Frédéric Rees
email: frederic.rees@inrae.fr

Marion Gauthier

Romain Barillot

Céline Richard-Molard

Alexandra Jullien

Claire Chenu

Christophe Pradal

Bruno Andrieu

Quantitative importance of various rhizodeposition processes: lessons from a mechanistic functional-structural root model

Keywords: FSPM, root exudation, mucilage, border cells, root hairs, wheat

Introduction

Rhizodeposits, defined here as any organic materials released by living roots, can represent up to 15% of net amount of carbon (C) photosynthesized by a plant (Pausch and Kuzyakov, 2018). Rhizodeposits have been shown to play an important role in plant growth through various mechanisms (e.g. by favoring water uptake through mucilage secretion, or nutrient uptake through the exudation of soluble compounds), and to represent a major contributor to the formation of stable soil organic matter -and thus to the mitigation of climate change through soil C sequestration. Decomposing rhizodeposition into its elementary processes (e.g. exudation, mucilage secretion, cells sloughing) is required for a better understanding of the response of rhizodeposition to various environmental constraints and of its associatedbenefits. While many works have attempted to quantify the exudation of soluble organic compounds at the scale of a plant or the net rhizodeposition of C in soils, very few studies have been able to estimate the amount of mucilage or sloughed cells released by a whole root system over time. Consequently, while it has been claimed that the amount of soluble organic C is much higher than the amount of C released as mucilage or sloughed cells by roots (Nguyen, 2003;Rees et al. 2005), the relative importance of distinct rhizodeposition processes in plant's and soil's C balance remains largely unknown. While the comparison of mucilage secretion and citrate exudation within a FSPM has been recently attempted (Landl et al. 2021), no model has been developed so far for simulating such rhizodeposition processes as part of plant's C management. The present work aimed to give a better assessment of the contribution of distinct rhizodeposition processes to the total amount of organic C released by roots, for different plant species, growth stages and environments.

Modelling approach

We used the recently developed root model RhizoDep, which aims to simulate rhizodeposition over time and space as a function of plant's environment. Its originality lies in the calculation of a full C balance in each part of the root system, which enables to simulate physiological processes (e.g. growth, maintenance and rhizodeposition) in a plastic way, depending on the variations of root sugar concentrations (Figure 1). Three distinct rhizodeposition processes have been implemented: the exudation of soluble organic compounds, the secretion of mucilage and the sloughing of root cells. The present version also explicitly considers the dynamics of root hairs, as it may significantly influence rhizodeposition processes, e.g. by changing root external surface. Model's parameters were taken from literature or recalculated from published data. The amount of C allocated to the root system over time, which is the major input variable of the model, was either estimated from literature data or simulated by the wheat FSPM CN-Wheat (Barillot et al. 2016;Gauthier et al. 2020).

Simulations & first results

We simulated the amount of C released by roots from species that contrast in terms of root architecture and rhizodeposition (e.g. wheat, oilseed rape, faba bean). Simulations were done for different growth stages and environmental conditions (e.g. nitrogen availability, light availability and soil mechanical impedance). Our results show that the exudation of soluble compounds is predominant compared to other rhizodeposition processes, but the secretion of mucilage and the release of cells are substantially increased in certain situations. Mucilage secretion or cells sloughing had until now been investigated only at the scale of a single root and over periods of a few hours. Our work shows that FSPM-modelling offers a unique opportunity to integrate these processes at plant's scale over the whole growth period, while considering various environmental influences on root growth and rhizosphere processes.


Laura Mary : Investigation

Mélanie Fradin

Laurent Pasquier

Chloé Quelin

Philippe Loget

Maela Le Lous

Gwenaëlle Le Bouar

Sylvie Nivot-Adamiak

Anna Lokchine

Christèle Dubourg

Vincent Jauffret

Bénédicte Nouyou

Catherine Henry

Erika Launay

Sylvie Odent

Le Lous

Le Bouar

Belaud- Rotureau Ma

Role of chromosomal imbalances in the pathogenesis of DSD: a retrospective analysis of 115 prenatal samples

Keywords:

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

Introduction

Differences of sex development (DSDs) are congenital variations characterized by a discrepancy between chromosomal, gonadal, and genital sex development. Prevalence of DSDs differs greatly depending on the variation considered: from 1 in 20,000 births for 46,XY complete gonadal dysgenesis or 46,XX testicular DSD to 1 birth in 250 boys for hypospadias [START_REF] Atlas | Targeting the Non-Coding Genome for the Diagnosis of Disorders of Sex Development[END_REF]. Identification of a DSD often lead to a lifelong multidisciplinary medical management, with significant impact on medical, psychological and reproductive life of the patients [START_REF] Hughes | Consensus statement on management of intersex disorders[END_REF]. Moreover, delay in diagnosing DSDs may burden a patient's life prognosis, since some DSDs increase the risk of gonadal malignancies [START_REF] Cools | Caring for individuals with a difference of sex development (DSD): a Consensus Statement[END_REF].

The genetic landscape underlying these differences depends on the type of DSD considered.

Gonosomal aneuploidies account for 33% to almost 50% of all DSD patients [START_REF] Globa | Disorders of Sex Development in a Large Ukrainian Cohort: Clinical Diversity and Genetic Findings[END_REF][START_REF] Mazen | Advances in genomic diagnosis of a large cohort of Egyptian patients with disorders of sex development[END_REF]. Variations in gonadal development are frequently due to SRY translocation on one X chromosome (50-90%, [START_REF] Mazen | Advances in genomic diagnosis of a large cohort of Egyptian patients with disorders of sex development[END_REF]). A large amount of the remaining DSDs are monogenic, with whole exome sequencing (WES) reaching a diagnosis efficiency of around 40-60% in these forms J o u r n a l P r e -p r o o f of DSD [START_REF] Globa | Disorders of Sex Development in a Large Ukrainian Cohort: Clinical Diversity and Genetic Findings[END_REF][START_REF] Mazen | Advances in genomic diagnosis of a large cohort of Egyptian patients with disorders of sex development[END_REF]. In addition to point variations, copy number variants (CNVs) and large chromosomal imbalances are rarely identified in cohorts of isolated DSD patients (4%, [START_REF] Mazen | Advances in genomic diagnosis of a large cohort of Egyptian patients with disorders of sex development[END_REF]), but can hit 30% when syndromic DSDs are also considered [START_REF] Ledig | Array-CGH analysis in patients with syndromic and non-syndromic XY gonadal dysgenesis: evaluation of array CGH as diagnostic tool and search for new candidate loci[END_REF]. However, deciphering the sexual development complexity remains challenging. Pathogenic CNVs identified in syndromic DSD patients may help to (1) identify new genetic mechanisms in known DSD genes, (2) highlight new DSD genes, (3) help to assess the role of the non-coding genome in DSDs.

In this study, we aimed at describing the CNV burden in DSD patients extracted from a cohort of 4657 prenatal samples addressed for a polymalformative syndrome, and review the role of pathogenic CNVs in DSDs.

Patients and Methods

Patients

We retrospectively analyzed clinical and genetic data from fetal consecutive samples addressed to our laboratory between 2010 and 2021. A total of 4657 cases with polymalformative syndromes diagnosed through prenatal ultrasound examination were considered. Genital malformations were diagnosed and classified according to the Chicago DSD consensus [START_REF] Hughes | Consensus statement on management of intersex disorders[END_REF]. Fetuses or neonates presenting with DSD and carrying at least one pathogenic chromosomal imbalance were retained for inclusion in this study. CNVs' classification was established based on the recommendations of the French array-CGH (aCGH) network (AChro-Puce https://acpa-achropuce.com/). DSD linked to gonosomal aneuploidies, trisomy for chromosomes 13, 18 and 21, samples without clinical data, and patients carrying only CNVs considered benign were dismissed. Standardized autopsy procedures were performed, including skeletal X-rays, macroscopic examinations and histology analyses. All samples were obtained with informed consent according to the protocols approved by the local ethics committee (Rennes hospital).

Methods

J o u r n a l P r e -p r o o f

Karyotype: RHG-banding karyotypes were performed on cultured amniocytes or on chorionic villi (for all the fetuses, except P6 and P9) and cultured lymphocytes obtained from peripheral blood (for newborn and parents) according to standard cytogenetic procedures.

