Time course analysis of RNA stability in human placenta

<p>Abstract</p> <p>Background</p> <p>Evaluation of RNA quality is essential for gene expression analysis, as the presence of degraded samples may influence the interpretation of expression levels. Particularly, qRT-PCR data can be affected by RNA integrity and stability. To explore systematically how RNA quality affects qRT-PCR assay performance, a set of human placenta RNA samples was generated by two protocols handlings of fresh tissue over a progressive time course of 4 days. Protocol A consists of a direct transfer of tissue into RNA-stabilizing solution (RNAlater™) solution. Protocol B uses a dissection of placenta villosities before bio banking. We tested and compared RNA yields, total RNA integrity, mRNA integrity and stability in these two protocols according to the duration of storage.</p> <p>Results</p> <p>A long time tissue storage had little effect on the total RNA and mRNA integrity but induced changes in the transcript levels of stress-responsive genes as TNF-alpha or COX2 after 48 h. The loss of the RNA integrity was higher in the placental tissues that underwent a dissection before RNA processing by comparison with those transferred directly into RNA later™ solution. That loss is moderate, with average RIN (RNA Integration Numbers) range values of 4.5–6.05, in comparison with values of 6.44–7.22 in samples directly transferred to RNAlater™ (protocol A). Among the house keeping genes tested, the B2M is the most stable.</p> <p>Conclusion</p> <p>This study shows that placental samples can be stored at + 4°C up to 48 h before RNA extraction without altering RNA quality. Rapid tissue handling without dissection and using RNA-stabilizing solution (RNAlater™) is a prerequisite to obtain suitable RNA integrity and stability.</p

Whole genome sequencing for USH2A-associated disease reveals several pathogenic deep-intronic variants that are amenable to splice correction

A significant number of individuals with a rare disorder such as Usher syndrome (USH) and (non-)syndromic autosomal recessive retinitis pigmentosa (arRP) remain genetically unexplained. Therefore, we assessed subjects suspected of USH2A-associated disease and no or mono-allelic USH2A variants using whole genome sequencing (WGS) followed by an improved pipeline for variant interpretation to provide a conclusive diagnosis. One hundred subjects were screened using WGS to identify causative variants in USH2A or other USH/arRP-associated genes. In addition to the existing variant interpretation pipeline, a particular focus was put on assessing splice-affecting properties of variants, both in silico and in vitro. Also structural variants were extensively addressed. For variants resulting in pseudoexon inclusion, we designed and evaluated antisense oligonucleotides (AONs) using minigene splice assays and patient-derived photoreceptor precursor cells. Biallelic variants were identified in 49 of 100 subjects, including novel splice-affecting variants and structural variants, in USH2A or arRP/USH-associated genes. Thirteen variants were shown to affect USH2A pre-mRNA splicing, including four deep-intronic USH2A variants resulting in pseudoexon inclusion, which could be corrected upon AON treatment. We have shown that WGS, combined with a thorough variant interpretation pipeline focused on assessing pre-mRNA splicing defects and structural variants, is a powerful method to provide subjects with a rare genetic condition, a (likely) conclusive genetic diagnosis. This is essential for the development of future personalized treatments and for patients to be eligible for such treatments

Comparing HLA Shared Epitopes in French Caucasian Patients with Scleroderma

Although many studies have analyzed HLA allele frequencies in several ethnic groups in patients with scleroderma (SSc), none has been done in French Caucasian patients and none has evaluated which one of the common amino acid sequences, 67FLEDR71, shared by HLA-DRB susceptibility alleles, or 71TRAELDT77, shared by HLA-DQB1 susceptibility alleles in SSc, was the most important to develop the disease. HLA-DRB and DQB typing was performed for a total of 468 healthy controls and 282 patients with SSc allowing FLEDR and TRAELDT analyses. Results were stratified according to patient’s clinical subtypes and autoantibody status. Moreover, standardized HLA-DRß1 and DRß5 reverse transcriptase Taqman PCR assays were developed to quantify ß1 and ß5 mRNA in 20 subjects with HLA-DRB1*15 and/or DRB1*11 haplotypes. FLEDR motif is highly associated with diffuse SSc (χ2 = 28.4, p<10−6) and with anti-topoisomerase antibody (ATA) production (χ2 = 43.9, p<10−9) whereas TRAELDT association is weaker in both subgroups (χ2 = 7.2, p = 0.027 and χ2 = 14.6, p = 0.0007 respectively). Moreover, FLEDR motif- association among patients with diffuse SSc remains significant only in ATA subgroup. The risk to develop ATA positive SSc is higher with double dose FLEDR than single dose with respectively, adjusted standardised residuals of 5.1 and 2.6. The increase in FLEDR motif is mostly due to the higher frequency of HLA-DRB1*11 and DRB1*15 haplotypes. Furthermore, FLEDR is always carried by the most abundantly expressed ß chain: ß1 in HLA DRB1*11 haplotypes and ß5 in HLA-DRB1*15 haplotypes

