Corentin Bochaton
email: corentin.bochaton@mnhn.fr

Vivian De Buffrénil

Michel Lemoine

Salvador Bailon

Ivan Ineich

Body location and tail regeneration effects on osteoderms morphologyare they useful tools for systematic, paleontology and skeletochronology in diploglossine lizards (Squamata, Anguidae)?

Keywords: Aging, Anatomy, Galliwasp, Histology, Osteology

Although diploglossine osteoderms were mentioned in several systematic and paleontological studies, their morphological variability in single specimens or within species remains paradoxically undescribed. This is mainly the effect of the lack of attention paid hitherto to the morphological and histological characteristics of the tail osteoderms. This study demonstrated Bochaton 2 that a previously undescribed morphological variability exists in these osteoderms, especially in those resulting from tail regeneration. Indeed, regenerated osteoderms display a plesiomorphic anguid morphology that was previously considered to be absent in Diploglossinae. We also provide the first histological description of diploglossine osteoderms and new information about the obvious differences in growth dynamic between regenerated and non-regenerated osteoderms.

These new data raise questions about the usefulness of diploglossine osteoderms in systematic, paleontological and skeletochronological studies. Our study shows that the exact position on the trunk or on a regenerated or non-regenerated tail of each studied osteoderm must be known in order to avoid mistakes related to their important inter-and intraspecies variability.

Introduction

Several squamate families are known to present bony plates, osteoderms, that occur within the dermis under epidermal horny scales on the head or the whole body [START_REF] Moss | Comparative histology of dermal sclerifications in reptiles[END_REF][START_REF] Vickaryous | The integumentary skeleton of tetrapods: origin, evolution, and development[END_REF]. Among these families, members of the clade Anguidae display osteoderms on most of their head and body. These osteoderms differ from those of Scincidae by being composed of a single element while scincid osteoderms are compound [START_REF] Hoffstetter | Observation sur les ostéodermes et la classification des anguidés actuels et fossiles (Reptiles, Sauriens)[END_REF]. Among the anguids, two distinct types of osteoderms were described [START_REF] Hoffstetter | Observation sur les ostéodermes et la classification des anguidés actuels et fossiles (Reptiles, Sauriens)[END_REF]. The first is mainly encountered in gerrhonotines (Gerrhonotinae + Anguinae sensu [START_REF] Pyron | A phylogeny and revised classification of Squamata, including 4161 species of lizards and snakes[END_REF] whose members bear a rigid armor formed of osteoderms strongly linked to each other. In this case, osteoderms are beveled along their lateral edges, which increases contact surfaces between osteoderms and thus, strengthens the shield that they form [START_REF] Hoffstetter | Observation sur les ostéodermes et la classification des anguidés actuels et fossiles (Reptiles, Sauriens)[END_REF][START_REF] Meszoely | North American Fossil Anguid Lizards[END_REF].

The second type of osteoderms is encountered in Diploglossinae (sensu [START_REF] Pyron | A phylogeny and revised classification of Squamata, including 4161 species of lizards and snakes[END_REF] and in Anguis Linnaeus, 1758, the armor of which is formed of rounded, non-beveled osteoderms allowing shield flexibility [START_REF] Hoffstetter | Observation sur les ostéodermes et la classification des anguidés actuels et fossiles (Reptiles, Sauriens)[END_REF][START_REF] Meszoely | North American Fossil Anguid Lizards[END_REF]. This last condition is supposed to be apomorphic within Anguidae (Meszoely1970). In addition, osteoderm morphology in the three diploglossine genera (Celestus Gray, 1839;Diploglossus Wiegmann, 1834 andOphiodes Wagler, 1828) has been especially well described and studied by [START_REF] Strahm | Osteoderms in the anguid lizard subfamily diploglossinae and their taxonomic importance[END_REF] who proposed a character for the distinction of Diploglossus and Celestus: the occurrence of a cloudlike radix system of nutritive canals under the gliding surface of the osteoderms which is visible without special preparation due to the thinness of the osteoderm. As a consequence osteoderms were commonly used in anguid systematics [START_REF] Hoffstetter | Observation sur les ostéodermes et la classification des anguidés actuels et fossiles (Reptiles, Sauriens)[END_REF][START_REF] Strahm | Osteoderms in the anguid lizard subfamily diploglossinae and their taxonomic importance[END_REF][START_REF] Gauthier | Fossil xenosaurid and anguid lizards from the early Eocene Wasatch Formation, southeast Wyoming, and a revision of the Anguioidea[END_REF][START_REF] Mead | Pleistocene lizards (Reptilia: Squamata) from San Josecito cave, nuevo leon, Mexico[END_REF].

However, the taxonomic significance of anguid osteoderms suffers obvious limitations because it refers exclusively to trunk osteoderms and takes no account of osteoderm morphology from other regions of the body. Moreover, it is commonly known that anguids, like other lizards, can regenerate their autotomized tail [START_REF] Bryant | Tail regeneration in the lizards Anguis fragilis and Lacerta dugesii[END_REF][START_REF] Guibé | L'autotomie et la régénération[END_REF], and that scale morphology may strongly differ between regenerated and non-regenerated parts of the tail in several taxa [START_REF] Woodland | Some observations on caudal autotomy and regeneration in the gecko (Hemidactylus flaviviridis Rüppell), with notes on the tails of Sphenodon and Pygopus[END_REF][START_REF] Bustard | Australian lizards[END_REF], including Anguis [START_REF] Ali | Studies on the anatomy of the tail in sauria and rhynchocephalia IV. Anguis fragilis[END_REF][START_REF] Bryant | Tail regeneration in the lizards Anguis fragilis and Lacerta dugesii[END_REF]. In Anguidae, osteoderm histology and ultrastructure were described in detail in Anguis fragilis Linnaeus, 1758 by [START_REF] Zylberberg | New data on the structure and the growth of the osteoderms in the reptile Anguis fragilis L. (Anguidae, Squamata)[END_REF], the same authors also described osteoderms from other taxa, for instance in the gecko Tarentola (Levrat-Calviac and [START_REF] Levrat-Calviac | The structure of the osteoderms in the gekko: Tarentola Mauritanica[END_REF]; however, possible peculiarities of regenerated osteoderms were not considered. As a consequence, potential intraspecific variability in anguid osteoderm morphology and structure remains largely unknown, a situation that can be especially problematic in paleontology.

The aim of this study is to further document intra-individual and intra-generic variability of osteoderm morphology and structure in diploglossine lizards. Our intention is to consider the influence of growth and regeneration on these characteristics, and assess the final relevance of osteoderm description in systematic and paleontological studies.

Materials and Methods

The biological sample comprised 82 anguid osteoderms collected from 17 specimens and including 52 diploglossine osteoderms and 30 osteoderms from other subfamilies (Gerrhonotinae -genera Barisia Gray, 1838;Gerrhonotus Wiegmann, 1828and Elgaria Gray, 1838-and Anguinae -genera: Ophisaurus Daudin, 1803 and Anguis-).

