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DNA hybridization-capture techniques allow researchers to focus their sequencing efforts on preselected genomic regions. This feature is especially useful when analys- ing ancient DNA (aDNA) extracts, which are often dominated by exogenous environ- mental sources. Here, we assessed, for the first time, the performance of hyRAD as an inexpensive and design-free alternative to commercial capture protocols to obtain authentic aDNA data from osseous remains. HyRAD relies on double enzymatic re- striction of fresh DNA extracts to produce RNA probes that cover only a fraction ofthe genome and can serve as baits for capturing homologous fragments from aDNA li- braries. We found that this approach could retrieve sequence data from horse remains coming from a range of preservation environments, including beyond radiocarbon range, yielding up to 146.5-fold on-target enrichment for aDNA extracts showing ex- tremely low endogenous content (20%¿30%), while the fraction of endogenous reads mapping on- and off-target was relatively insensi- tive to the original endogenous DNA content. Procedures based on two instead of a single round of capture increased on-target coverage up to 3.6-fold. Additionally, we used methylation-sensitive restriction enzymes to produce probes targeting hypo- methylated regions, which improved data quality by reducing post-mortem DNA dam- age and mapping within multicopy regions. Finally, we developed a fully automated hyRAD protocol utilizing inexpensive robotic platforms to facilitate capture process- ing. Overall, our work establishes hyRAD as a cost-effective strategy to recover a set of shared orthologous variants across multiple ancient samples., This project received funding from: the University Paul Sabatier IDEX Chaire d’Excellence (OURASI); the CNRS Programme de Recherche Conjoint (PRC); the CNRS International Research Project (IRP AMADEUS); the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 797449; the Russian Foundation for Basic Research, project No. 19-59-15001 “Horses and their importance in the life of the ancient population of Altai and adjacent territories: interdisciplinary research and reconstruction”; and the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement 681605).

Alongside horses, donkeys and their first-generation hybrids represent members of the Equidae family known for their social, economic and symbolic importance in protohistoric and historical France. However, their relative importance and their respective roles in different regions and time periods are difficult to assess based on textual, iconographic and archaeological evidence. This is both due to incomplete, partial and scattered historical sources and difficulties to accurately assign fragmentary archaeological remains at the proper taxonomic level. DNA- based methods, however, allow for a robust identification of the taxonomic status of ancient equine osseous material from minimal sequence data. Here, we leveraged shallow ancient DNA sequencing and the dedicated Zonkey computational pipeline to obtain the first baseline distribution for horses, mules and donkeys in France from the Iron Age to the Modern period. Our collection includes a total of 873 ancient specimens spanning 128 sites and comprising 717 horses, 100 donkeys, 55 mules and a single hinny individual. While horses were ubiquitous and the most dominant species identified, our dataset reveals the importance of mule breeding during Roman times, especially between the 1st and 3rd centuries CE (Common Era), where they represented between 20.0% and 34.2% of equine assemblages. In contrast, donkeys were almost absent from northern France as- semblages during the whole Roman period, but replaced mules in rural and urban commercial and economic centers from the early Middle Ages. Our work also identified donkeys of exceptional size during Late Antiquity, which calls for a deep reassessment of the true morphological space of past equine species. This study confirmed the general preference toward horses throughout all time periods investigated but revealed dynamic manage- ment strategies leveraging the whole breadth of equine resources in various social, geographic and temporal contexts., Pierre Clavel’s PhD position is funded by the CNRS MITI interdisciplinary programme (Mission pour les Initiatives Transverses et Interdisciplinaires). Xuexue Liu was supported by the European Union’s Horizon 2020 research and Innovation programme under the Marie Skłodowska-Curie grant agreement 101027750. Andaine Seguin-Orlando acknowledges IAST for funding from ANR under grant ANR-17-EURE-0010 (Investissements d’Avenir programme). This work was supported by the France G ́enomique Appel `a Grand Projet (ANR-10-INBS-09-08, BUCEPHALE project); the Initiative d’Excellence Chaires d’attractivit ́e, Universit ́e de Toulouse (OURASI) and the Villum Fonden miGENEPI research project. This project has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement No. 681605)