Chromosomal microarray analysis (CMA): Oligonucleotide aCGH was performed using the Agilent Human Genome CGH microarray 44K, 60K, 105K or 180K (depending on the year the sample was received), with a genome-wide median probe spacing 43 kb, 41 kb, 22 kb and 13 kb respectively (Agilent Technologies, Santa Clara, CA, USA). The experiments were performed according to the manufacturer's protocol. Patients' gDNAs were obtained from chorionic villi, amniotic fluid, peripheral blood or frozen tissues using Qiagen DNeasy Blood and Tissue kit (Qiagen, Hilden, Germany) before 2016 and a Prepito instrument (PerkinElmer Inc., Turku, Finland) after this year. A graphic overview and analysis of the data were obtained using Agilent softwares. The probes were mapped using the GRCh37 genome version. FISH: FISH analyses using in-house BAC probes were used to confirm chromosomal imbalances and to study their inheritance, when parental samples were available (probes available on demand).

Classification of the variants was based on data found in the literature and international CNV databases (DECIPHER, DGV, gnomAD), and on inheritance and gene content of the CNV, in accordance with recommendations by the French aCGH network (AChro-Puce).

Results

Genital malformations were present in approximately 2.5% (115) of the 4657 patients. Of note, in our cohort, 3 patients without genital anomalies carried pathogenic CNV encompassing genes previously reported in DSD (SOX9, SOX3, GATA4, not detailed in this work). An inclusion chart is presented in Figure 1a. Global description of the CNVs observed in the reported patients is summarized in Figure 1b.

Fourteen patients were retained for inclusion in this work. A termination of pregnancy (TOP) was decided in 13/14 cases due to severe malformations, and one pregnancy ended with a living birth.

J o u r n a l P r e -p r o o f

Autopsy procedures were performed on 10 fetuses and 3 others benefited only from full external examination. Pediatricians evaluated the newborn patient shortly after birth and at 3 months of age.

Nine patients had a XY chromosomal sex (P2 to P9, P12) and 5 patients a XX chromosomal sex (P1, P10, P11, P13 and P14). Chromosome 9 anomalies accounted for 28% of the CNVs found in our patients (4/14). Chromosomal imbalances were also observed for chromosomes 3, 4 and 11, in 14% of the patients each (2/14). All patients presented with a syndromic DSD. Complete phenotypes of the patients (including non-genital/gonadal malformations) are summarized in Table 1. For 46,XY DSD patients, the clinical findings were mainly micropenis, isolated (P3, P6 to P8) or associated with hypospadias (P4, P5, P12). Cryptorchidism and isolated hypospadias was observed in one case each (P2, P9). None of them presented any Müllerian remnants. 46,XX DSD presented with more variable phenotypes, ranging from isolated hypoplastic ovaries (P14) to almost complete sex reversal with ovotestis (P1). The 3 other 46,XX DSD patients presented with Müllerian malformations (P11 and P13) or agenesis (P10). Familial segregation of the anomalies are reported in Table 1 when obtained.

Patients are presented according to the size of their imbalance(s) (larger or smaller than 5Mb, sSMC (small supernumerary chromosome marker)) and then by patient's numerical order.

Imbalances >5Mb

P1 presented a XX DSD with a urethral opening under the genital tubercle, fused labioscrotal swellings and absent vaginal introitus nor Müllerian remnants. Histologic examination of the gonads revealed a left gonad being an ovotestis: a fibrous gonad with a region of testicular-like architecture with albuginea and seminiferous tubes containing Leydig cells, near an ovary-like region with isolated germ cells in an undifferentiated stroma (Figure 2a). The architecture of the right gonad was closer to a testis with fibrous modifications (Figure 2b). This case presented with mosaicism for complete trisomy 9. The mosaicism was determined to be around 30% through CMA on amniotic fluid. A karyotype was performed on an umbilical cord sample, highlighting a 14% mosaicism, confirmed by FISH analyses on cultured fibroblasts using a chromosome 9 centromeric probe associated with a CDKN2A probe.

