Eugene V Koonin
email: koonin@ncbi.nlm.nih.gov

# Valerian

V Dolja

Mart Krupovic

The healthy human virome: from virus-host symbiosis to disease

Viruses are ubiquitous, essential components of any ecosystem, and of multicellular organism holobionts. Numerous viruses cause acute infection, killing the host or being cleared by immune system. In many other cases, viruses coexist with the host as symbionts, either temporarily or for the duration of the host's life. Apparently, virus-host relationships span the entire range from aggressive parasitism to mutualism. Here we attempt to delineate the healthy human virome, that is, the entirety of viruses that are present in a healthy human body. The bulk of the healthy virome consists of bacteriophages infecting bacteria in the intestine and other locations. However, a variety of viruses, such as anelloviruses and herpesviruses, and the numerous endogenous retroviruses, persist by replicating in human cells, and these are our primary focus. Crucially, the boundary between symbiotic and pathogenic viruses is fluid such that members of the healthy virome can become pathogens under changing conditions.

Introduction

Billions of years of perennial and ubiquitous co-evolution between viruses and cells have produced a broad spectrum of virus-host interaction regimes, ranging from aggressive antagonism to commensalism, whereby viruses coexist with their hosts without harming them, at least, in the short term, and even to mutualism when a virus is beneficial and can be essential to the host [START_REF] Koonin | A virocentric perspective on the evolution of life[END_REF][START_REF] Forterre | The major role of viruses in cellular evolution: facts and hypotheses[END_REF][START_REF] Roossinck | Symbiosis: Viruses as Intimate Partners[END_REF][START_REF] Koonin | Evolutionary entanglement of mobile genetic elements and host defence systems: guns for hire[END_REF].

The relationship between a particular virus and its host can be rarely, if ever, defined by a single regime. Rather, the mode of virus-host interaction is a function of multiple factors, including the environmental conditions, host and virus population structure, the immunological status of the host and many more.

Virus-host relationships that do not result in the demise of the host cell have been described across the virosphere [START_REF] Roossinck | Symbiosis: Viruses as Intimate Partners[END_REF][START_REF] Koonin | Evolutionary entanglement of mobile genetic elements and host defence systems: guns for hire[END_REF]. Hence the concept of the 'normal' or 'healthy' virome, in principle, is applicable to any organism, including humans. Indeed, although most of the best characterized human viruses cause acute infections and are associated with diseases, a healthy human organism is host to a much greater variety of viruses [START_REF] Rascovan | Metagenomics and the Human Virome in Asymptomatic Individuals[END_REF][START_REF] Adiliaghdam | Illuminating the human virome in health and disease[END_REF][START_REF] Kumata | A tissue level atlas of the healthy human virome[END_REF]. The substantial majority of these infect bacteria that inhabit the human intestine, but a number of viruses actually reproduce in human cells without causing disease, at least, in the short term.

In this brief review, we systematically discuss the healthy human virome and emphasize that the boundary between "normal" and pathogenic (that is, causally associated with clinically manifested disease) viromes is blurred. Indeed, the same virus can be either a symbiont (with either no perceptible fitness effect on the host, that is, effectively, a commensal, or with a beneficial effect) or a pathogen depending on the conditions such as the health status and developmental stage of the host. We further stress that the current knowledge of symbiotic viruses lags far behind that of the pathogenic viruses.

The healthy human phageome

The human intestine, by far the richest microbial habitat in the body, contains about 10 14 bacterial and archaeal cells at any given moment [START_REF] Shkoporov | The Human Gut Virome Is Highly Diverse, Stable, and Individual Specific[END_REF]. As many microbial communities, the human microbiome hosts a broad variety of viruses, in which tailed bacteriophages (class Caudoviricetes within the realm Duplodnaviria) comprise the overwhelming majority [START_REF] Shkoporov | The Human Gut Virome Is Highly Diverse, Stable, and Individual Specific[END_REF][START_REF] Manrique | Healthy human gut phageome[END_REF], albeit with a considerable contribution by ssDNA phages of the families Microviridae [START_REF] Wang | Gut virome of mammals and birds reveals high genetic diversity of the family Microviridae[END_REF][START_REF] Krupovic | Microviridae goes temperate: microvirus-related proviruses reside in the genomes of Bacteroidetes[END_REF][START_REF] Minot | Rapid evolution of the human gut virome[END_REF] and Inoviridae [START_REF] Roux | Cryptic inoviruses revealed as pervasive in bacteria and archaea across Earth's biomes[END_REF] (realm Monodnaviria). Systematic metagenomics surveys of the human phageome identify thousands of phages but show that relatively few are common components of the healthy gut phageome. Thus, one of the most detailed metagenomics analyses resulted in the identification of only 23 phages that were shared by >50% of the tested individuals [START_REF] Manrique | Healthy human gut phageome[END_REF]. Strikingly, the most prevalent human-associated phage (and most prominent component of the healthy human virome altogether), the crAssphage, has been discovered only through metagenomics [START_REF] Dutilh | A highly abundant bacteriophage discovered in the unknown sequences of human faecal metagenomes[END_REF]. This initial finding has been followed by the characterization of an expansive group of crAss-like phages, also by metagenome analysis [START_REF] Yutin | Discovery of an expansive bacteriophage family that includes the most abundant viruses from the human gut[END_REF][START_REF] Yutin | virus genomes from the human gut dramatically expanding the diversity of crAss-like phages and demonstrating that this group of phages dominates the human "microbiovirome". Some subgroups of crAss-like phages are shown to possess unique gene organizations, with unprecedented densities of self-splicing introns and intein, and alternative genetic codes[END_REF]. Subsequently, some crAss-like phages have been grown in cultures of the respective host bacteria, members of the phylum Bacteroidetes [START_REF] Guerin | Biology and Taxonomy of crAss-like Bacteriophages, the Most Abundant Virus in the Human Gut[END_REF][START_REF] Drobysheva | Structure and function of virion RNA polymerase of a crAss-like phage[END_REF]. Given the difficulty of growing the human intestine-associated bacteria-phage systems in the laboratory, the actual size and diversity of the healthy human phageome remains to be discovered. Importantly, substantial changes in the human phageome have been associated with various diseases conditions, including infection with human and simian immunodeficiency viruses, and in some cases, the phageome perturbation was, apparently, decoupled from the changes in the microbiome [START_REF] Norman | Disease-specific alterations in the enteric virome in inflammatory bowel disease[END_REF][START_REF] Monaco | Altered Virome and Bacterial Microbiome in Human Immunodeficiency Virus-Associated Acquired Immunodeficiency Syndrome[END_REF][START_REF] Handley | SIV Infection-Mediated Changes in Gastrointestinal Bacterial Microbiome and Virome Are Associated with Immunodeficiency and Prevented by Vaccination[END_REF].