International audience; Recent developments in High-Throughput DNA sequencing (HTS) technologies and ancient DNA (aDNA) research have opened access to the characterization of the microbial communities within past populations. Most studies have, however, relied on the analysis of dental calculus as one particular material type particularly prone to the molecular preservation of ancient microbial biofilms and potential of entire teeth for microbial characterization, both of healthy communities and pathogens in ancient individuals, remains overlooked. In this study, we used shotgun sequencing to characterize the bacterial composition from historical subjects showing macroscopic evidence of oral pathologies. We first carried out a macro-scopic analysis aimed at identifying carious or periodontal diseases in subjects belonging to a French rural population of the 18th century AD. We next examined radiographically six subjects showing specific, characteristic dental pathologies and applied HTS shotgun sequencing to characterize the microbial communities present in and on the dental material. The presence of Streptococcus mutans and also Rothia dentocariosa, Actinomyces visco-sus, Porphyromonas gingivalis, Tannerella forsythia, Pseudoramibacter alactolyticus, Olse-nella uli and Parvimonas micra was confirmed through the presence of typical signatures of post-mortem DNA damage at an average depth-of-coverage ranging from 0.5 to 7X, with a minimum of 35% (from 35 to 93%) of the positions in the genome covered at least once. Each sampled tooth showed a specific bacterial signature associated with carious or peri-odontal pathologies. This work demonstrates that from a healthy independent tooth, without visible macroscopic pathology, we can identify a signature of specific pathogens and deduce the oral health status of an individual.

Recessive dystrophic epidermolysis bullosa (RDEB) is caused by mutations in the COL7A1 gene encoding type VII collagen. Variations in severity between the different clinical forms of RDEB likely depend on the nature and location of COL7A1 mutations, but observed intrafamilial phenotypic variations suggest additional genetic and/or environmental factors. Candidate modifier genes include MMP1, encoding matrix metalloproteinase 1, the first gene implicated in RDEB before its primary role in the disease was excluded. Type VII collagen is a substrate of MMP1 and an imbalance between its synthesis and degradation could conceivably worsen the RDEB phenotype. Here, we studied a previously described family with three affected siblings of identical COL7A1 genotype but displaying great sibling-to-sibling variations in disease severity. RDEB severity did not correlate with type VII collagen synthesis levels, but with protein levels at the dermal-epidermal junction, suggesting increased degradation by metalloproteinases. This was supported by the presence of increased transcript and active MMP1 levels in the most severely affected children, who carried a known SNP (1G/2G) in the MMP1 promoter. This SNP creates a functional Ets binding site resulting in transcriptional upregulation. We next studied a French cohort of 31 unrelated RDEB patients harboring at least one in-frame COL7A1 mutation, ranging from mild localized RDEB to the severe Hallopeau-Siemens form. We found a strong genetic association between the 2G variant and the Hallopeau-Siemens disease type (odds ratio: 73.6). This is the first example of a modifier gene in RDEB and has implications for its prognosis and possible new treatments.

Sex-testing using molecular genetic technique is routinely used in the fields of forensics, population genetics and conservation biology. However, none of the assay used so far allows a non-ambiguous and successful sex determination for human and non-human primate species. The most widely used method, AMELY/X, and its alternatives suffer from a set of drawbacks in humans and can rarely be used in New World primate species. Here, we designed a new sex-typing assay using a multiplexed PCR amplification of UTX and UTY-homologous loci and combined male-specific SRY locus. This method was successfully tested on 1048 samples, including 82 non-human primates from 45 Anthropoidea and Lemuriformes species and 966 human samples from 24 populations (Africans, Europeans, and South Americans). This sex-typing method is applicable across all primate species tested from Hominoidea to Indriidae, and also on various populations with different background origins; it represents a robust and cheap sex-typing assay to be used both by the anthropologist and primatologist communities.