Most of the diploglossine osteoderms (36) were directly extracted from six dry or alcohol (MCZ 131518), and Ophiodes striatus (Spix, 1824) (SMF 99342). Osteoderm sampling was made with a scalpel and the position of each osteoderm on the body was recorded. Since the morphology of trunk osteoderms was already well-known [START_REF] Strahm | Osteoderms in the anguid lizard subfamily diploglossinae and their taxonomic importance[END_REF] we only sampled two trunk osteoderms on each specimen in order to check previously published observations. These osteoderms were all extracted on the lateral part of the trunk just behind the forelimb. In order to take account of the impact of regeneration processes on osteoderm morphology we first checked the regenerated or non-regenerated condition of the studied tail parts on each alcohol preserved specimens using the MECADEV (CNRS-MNHN) radiography platform. Our observations showed that these six specimens had regenerated their tail to variable extents. For some of them this condition (regenerated tail) could not be assessed on the sole basis of external tail morphology and scale pattern. Consequently, osteoderms were extracted from the proximal (non-regenerated) and distal (regenerated) thirds of the lateral part of the tail on the basis of their x-ray images. Additional osteoderms from the mid-length of the tail (nonregenerated on D. monotropis and regenerated on C. occiduus) were collected only on the MNHN-RA specimens. Our D. plei specimen (MCZ 131518) was a dry skeleton an only regenerated tail and some trunk osteoderms were observable. In the alcohol preserved specimens two osteoderms were removed in each of the three of four sampled body parts in order to cross-section one of them for histological observations. Consequently, among the sampled osteoderms, 16 were photographed before being embedded in a polyester resin, sectioned along their transversal axes, mounted on a glass slide and then polished to obtain ground sections some 100 µm thick. The slides were observed with a compound microscope in natural and polarized light. Unfortunately, we were unable to crosssection all the other sampled osteoderms that were already mounted on microscope slides during the studies of R. [START_REF] Hoffstetter | Observation sur les ostéodermes et la classification des anguidés actuels et fossiles (Reptiles, Sauriens)[END_REF].

The nomenclature used follows the terminologies defined by [START_REF] Buffrénil | The histological structure of glyptosaurine osteoderms (Squamata: Anguidae), and the problem of osteoderm development in Squamates[END_REF] and [START_REF] Strahm | Osteoderms in the anguid lizard subfamily diploglossinae and their taxonomic importance[END_REF]. Following these terms, the osteoderms present a superficial (=external sensu [START_REF] Strahm | Osteoderms in the anguid lizard subfamily diploglossinae and their taxonomic importance[END_REF] surface and a basal (=internal sensu [START_REF] Strahm | Osteoderms in the anguid lizard subfamily diploglossinae and their taxonomic importance[END_REF] surface. This superficial surface is divided into an anterior portion called the gliding surface that lie below the osteoderm of the previous row and a posterior portion that is connected to the apex of the epidermal scale (Fig. 1: A).

Results

External morphology

Non-regenerated osteoderms Trunk osteoderms (Fig. 1; Table . 1) External morphology of trunk osteoderms is similar to that described in previous studies: they are rounded in superficial view, flat in transversal section (Fig. 1: F), thin (thickness/length ratio is between 5 to 9%), and display a crescentic gliding surface with a posterior projection, but lack beveled lateral edges [START_REF] Hoffstetter | Observation sur les ostéodermes et la classification des anguidés actuels et fossiles (Reptiles, Sauriens)[END_REF][START_REF] Strahm | Osteoderms in the anguid lizard subfamily diploglossinae and their taxonomic importance[END_REF]. As previously reported, Ophiodes and Diploglossus osteoderms have a "cloud-like" radix system on their anterior portion or gliding surface which is absent in Celestus [START_REF] Strahm | Osteoderms in the anguid lizard subfamily diploglossinae and their taxonomic importance[END_REF]. This character has been considered as ontogenetically variable [START_REF] Wilson | Distributional and taxonomic comments on some members of the Honduran herpetofauna[END_REF], which is in agreement with our observations because radix system on our smallest specimen of Diploglossus is less developed (D. delasagra MNHN-RA 2859).

Osteoderms of the proximal part of the tail (Fig. 2; Table . 1) Osteoderms of the proximal third of the tail display slight differences with those from the trunk: they are less flat in transversal section, more transversally arched (Fig. 2: F) and thicker (thickness/length ratio between 9 to 13%). Additional differences occur in Celestus where the proximal third of tail osteoderms bear a cloud-like radix system that does not occur on trunk osteoderms. Whereas in Ophiodes these osteoderms are slender than trunk osteoderms, and also present a reduction of their gliding surface, a smaller posterior projection and a less developed radix system.

Osteoderms of the mid-length of the tail (Fig. 3; Table . 1) Because most of our specimens have a regenerated tail forming more than two-thirds of the full length of the tail we could only observe osteoderms from the mid-length of a non-regenerated tail on two specimens (D. monotropis -MNHN-RA 1906.0229-and C. bivittatus). These osteoderms present different morphologies. The D. monotropis osteoderm (Fig. 3: A) has a subcircular shape in superficial view, very similar to the more proximal ones, but is divided into two smaller osteoderms of rectangular shape only partially fused together in superficial view that are nested together by two small hooks on the basal side of the osteoderm. This division may represent the transition from an ovoid shape for the proximal ones to a rectangular shape for the distal ones but unfortunately, morphology of the non-regenerated distal osteoderms remains unknown to us. The C. bivittatus osteoderm (Fig. 3: B) is even more distinct. Its general shape is strongly arched in transversal section and it presents an important reduction of its gliding surface that is void of radix system and posterior projection. Both these osteoderms are thin (thickness/length ratio of 7 and 9%, respectively).

Regenerated osteoderms

Osteoderms of the mid-length of the tail (Fig. 4; Table . 1) In three of our specimens (D. monotropis -SMF 93787-, D. plei and C. occiduus) the second third of the tail was regenerated. The morphology of these osteoderms is very different from that previously described since they bear a pit-like ornamentation on their posterior portion and are all beveled along their lateral edges. This last character shows that these osteoderms are more tightly imbricated and so form a less flexible shield than the osteoderms of the trunk and nonregenerated part of the tail. This condition is considered as plesiomorphic within Anguidae [START_REF] Meszoely | North American Fossil Anguid Lizards[END_REF] and was absent in the non-regenerated osteoderms. However, their morphology is still highly variable. In MCZ 131518 (D. plei) and MNHN-RA 1227 (C. occiduus) (Fig. 4: A & B) the osteoderms are oval in superficial view, and thicker than non-regenerated ones (thickness/length ratio between 17 to 25%). In SMF 93787 (D. monotropis) (Fig. 4: C), the osteoderms seem to result from the fusion of two rectangular osteoderms (as shown by the presence of a superficial furrow on their longitudinal axe) to form simple osteoderms beveled along their lateral edges. However, histological observations reveal that this osteoderm formed as a whole, in one piece and that it was probably newly formed (see below: Table . 1); its morphology may thus reflect an early developmental stage.