Whole genome sequencing for USH2A-associated disease reveals several pathogenic deep-intronic variants that are amenable to splice correction

A significant number of individuals with a rare disorder such as Usher syndrome (USH) and (non-)syndromic autosomal recessive retinitis pigmentosa (arRP) remain genetically unexplained. Therefore, we assessed subjects suspected of USH2A-associated disease and no or mono-allelic USH2A variants using whole genome sequencing (WGS) followed by an improved pipeline for variant interpretation to provide a conclusive diagnosis. One hundred subjects were screened using WGS to identify causative variants in USH2A or other USH/arRP-associated genes. In addition to the existing variant interpretation pipeline, a particular focus was put on assessing splice-affecting properties of variants, both in silico and in vitro. Also structural variants were extensively addressed. For variants resulting in pseudoexon inclusion, we designed and evaluated antisense oligonucleotides (AONs) using minigene splice assays and patient-derived photoreceptor precursor cells. Biallelic variants were identified in 49 of 100 subjects, including novel splice-affecting variants and structural variants, in USH2A or arRP/USH-associated genes. Thirteen variants were shown to affect USH2A pre-mRNA splicing, including four deep-intronic USH2A variants resulting in pseudoexon inclusion, which could be corrected upon AON treatment. We have shown that WGS, combined with a thorough variant interpretation pipeline focused on assessing pre-mRNA splicing defects and structural variants, is a powerful method to provide subjects with a rare genetic condition, a (likely) conclusive genetic diagnosis. This is essential for the development of future personalized treatments and for patients to be eligible for such treatments.</p

Whole genome sequencing for USH2A-associated disease reveals several pathogenic deep-intronic variants that are amenable to splice correction

A significant number of individuals with a rare disorder such as Usher syndrome (USH) and (non-)syndromic autosomal recessive retinitis pigmentosa (arRP) remain genetically unexplained. Therefore, we assessed subjects suspected of USH2A-associated disease and no or mono-allelic USH2A variants using whole genome sequencing (WGS) followed by an improved pipeline for variant interpretation to provide a conclusive diagnosis. One hundred subjects were screened using WGS to identify causative variants in USH2A or other USH/arRP-associated genes. In addition to the existing variant interpretation pipeline, a particular focus was put on assessing splice-affecting properties of variants, both in silico and in vitro. Also structural variants were extensively addressed. For variants resulting in pseudoexon inclusion, we designed and evaluated antisense oligonucleotides (AONs) using minigene splice assays and patient-derived photoreceptor precursor cells. Biallelic variants were identified in 49 of 100 subjects, including novel splice-affecting variants and structural variants, in USH2A or arRP/USH-associated genes. Thirteen variants were shown to affect USH2A pre-mRNA splicing, including four deep-intronic USH2A variants resulting in pseudoexon inclusion, which could be corrected upon AON treatment. We have shown that WGS, combined with a thorough variant interpretation pipeline focused on assessing pre-mRNA splicing defects and structural variants, is a powerful method to provide subjects with a rare genetic condition, a (likely) conclusive genetic diagnosis. This is essential for the development of future personalized treatments and for patients to be eligible for such treatments.</p

Whole genome sequencing for USH2A-associated disease reveals several pathogenic deep-intronic variants that are amenable to splice correction