Symbiotic viruses replicating in human cells

Over many decades, diverse viruses have been isolated from healthy humans more or less by chance. In the last few years, systematic surveys of the healthy human virome became possible thanks to the advances of metagenomics [START_REF] Adiliaghdam | Illuminating the human virome in health and disease[END_REF][START_REF] Kumata | A tissue level atlas of the healthy human virome[END_REF]. Below we briefly discuss the viruses that have been shown, either by traditional virology approaches or by metagenomics (Figure 1a), to be commonly present in healthy humans without causing apparent disease, following the main divisions of the current virus taxonomy [START_REF] Koonin | A comprehensive analysis of the evolutionary relationships among the major groups of viruses. A megataxonomy of viruses[END_REF][START_REF]International Committee on Taxonomy of Viruses Executive C: The new scope of virus taxonomy: partitioning the virosphere into 15 hierarchical ranks[END_REF]. The key aspects of the association of these viruses with the human organism are summarized in Figure 1.

Realm Riboviria

Kingdom Orthornavira

The realm Riboviria consists of viruses with RNA genomes as well as viruses with DNA genomes that employ reverse transcription in their replication cycles. The kingdom Orthornavira encompasses RNA viruses that share homologous RNA-dependent RNA polymerases. Many RNA viruses have been isolated or detected by metatranscriptomics in healthy humans but few can be confidently identified as symbionts replicating in human cells (Figure 1a).

Perhaps, the most notable apparent human symbionts among the orthornaviruses are pegiviruses (family Flaviviridae) [START_REF] Stapleton | The GB viruses: a review and proposed classification of GBV-A, GBV-C (HGV), and GBV-D in genus Pegivirus within the family Flaviviridae[END_REF][START_REF] Hartlage | The Strange, Expanding World of Animal Hepaciviruses[END_REF]. The prototypical pegivirus has been initially tentatively identified as hepatitis G virus [START_REF] Linnen | Molecular cloning and disease association of hepatitis G virus: a transfusion-transmissible agent[END_REF], but no association with hepatitis has been subsequently confirmed [START_REF] Alter | Acute non-A-E hepatitis in the United States and the role of hepatitis G virus infection. Sentinel Counties Viral Hepatitis Study Team[END_REF].

In the family tree of flavivirids, pegiviruses tightly cluster with hepaciviruses which include hepatitis C virus (HCV), a major human pathogen [START_REF] Stapleton | The GB viruses: a review and proposed classification of GBV-A, GBV-C (HGV), and GBV-D in genus Pegivirus within the family Flaviviridae[END_REF][START_REF] Hartlage | The Strange, Expanding World of Animal Hepaciviruses[END_REF]. However, unlike HCV, pegiviruses have not been linked to any pathology. Pegivirus infection in humans is common, with the incidence of about 5%, and pegiviruses readily grow in human cell cultures, leaving no doubt that these are bona fide human viruses [START_REF] Hartlage | The Strange, Expanding World of Animal Hepaciviruses[END_REF]. Notably, pegivirus infection appears to be associated with benign clinical outcome in AIDS patients [START_REF] Schwarze-Zander | Role of GB virus C in modulating HIV disease[END_REF][START_REF] Greenhalgh | Fighting the Public Health Burden of AIDS With the Human Pegivirus[END_REF] indicating that these apparent viral symbionts of humans could benefit the host via protection from other viruses.

A recent, intriguing addition to the human virome are statoviruses (for STool-Associated TOmbuslike viruses) that were originally identified in multiple metagenomes from humans, macaques, cows and mice [START_REF] Janowski | Statoviruses, A novel taxon of RNA viruses present in the gastrointestinal tracts of diverse mammals[END_REF], followed by detection in nasal-throat swabs of humans with acute respiratory disease [START_REF] Kha | The Virome of Acute Respiratory Diseases in Individuals at Risk of Zoonotic Infections[END_REF]. Although the actual hosts of statoviruses remain unknown, RdRP phylogeny, where statovirus RdRPs cluster with a variety of unclassified tombus-like viruses from invertebrate holobionts (that is, a host together with the entirety of associated symbionts and parasites) [START_REF] Wolf | Origins and Evolution of the Global RNA Virome[END_REF],

suggests that statoviruses are associated with either mammalian diet (e.g., plants) or, more likely, some protist symbionts or parasites.

Another widespread group of putative members of the healthy human virome are members of Picobirnaviridae. Picobirnaviruses are commonly detected in mammalian, including human, intestines and have not been convincingly linked to any diseases although are suspected to be associated with diarrhea [START_REF] Ganesh | Picobirnavirus infections: viral persistence and zoonotic potential[END_REF][START_REF] Ganesh | Animal picobirnavirus[END_REF]. Picobirnaviruses have never been grown in cell cultures, and their true hosts remain unknown. The presence of highly conserved ribosome-binding sites (Shine-Dalgarno sequences), which are a hallmark of prokaryotic mRNAs, has led to the suggestion that picobirnaviruses infect bacteria in mammalian microbiomes [START_REF] Krishnamurthy | Extensive conservation of prokaryotic ribosomal binding sites in known and novel picobirnaviruses[END_REF].

In addition, certain food-derived plant viruses are present in the human gastrointestinal tract and in feces, sometimes in substantial amounts. The list of such viruses is topped by pepper mild mottle virus (PMMoV), a tobamovirus commonly found in pepper, pepper-derived products and their consumers all over the world [START_REF] Zhang | RNA viral community in human feces: prevalence of plant pathogenic viruses[END_REF]. Remarkably, PMMoV retains infectivity after passing through the human alimentary tract. Due to its stability and wide presence in human feces, PMMoV is used as a surrogate marker of fecal contamination of water [START_REF] Symonds | Pepper mild mottle virus: Agricultural menace turned effective tool for microbial water quality monitoring and assessing (waste)water treatment technologies[END_REF]. However, given that there is no evidence of plant virus replication in vertebrate cells, any substantial role of plant viruses in human health is an extremely remote possibility.

Kingdom Pararnavira

This virus kingdom includes viruses that employ reverse transcription in their replication cycles. Over 3,000 of human endogenous retroviruses (HERVs) are integrated into the host genome, comprising about 8% of human DNA [START_REF] Tokuyama | ERVmap analysis reveals genome-wide transcription of human endogenous retroviruses[END_REF]. Accordingly, the HERVs play important and diverse roles in human biology that cannot be discussed in this brief review in any detail (for recent reviews, see [START_REF] Johnson | Origins and evolutionary consequences of ancient endogenous retroviruses[END_REF][START_REF] Chiu | Endogenous Retroviruses Drive Resistance and Promotion of Exogenous Retroviral Homologs[END_REF][START_REF] Cullen | Endogenous Retroviruses Walk a Fine Line between Priming and Silencing[END_REF][START_REF] Alcazer | Human Endogenous Retroviruses (HERVs): Shaping the Innate Immune Response in Cancers[END_REF]). Most of the HERVs appear to descend from ancient integration events so that the virus genes are disrupted and rearranged. The legacy of these ancient HERVs are genes encoding virus structural proteins (Gag and Env) that have been recruited for a variety of physiological functions [START_REF] Koonin | The depths of virus exaptation[END_REF], the best known being syncitins, the placental trophoblast receptors [START_REF] Johnson | Origins and evolutionary consequences of ancient endogenous retroviruses[END_REF]. However, some members of the youngest group of HERVs, known as HERV-K or HML2, that are thought to have invaded the human genome less than a million years ago form virus particles, particularly, in early embryogenesis [START_REF] Garcia-Montojo | Human endogenous retrovirus-K (HML-2): a comprehensive review[END_REF][START_REF] Xue | Human Endogenous Retrovirus K (HML-2) in Health and Disease[END_REF]. The HERV-K viruses, at least, are bona fide members of the healthy human virome. Many of the other HERVs are expressed as well and are implicated in a variety of functions including modulation of innate immunity, even though functional virus proteins are usually not produced [START_REF] Grandi | Human Endogenous Retroviruses Are Ancient Acquired Elements Still Shaping Innate Immune Responses[END_REF]. In particular, it has been reported that expression of one of HERV-K suppresses the spread of invasive melanoma [START_REF] Singh | Human Endogenous Retrovirus K Rec forms a Regulatory Loop with MITF that Opposes the Progression of Melanoma to an Invasive Stage[END_REF]. However, potential associations between HERVs and various diseases have been reported as well. Thus, the relationship between the HERVs and the human host is a typical symbiosis, with both beneficial and potential deleterious effects on the host (Figure 1b).