Osteoderms of the distal part of the tail (Fig. 5; Table 1) Two clearly different osteoderm morphotypes were observed in the distal third of regenerated tails of our specimens. The first one, in D. plei and C. occiduus, is strictly similar to the osteoderms previously described in the mid-length of the tail of the same specimens (Fig. 5: A

& B).
The second type was observed in D. monotropis, C. bivittatus and O. striatus (Fig. 5: C-F). These osteoderms are rectangular instead of ovoid in superficial view, beveled on their medial or lateral sides and their posterior portion is ornamented with ridges or small pits. They are thin (thickness/length ratio between 7 to 13%) and lack "cloud-like" radix system on their gliding surface. This gliding surface is broad in the largest D. monotropis, but proportionally much smaller in all the other specimens, which suggest that this character could be ontogenetically variable.

The two morphotypes do not reflect ontogenetic variability because both are present in "adult" osteoderms (see below). They could possibly be linked to osteoderm position on the tail portion that was regenerated after autotomy, but this remains hypothetical.

Osteoderm histology

Non-regenerated osteoderms All non-regenerated osteoderms from the trunk and tail display a similar histological structure.

They are made of three distinct tissue types, located, respectively, in superficial and basal regions of the osteoderms, and in the core the osteoderms (Fig. 6):

1) The superficial cortex is composed of a tissue type intermediate between parallel-fibered and lamellar bone (Fig. 7 cortex a) and is the thinnest cortex of the osteoderm (less than one fifth of the osteoderms' maximal thickness) (Fig. 7: A). In polarized and natural light this cortex shows an alternation of very thin dark and slightly thicker light strata decreasing in thickness from cortical depth to the periphery of the osteoderms. A similar structure but with far thicker strata is also observed in the deep cortex of the osteoderms and will further described below. Some flat osteocyte lacunae occur in this tissue. The outer surface of this cortex displays evidence of extensive superficial resorption and, in four of our five specimens, this cortex was either partly or entirely eroded (Fig. 7: D). This resorptive process is not followed by secondary reconstruction deposits.

2) The core of each osteoderm is composed of a thin cortex of woven-fibered bone (Fig. 7 cortex b) the thickness of which represents less than one fourth of osteoderms' maximal thickness (Fig. 7: A). Remnants of primary bone tissue in this cortex are monorefringent in polarized light and show high density of rounded osteocyte lacunae, distributed unevenly within the bone matrix. This tissue also displays evidence of strong remodeling, in the form of numerous large resorption bays, the walls of which are partly reconstructed by secondary endosteal deposits of lamellar bone tissue, separated from primary deposits by resorption lines (Fig. 7: B (arrows)). This central bone cortex is bordered in upper (superficial) and lower (basal) positions by two quite distinct bone formations.

3) The deep (basal) cortex (basal plate sensu [START_REF] Buffrénil | The histological structure of glyptosaurine osteoderms (Squamata: Anguidae), and the problem of osteoderm development in Squamates[END_REF] (Fig. 7 cortex c) is composed of parallel-fibered bone extending on one third to one half of osteoderms' maximal thickness (Fig. 7: A). As in the superficial cortex, this cortex shows in polarized and natural light an alternation of thin dark and thicker light strata. Decrease in thickness of these strata from cortical depth (where they are some 20 µm thick) to periphery (where stratum thickness is less than 10 µm) indicates that they represent cyclic growth marks (later designated as CGMs) rather than bone lamellae (Fig. 7: C). These strata also display an irregular birefringence in polarized light, also supporting this conclusion. The more important thickness of these CGMs, as compare to these observed in the superficial cortex, indicate that bone accretion over the basal cortex was more active than on the superficial cortex (Fig. 7: B). In both cortexes (superficial and basal) osteocyte lacunae are flattened and less densely distributed than in the core region. Their morphology and their occurrence in both light and dark strata, with no change in their orientation, are further evidence in favor of the parallel-fibered bone tissue type. Many obliquely oriented Sharpey's fibers (collagen fibers that anchor the osteoderm to outer adjacent structures -Francillon-Vieillot et al., 1989-) of variable length cross this cortex (Fig. 7: C).

The precise number of CGMs in each osteoderm was uneasy to assess because the deep cortical cortexes was often resorbed. Despite these limitations, the number of counted growth lines was relatively stable in the non-regenerated osteoderms of a single specimen (Table 2). The maximum number of CGMs observed in the basal plate of non-regenerated osteoderms, 13 CGMs, was consistent with a yearly deposit of these marks since available data attribute a maximal longevity more than 12.3 years for Diploglossus and Celestus (de [START_REF] Magalhaes | A database of vertebrate longevity records and their relation to other life-history traits[END_REF][START_REF] Henderson | Natural History of West Indian Reptiles and Amphibians[END_REF]. In addition, the specimen presenting the highest number of CGMs, D. monotropis (SMF 93787), was kept in captivity until its death and so may had reached an advanced age. Therefore the number of CGMs in the basal cortex was likely to correspond to the age, in years, of the animal (one CGM by year).

In the osteoderms of SMF 93787 (D. monotropis) (Fig. 7: A-E) the deep cortex is superficially bordered by an additional cortex of similar structure but with blurred cyclic growth marks and lesser birefringence (Fig. 7 cortex d). Some broadly spaced CGMs can nevertheless be observed, together with densely-packed bundles of Sharpey's fiber obliquely orientated. The density of Sharpey's fibers could indicate a preferential anchoring area, and suggests the local occurrence of a particular mechanical context. Moreover, the broad spacing of the he CGMs is likely to reflect a local resumption of growth (possibly due to traction of Sharpey's fibers?) after a previous stage of growth slackening.

In the lateral parts of the osteoderms the CGMs of the basal plate are in continuity with those of the superficial cortex, a situation suggesting that a unique, continuous osteogenetic process, creating the superficial cortex, occurs all around the osteoderm.

With reference to the histological observations developed above, the growth of the osteoderms can be reconstructed as follows: Their oldest part (core of osteoderms), composed of wovenfibered tissue, could be produced by a metaplastic process that directly transforms the dermis into bone, as it occurs in lepidosaurian [START_REF] Zylberberg | New data on the structure and the growth of the osteoderms in the reptile Anguis fragilis L. (Anguidae, Squamata)[END_REF][START_REF] Buffrénil | An enamel-like tissue, osteodermine, on the osteoderms of a fossil anguid (Glyptosaurinae) lizard[END_REF] and crocodylian [START_REF] Vickaryous | Development of the dermal skeleton in Alligator mississippiensis (Archosauria, Crocodylia) with comments on the homology of osteoderms[END_REF] osteoderms. After this initial stage, classical osteoblastic osteogenesis takes place, and produces the deep and superficial cortices. Cortical growth is clearly more active on the basal osteoderm surface, as shown by the spacing of the CGMs.