A significant number of individuals with a rare disorder such as Usher syndrome (USH) and (non-)syndromic autosomal recessive retinitis pigmentosa (arRP) remain genetically unexplained. Therefore, we assessed subjects suspected of USH2A-associated disease and no or mono-allelic USH2A variants using whole genome sequencing (WGS) followed by an improved pipeline for variant interpretation to provide a conclusive diagnosis. One hundred subjects were screened using WGS to identify causative variants in USH2A or other USH/arRP-associated genes. In addition to the existing variant interpretation pipeline, a particular focus was put on assessing splice-affecting properties of variants, both in silico and in vitro. Also structural variants were extensively addressed. For variants resulting in pseudoexon inclusion, we designed and evaluated antisense oligonucleotides (AONs) using minigene splice assays and patient-derived photoreceptor precursor cells. Biallelic variants were identified in 49 of 100 subjects, including novel splice-affecting variants and structural variants, in USH2A or arRP/USH-associated genes. Thirteen variants were shown to affect USH2A pre-mRNA splicing, including four deep-intronic USH2A variants resulting in pseudoexon inclusion, which could be corrected upon AON treatment. We have shown that WGS, combined with a thorough variant interpretation pipeline focused on assessing pre-mRNA splicing defects and structural variants, is a powerful method to provide subjects with a rare genetic condition, a (likely) conclusive genetic diagnosis. This is essential for the development of future personalized treatments and for patients to be eligible for such treatments

Conséquences placentaires associées au diabète et macrosomie fœtale

Malgré un contrôle correct de la glycémie au cours de la grossesse, l’incidence de la macrosomie demeure élevée chez les femmes ayant développé un diabète prégestationnel. L’excès de croissance foetale signifie un risque élevé de morbidité néonatale et à plus long terme une fréquence élevée de diabète ultérieur ou de syndrome métabolique. La croissance fœtale est régulée par l’équilibre ente les besoins fœtaux en nutriments, déterminés par la composante génétique du fœtus et l’offre en nutriments dépendante de la capacité placentaire. En cas de diabète, cet équilibre est rompu. Le premier mécanisme invoqué est le changement métabolique de l’environnement maternel et utérin occasionné par l’hyperglycémie, se traduisant par une hyperinsulinémie, des altérations des paramètres lipidiques, des taux d’hormones et de facteurs de croissance maternels. Le second mécanisme implique le placenta, véritable réseau à l’interface mère/fœtus régulateur du transfert de nutriments au foetus. Le placenta est également un sécréteur d’un large éventail d’hormones et de facteurs de croissance essentiels au bon déroulement de la grossesse et au développement du fœtus. Des études récentes font état d’un transfert accru de nutriments lors des grossesses diabétiques conséquence d’une sur expression d’un grand nombre de gènes placentaires dont les transporteurs de nutriments, la leptine et des médiateurs de l’inflammation. Ces modifications sont observées même pour des modifications mineures de la glycémie. Selon Deshoye (1), la macrosomie survient lorsque l’hyperglycémie fœtale dépasse la capacité placentaire d’épurer le fœtus d’un excédent de glucose. Dans ce contexte, la macrosomie serait le résultat d’un défaut placentaire, de son incapacité à s’adapter aux bouleversements maternels et fœtaux. En cas de diabète ces phénomènes sont exacerbés. Le troisième mécanisme intéresse le foetus et son capital d’adaptation. dépendant du génome et notamment de l’expression de gènes soumis à empreinte. La grossesse peut être comprise comme une période dynamique d’adaptation du foetus et du placenta aux modifications maternelles

Etude des marqueurs de risque génétique pour le diabète de type 1 dans les familles suivies au centre de dépistage du prédiabète de type 1 de Lille

LILLE2-BU Santé-Recherche (593502101) / SudocPARIS-BIUM (751062103) / SudocSudocFranceF

POLYMORPHISME DU GENE DU RECEPTEUR DES PRODUITS FINAUX DE GLYCATION ET DIABETE DE TYPE I (EVALUATION DANS LA NEPHROPATHIE DIABETIQUE)

LILLE2-BU Santé-Recherche (593502101) / SudocPARIS-BIUM (751062103) / SudocSudocFranceF

Facteurs prédictifs de diabète de type 1 chez les enfants apparentés à des sujets diabétiques

LILLE2-BU Santé-Recherche (593502101) / SudocSudocFranceF