potassium channel which potential implication in DS cardiopathy is a new way of investigations for understanding the variability of this DS trait. The second 50 SNPs set covers the 7.9 sub-telomeric mégabases of chromosome 21. We could not observe significant differences between recombination distributions in context of normal and non-disjoined chromosomes. Nevertheless, the presence of a unique recombination event in a 2 Mb region, located at 2 Mb from the telomere, seems to be critical for the generation of non-disjoined chromosome 21.MONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

Région juxtacentrométrique du chromosome 21 et plasticité génomique

MONTPELLIER-BU Médecine UPM (341722108) / SudocPARIS-BIUP (751062107) / SudocMONTPELLIER-BU Médecine (341722104) / SudocSudocFranceF

Identification of Cis-regulating sequences involved in the activity of the lamin B2 origin of DNA replication

Les éléments agissants en cis nécessaires à l activité des origines de réplication de l ADN chez les métazoaires sont peu connus. Nous avons étudié le rôle des ces éléments sur l activation de l origine de réplication humaine associée au gène de la Lamine B2 (LaminB2-Ori). Nous avons généré des clones de cellules HeLa à partir de l intégration stable d un fragment d ADN de 1.2 kb, contenant le site d initiation de la réplication et un îlot CpG. Nous avons étudié l initiation de la réplication par PCR sur les ADN naissants provenant de cellules asynchrones. Dans la majorité des clones, l activité de cette origine ectopique était comparable à celle de l origine endogène. Dans quelques cas, ce fragment montrait une activité réplicative réduite. Pour réduire la variabilité de l activité due à l environnement chromatinien du site d insertion, nous avons développé un outil d insertion site-spécifique via la recombinase Cre de cassettes contenant la LaminB2-Ori ectopique. Cette méthode présente une homogénéité de l activité de la LaminB2-Ori ectopique et par conséquent permet de quantifier l activité de mutants de cette Ori. Une dissection des éléments en cis en proximité de l origine de la Lamine B2 ectopique a été réalisée. Les résultats indiquent que la séquence centrale de l origine, où se fixe le complexe ORC, est nécessaire à l initiation de la réplication et que l îlot CpG, les sites de fixation des facteurs de transcription et la présence de gènes activement transcrits influencent l initiation de la réplication. L ensemble des résultats suggère que la LaminB2-Ori possède une organisation modulaire de séquences en cis. Cette structure peut imposer une configuration chromatinienne spécifique ou favoriser quelques modifications épigénétiques qui promeuvent l initiation de la réplication et que l activation de la LaminB2-Ori domine sur les séquences environnantesThe cis-acting elements necessary for the activity of DNA replication origins in metazoan are poorly understood. We studied the role of this elements on the activation of the human origin of replication associated to the Lamin B2 gene. We generated HeLa clones of stable integration of a 1.2 kb DNA segment, comprising the start site of DNA replication and the CpG island. We assessed the initiation of DNA replication by PCR on nascent DNA isolated from asynchronously cells. In the majority of clones the activity of this ectopic origin was comparable to the endogenous origin. In some cases this segment shows a reduced replication activity. For reducing the variability due to the chromatin environment of the insertion site, we developed a toll of site-specific integration mediated by the Cre recombinase of cassettes containing the ectopic LaminB2-Ori. This method shows homogeneity of activity of the ectopic LaminB2-Ori, and by consequence the possibility to quantify the mutants Ori activity. A dissection of the cis-elements in the closed proximity of the ectopic Lamin B2 origin was carried out. The results indicate that core sequence of the origin which is bound by the ORC complex is required for the initiation of the replication and that the CpG island, binding sites for transcription factors and the presence of actively transcribed genes positively influence the replication initiation. Altogether these results suggest that the LaminB2-Ori shows a modular organization of cis-acting sequences. This structure may impose a specific chromatin configuration or favorite some epigenetic modifications that prompt the initiation of replication and that LamB2-Ori activation is dominant over the surrounding sequencesMONTPELLIER-BU Sciences (341722106) / SudocSudocFranceItalyFRI

A new 5' sequence associated with mouse L1 elements is representative of a major class of L1 termini.

International audienc

Global analysis of DNA methylation and transcription of human repetitive sequences.

International audienceHalf of the human genome consists of repetitive DNA sequences. Recent studies in various organisms highlight the role of chromatin regulation of repetitive DNA in gene regulation as well as in maintainance of chromosomes and genome integrity. Hence, repetitive DNA sequences might be potential "sensors" for chromatin changes associated with pathogenesis. Therefore, we developed a new genomic tool called RepArray. RepArray is a repeat-specific microarray composed of a representative set of human repeated sequences including transposon-derived repeats, simple sequences repeats, tandemly repeated sequences such as centromeres and telomeres. We showed that combined to anti-methylcytosine immunoprecipitation assay, the RepArray can be used to generate repeat-specific methylation maps. Using cell lines impaired chemically or genetically for DNA methyltransferases activities, we were able to distinguish different epigenomes demonstrating that repeats can be used as markers of genome-wide methylation changes. Besides, using a well-documented system model, the thermal stress, we demonstrated that RepArray is also a fast and reliable tool to obtain an overview of overall transcriptional activity on whole repetitive compartment in a given cell type. Thus, the RepArray represents the first valuable tool for systematic and genome-wide analyses of the methylation and transcriptional status of the repetitive counterpart of the human genome

Genome-wide association analysis in dilated cardiomyopathy reveals two new players in systolic heart failure on chromosomes 3p25.1 and 22q11.23

Aims: Our objective was to better understand the genetic bases of dilated cardiomyopathy (DCM), a leading cause of systolic heart failure. Methods and results: We conducted the largest genome-wide association study performed so far in DCM, with 2719 cases and 4440 controls in the discovery population. We identified and replicated two new DCM-associated loci on chromosome 3p25.1 [lead single-nucleotide polymorphism (SNP) rs62232870, P = 8.7 x 10(-11) and 7.7 x 10(-4) in the discovery and replication steps, respectively] and chromosome 22q11.23 (lead SNP rs7284877, P = 3.3 x 10(-8) and 1.4 x 10(-3) in the discovery and replication steps, respectively), while confirming two previously identified DCM loci on chromosomes 10 and 1, BAG3 and HSPB7. A genetic risk score constructed from the number of risk alleles at these four DCM loci revealed a 3-fold increased risk of DCM for individuals with 8 risk alleles compared to individuals with 5 risk alleles (median of the referral population). In silico annotation and functional 4C-sequencing analyses on iPSC-derived cardiomyocytes identify SLC6A6 as the most likely DCM gene at the 3p25.1 locus. This gene encodes a taurine transporter whose involvement in myocardial dysfunction and DCM is supported by numerous observations in humans and animals. At the 22q11.23 locus, in silico and data mining annotations, and to a lesser extent functional analysis, strongly suggest SMARCB1 as the candidate culprit gene. Conclusion: This study provides a better understanding of the genetic architecture of DCM and sheds light on novel biological pathways underlying heart failure. [GRAPHICS