Regenerated osteoderms

Regenerated osteoderms display a histological organization very similar to the non-regenerated osteoderms yet with some differences including the occurrence of a fourth type of tissue in the largest osteoderms (Fig. 8). Their histological structure is also more variable because they may represent different growth stages, from those bearing no basal plate and only a woven-fibered core (Fig. 9: A) to some presenting a thick basal plate with numerous (most likely non-annual; see below) CGMs (Fig. 9: B). The four tissues encountered are the following:

1) The superficial cortex (Fig. 9 cortex a) of regenerated osteoderms present some differences with the superficial cortex of non-regenerated osteoderms. In the regenerated osteoderms, this cortex is thicker, the CGMs are wider and their number more variable, and osteocytes lacunae are far more numerous (Fig. 9: C) than in the non-regenerated osteoderms. In addition, unlike in the non-regenerated osteoderms, no strong resorption of this cortex was observed.

2) All regenerated osteoderms present a core of woven-fibered bone (Fig. 9 cortex b) strictly similar to that of non-regenerated osteoderms (Fig. 9: B-E). The difference is that this cortex is thicker since it can occupy more than half of the total thickness on the smaller osteoderms to one third on the large osteoderms.

3) The basal plate (Fig. 9 cortex b) is also similar to that of non-regenerated osteoderms. Its thickness is highly variable: thin in the small osteoderms with no clearly distinct CGMs (Fig. 9: A) and thick in larger osteoderms displaying many CGMs (Fig. 9: B). The main difference with non-regenerated osteoderms is about the number of growth lines. In the largest osteoderms the number of CGMs is significantly higher (up to a maximum of 25) than in the non-regenerated osteoderms of the same specimen (Fig. 9: B, Table 2); conversely, the number of growth marks is smaller in the smallest regenerated osteoderms (Fig. 9: A, C, Table 2). Unlike the situation of non-regenerated osteoderms, no cortex possibly related to growth resumption was observed. 4) On the biggest regenerated osteoderms (MNHN-RA 1906.0229, D. monotropis; and MNHN-RA 1227, C. occiduus) a fourth type of tissue occurs in the lateral margins of the sections (Fig. 9: D). This tissue displays irregular birefringence, a high density of rounded osteocytes lacunae and numerous, long Sharpey's fibers parallel to each other and nearly perpendicular to the orientation of the basal plate Sharpey's fibers. It represents woven-fibered tissue with extremely dense bundles of anchorage (Sharpey's) fiber bundles (Sharpey's fiber bone). It was not observed in the non-regenerated osteoderms but is similar to that described by [START_REF] Buffrénil | The histological structure of glyptosaurine osteoderms (Squamata: Anguidae), and the problem of osteoderm development in Squamates[END_REF] in the same area of glyptosaurine osteoderms. The occurrence of this tissue may be linked to the fact that all these osteoderms are beveled along their lateral edges and strongly linked to each other by sharpey's fiber. Thus its occurrence could also be an anguid plesiomorphic character.

Discussion

Our results clearly show that intra-individual and intra-generic morphological variability of diploglossine osteoderms is much greater than previously supposed. We reported morphology of regenerated and non-regenerated osteoderms of the tail and pointed out a probable reappearance of plesiomorphic anguid characters in regenerated osteoderms.

Morphological variability of diploglossine osteoderms

We reported a previously unknown intra-individual morphological variability in diploglossine osteoderms. Non-regenerated osteoderms from the trunk display all the characteristic features already described in literature; conversely tail osteoderms deviate from this morphology depending of their position on the tail. Osteoderms of the proximal third of the tail are similar to those from the trunk but less flat, more transversally arched and slightly thicker. This character should thus be re-defined and restricted as only applicable to trunk osteoderms. As previously reported by [START_REF] Wilson | Distributional and taxonomic comments on some members of the Honduran herpetofauna[END_REF], the expansion of the radix system seem partly subject to ontogenetic variability and was discarded by [START_REF] Savage | A new species of Celestus from west-central Panama, with consideration of the status of the genera of the Anguidae: Diploglossinae (Squamata)[END_REF] for this reason. However, no radix at all occurs on trunk osteoderms of Celestus specimens we observed (Fig. 1). Thus, we disagree with [START_REF] Savage | A new species of Celestus from west-central Panama, with consideration of the status of the genera of the Anguidae: Diploglossinae (Squamata)[END_REF] and believe that this character can still be useful if carefully used.

Osteoderms from the mid part of the tail present two different morphologies that strongly differ from the morphology of the trunk osteoderms. On D. monotropis (MNHN-RA 1906.229), the shape of these mid tail osteoderms is similar to the others osteoderms from trunk and proximal third of the tail but it's formed of two fused rectangular osteoderms linked by a nip on their basal side (Fig. 3: A). This could indicate the occurrence of osteoderm of different morphology in the more distal part of the non-regenerated tail but since all were regenerated in our material we were unable to observe it. The second morphology observed on C. bivittatus (SMF 79022) was even more different, the general shape was less rounded, the gliding surface reduced, the osteoderm is transversally arched rather than flat, and lack the cloud-like radix system and the posterior projection characteristic of Diploglossinae that occurred on all the others non-regenerated osteoderms (Fig. 3: B). This Celestus osteoderm did not bear any of the diploglossine characters except that it is not beveled along their lateral edges.

Histologically, all non-regenerated osteoderms present the same basic organization with three different types of tissues: a core of woven fibered bone, a basal plate of parallel-fibered bone presenting CGMs, and an superficial cortex composed of an intermediate tissue type between parallel-fibered and lamellar bone (Fig. 6 and7). A similar organization was observed in glyptosaurine osteoderms [START_REF] Buffrénil | An enamel-like tissue, osteodermine, on the osteoderms of a fossil anguid (Glyptosaurinae) lizard[END_REF] but their basal plate is made of lamellar bone and their superficial cortex includes a hyper-mineralized tissue that does not occur in diploglossines. Both these organizations differ from that of another anguid osteoderms: Anguis fragilis [START_REF] Zylberberg | New data on the structure and the growth of the osteoderms in the reptile Anguis fragilis L. (Anguidae, Squamata)[END_REF] that is simpler with only two types of tissue: a superficial cortex of woven-fibered bone and a basal plate of parallel fibered bone. These facts suggest that this simple organization could be an Anguis apomorphic character. However, diploglossine non-regenerated osteoderms share the absence of the Sharpey's fiber bone and a parallel-fibered basal plate with Anguis fragilis osteoderm, two characters that do not occur in glyptosaurine osteoderms.