Realm Monodnaviria

Realm Monodnaviria includes prokaryotic and eukaryotic viruses which encode homologous replication initiation endonucleases of the HUH superfamily or their inactivated derivatives, as in the case of polyomaviruses and papillomaviruses [START_REF] Koonin | A comprehensive analysis of the evolutionary relationships among the major groups of viruses. A megataxonomy of viruses[END_REF][START_REF] Kazlauskas | Multiple origins of prokaryotic and eukaryotic single-stranded DNA viruses from bacterial and archaeal plasmids[END_REF]. In addition to phages of the Microviridae and Inoviridae families mentioned above, several other representatives of Monodnaviria have been repeatedly identified as part of the healthy human virome. These include members of the families Parvoviridae, Genomoviridae, Smacoviridae, Papillomaviridae and Polyomaviridae (Figure 1a). The actual hosts for human-associated genomoviruses [START_REF] Krupovic | Genomoviridae: a new family of widespread singlestranded DNA viruses[END_REF] and smacoviruses [START_REF] Varsani | Smacoviridae: a new family of animal-associated single-stranded DNA viruses[END_REF] remain unknown but these viruses likely infect human-associated microbes rather than humans directly.

Parvoviruses

Parvoviruses from several genera have been detected in various samples from apparently healthy humans, including human bocaviruses (HBoV; genus Bocaparvovirus), adeno-associated viruses (AAVs; genus Dependoparvovirus), human parvovirus 4 (PARV4; genus Tetraparvovirus), parvovirus B19 (B19V; genus Erythroparvovirus) and several protoparvoviruses (genus Protoparvovirus) [START_REF] Matthews | Human parvovirus 4 'PARV4' remains elusive despite a decade of study[END_REF][START_REF] Qiu | Human Parvoviruses[END_REF][START_REF] Soderlund-Venermo | Emerging Human Parvoviruses: The Rocky Road to Fame[END_REF][START_REF] Cotmore | ICTV Virus Taxonomy Profile: Parvoviridae[END_REF]. These viruses display highly variable cell tropism and pathogenicity.

For instance, AAVs and PARV4 can infect cells from multiple tissue types and are not known to cause any disease. By contrast, HBoV is most commonly found in the respiratory and gastrointestinal tracts as well as in blood [START_REF] Allander | Human bocavirus[END_REF][START_REF] Guido | Human bocavirus: Current knowledge and future challenges[END_REF], and is associated with acute respiratory symptoms, especially in children [START_REF] Ma | Detection of human bocavirus in Japanese children with lower respiratory tract infections[END_REF]. However, the direct role of HBoV as a pathogen remains unclear. B19V invades red blood cell precursors in the bone marrow and is commonly considered as a human pathogen causing a wide range of pathological conditions, including the fifth disease in children, persistent anemia in immunocompromised patients, transient aplastic crises, hydrops fetalis in pregnant women, and arthropathy [START_REF] Qiu | Human Parvoviruses[END_REF]. Yet, in healthy adults, B19V infections are largely asymptomatic [START_REF] Woolf | Clinical manifestations of human parvovirus B19 in adults[END_REF], with the prevalence of up to 25% in healthy human skin biopsies [START_REF] Bonvicini | Parvovirus b19 DNA is commonly harboured in human skin[END_REF]. Thus, B19V is a conditional component of the "healthy" human virome that can turn into a pathogen in response to various factors (Figure 1b).

Polyomaviruses and Papillomaviruses

Monodnaviria includes class Papovaviricetes that consists of Polyomaviridae and Papillomaviridae, two families of viruses with small (5-8 Kbps), circular dsDNA genomes [START_REF] Moens | Biology, evolution, and medical importance of polyomaviruses: An update[END_REF][START_REF] Spriggs | Human Papillomavirus and the DNA Damage Response: Exploiting Host Repair Pathways for Viral Replication[END_REF]. Papovaviruses are thought to have evolved from parvoviruses [START_REF] Kazlauskas | Multiple origins of prokaryotic and eukaryotic single-stranded DNA viruses from bacterial and archaeal plasmids[END_REF], with polyomaviruses emerging in invertebrates and co-evolving with animals for at least half a billion years [START_REF] Buck | The Ancient Evolutionary History of Polyomaviruses[END_REF]. In humans, polyomaviruses lead a low-profile life styles characterized by low propagation levels, evasion of clearance by the immune system and asymptomatic infections in immunocompetent individuals [START_REF] Moens | Biology, evolution, and medical importance of polyomaviruses: An update[END_REF]. Apparently, human polyomaviruses have evolved mechanisms to limit their own reproduction levels in order to establish persistent infections [START_REF] Krump | Mechanisms of persistence by small DNA tumor viruses[END_REF]. A poster child human polyomavirus is John Cunningham virus (JCV) that establishes life-long latent infections [START_REF] Cortese | Progressive multifocal leukoencephalopathy and the spectrum of JC virus-related disease[END_REF]. The seroprevalence of JCV and other human polyomaviruses in adults can reach 90% or higher, with common coinfection by several polyomaviruses that are transmitted through direct contacts between humans or through contaminated objects. Contrary to their name and the oncogenic potential of the large T (tumor) antigen (the early virus protein involved in genome replication) in experimental settings, most of the human polyomaviruses are not oncogenic. The Merkel cell polyomavirus is the only one that has been convincingly identified as the etiological agent of the eponymous carcinoma, a skin cancer caused by malignant transformation of the skin neuroendocrine cells apparently facilitated by virus genome integration into the host chromosomes [START_REF] Moens | Biology, evolution, and medical importance of polyomaviruses: An update[END_REF][START_REF] Krump | Molecular mechanisms of viral oncogenesis in humans[END_REF].

