beDNA : un projet visant à la collection systématique d’échantillons humains archéologiques à vocation paléogénétique – une première expérimentation

La paléogénétique occupe désormais une place importante dans les problématiques archéologiques. Toutefois, les analyses d’ADN ancien peuvent être desservies, voire empêchées, par l’état de préservation des échantillons en raison de contamination par de l’ADN moderne ou de mauvaises conditions de stockage. Le projet beDNA, "banque d’échantillons et de Données Nationale Archéogénétique", souhaite donner les moyens d’analyses paléogénétiques futures, en proposant le stockage systématique d’échantillons des squelettes humains tenant compte des contraintes inhérentes à la préservation de l’ADN ancien. Le projet implique (1) un protocole systématique d’échantillonnage "propre" des restes humains sur le terrain commun à toutes les opérations archéologiques, (2) un espace de stockage dédié à ces échantillons adapté à la conservation de l’ADN ancien, (3) une base de données faisant le lien entre les sites et les échantillons conservés dans la banque, (4) l’approbation par l’État des demandes d’analyse d’échantillons après expertise. La phase de test du projet, initiée en septembre 2020 sur la région Île-de-France, nous a permis d’évaluer et d’ajuster le protocole d’échantillonnage sur le terrain et les dispositifs de transfert vers la banque. Cette note présente les étapes envisagées pour chaque échantillon, depuis les terrains jusqu’aux laboratoires d’analyse génétique, ainsi que le déroulement de sa phase test, en cours, et les premiers retours d’expérience.Palaeogenetics is becoming increasingly important in tackling archaeological issues. However, analyses of ancient DNA can be hampered or even prevented by the state of preservation of samples due to poor storage conditions, and because of contamination by modern DNA. The beDNA project for a national archaeological genetic data and sample bank (banque d’échantillons et de Données Nationale Archéogénétique) is developing the means to enable future palaeogenetic analyses by systematically storing human skeletal samples, with the constraints inherent to the preservation of ancient DNA taken into account. This project comprises (1) a systematic protocol for "clean" sampling of human remains to be common to all archaeological operations, (2) a dedicated storage space for samples, suited to aDNA preservation, (3) a database linking sites with the samples stored in the bank, (4) approval of sample analysis requests by authorities, after expert review. The test phase of the project, which began in September 2020 in the Île-de-France region, enabled us to evaluate and adjust both the sampling protocol in the field and the transfer process to the beDNA bank. This note describes the different stages envisaged for each sample, from the archaeological field to the genetics laboratory, as well as the development of the experimental phase and initial feedback from it

Rescue of Advanced Pompe Disease in Mice with Hepatic Expression of Secretable Acid α-Glucosidase.

Pompe disease is a neuromuscular disorder caused by disease-associated variants in the gene encoding for the lysosomal enzyme acid α-glucosidase (GAA), which converts lysosomal glycogen to glucose. We previously reported full rescue of Pompe disease in symptomatic 4-month-old Gaa knockout (Gaa-/-) mice by adeno-associated virus (AAV) vector-mediated liver gene transfer of an engineered secretable form of GAA (secGAA). Here, we showed that hepatic expression of secGAA rescues the phenotype of 4-month-old Gaa-/- mice at vector doses at which the native form of GAA has little to no therapeutic effect. Based on these results, we then treated severely affected 9-month-old Gaa-/- mice with an AAV vector expressing secGAA and followed the animals for 9 months thereafter. AAV-treated Gaa-/- mice showed complete reversal of the Pompe phenotype, with rescue of glycogen accumulation in most tissues, including the central nervous system, and normalization of muscle strength. Transcriptomic profiling of skeletal muscle showed rescue of most altered pathways, including those involved in mitochondrial defects, a finding supported by structural and biochemical analyses, which also showed restoration of lysosomal function. Together, these results provide insight into the reversibility of advanced Pompe disease in the Gaa-/- mouse model via liver gene transfer of secGAA.This work was supported by Genethon, the French Muscular Dystro-phy Association (AFM), and Spark Therapeutics. It was also sup-ported by the European Union’s Research and Innovation Programunder grant agreement number 667751 (to F.M.), the EuropeanResearch Council Consolidator Grant under grant agreement number617432 (to F.M.), and Marie Skłodowska-Curie Actions-IndividualFellowship (MSCA-IF) grant agreement number 797144 (to U.C.)S

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Kininogen Cleavage Assay: Diagnostic Assistance for Kinin-Mediated Angioedema Conditions.

Angioedema without wheals (AE) is a symptom characterised by localised episodes of oedema presumably caused by kinin release from kininogen cleavage. It can result from a hereditary deficiency in C1 Inhibitor (C1Inh), but it can present with normal level of C1Inh. These forms are typically difficult to diagnose although enhanced kinin production is suspected or demonstrated in some cases.We wanted to investigate bradykinin overproduction in all AE condition with normal C1Inh, excluding cases with enhanced kinin catabolism, and to propose this parameter as a disease biomarker.We retrospectively investigated high molecular weight kininogen (HK) cleavage pattern, using gel electrophoresis and immunorevelation. Plasma samples were drawn using the same standardised procedure from blood donors or AE patients with normal C1Inh conditions, normal kinin catabolism, and without prophylaxis.Circulating native HK plasma concentrations were similar in the healthy men (interquartile range: 98-175μg/mL, n = 51) and in healthy women (90-176μg/mL, n = 74), while HK cleavage was lower (p14.4% HK cleavage for men; 33.0% HK cleavage for women, with >98% specificity achieved for all parameters. In plasma from patients undergoing recovery two months after oestrogen/progestin combination withdrawal (n = 13) or two weeks after AE attack (n = 2), HK cleavage was not fully restored, suggesting its use as a post-attack assay.As a diagnostic tool, HK cleavage can offer physicians supportive arguments for kinin production in suspected AE cases and improve patient follow-up in clinical trials or prophylactic management

Gene therapy with secreted acid alpha-glucosidase rescues Pompe disease in a novel mouse model with early-onset spinal cord and respiratory defects.