Regenerated osteoderms are morphologically highly variable because they reflect different growth stages and positions on the tail (Fig. 4 and5). However, clear general differences with the non-regenerated osteoderms can be noted. They tend to be thicker and bear ornamentation formed of small punctures on their posterior portion. But the most striking difference is that the regenerated osteoderms are beveled along one or both of their lateral edges. Such morphology, indicative of a strong link between adjacent osteoderms, was not known to occur in diploglossine anguids. This strong link is histologically represented in our diploglossine osteoderms by a tissue very rich in Sharpey's fiber (Sharpey's fiber bone) occurring in the lateral borders of the osteoderms, that is absent in the non-regenerated osteoderms (Fig. 8 and9). This type of tissue was previously reported in other squamates [START_REF] Levrat-Calviac | The structure of the osteoderms in the gekko: Tarentola Mauritanica[END_REF][START_REF] Buffrénil | The histological structure of glyptosaurine osteoderms (Squamata: Anguidae), and the problem of osteoderm development in Squamates[END_REF][START_REF] Buffrénil | An enamel-like tissue, osteodermine, on the osteoderms of a fossil anguid (Glyptosaurinae) lizard[END_REF] but also in the xenarthan mammal Dasypus novemcinctus Linnaeus, 1758 by [START_REF] Vickaryous | Osteoderm morphology and development in the nine-banded Armadillo, Dasypus novemcinctus (Mammalia, Xenarthra, Cingulata)[END_REF]. The diploglossine body is entirely covered with osteoderms that usually lack such links that allow sliding between them and the movement of the animal (Hoffseteter, 1962). This condition is considered as apomorphic in regard of the plesiomorphic condition encountered in Gerrhonotinae [START_REF] Meszoely | North American Fossil Anguid Lizards[END_REF]. The occurrence of osteoderms beveled along their lateral edges in the regenerated diploglossine tail is consequently very surprising and seems to be a reversion to a more ancestral condition (atavism) after regeneration. [START_REF] Barbour | The squamation of Homoeosaurus[END_REF] hypothesized a similar phenomenon when comparing the scaling pattern of the regenerated tail of modern Sphenodon Gray, 1831 to scaling pattern of the fossil sphenodontid Homeosaurus maximiliani Wagner, 1853. In Ophisaurus gracilis (Gray, 1845), [START_REF] Boulenger | On the Scaling of the reproduced tail in lizards[END_REF] observed the occurrence of quadrangular scales on the trunk but cycloid scales on the regenerated tail. No observations were made about the morphology of the regenerated osteoderms from this last species but the shape of the regenerated scales could match with the regenerated osteoderms we described in Diploglossinae but could also be similar to the nonregenerated osteoderms of this family. Further investigations are needed to clearly assess the phylogenetic significance of these differences between regenerated and non-regenerated osteoderms.

The use of osteoderms in systematic and paleontology studies Many characters are traditionally used to separate anguid genera by their osteoderms (Hofftetter, 1962;[START_REF] Meszoely | North American Fossil Anguid Lizards[END_REF] including thickness, length/width ratio, expansion and shape of the gliding surface and, the most important, the occurrence or absence of a lateral bevel. However, this study points out that these characters are prone to intra-individual and intra-generic variability depending of the position, age and regenerated or non-regenerated condition of the osteoderms. These biases can be easily avoided by using trunk osteoderms of adult specimens.

Observation of the osteoderms used in some classical systematic studies (Hofftetter, 1962;[START_REF] Meszoely | North American Fossil Anguid Lizards[END_REF]Strahm and Schwatz, 1977), reveals that only trunk osteoderms of adult specimens were taken into account. That is why we do not question the reliability of the preexisting characters established by these studies. However, this question becomes far more problematic in paleontological studies when isolated osteoderms of unknown body localization are considered. Fortunately, our result does not demonstrate that such osteoderms cannot be used at all, and we found that combination of characters still allow a reliable identification of diploglossine osteoderms.

-diploglossine osteoderms from trunk and proximal part of the tail: rounded shape, flat, not beveled along their lateral edges, present a crescent shape gliding surface with a posterior peak.

Celestus: absence of a cloud-like radix system on the gliding surface. We believe that the distinction between osteoderms from the trunk and proximal part of the tail is difficult and thus we only consider here characters applicable to both of them. The other diploglossine genera (Diploglossus and Ophiodes) cannot be distinguished on the basis of morphology of isolated osteoderms. These osteoderms are similar to Anguis osteoderms but Anguis lack posterior projection of the gliding surface.

-diploglossine osteoderms from regenerated tail: ovoid shape never clearly rectangular, gliding surface broadly extended on one of both of the lateral margins and never of band appearance, beveled on one of both of the lateral edges, posterior portion ornamented with small ridges or pits, possible occurrence of a posterior projection and lack of medial keel. However, some Ophisaurus osteoderms presents a morphology that cannot be distinguished from diploglossine regenerated osteoderm.

Consequently, although some combinations of characters seem still typical of diploglossines, the important morphological variability of regenerated osteoderms makes their identification complicated because they can provide ambiguous taxonomic conclusions. Therefore, as previously suggested for other taxa [START_REF] Hill | New data on the anatomy and relationships of the Paleocene crocodylian Akanthosuchus langstoni[END_REF][START_REF] Burns | Taxonomic Utility of Ankylosaur (Dinosauria, Ornithischia) Osteoderms: Glyptodontopelta mimus Ford, 2000: A Test Case[END_REF], we recommend avoiding the use of isolated osteoderms (not located on a particular body part) in systematic studies and especially in paleontological studies.

Use of osteoderms in skeletochronology

We observed that, in polarized light, the basal plate shows an alternation of light and dark strata progressively decreasing in thickness towards bone periphery. Such deposits are undoubtedly cyclic growth marks reflecting cyclical decreases in individual growth rate but the histological variability of the osteoderms raises question about the possibility of their use for estimating the age of the specimen using skeletochronology [START_REF] Castanet | L'enregistrement de la croissance cyclique par le tissu osseux chez les vertébrés poïkilothermes: données comparatives et essai de synthèse[END_REF][START_REF] Castanet | Les marques de croissance osseuse comme indicateurs de l'âge chez les lézards[END_REF]. Our observations show that the number of growth lines is roughly stable in the non-regenerated osteoderms of a single specimen (Table 2) and that they could possibly be yearly deposits. Nonregenerated osteoderms seem therefore potentially useful for skeletochronology. However, the number of CGMs observed in regenerated osteoderms is every time very different depending of the "maturity" of the regenerated osteoderm (Table . 2). In addition, our observations show a clear difference in growth cyclicality between non-regenerated and regenerated osteoderms. Taking account of this bias, it is clear that regenerated osteoderms cannot in any manner be used to estimate the age of a specimen.