Papillomaviruses are the closest, albeit apparently somewhat 'younger' relatives of polyomaviruses that likely emerged in vertebrates ~350 mya, but their life style is distinct. The majority of the ~200 known human papillomaviruses (HPVs) belong to Beta and Gamma types (Betapapillomavirus and Gammapapillomavirus genera, respectively), and cause unapparent productive infections or low-grade disease of the skin or mucosal epithelium (e.g., warts and condylomas) that are normally cleared by the immune system [START_REF] Doorbar | Human papillomavirus molecular biology and disease association[END_REF][START_REF] Graham | The human papillomavirus replication cycle, and its links to cancer progression: a comprehensive review[END_REF]. The virus replication cycle is tightly linked to epithelial differentiation and is orchestrated by a regulatory network that involves coordination between the cell cycle, virus DNA replication and transcription, and RNA splicing [START_REF] Graham | The human papillomavirus replication cycle, and its links to cancer progression: a comprehensive review[END_REF][START_REF] Chiu | Plasmid Partitioning by Human Tumor Viruses[END_REF]. Most of the infections by the 'high-risk', Alpha HPV types (HPV16 and HPV18 being most prevalent; genus Alphapapillomavirus) in women result in more prolonged cervical infections, 80-90% of which are asymptomatic and are eventually cleared by the immune system [START_REF] Doorbar | Human papillomavirus molecular biology and disease association[END_REF][START_REF] Graham | The human papillomavirus replication cycle, and its links to cancer progression: a comprehensive review[END_REF]. However, a small fraction of such infections, primarily due to defects in the immune response, progress to the formation of persistent papillomas and, in a minority of cases, to cervical cancer. This small fraction of HPV infections, nevertheless, accounts for close to 100% of cervical cancers that affect over 500,000 women annually [START_REF] Serrano | Epidemiology and burden of HPV-related disease[END_REF]. On much rarer occasions, the high-risk HPV also cause other types of carcinomas [START_REF] Araldi | The human papillomavirus (HPV)-related cancer biology: An overview[END_REF]. Carcinogenesis is a dead end for HPV because transformed cells produce no infectious virus. Therefore, despite the carcinogenic potential of high-risk HPVs, by and large, these common components of human virome should be considered symbionts, as demonstrated by the protection from skin cancer caused by immunity to ubiquitous skin-infecting HPVs [START_REF] Strickley | Immunity to commensal papillomaviruses protects against skin cancer[END_REF] (Figure 1b).

Anelloviruses

Members of the family Anelloviridae are among the most enigmatic components of the healthy human virome, both in terms of their evolutionary origin and the impact on human health.

Anelloviruses have small icosahedral virions that encapsidate tiny (3 to 3,5 kb) circular ssDNA genomes [START_REF] Kaczorowska | Human anelloviruses: diverse, omnipresent and commensal members of the virome[END_REF], but unlike the ssDNA viruses in the realm Monodnaviria, do not encode recognizable homologs of the rolling circle replication endonuclease. Moreover, no homologs outside the family have been detected for any of the anellovirus proteins. Hence, anelloviruses are currently not included in the realm Monodnaviria and their provenance remains unclear [START_REF] Koonin | A comprehensive analysis of the evolutionary relationships among the major groups of viruses. A megataxonomy of viruses[END_REF][START_REF] Kazlauskas | Multiple origins of prokaryotic and eukaryotic single-stranded DNA viruses from bacterial and archaeal plasmids[END_REF].

The entire human population is believed to be infected with anelloviruses, and there is no convincing evidence of viral clearance from infected individuals [START_REF] Kaczorowska | Human anelloviruses: diverse, omnipresent and commensal members of the virome[END_REF][START_REF] Webb | Torque teno viruses in health and disease[END_REF]. The infections occur at an early age and so far have not been convincingly associated with any disease. The virus load appears to be controlled by the immune system because virus levels increase with the level of host immunosuppression [START_REF] Focosi | Torquetenovirus: the human virome from bench to bedside[END_REF][START_REF] Blatter | Anellovirus loads are associated with outcomes in pediatric lung transplantation[END_REF]. Asymptomatic anellovirus infections are also common in other mammals [START_REF] Webb | Torque teno viruses in health and disease[END_REF][START_REF] Fahsbender | Diverse and highly recombinant anelloviruses associated with Weddell seals in Antarctica[END_REF][START_REF] De Souza | Discovery of novel anelloviruses in small mammals expands the host range and diversity of the Anelloviridae[END_REF][START_REF] Crane | Novel anelloviruses identified in buccal swabs of Antarctic fur seals[END_REF], suggesting extensive coevolution of anelloviruses with mammalian hosts.

Although the potential impact of anelloviruses on human health remains a matter of debate, it has been suggested that they positively influence human physiology by shaping the immunity during early development [START_REF] Kaczorowska | Human anelloviruses: diverse, omnipresent and commensal members of the virome[END_REF]. Thus, anelloviruses might be the most 'friendly', genuinely symbiotic component of the human virome.

Realm Duplodnaviria

The realm Duplodnaviria includes viruses with dsDNA genomes that are encapsidated in icosahedral capsids consisting of a distinct type of capsid protein, displaying the HK97 fold (after the first phage for which the capsid protein structure was solved), with the help of the corresponding variety of packaging ATPase, known as the terminase [START_REF] Koonin | A comprehensive analysis of the evolutionary relationships among the major groups of viruses. A megataxonomy of viruses[END_REF]. This realm includes the bulk of the viruses associated with the human microbiome, namely, the numerous tailed bacteriophages, as well as a major component of the virome associated with human cells, the herpesviruses. Some of the human herpesviruses (Herpesviridae) infect a variety of cell types and cause life-long latent infections [START_REF] Connolly | The structural basis of herpesvirus entry[END_REF]. Of the 9 human herpesviruses identified so far, herpes simplex virus 1 (HSV1), human cytomegalovirus (HCMV), Epstein-Barr virus (EBV) and varicella zoster virus (VZV) are highly prevalent in the human population. Depending on the geographic location, socioeconomic status and, in the case of VZV, vaccination levels, the incidence of these viruses reaches up to 96% [START_REF] Cohen | Epstein-Barr virus infection[END_REF][START_REF] Gupta | Genital herpes[END_REF][START_REF] Zuhair | Estimation of the worldwide seroprevalence of cytomegalovirus: A systematic review and meta-analysis[END_REF]. The health impact of these viruses (if any) depends on the age and immune system status of the infected person, with ethnicity, gender and genotype being additional significant contributors.

The most stealthy of the human herpes viruses are apparently EBV and HCMV. If acquired in childhood, both of these viruses typically cause asymptomatic infections, mainly, in Blymphocytes in the case of EBV [START_REF] Taylor | The immunology of Epstein-Barr virus-induced disease[END_REF] or in several cell types in the case of HCMV [START_REF] Connolly | The structural basis of herpesvirus entry[END_REF]. Such silent infections, however, even if not manifested in disease, cause a range of effects at the molecular, cellular, tissue and organism levels. In the case of EBV, the gene expression pattern of infected Bcells is reprogrammed [START_REF] Moustafa | The blood DNA virome in 8,000 humans[END_REF] and the proportion of plasma cells in blood appears to be increased [START_REF] Kumata | A tissue level atlas of the healthy human virome[END_REF].