International audience; Madagascar is located at the crossroads of the Asian and African worlds and is therefore of particular interest for studies on human population migration. Within the large human diversity of the Great Island, we focused our study on a particular ethnic group, the Antemoro. Their culture presents an important Arab-Islamic influence, but the question of an Arab biological inheritance remains unresolved. We analyzed paternal (n=129) and maternal (n=135) lineages of this ethnic group. Although the majority of Antemoro genetic ancestry comes from sub-Saharan African and Southeast Asian gene pools, we observed in their paternal lineages two specific haplogroups (J1 and T1) linked to Middle Eastern origins. This inheritance was restricted to some Antemoro sub-groups. Statistical analyses tended to confirm significant Middle Eastern genetic contribution. This study gives a new perspective to the large human genetic diversity in Madagascar.

Background Retracing the genetic histories of the descendant populations of the Slave Trade (16th-19th centuries) is particularly challenging due to the diversity of African ethnic groups involved and the different hybridisation processes with Europeans and Amerindians, which have blurred their original genetic inheritances. The Noir Marron in French Guiana are the direct descendants of maroons who escaped from Dutch plantations in the current day Surinam. They represent an original ethnic group with a highly blended culture. Uniparental markers (mtDNA and NRY) coupled with HTLV-1 sequences (env and LTR) were studied to establish the genetic relationships linking them to African American and African populations. Results All genetic systems presented a high conservation of the African gene pool (African ancestry: mtDNA = 99.3%; NRY = 97.6%; HTLV-1 env = 20/23; HTLV-1 LTR = 6/8). Neither founder effect nor genetic drift was detected and the genetic diversity is within a range commonly observed in Africa. Higher genetic similarities were observed with the populations inhabiting the Bight of Benin (from Ivory Coast to Benin). Other ancestries were identified but they presented an interesting sex-bias. Whilst male origins spread throughout the north of the bight (from Benin to Senegal), female origins were spread throughout the south (from the Ivory Coast to Angola). Conclusions The Noir Marron are unique in having conserved their African genetic ancestry, despite major cultural exchanges with Amerindians and Europeans through inhabiting the same region for four centuries. Their maroon identity and the important number of slaves deported in this region have maintained the original African diversity. All these characteristics permit to identify a major origin located in the former region of the Gold Coast and the Bight of Benin; regions highly impacted by slavery, from which goes a sex-biased longitudinal gradient of ancestry.

Two Bolivian samples belonging to the two main Andean linguistic groups (Aymaras and Quechuas) were studied for mtDNA and Y-chromosome uniparental markers to evaluate sex-specific differences and give new insights into the demographic processes of the Andean region. mtDNA-coding polymorphisms, HVI-HVII control regions, 17 Y-STRs, and three SNPs were typed in two well-defined populations with adequate size samples. The two Bolivian samples showed more genetic differences for the mtDNA than for the Y-chromosome. For the mtDNA, 81% of Aymaras and 61% of Quechuas presented haplogroup B2. Native American Y-chromosomes were found in 97% of Aymaras (89% hg Q1a3a and 11% hg Q1a3*) and 78% of Quechuas (100% hg Q1a3a). Our data revealed high diversity values in the two populations, in agreement with other Andean studies. The comparisons with the available literature for both sets of markers indicated that the central Andean area is relatively homogeneous. For mtDNA, the Aymaras seemed to have been more isolated throughout time, maintaining their genetic characteristics, while the Quechuas have been more permeable to the incorporation of female foreigners and Peruvian influences. On the other hand, male mobility would have been widespread across the Andean region according to the homogeneity found in the area. Particular genetic characteristics presented by both samples support a past common origin of the Altiplano populations in the ancient Aymara territory, with independent, although related histories, with Peruvian (Quechuas) populations. Fil: Gayà Vidal, Magdalena. Universidad de Barcelona; España Fil: Moral, Pedro. Universidad de Barcelona; España Fil: Saenz Ruales, Nancy. Université Paul Sabatier; Francia Fil: Gerbault, Pascale. Universite de Toulouse Iii Paul Sabatier; Francia Fil: Tonasso, Laure. Université Paul Sabatier; Francia Fil: Villena, Mercedes. Universidad Mayor de San Andres; Bolivia Fil: Vasquez, René. Universidad Autonoma Tomas Frias; Bolivia Fil: Bravi, Claudio Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; Argentina Fil: Dugoujon, Jean Michel. Université Paul Sabatier; Francia