To conclude, our results show that diploglossine osteoderms display an important morphological variability at both intra-individual and intra-specific levels according to their location on the body (trunk, non-regenerated tail or regenerated tail) and age. Tail regeneration strongly modifies the morphology of diploglossine osteoderms that present a plesiomorphic anguid condition after regeneration that is absent in non-regenerated ones. These new data make the use of osteoderms in systematic and paleontological studies subject to caution when a doubt exists about their exact localization on the body and about their regenerated or non-regenerated condition. We have also shown that regenerated and non-regenerated tail osteoderms probably do not follow the same growth rates and that the application of skeletochronology should be limited to non-regenerated osteoderms.

Acknowledgment

  preserved museum specimens belonging to the Muséum national d'Histoire naturelle (MNHN-RA, Paris, France), the Senckenberg Museum of Frankfurt (SMF, Germany) and the Museum of Comparative Zoology of Harvard (MCZ, USA): Celestus bivittatus (Boulenger, 1895) (SMF 79022), Celestus occiduus (Shaw, 1802) (MNHN-RA 1227), Diploglossus monotropis (Kuhl, 1820) (MNHN-RA 1906.0229 and SMF 93787), Diploglossus plei Duméril and Bibron, 1839

  Additionally, we observed previously prepared trunk osteoderms from MNHN-RA diploglossine specimens were also observed: C. striatus Gray, 1838 (synonym of Celestus stenurus (Cope, 1862) ) (MNHN-RA 1897.0010), D. monotropis (MNHN-RA 1906.0229), D. delasagra (Cocteau, 1838 in Cocteau and Bibron, 1843) (MNHN-RA 2859) and Ophiodes vertebralis Bocourt, 1881 (MNHN-RA 7144), as well as osteoderms from other anguid genera: Anguis fragilis (MNHN-RA 1922.0089), Barisia imbricata (Wiegmann, 1828) (MNHN-RA 6136), Gerrhonotus liocephalus Wiegmann, 1828 (MNHN-RA 1637), Elgaria multicarinata (Blainville, 1835) (MNHN-RA 2002), Ophisaurus harti (Boulenger, 1899) (MNHN-RA 1908.0006), Ophisaurus koellikeri (Günther, 1873) (MNHN-RA 1912.0469), and Ophisaurus ventralis (Linnaeus, 1766) (MNHN-RA 7046).

Figure 1 :Figure 2 :

 12 Figure 1: Trunk osteoderms in superficial view: A: Diploglossus monotropis (MNHN-RA

Figure 3 :

 3 Figure 3: Osteoderms of the mid-length of the non-regenerated tail: A: Diploglossus monotropis

Figure 4 :

 4 Figure 4: Osteoderm of the mid-length of the regenerated tail in superficial view: A: Celestus

Figure 5 :

 5 Figure 5: Superficial view of osteoderm of the distal third of the regenerated tail: A: Celestus

Figure 6 :

 6 Figure 6: Schematic representation of a section of a non-regenerated osteoderm. a: Superficial

Figure 7 :

 7 Figure 7: A-B-C-E-F: Non-regenerated proximal third of the tail osteoderm (Diploglossus

  plate with alternation of light and dark strata (CGMs) decreasing in size (C-F). d: Secondary deposit under the basal plate with less marked and more spaced strata, and high density of horizontally orientated Sharpey's fibers (E: arrows).

Figure 8 :

 8 Figure 8: Schematic representation of a section of a large regenerated tail osteoderm. a:


Arnaud Lamy

Celine Vial

Sandrine Costa

come

Brakes and levers for horsemeat consumption in France

in 2019

Results:

Conclusions & further work:

Our work in 2020/2021 focuses on the intentions to buy of horse meat, both for eating at home and eating out. Moreover, we are interested in the representations and practices of chefs, to understand why horsemeat is used so rarely in restaurants.

Shared outcomes from the survey and the interviews:

Consumers of horsemeat, as well as non-consumers, have a low level of knowledge on this subject. The main knowledge concerns the product, such as its color, texture and nutritional qualities (proteins, iron). Culinary knowledge is limited (grilled for steaks or tartar for raw meat). The production methods for this meat are almost unknown.

Among consumers, many consume it since childhood, in the family circle. They reproduce these habits in adulthood with few nuances. Consumption is generally lower than in the previous generation, and events or lifestyles (getting married, moving home, ...) influence the maintenance of a regular consumption.

For many non-consumers, the main reason for non-consuming is the status of the horse as an animal too close to humans to be eaten. Non-consumers are not only people who are put off by the idea of consuming horse meat for moral reasons. Rather, a number of them do not consume it for cultural (eating habits and routines), economic (the price of this meat), and/or commercial reasons (availability in usual purchase places, absence of this meat in restaurants).

Key figures:

16% consumed once horsemeat 43% never consumed horsemeat 41% already consumed several times horsemeat Among 916 meat eaters: Among these few or no consumers (542 individuals):

61% do not consume for moral reasons (love the animal too much to be able to eat it) 39% do not consume for reasons related to eating habits (color, taste), supply or price CHRISTIAN HARTMANN /REUTERS © Our results show the influence of cultural, psychological, economic and marketing factors around the consumption of horsemeat. Moreover, non-consumers are not necessarily polarized by moral concerns, which highlights the potential for an increase in the number of consumers, provided that appropriate marketing measures would be implemented.

1

  MOISA, INRAE, CIHEAM-IAMM, CIRAD, Montpellier Supagro, Univ Montpellier, Montpellier, France 2 IFCE, pôle développement innovation et recherche, 61310 Exmes, France This research aims to identify the factors that can encourage n=1005) about horsemeat consumption and representations of horses Literature review about horsemeat consumption, meat consumption determinants and human-animal relationship In-depth interviews (n=41) about horsemeat status, benefits and barriers of horsemeat consumption Context: Horsemeat consumption: morally taboo in France until mid-19 th century, taboo nowadays in many countriesSince the mid-20 th century, the consumption has been collapsing(FranceAgriMer, 2015) 1% of red meat volume purchased in France(FranceAgriMer, 2019) Two-third of French people do not consume this meat(Cazes-Valette, 2015)