Major immunity stimulation effects, a likely result of a balance reached over long virus-host coevolution process, are well established for HCMV. In particular, ~10% of the CD4 + and CD8 + T cells in latently infected, otherwise healthy adults are HCMV-specific. Likewise, HCMV causes expansion of adaptive CD57 + natural killer (NK) cells targeting virus-infected cells [START_REF] Semmes | Cytomegalovirus as an immunomodulator across the lifespan[END_REF]. These powerful arms of the immune system, however, fail to clear the virus due to its armament of immunoevasins, HCMV-encoded proteins involved in modulation of host immunity [START_REF] Berry | Modulation of innate and adaptive immunity by cytomegaloviruses[END_REF].

Extensive immunity stimulation in HCMV-seropositive individuals appears to enhance responsiveness and protection against heterologous viruses rather than compromise host immunity [START_REF] Berry | Modulation of innate and adaptive immunity by cytomegaloviruses[END_REF]. These potentially positive effects notwithstanding, damaging consequences of herpesvirus infections appear to be fairly common and vary from mild illness to a variety of life-threatening conditions. Thus, HCMV congenital (in utero) infections that occur at up to 2% of childbirths frequently cause neurodevelopmental defects or leukemia [START_REF] Semmes | Cytomegalovirus as an immunomodulator across the lifespan[END_REF]. At the other life extremity, in seniors, HCMV seropositivity is associated with increased risks of cancer, cardiovascular disease, and ultimately, mortality.

The delicate, life-long EBV-host balance arising from most childhood infections is also fragile: if infection occurs in even non-immunocompromised young adults, it results in infectious mononucleosis ('kissing disease') [START_REF] Fuentes-Panana | Beta and Gamma Human Herpesviruses: Agonistic and Antagonistic Interactions with the Host Immune System[END_REF]. Similarly, EBV is etiologically linked to Hodgkin's and Burkitt's lymphomas and nasopharyngeal carcinoma which are common in Southern Chinese and Eskimo people, as well as to a host of other diseases in immunocompromised individuals [START_REF] Taylor | The immunology of Epstein-Barr virus-induced disease[END_REF]. A different pathology pattern is characteristic of VZV infections that cause varicella (chickenpox) in children followed by prolonged latency that, in about 15% cases, leads to virus reactivation in elderly people with weakened immune control, causing zoster (postherpetic neuralgia) [START_REF] Gershon | Varicella zoster virus infection[END_REF].

The HSV1 infections exhibit another distinct pattern of virus-host interactions. This virus is normally acquired in childhood causing mainly oral infections; its seroprevalence varies from 70% in developed nations to 100% in developing nations [START_REF] Gupta | Genital herpes[END_REF][START_REF] Saleh | Herpes Simplex Type 1[END_REF]. Upon infecting epithelial cells, HSV1 is transmitted to axons and establishes life-long latency in dorsal root ganglia that is periodically manifested in reactivation leading to recurrent acute infections or asymptomatic virus shedding.

Similar to other human herpesviruses, HSV1 efficiently evades antiviral innate immune responses mediated by Toll-like and other pathogen recognition receptors. The underlying mechanisms of HSV1 immunoevasion involve multiple virus proteins targeting diverse innate immunity signaling pathways [START_REF] Zhu | The Race between Host Antiviral Innate Immunity and the Immune Evasion Strategies of Herpes Simplex Virus 1[END_REF].

Thus, human herpesviruses display a striking variety of cell tropisms and infection patterns that emerged over long-term virus-human co-evolution and co-adaptation. Some herpesviruses, in particular HCMV and EBV, can reach a perfect balance with the human host and often persist for the host's lifetime without causing any pathology. However, because of the complexity of virushost interactions that involve a variety of genetic, environmental and socioeconomic factors, this balance is fragile and can be broken in many situations resulting in morbidity or even mortality (Figure 1b). Therefore, herpesvirus-host co-existence that involves the majority of the human population blurs the very concept of a 'healthy human virome'.

Realm Varidnaviria

This realm includes a broad variety of viruses with dsDNA genomes and icosahedral capsids built of protein unrelated to the capsid proteins of duplodnaviruses [START_REF] Koonin | A comprehensive analysis of the evolutionary relationships among the major groups of viruses. A megataxonomy of viruses[END_REF].Unlike the members of Duplodnaviria, varidnaviruses are minor components of the healthy human virome, at best.

Several intriguing reports have appeared on the detection of large and giant viruses of the class

Nucleocytoviricota in healthy humans [START_REF] Colson | The role of giant viruses of amoebas in humans[END_REF]. Perhaps, the most notable of these findings is the detection in human blood of several members of Marseilleviridae one of which has been reported to grow in T lymphocytes [START_REF] Popgeorgiev | Marseillevirus-like virus recovered from blood donated by asymptomatic humans[END_REF]. Additionally, several viruses from both Marseilleviridae and Mimiviridae have been isolated from human stools or detected in human-associated metagenomes [START_REF] Colson | Evidence of the megavirome in humans[END_REF]. Furthermore, the presence of mimiviruses in peripheral blood mononuclear cells of patients with atypical pneumonia has been reported, and a role for these viruses in the pneumonia pathogenesis has been suggested [START_REF] Colson | Giant viruses of amoebae as potential human pathogens[END_REF]. Unexpectedly, DNA of Acanthocystis turfacea chlorella virus 1, a member of Phycodnaviridae, has been detected in nearly half of the tested oropharyngeal samples from healthy humans, and also has been reported to persist in mouse macrophages [START_REF] Petro | Response of Mammalian Macrophages to Challenge with the Chlorovirus Acanthocystis turfacea Chlorella Virus 1[END_REF].

However, so far, many of the reports on the presence of members of Nucleocytoviricota in human samples and, especially, their ability to replicate in human cells have been disavowed in followup studies [START_REF] Abrahao | Lack of evidence of mimivirus replication in human PBMCs[END_REF]. Thus, the status of these viruses as components of the healthy human virome except, perhaps, as occasional contaminants, remains dubious.

Conclusions

The healthy virome of any organism, and especially humans, clearly, is an important component of the holobiont that makes a major contribution to the health status of the host. However, the very concept of a healthy virome is nebulous and fluid because it is virtually impossible to ascertain that any virus would not cause disease under any conditions. A strong case in point are the herpesviruses that are nearly ubiquitous in the human population, remaining symbionts in most individuals most of the time, but consistently cause disease, in some cases, devastating, in immunocompromised individuals. Conversely, it appears plausible that any virus can become beneficial to the host through protection from other viruses, general stimulation of immunity as in the striking case of β-HPV protecting human hosts from skin cancer, or recruitment of virus genes for host functions. The numerous HERVs integrated in the human genome can be considered the paradigm of virus-host symbiosis. Generally, there is no doubt that many viruses evolved multiple mechanisms to manipulate the host innate and adaptive immunity pathways, ensuring virus persistence and controlling the damage to the host, as most conspicuously exemplified by the latent herpesviruses that are virtually ubiquitous in the human population.