Pompe disease (PD) is a neuromuscular disorder caused by deficiency of acidalpha-glucosidase (GAA), leading to motor and respiratory dysfunctions. Available Gaa knock-out (KO) mouse models do not accurately mimic PD, particularly its highly impaired respiratory phenotype. Here we developed a new mouse model of PD crossing Gaa KOB6;129 with DBA2/J mice. We subsequently treated Gaa KODBA2/J mice with adeno-associated virus (AAV) vectors expressing a secretable form of GAA (secGAA). Male Gaa KODBA2/J mice present most of the key features of the human disease, including early lethality, severe respiratory impairment, cardiac hypertrophy and muscle weakness. Transcriptome analyses of Gaa KODBA2/J, compared to the parental Gaa KOB6;129 mice, revealed a profoundly impaired gene signature in the spinal cord and a similarly deregulated gene expression in skeletal muscle. Muscle and spinal cord transcriptome changes, biochemical defects, respiratory and muscle function in the Gaa KODBA2/J model were significantly improved upon gene therapy with AAV vectors expressing secGAA. These data show that the genetic background impacts on the severity of respiratory function and neuroglial spinal cord defects in the Gaa KO mouse model of PD. Our findings have implications for PD prognosis and treatment, show novel molecular pathophysiology mechanisms of the disease and provide a unique model to study PD respiratory defects, which majorly affect patients. This work was supported by Genethon, the French Muscular Dystrophy Association (AFM), the European Commission (grant nos. 667751, 617432, and 797144), and Spark Therapeutics.This work was supported by Genethon and the French Muscular Dystrophy Association (AFM, to F.M.). It was also supported by the European Union’s research and innovation program under grant agreement no. 667751 (to F.M.), the European Research Council Consolidator Grant under grant agreement no. 617432 (to F.M.), Marie Skodowska-Curie Actions Individual Fellowship (MSCA-IF) grant agreement no. 797144 (to U.C.), and by Spark Therapeutics under a sponsored research agreement.S

Quantification of HK molecular pattern in control male and female samples.

<p><b>A, B</b>: Purified human HK (20, 40, 80, and 110ng) and 0.5μL of plasma from five male healthy donors, (H1 to H5) (A), and five females (H6 to H10) (B) were subjected to SDS-PAGE. The signal of HK native chain (N-HK), light chain (LC-HK), and cleaved light chain (cLC-HK) were quantified by densitometry and N-HK concentration was evaluated by the displayed linear regression. <b>C, D</b>: Box-plot displaying 5–95% range (<i>italic</i> fonts), median (<b>bold</b> fonts), and interquartile values of plasma concentration of N-HK (C) and HK cleavage (D), measured as in panel A. densito: densitometry in 10⁷ arbitrary units; NS: non-significant; ****: <i>p</i><10⁻⁴ (Mann-Whitney-Wilcoxon test).</p

HK concentration in samples from nC1Inh-AE patients taking oral contraception.

<p>High-molecular-weight kininogen (HK) molecular pattern of plasma samples from healthy women taking or not estrogen/progestin combination (OP; Healthy+OP, n = 32; Healthy-OP, n = 30) and from nC1Inh-AE patients taking OP and ≥2 months after OP withdrawal (nC1INh-AE+/-OP, n = 13). <b>A</b>: Analysis of plasma samples (0.5μL/lane) from six patients (O1–O6) and the corresponding figures. N-HK concentration of P1 and P2 was evaluated by the displayed linear regression, kinin-forming activity was determined as in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163958#pone.0163958.ref026" target="_blank">26</a>], pathological values were labelled in <b>bold</b> fonts. <b>B, C</b>: Box-plot displaying 5–95% range (<i>italic</i> fonts), median (<b>bold</b> fonts), and interquartile values of the plasma concentration of N-HK (B) and cleaved HK species (C), measured as in panel A. <b>D, E</b>: Details of the paired samples of panel B and C respectively analysed using the Wilcoxon matched-pairs signed rank test. densito: densitometry in 10⁷ arbitrary units; <i>NS</i>: non-significant; *: <i>p</i><0.05; **: <i>p</i><0.01; ***: <i>p</i><0.001; ****: <i>p</i><10⁻⁴ (non-parametric tests); N-HK, LC-HK, cLC-HK: native chain, light chain, cleaved light chain of HK respectively; nC1Inh-AE: angioedema with normal C1 inhibitor.</p

Threshold of N-HK concentration and HK cleavage.

<p><b>A, B</b>: ROC curve analysis between healthy men and nC1Inh-AE male patients of N-HK concentration (A) and HK molecular species (B), displayed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163958#pone.0163958.g002" target="_blank">Fig 2B and 2C</a>, respectively. The arrows point to the dots corresponding to the maximum of the sum {sensitivity + specificity} and the corresponding thresholds. <b>C</b>: Figures associated with the ROC curve analysis shown in A and B along with their calculated 95% confidence levels (95% CI). <b>D, E, F</b>: as in A, B, C for indicators on women displayed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163958#pone.0163958.g002" target="_blank">Fig 2E and 2F</a>. AUC: area under the curve; PPV: predictive positive value; NPV: negative predictive value; N-HK: native single-chain of HK.</p