Recessive dystrophic epidermolysis bullosa (RDEB) is caused by loss-of-function mutations in COL7A1 encoding type VII collagen which forms key structures (anchoring fibrils) for dermal-epidermal adherence. Patients suffer since birth from skin blistering, and develop severe local and systemic complications resulting in poor prognosis. We lack a specific treatment for RDEB, but ex vivo gene transfer to epidermal stem cells shows a therapeutic potential. To minimize the risk of oncogenic events, we have developed new minimal self-inactivating (SIN) retroviral vectors in which the COL7A1 complementary DNA (cDNA) is under the control of the human elongation factor 1alpha (EF1alpha) or COL7A1 promoters. We show efficient ex vivo genetic correction of primary RDEB keratinocytes and fibroblasts without antibiotic selection, and use either of these genetically corrected cells to generate human skin equivalents (SEs) which were grafted onto immunodeficient mice. We achieved long-term expression of recombinant type VII collagen with restored dermal-epidermal adherence and anchoring fibril formation, demonstrating in vivo functional correction. In few cases, rearranged proviruses were detected, which were probably generated during the retrotranscription process. Despite this observation which should be taken under consideration for clinical application, this preclinical study paves the way for a therapy based on grafting the most severely affected skin areas of patients with fully autologous SEs genetically corrected using a SIN COL7A1 retroviral vector.

International audience; More than a decade of mitochondrial DNA (mtDNA) studies have given the 'Polynesian motif' renowned status as a marker for tracing the late-Holocene expansion of Austronesian speaking populations. Despite considerable research on the Polynesian motif in Oceania, there has been little equivalent work on the western edge of its expansion - leaving major issues unresolved regarding the motif's evolutionary history. This has also led to considerable uncertainty regarding the settlement of Madagascar. In this study, we assess mtDNA variation in 266 individuals from three Malagasy ethnic groups: the Mikea, Vezo, and Merina. Complete mtDNA genome sequencing reveals a new variant of the Polynesian motif in Madagascar; two coding region mutations define a Malagasy-specific sub-branch. This newly defined 'Malagasy motif' occurs at high frequency in all three ethnic groups (13-50%), and its phylogenetic position, geographic distribution, and estimated age all support a recent origin, but without conclusively identifying a specific source region. Nevertheless, the haplotype's limited diversity, similar to those of other mtDNA haplogroups found in our Malagasy groups, best supports a small number of initial settlers arriving to Madagascar through the same migratory process. Finally, the discovery of this lineage provides a set of new polymorphic positions to help localize the Austronesian ancestors of the Malagasy, as well as uncover the origin and evolution of the Polynesian motif itself.

The genetic basis of androstenone anosmia has been well studied due to androstenone's putative role as a human sex pheromone and its presence in pork meat. Polymorphisms have been identified on the olfactory receptor gene OR7D4, which significantly affect perception of androstenone pleasantness and intensity in several Western populations. This study aims to investigate androstenone sensitivity and the influence of OR7D4 polymorphisms in non-Western populations. Androstenone perception was tested in 132 individuals from Madagascar using a double three-alternative choice test with two concentrations of androstenone (0.17 and 1.7 µg/ml). We found that Malagasy populations described this molecule in a similar way to European populations, and 21% of the sample was not able to smell androstenone. In contrast to previous studies, there was no significant evidence of the influence of rs61729907: C>T (R88W) and rs5020278: C>T polymorphisms (T133M) on androstenone sensitivity in Malagasy populations. We found, however, a significant effect of the polymorphism rs61732668 (P79L) and a significant difference in androstenone perception between populations in different locations across Madagascar. This study indicates the existence of population-specific factors in androstenone sensitivity, suggesting that population history has a role in shaping an individual's smell and flavor preferences and food preferences in general.