The healthy virome is obviously heterogeneous and consists of 3 distinct components (Figure 1a): i) viruses that systematically enter the human organism, primarily, with food, but do not replicate in humans, ii) viruses infecting prokaryotes and, possibly, unicellular eukaryotes that comprise the healthy human microbiome, and iii) viruses that actually replicate and persist in human cells. With the advances of metagenomics, the human "microbiovirome" has become a subject of intense studies that continue bringing discoveries of new bacteriophage groups. In contrast, the "true" healthy human virome is poorly understood, with many questionable sightings of diverse viruses but little solid evidence on persistence mechanisms. On the whole, and in contrast to the diseaseassociated virome, the healthy human virome appears to be dominated by DNA viruses, in particular, anelloviruses and herpesviruses, that are substantially more common than RNA viruses in healthy humans. A thorough investigation of this component of the healthy virome can be expected to enhance our understanding of virus-host interactions and have major implications for human health.

Figure 1 .

 1 Figure 1. The healthy human virome. (a) Taxa of viruses found in healthy humans. Taxa of viruses replicating in human cells are shown on the left, whereas those of viruses infecting human-associated microbes or associated with food sources are shown on the right (the actual or suspected hosts are listed next to the corresponding taxa, with the uncertain assignments indicated with broken lines). Depicted virus taxa are represented with virion structures which were retrieved from VIPERdb (viperdb.scripps.edu) or the Electron Microscopy Data Bank (https://www.ebi.ac.uk/pdbe/emdb/). When the structure of a virus representing a particular taxon was not available, a structurally related member was chosen instead. Virus taxa are colored according to their realm affiliation (the key is provided at the bottom). (b) Fluidity of the healthy human virome. The (potential) beneficial effects of the healthy human virome are indicated next to the corresponding virus structures. Question marks denote uncertainty. Upon changes in the health status/immunosuppression, viruses that cause asymptomatic infections or are beneficial in healthy individuals proliferate and can cause diseases including severe ones. The figure illustrates the general tendency of increased virus load under immunosuppression/disease conditions and should not be interpreted as a quantitative representation of the changes for any depicted group of viruses.

Acknowledgements E.V.K. is supported by the Intramural Research Program of the National Institutes of Health (National Library of Medicine). M.K was supported by l'Agence Nationale de la Recherche grant ANR-20-CE20-0009.


MD Chloé Angelini
email: chloe.angelini@icloud.com

Marie Thibaud

Nathalie Aladjidi

Pierre Bessou

Sébastien Cabasson

Cindy Colson

Caroline Espil-Taris

Cyril Goizet

Marie Husson

Fanny Morice-Picard

Annachiara De Sandre-Giovannoli

Jean-Michel Pédespan

Expanding the Spectrum of Neurological Manifestations in Cutis Laxa, Autosomal Recessive, Type IIIA

Keywords:

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Introduction

Cutis laxa represents a spectrum of clinical entities, including lax and hyperelastic skin with visible veins. It is caused by defective elastin synthesis, structural abnormalities of the extracellular matrix, and also defects in metabolic pathways. These diseases may be acquired or inherited. Inherited cutis laxa are very rare diseases and show different inheritance patterns (autosomal dominant, autosomal recessive, and X-linked recessive inheritance). [START_REF] Mohamed | Cutis laxa[END_REF] To date, 13 different forms of inherited cutis laxa syndromes have been described. [START_REF] Ritelli | Clinical and molecular characterization of an 18-month-old infant with autosomal recessive cutis laxa type 1C due to a novel LTBP4 pathogenic variant, and literature review[END_REF] Cutis laxa type IIB (OMIM#612940), type IIIA (OMIM#219150), and type IIIB (OMIM#614438), are known as De Barsy syndrome, and are associated with pathogenic variants in PYCR1 and ALDH18A1, two genes playing an important role in proline metabolism. [START_REF] Wolthuis | Cutis laxa, fat pads and retinopathy due to ALDH18A1 mutation and review of the literature[END_REF] PYCR1 encodes for pyrroline-5-carboxylate reductase, an enzyme involved in the last steps of proline synthesis from glutamate. [START_REF] Coutelier | Alteration of ornithine metabolism leads to dominant and recessive hereditary spastic paraplegia[END_REF] PYCR1 pathogenic variants can lead to cutis laxa, autosomal recessive, type IIB and type IIIB, with a phenotypic overlap, and are characterized by a cutis laxa of variable severity, abnormal growth, developmental delay, and associated skeletal abnormalities.

ALDH18A1 encodes for delta-1-pyrroline-5-carboxylate synthase (P5CS), an enzyme that catalyzes the reduction of glutamate to delta1-pyrroline-5-carboxylate, and plays a key role in the de novo biosynthesis of proline, ornithine, and arginine. 5 ALDH18A1 pathogenic variants are responsible for cutis laxa, autosomal recessive, type IIIA, cutis laxa autosomal dominant 3 (OMIM#616603), and also spastic paraplegia 9A, autosomal dominant (OMIM#601162), and 9B, autosomal recessive (OMIM#616586). There is a phenotypic overlap between these four syndromes, with variable neurologic symptoms.

Cutis laxa, autosomal recessive, type IIIA, is a rare genetic condition. [START_REF] Kivuva | De Barsy syndrome: a review of the phenotype[END_REF][START_REF] Bicknell | A missense mutation in ALDH18A1, encoding Delta1-pyrroline-5carboxylate synthase (P5CS), causes an autosomal recessive neurocutaneous syndrome[END_REF] Only about 20 patients have been described in the literature. [START_REF] Lefebvre | Extending the ALDH18A1 clinical spectrum to severe autosomal recessive fetal cutis laxa with corpus callosum agenesis[END_REF] Its clinical spectrum includes abundant and wrinkled skin, skeletal anomalies, neurodevelopmental disorders, cataract and corneal clouding, and, in some cases, severe neurological symptoms such as major developmental delay, hypotonia in infancy, hyperreflexia, athetoid movements, and seizures. [START_REF] De Barsy | Dwarfism, oligophrenia and degeneration of the elastic tissue in skin and cornea. A new syndrome?[END_REF][START_REF] Hoefnagel | Congenital athetosis, mental deficiency, dwarfism and laxity of skin and ligaments[END_REF] The cellular phenotype is characterized by diminished production of collagen types I and III, altered elastin ultrastructure, and diminished cell proliferation. 11 Cutis laxa, autosomal dominant 3, is characterized by excessive skin with visible veins, cataract or corneal clouding, clenched fingers, pre-and postnatal growth retardation, and moderate intellectual disability. Some patients may also present muscular hypotonia with brisk muscle reflexes and cranial vessel tortuosity with foramen magnum stenosis. The phenotype seems to be less severe than cutis laxa, autosomal recessive, type IIIA, but with a greater neurological involvement, with hypertonia, spasticity, and brain magnetic resonance imaging (MRI) abnormalities, such as tortuous intracranial vessels, widened perivascular spaces, and white matter signal anomalies on T2 and fluid-attenuated inversion recovery (FLAIR) sequences. [START_REF] Fischer-Zirnsak | Recurrent de novo mutations affecting residue Arg138 of pyrroline-5-carboxylate synthase cause a progeroid form of autosomal-dominant cutis laxa[END_REF] Spastic paraplegia 9A is a neurologic disorder with progressive spasticity appearing from adolescence to adulthood. Patients present with lower limbs spasticity, and may have additional features, such as cataract, cerebellar signs, and urinary urgency. [START_REF] Coutelier | Alteration of ornithine metabolism leads to dominant and recessive hereditary spastic paraplegia[END_REF] Spastic paraplegia 9B tends to be more severe, with childhood onset spastic paraplegia, delayed psychomotor development, intellectual disability, and severe motor impairment. [START_REF] Coutelier | Alteration of ornithine metabolism leads to dominant and recessive hereditary spastic paraplegia[END_REF] We describe here a 13-month-old patient, with a homozygous missense pathogenic variant in ALDH18A1. He had a very severe phenotype, with strong neurological involvement. This description widens the phenotypic spectrum associated with mutations of this gene.