Aurélien Justet

Dymph Klay

Raphaël Porcher

Vincent Cottin

Kais Ahmad

Maria Molina Molina

Hilario Nunes

Martine Reynaud-Gaubert

Jean Marc Naccache

Effrosyni Manali

Maria Molina Molina

Antoine Froidure

Stéphane Jouneau

Lidwine Wemeau

Claire Andrejak

Anne Gondouin

Sandrine Hirschi

Elodie Blanchard

Benjamin Bondue

Philippe Bonniaud

Cécile Tromeur

Grégoire Prévot

Sylvain Marchand-Adam

Manuela Funke-Chambour

Anne Sophie Gamez

Ibrahima Ba

Spyridon Papiris

Jan Grutters

Bruno Crestani

Coline Van Moorsel

C Kannengiesser

Raphaël Borie

Raphael Borie
email: raphael.borie@aphp.fr

Safety and efficacy of pirfenidone and nintedanib in patients with Idiopathic Pulmonary Fibrosis and carrying a telomere related gene mutation

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

12

Cliniques Universitaires Saint-Luc, Service de Pneumologie -Bruxelles (France), 13 Center for rare pulmonary disease , Centre Hospitalier Universitaire de Rennes, Service de Pneumologie, -IRSET (Institut de recherche en santé, environnement et travail) -UMR_S 1085, Université de Rennes 1, -Rennes (France), 14 Reference center for rare pulmonary diseases Service de Pneumologie, CHRU de Lille -Lille (France), 15 Center for rare pulmonary disease Service de Pneumologie, Hôpital d'Amiens, Université de Picardie Jules Verne -Amiens (France), 16 Center for rare pulmonary disease CHU de Besançon, Service de Pneumologie -Besançon (France), 17 Center for rare pulmonary disease Service de Pneumologie, Groupe de Transplantation Pulmonaire, Hôpitaux Universitaires de Strasbourg -Strasbourg (France), 18 Center for rare pulmonary disease CHU de Bordeaux, Service de Pneumologie-Pessac (France) 19 Hôpital Erasme -ULB -Brussel (Belgium), 20 Reference center for rare pulmonary diseases Service de Pneumologie -Dijon (France), 21 CHU de la Cavale Blanche, Département de médecine interne et de pneumologie -Brest (France), 22 Center for rare pulmonary diseases Service de Pneumologie, Hôpital Larrey -Toulouse (France), 23 Center for rare pulmonary diseases , CHU de Tours, Service de Pneumologie et Explorations Fonctionnelles Respiratoires -Tours (France), 24 Service universitaire de pneumologie -Bern (Switzerland), 25 Center for rare pulmonary diseases, Département de Pneumologie et Addictologie, Hôpital Arnaud de Villeneuve, CHU Montpellier -Montpellier (France), 26 Department of Genetics, APHP, Hôpital Bichat-Paris (France),

To the Editor

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive and deadly interstitial lung disease (ILD). Over the past decade, familial occurrence of IPF led to the identification of genetic susceptibility traits [START_REF] Martinez | Idiopathic pulmonary fibrosis[END_REF]. Germline pathogenic variations in telomeres related genes (TRG) such as TERT, TERC TINF2, DKC1, RTEL1, PARN, NAF1, ZCCHC8, NHP2, and NOP10 have been detected in 20-30% of patients with familial pulmonary fibrosis (FPF) and in 1-5% of sporadic IPF [START_REF] Borie | The genetics of interstitial lung diseases[END_REF] [3], [START_REF] Kannengiesser | First heterozygous NOP10 mutation in Familial Pulmonary Fibrosis[END_REF]. In comparison with IPF patients, carriers of a TRG mutation are significantly younger and show an accelerated decline of FVC [START_REF] Newton | Telomere-related lung fibrosis is diagnostically heterogeneous but uniformly progressive[END_REF][6] [START_REF] Justet | Safety and efficacy of pirfenidone in patients carrying telomerase complex mutation[END_REF]. Two drugs, pirfenidone and nintedanib, have been shown to reduce the decline of forced vital capacity (FVC) in IPF patients [START_REF] King | A phase 3 trial of pirfenidone in patients with idiopathic pulmonary fibrosis[END_REF], [START_REF] Richeldi | Efficacy and safety of nintedanib in idiopathic pulmonary fibrosis[END_REF]. So far, two studies reported on the safety and effectiveness of pirfenidone in patients with a TRG mutation [START_REF] Justet | Safety and efficacy of pirfenidone in patients carrying telomerase complex mutation[END_REF], [START_REF] Dressen | Analysis of protein-altering variants in telomerase genes and their association with MUC5B common variant status in patients with idiopathic pulmonary fibrosis: a candidate gene sequencing study[END_REF] whereas no study investigated nintedanib in this specific population. Thus, the aim of this retrospective study was to assess safety and efficacy of nintedanib and pirfenidone in IPF patients with a TRG mutation.

Patients from specialized European interstitial lung disease (ILD) centers from

France (OrphaLung network), Netherlands, Spain, Greece, Belgium, and Switzerland were included in the study if they fulfilled the following criteria:1) a multidisciplinary team diagnosis of IPF, 2) carrier of a TRG variant interpreted as pathogenic and called as mutation in the remaining manuscript, and 3) received at least one dose of pirfenidone or nintedanib. Demographical data, clinical status, treatment continuation (as assessed by the physician in charge), adverse events (clinical and biological) and all lung function tests (LFT) results available were collected, at diagnosis, at antifibrotic treatment initiation and during treatment follow up. Time zero was set to the date of first antifibrotic treatment.

In a first analysis, the impact of the first antifibrotic treatment on the evolution of FVC was analyzed by modeling the longitudinal FVC measurement with a mixed-effects model. The evolution after treatment was then compared to what a model fit with pretreatment data only would predict. Confidence intervals were obtained by nonparametric bootstrap. In a second analysis, the post-treatment evolution was compared between groups of patients receiving pirfenidone or nintedanib using inverse probability of treatment weighting. The evolution of FVC was then compared between groups using a weighted linear mixed effects model, where the treatment effect on the slope of FVC was represented by the time by group interaction. All analyses were carried out using the R statistical software version 3. The median transplant-free survival was 64.9 months (3.0 months to 117.2 months) and the median duration of treatment was 22.0 months (13.5 months to 36.5 months) without significant difference between the 2 groups (figure 1A). No patient from this analysis received danazole. While being treated, 12 patients experienced an acute exacerbation, 9 patients received a lung transplantation and 22 patients died. All deaths were caused by respiratory insufficiency due to progression of lung fibrosis.

During the follow up, 9 patients treated by pirfenidone (25.9%), 12 patients treated by nintedanib (32.4%) stopped the treatment due to gastro intestinal disorders. Three patients (3.7%) 2 with pirfenidone and 1 with nintedanib, showed an increase in liver enzymes , leading to treatment termination. Three patients treated with pirfénidone (5.6%) showed a skin related side effect leading to treatment cessation. Whilst 27 (30.3%) patients presented initially with blood abnormalities in the context of TRG mutation, none of the 89 patient experienced hematological adverse event.