Ultraviolet (UV) radiation from the sun is widely considered as a major cause of human skin photoaging and skin cancer. Granzyme B (GrB) and perforin (PFN) are two proteins contained in granules and implicated in one of the mechanisms by which cytotoxic lymphocytes and natural killer cells exert their cytotoxicity against virus-infected, alloreactive, or transformed cells. The distribution of GrB and PFN in the skin has received little attention. However, Berthou and co-workers (Berthou, C., Michel, L., Soulie, A., Jean-Louis, F., Flageul, B., Dubertret, L., Sigaux, F., Zhang, Y., and Sasportes, M. (1997) J. Immunol. 159, 5293-5300) described that, whereas freshly isolated epidermal cells did not express GrB or PFN, keratinocyte growth to confluence was associated with GrB and PFN mRNA and protein synthesis. In this work, we have investigated the possible role of UV-B on GrB and PFN expression in keratinocytes. We found that UV-B induces GrB and PFN expression in these cells through redox-, epidermal growth factor receptor-, and mitogen-activated protein kinase-dependent signaling. Furthermore, under UV irradiation, keratinocytes acquire a significant cytotoxicity, which is GrB and PFN dependent, toward a variety of cellular targets including transformed T-lymphocytes, melanocytes, and keratinocytes. This phenomenon may have important functional consequences in the regulation of skin inflammatory response and in the emergence of cancer skin.

Loss-of-function mutations in the gene encoding type VII collagen, COL7A1, are the molecular basis of the blistering skin disorder, recessive dystrophic epidermolysis bullosa (RDEB). COL7A1 maps to a region of the short arm of chromosome 3 that has been found to be deleted in many types of malignancies. We have characterized the first case of a large genomic deletion in chromosome 3p21.31 that removes COL7A1 entirely in an RDEB patient. This interstitial deletion spans 255 to 520 kb and encompasses 9 to 15 genes, but seems to have no pathological consequences other than RDEB. We show that the second, hemizygous allele of COL7A1 in this patient bears a base substitution within exon 94, c.7245G>A. This translates into an amino acid substitution, p.M2415I, and leads to a complex splicing abnormality that allows marginal levels of functional mRNA and protein to be synthesized. We propose that the leakiness of the splicing defect enables the partial rescue of collagen VII deficiency. This is consistent with the diagnosis of the moderately severe form of RDEB in the proband, at variance with the most severe form, RDEB Hallopeau-Siemens, that would arise from complete collagen VII deficiency. © 2006 Wiley-Liss, Inc.

Chk2 is a key player of the DNA damage signalling pathway. To identify new regulators of this kinase, we performed a yeast two-hybrid screen and found that Chk2 associated with the B′ regulatory subunit of protein phosphatase PP2A. In vitro GST-Chk2 pulldowns demonstrated that B′γ isoforms bound to Chk2 with the strongest apparent affinity. This was confirmed in cellulo by co-immunoprecipitation after overexpression of the respective partners in HEK293 cells. The A and C subunits of PP2A were present in the complexes, suggesting that Chk2 was associated with a functionnal PP2A. In vitro kinase assays showed that B′γ3 was a potent Chk2 substrate. This phosphorylation increased the catalytic phosphatase activity of PP2A measured on MAP kinase-phosphorylated myelin basic protein as well as on autophosphorylated Chk2. Finally, we demonstrated that overexpressing B′γ3 in HEK293 suppressed the phosphorylation of Chk2 induced by a genotoxic treatment, suggesting that PP2A may counteract the action of the checkpoint kinase in living cells.

Until the identification of ABCA12 as the causative gene, prenatal diagnosis (PD) for harlequin ichthyosis (HI) had been performed by electron microscopic observation of fetal skin biopsy samples. We report the first case of HI DNA-based PD. Direct sequence analysis of ABCA12 revealed that the deceased proband was a compound heterozygote for two novel mutations. The maternal nonsense mutation p.Ser1249Term likely leads to nonsense-mediated messenger RNA decay. The paternal mutation c.7436G>A affects the last codon of exon 50 and was expected to be a splice site mutation. For their third pregnancy, the parents requested PD. Direct sequence analysis of fetal genomic DNA from amniotic fluid cells at 17 weeks gestation revealed the fetus was a compound heterozygote for both mutations. The parents requested the pregnancy to be terminated. Analysis of ABCA12 transcripts of cultured keratinocytes from the abortus showed the presence of six abnormally spliced products from the allele carrying the splice site mutation. Four of them lead to premature termination codons whereas the two others produced shortened proteins missing 21 and 31 amino acids from the second ATP-binding cassette. This report provides evidence for residual ABCA12 expression in HI, and demonstrates the efficiency of early DNA-based PD of HI.