Clinical Report

The patient was the first child of a consanguineous couple of first cousins, who were both from Senegal. No medical relevant history was found in their families.

During pregnancy appeared severe intrauterine growth retardation (IUGR), associated to oligoamnios. Amniocentesis was done to perform a genetic work up, including an array-comparative genomic hybridization and a karyotype, both were normal.

He was born at 34 gestational weeks by a cesarean section, planned because of IUGR. He needed breathing assistance immediately after birth, with noninvasive ventilation, during 3 hours. He weighed 1.66 kg (-3.9 standard deviation [SD]), measured 40 cm (-5SD), and his head circumference was 31 cm (-3.8 SD).

Examination at birth revealed a large fontanel, adductus thumbs, global hypotonia, and a major systolic cardiac murmur. Transfontanellar ultrasound was normal, as well as an electroencephalogram, an abdominal ultrasound, and skeletal X-rays. Cardiac ultrasound found a large ventricular septal defect and a large atrial septal defect. Ophthalmological examination with eye fundoscopy revealed bilateral corneal clouding.

Neurological examination worsened with months and he presented with four limbs spasticity, permanent tremor, and eye twitching. He had also severe dystonia and four limbs contractures that led to hip and shoulder dislocations (►Fig. 1). Adductus thumbs as well as calcaneus valgus feet were persistent.

He presented at age 5 months partial seizures with focal onset and motor manifestations (focal myoclonia), recorded on electroencephalogram with multifocal spikes, and slow waves with very slow background activity and absence of sleep spindles (►Fig. 2). He was from then effectively treated with oxcarbazepine.

A brain MRI was performed at age 8 months (►Fig. 3). Diffuse supra-and infratentorial cortical atrophy was found, with brain stem contours distortions, associated to a thin corpus callosum and large ventricles due to gray matter paucity. A severe dysmyelination was observed with abnormal spectroscopy (lactate increased and N-acetyl aspartate decreased). Basal ganglia were hardly identified. Willis polygon vessels were abnormal, and described as megadolichovessels.

He exhibited a severe failure to thrive during follow-up. He presented with multiple regurgitations and feeding was performed with a nasogastric tube. At age 13 months, he weighed 3.46 kg (-6.0 SD), measured 50 cm (-10.1 SD), and head circumference was 37 cm (-8.2 SD).

His skin was very wrinkled and abundant. He had three episodes of skin abscesses, with persistent axillar adenopathies. He also had several episodes of respiratory distress without opportunist infections. Humoral and cellular immune analyses were normal. Blood lactate assay was normal at 1.5 mmol/L. Dysmorphia was marked, with a triangular face, microcephaly, small mouth, retrognathism, and posteriorly rotated ears (►Fig. [START_REF] Mohamed | Cutis laxa[END_REF].

He died at age 16 months, after 2 weeks of resopiratory infection and global worsening of his general state.

Molecular Analysis

Deoxyribonucleic acid was extracted from peripheral blood samples of the patient and his parents. The targeted nextgeneration sequence analysis of a 82 genes panel including genes involved in premature aging and laminopathies was performed for the proband (gene list available on orphanet, https://www.orpha.net/consor/cgi-bin/ClinicalLabs_Search. php?lng=FR&data_id=128241&search=ClinicalLabs_Search_ Simple&data_type=Test&title=Diagnostic%20des%20lamino-pathies%20et%20du%20vieillissement%20prematur%DA%20 Panel&MISSING%20CONTENT=Diagnostic-des-laminopathies-et-du-vieillissement-prematur-Panel-).

A SureSelectXT 12 to 24 Mb library (Agilent Technologies, Santa Clara, California, United States) was used for enrichment of targeted sequences using the SureSelect Target Enrichment System from Agilent. The Ion Proton platform (Thermo Fisher Scientific, United States) was used for highthroughput sequencing. Then, raw data were converted to Fastq files and aligned to the reference sequence of the human genome (University of California Santa Cruz, hg19/ GRCh37), using the Torrent Suite software (Thermo Fisher).

A variant calling format file and binary alignment map and index files were then obtained and used for variant annotation using the in-house software VarAFT (Variant Annotation and Filtration Tool). [START_REF] Desvignes | VarAFT: a variant annotation and filtration system for human next generation sequencing data[END_REF] Sequence reads and variants' visualization was performed using IGV (Integrative Genomics Viewer). [START_REF] Robinson | Integrative genomics viewer[END_REF] The variants were classified according to the American College of Medical Genetics and Genomics classification. [START_REF] Richards | Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology[END_REF] This targeted next-generation sequencing led to the identification of a homozygous pathogenic variant in ALDH18A1, NM_002860.3: exon 13: c.1499G > T, p. (Gly500Val) (ClinVar VCV000638645.1). This variant was absent from Human Gene Mutation Database (HGMD), GnomAD, ExAC, and 1000 Genomes databases. All of the in silico prediction programs, including Mutation Taster, UMD-Predictor, and Human Splicing Finder predicted this variant to be deleterious. Combined Annotation Dependent Depletion score was assessed at 27.8, a high score, supporting predictions of the programs. This variant was classified as likely pathogenic (PM1, PM2, PM3, PP3) according to the American College of Medical Genetics and Genomics classification. [START_REF] Richards | Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology[END_REF] Sanger sequencing of ALDH18A1 exon 13 proofed this variant in a homozygous state in the index patient and revealed it in a heterozygous state in the parents, consistent with autosomal recessive inheritance.