To assess anti fibrotic efficacy, we collected 581 LFT results. The longitudinal change in FVC (liters), modeled in a linear mixed effects model, was significantly reduced compared to the predicted evolution of the FVC (Figure 1). The mean FVC decline was 39 mL / month (95% CI 23 to 55 mL/month) before treatment, and 22 mL / month (95% CI17 to 28 mL/month) in the next 30 months after treatment initiation (p = 0.026).

We compared the slope of FVC in patients receiving pirfenidone and nintedanib, with a weighting on the propensity score to correct any differences in baseline characteristics. After adjustment for confounders, the slope of FVC in the 30 months following treatment was 15 mL/month (5 to 24) with nintedanib and 25 mL/month (17 to 32) with pirfenidone (p=0.12).

In this multicenter European retrospective study, we observed that antifibrotic treatment was associated with a reduced decline of FVC in IPF patients with TRG mutations. We did not observe any unexpected adverse event, neither any difference between pirfenidone and nintedanib in terms of efficacy.

As previously reported in patients with TRG mutations [START_REF] Dressen | Analysis of protein-altering variants in telomerase genes and their association with MUC5B common variant status in patients with idiopathic pulmonary fibrosis: a candidate gene sequencing study[END_REF], [START_REF] Justet | Safety and efficacy of pirfenidone in patients carrying telomerase complex mutation[END_REF], patients in this cohort were younger and showed a more rapid decline of lung function as compared to previous cohorts of sporadic IPF patients [START_REF] Guenther | The European IPF registry (eurIPFreg): baseline characteristics and survival of patients with idiopathic pulmonary fibrosis[END_REF]. Because of specific hematological and liver diseases associated with TRG mutation [START_REF] Armanios | Telomerase mutations in families with idiopathic pulmonary fibrosis[END_REF], we were concerned about an increased risk of liver toxicity and hematological adverse events [START_REF] King | A phase 3 trial of pirfenidone in patients with idiopathic pulmonary fibrosis[END_REF][START_REF] Richeldi | Efficacy and safety of nintedanib in idiopathic pulmonary fibrosis[END_REF]. Fortunately, our data suggest that the side effect profile of pirfenidone or nintedanib in patients with TRG mutation is similar with those observed in the general IPF population [START_REF] King | A phase 3 trial of pirfenidone in patients with idiopathic pulmonary fibrosis[END_REF][START_REF] Richeldi | Efficacy and safety of nintedanib in idiopathic pulmonary fibrosis[END_REF].

Twenty-seven patients (30.3%) had to stop the treatment due to a side effect, mostly gastro-intestinal. These results correspond to the usual proportion of patient stopping the antifibrotic drugs in reported retrospective and prospective cohorts of IPF patients [START_REF] Guenther | The European IPF registry (eurIPFreg): baseline characteristics and survival of patients with idiopathic pulmonary fibrosis[END_REF], [START_REF] Jo | Baseline characteristics of idiopathic pulmonary fibrosis: analysis from the Australian Idiopathic Pulmonary Fibrosis Registry[END_REF], [START_REF] Cottin | Long-term safety of pirfenidone: results of the prospective, observational PASSPORT study[END_REF], [START_REF] Crestani | Long-term safety and tolerability of nintedanib in patients with idiopathic pulmonary fibrosis: results from the openlabel extension study, INPULSIS-ON[END_REF].

We observed that antifibrotic drugs were associated with a reduced decline of FVC in IPF patients carrying TRG mutations. Our results are in line with a post hoc analysis of INSPIRE, CAPACITY, and ASCEND trials in patients carrying a rare TRG variant.

Patients with a rare TRG variant had a more rapid decline of FVC than IPF patients without a TRG variant [START_REF] Dressen | Analysis of protein-altering variants in telomerase genes and their association with MUC5B common variant status in patients with idiopathic pulmonary fibrosis: a candidate gene sequencing study[END_REF], though pirfenidone was still associated with a reduced decline of FVC compared to placebo in this subpopulation [START_REF] Dressen | Analysis of protein-altering variants in telomerase genes and their association with MUC5B common variant status in patients with idiopathic pulmonary fibrosis: a candidate gene sequencing study[END_REF].

Our study is the first to assess the benefit of nintedanib in this specific population, highlighted by the number of rare and unique mutation, and our results support the safety and efficacy of nintedanib in these patients.

This study has several limitations. Treatment continuation was self-reported by the patient, and we cannot ensure the presence of unreported adverse events due to the retrospective nature of the study. In addition, due to the limited number of patients included, we were neither able to evaluate the efficacy of antifibrotic according to the nature of the mutation status nor to compare the efficacy of pirfenidone and nintedanib. With respect to the very limited number of patients who switched from pirfenidone to nintedanib, we didn't assess efficacy or safety of the second line of anti-fibrotic treatment. Finally and most importantly, we compared the observed decline of FVC to predicted decline, a comparison which can be suffer many biases, and which cannot replace a randomized control trial .

In conclusion, this study suggests that pirfenidone and nintedanib can be used safely in IPF patients with a TRG mutation and that both drugs reduce FVC decline. These results should be confirmed in a larger prospective study.

Figure legend

  6.0 (The R Foundation for Statistical Computing, Vienna, Austria), with the Hmisc, lme4, WeMix and boot packages. We identified 89 patients with IPF, carrier of a TRG mutation: TERT (n=65), TERC (n=16), RTEL1 (n=5) or PARN (n=3). The mean age at diagnosis was 59.8 ± 9.4 years. At treatment initiation, median FVC was 84.3 % [70.0-94.2] of the predicted value and median DLCO was 45.8 [38.0-53.0]. At treatment initiation, patients treated with pirfenidone (n=55) or nintedanib (n=34) were similar in terms of age, smoking, mutation status, delay between diagnosis and treatment initiation or disease severity.

Figure 1 :

 1 Figure 1: a) Treatment continuation of pirfenidone and nintedanib This figure shows the percentage of patients treated by pirfenidone (left panel) or nintedanib (right panel). Blue column represents the percentage of patients treated by antifibrotic treatment at full dose (blue background) or reduced dose (hatched background). Red column represents the percentage of patients who stopped the treatment due to disease progression (including patients that died or received a lung transplantation) -(red background) or to a side effect (white background). At the bottom of the figures, the number of data available for each time. b) Longitudinal FVC decline of IPF patients carrying TRG mutation treated by nintedanib or pirfenidone The blue curves shows the FVC data collected for each patient, with a flexible model of the mean estimated by splines, in a mixed effects model. The curve represented by red dashes shows the predicted evolution of the FVC obtained with data before treatment only (until time zero)

Acknowledgement

We thank Pr Camille Taille, Dr Clairelyne Dupin (Bichat Hospital, Paris) and Dr Julie Traclet (Louis Pradel Hospital, Lyon) for their efficient collaboration and for their help to collect the data. Dr Cottin is a member of ERN-LUNG.

Supplementary Table 1: Pathogenic telomerase related gene mutations