Discussion

We present a patient with a cutis laxa, autosomal recessive, type IIIA. This syndrome is rare with only about 20 patients described to date. His clinical phenotype is similar to the other cases described. [START_REF] Lefebvre | Extending the ALDH18A1 clinical spectrum to severe autosomal recessive fetal cutis laxa with corpus callosum agenesis[END_REF] However, few data are available regarding neuroimaging. Only eight patients with autosomal recessive variants underwent a brain MRI, which highlighted white matter anomalies for most of patients, a thin corpus callosum in three, and vascular tortuosity in one. [START_REF] Wolthuis | Cutis laxa, fat pads and retinopathy due to ALDH18A1 mutation and review of the literature[END_REF][START_REF] Baumgartner | Δ1-pyrroline-5carboxylate synthase deficiency: neurodegeneration, cataracts and connective tissue manifestations combined with hyperammonaemia and reduced ornithine, citrulline, arginine and proline[END_REF] One of the patients described had a normal brain MRI. [START_REF] Fischer | Severe congenital cutis laxa with cardiovascular manifestations due to homozygous deletions in ALDH18A1[END_REF] De novo ALDH18A1 variants lead to a cutis laxa, autosomal dominant 3 syndrome, with a milder phenotype, but with a higher risk of developing hypertonia and spasticity. [START_REF] Fischer-Zirnsak | Recurrent de novo mutations affecting residue Arg138 of pyrroline-5-carboxylate synthase cause a progeroid form of autosomal-dominant cutis laxa[END_REF] A recent review from Sinnige et al describes three patients with de novo variants in ALDH18A1, and sums up the different neuroradiological findings in the literature. [START_REF] Sinnige | Imaging in cutis laxa syndrome caused by a dominant negative ALDH18A1 mutation, with hypotheses for intracranial vascular tortuosity and wide perivascular spaces[END_REF] Eleven patients are reported with dominant negative variants, and the main findings were tortuous intracranial vessels, widened perivascular spaces, and white matter signal anomalies on T2 and FLAIR sequences.

In our patient, tortuous intracranial vessels were noted and were predominant on Willis polygon circle. This is concordant with the description in the Sinnige et al review, where tortuous vessels were present mostly on arteries, only two patients were described with venous tortuosity, and only on the cerebral and basilar arteries. [START_REF] Sinnige | Imaging in cutis laxa syndrome caused by a dominant negative ALDH18A1 mutation, with hypotheses for intracranial vascular tortuosity and wide perivascular spaces[END_REF] Martinelli et al hypothesized that this tortuosity could be secondary to abnormal proline metabolism, affecting the production of proline-rich connective tissue proteins such as elastin and collagen and then leading to defective interactions between the extracellular matrix and vascular smooth muscle. [START_REF] Martinelli | Understanding pyrroline-5-carboxylate synthetase deficiency: clinical, molecular, functional, and expression studies, structure-based analysis, and novel therapy with arginine[END_REF] The data on our patient enlarge the spectrum of neuroimaging findings and provides an additional global description of a patient with cutis laxa, autosomal recessive, type IIIA. A link is thus established between the neurological phenotypes of autosomal dominant 3 and recessive type IIIA cutis laxa syndromes.

Moreover, this variant has never been described before. We performed an in silico analysis to establish its pathogenicity and also provide an explanation of the patient's clinical symptoms, which belong both to autosomal recessive type IIIA and autosomal dominant 3 cutis laxa.

P5CS, the enzyme encoded by this gene, is a single-chain homo-oligomeric protein, and is composed of two main domains, glutamate 5-kinase (G5K) and glutamyl-5-phosphate reductase (G5PR). [START_REF] Panza | ALDH18A1 gene mutations cause dominant spastic paraplegia SPG9: loss of function effect and plausibility of a dominant negative mechanism[END_REF][START_REF] Magini | P5CS expression study in a new family with ALDH18A1-associated hereditary spastic paraplegia SPG9[END_REF] The G5PR domain is divided into four parts: a cofactor binding domain, a catalytic domain, and two oligomerization domains. The oligomerization domains are close one to another in a tertiary structure, and the cofactor binding and catalytic domains interact with another P5CS in a quaternary structure, thanks to inter-subunit salt bridges.

No hotspot of pathogenic variants has been described for cutis laxa autosomal recessive type IIIA, as attested by the Clinvar and HGMD. For cutis laxa autosomal dominant 3, a predominance of a missense variant on residue Arg138 has been observed. [START_REF] Fischer-Zirnsak | Recurrent de novo mutations affecting residue Arg138 of pyrroline-5-carboxylate synthase cause a progeroid form of autosomal-dominant cutis laxa[END_REF] Mainly missense variants are described for the four pathologies linked to ALDH18A1. The position of our variant, p.Gly500, is considered as intolerant to missense variations in the Metadome database (score at 0.38). 21 Our variant is located in the cofactor binding domain of G5PR, and in quaternary structure sits at the junction of the cofactor binding domain, the catalytic domain, and the oligomerization domain of the G5PR part of the protein, as shown in Magini et al publication. [START_REF] Magini | P5CS expression study in a new family with ALDH18A1-associated hereditary spastic paraplegia SPG9[END_REF] We hypothesize that the amino acid change due to our variant disturbs the global equilibrium of the oligomerization.

However, these in silico analyses cannot account fully for the pathogenicity of the patient's variant, or explain the phenotypic overlap between autosomal recessive and dominant cutis laxa. While the patient's parents are symptomfree, it would have been interesting to perform functional studies in skin biopsies of the patient and his parents. Evaluation of proline biosynthesis after loading [START_REF] Desvignes | VarAFT: a variant annotation and filtration system for human next generation sequencing data[END_REF] C 5 -labeled glutamine, and comparison of the flux between controls, parents, and patient would have provided interesting data. It may have shown a significant reduction in proline biosynthesis in the patient, consistent with an enzymatic deficiency of P5CS. [START_REF] Coutelier | Alteration of ornithine metabolism leads to dominant and recessive hereditary spastic paraplegia[END_REF] Results for the parents might be harder to interpret as they are symptom-free, but a slight decrease in flux could be interpreted as a partial dominant negative effect of the variants.

In conclusion, this work suggests the phenotypic overlap between the four syndromes due to ALDH18A1 pathogenic variants, and underscores the need to perform a brain MRI in every patient with ALDH18A1-related disorders, whatever the type of variants highlighted.

Fig. 2

 2 Fig. 2 Electroencephalogram data. Standard parameters, 70 Hz, 70 µV/cm. Sleep interictal electroencephalogram showed active and irregular discharge of multifocal spikes, slow waves with abnormal (very slow) background activity, and absence of sleep spindles. No cluster of spasms were recorded after a focal seizure.

Fig. 1

 1 Fig.1Clinical presentations: photographs aged 11 months (A, B, C) and X-rays (D: chest X-rays and E: pelvis X-rays).Observe the dysmorphia with triangular face, low and posteriorly rotated ears, anteverted nares, and small mouth with thin vermilion lips. Skin is wrinkled, abundant, and dry with cracks on the face. Note four limb contractures with dislocated shoulders and hips, also present on X-rays.

Fig. 3

 3 Fig. 3 Brain magnetic resonance imaging (MRI). Axial T2-weighed sequences (A and B), sagittal T2-weighed sequences (C) and threedimensional (3D) time-of-flight (TOF) (D). Observe the supra-and infratentorial cortical atrophy and the thin corpus callosum. Dysmyelination can be observed in B. Interesting features to note are megadolichovessels of Willis polygone (A, C, D).

Neuropediatrics Vol. 51 No. 4/2020 Expanding the Spectrum of Neurological Manifestations in Cutis Laxa Angelini et al.

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Neuropediatrics Vol. 51 No. 4/2020

Conflict of Interest

None declared.