J o u r n a l P r e -p r o o f

Karyotype on placental sample found a complete homogeneous trisomy 9, leading to the diagnosis of fetoplacental mosaicism. Genitourinary variations are observed in almost half cases of complete trisomy 9, including XX sex reversal [START_REF] Stoll | Trisomy 9 mosaicism in two girls with multiple congenital malformations and mental retardation[END_REF]. XX sex reversal was also reported in trisomy 9 mosaic syndrome, despite the fact that cryptorchidism in XY individuals is the most frequent DSD observed ( [START_REF] Li | Trisomy 9 mosaic syndrome: Sixteen additional patients with new and/or less commonly reported features, literature review, and suggested clinical guidelines[END_REF][START_REF] Solomon | Trisomy 9 mosaicism and XX sex reversal[END_REF]. P1 also presented with a de novo 567kb-sized 13q13.3 deletion, disrupting 5 genes, including a read-through transcript containing SOHLH2 (OMIM*616066). This late gene is suspected to play a role in oogenesis and is considered as a candidate gene for premature ovarian failure (POF), but DSD were not reported in animal models [START_REF] Choi | Germ cell-specific transcriptional regulator sohlh2 is essential for early mouse folliculogenesis and oocyte-specific gene expression[END_REF]. The other 4 genes were not related to gonadal or genital development. Finally, such deletions were reported neither in the literature nor in general populations' databases, and we considered this CNV as a variant of unknown significance (VOUS), without significant role in the patient's genital phenotype.

Three patients (P2, P3 and P4) with a XY DSD had partial chromosome 9 trisomy. It is of note that P2 was the only living birth in our cohort. These chromosomal rearrangements were linked to the presence of a derivative chromosome from a balanced de novo (P4) or maternal (P3) reciprocal translocation. No paternal karyotype was performed for P2 and the mother had a normal karyotype.

Except for P2, a second pathogenic CNV was present in these patients. P2 was a XY syndromic DSD patient diagnosed shortly after birth with bilateral cryptorchidism. He presented with an almost complete 9p trisomy (9p24.3p11.2, 45.2Mb) due to a chromosome 15 derivative from a reciprocal (9;15) translocation. P3 was a syndromic XY DSD patient with micropenis. He showed a partial proximal 9p trisomy (9p24.3q21.13, 77.7Mb) and a 14q32.2q32.33 trisomy (3.3Mb) due to a supernumerary chromosome 9 derivative from a balanced maternal reciprocal translocation (9;14). P4 was a syndromic XY DSD patient with micropenis and hypospadias. He had a terminal 9q trisomy (9q33.3q34.3, 14.9Mb) and a 5.8Mb-sized 11q24.3q25 deletion linked to a chromosome 11 derivative of a de novo reciprocal translocation (9;11). Hypoplastic genitalia are reported in XY patients bearing pure 9p trisomy or partial distal 9q trisomy [START_REF] Sato | Pure 9p duplication syndrome with aplasia of the middle phalanges of the fifth fingers[END_REF]. Numerous genes are implicated in the imbalances diagnosed in patients P1 to P4, but two genes are known to be implicated in gonadal J o u r n a l P r e -p r o o f development, namely DMRT1 (9p24.3, OMIM*602424) and NR5A1 (9q33.3, OMIM*612965). Both genes are sensitive to haploinsufficiency, but only NR5A1 is triplosensitive. Duplications encompassing DMRT genes cluster were both reported in ClinVar and DECIPHER as likely benign or VOUS. However, Dmrt1 copy number variants are suspected to play a role in sex determination in non-mammalian species ( [START_REF] Lambeth | Over-expression of DMRT1 induces the male pathway in embryonic chicken gonads[END_REF] andDiscussion). P6 presented with a syndromic XY DSD with micropenis, and the association of de novo terminal 4p duplication (4p16.3p15.2, 27Mb) and terminal 4q deletion (4q32.3q35.2, 25Mb). The duplication encompassed the Wolf-Hirschhorn Syndrome (WHS, OMIM#194190) critical region. The deletion involved at least 60 genes, including NSD2 (OMIM*602952) and NELFA (OMIM*606026) that were linked to WHS core features, including DSD [START_REF] Battaglia | Wolf-Hirschhorn syndrome: A review and update[END_REF]. The association of both 4p duplication and 4q deletion was reported in the literature in case of recombinant of chromosome 4 pericentric inversion [START_REF] Maurin | Molecular cytogenetic characterization of a 4p15.1-pter duplication and a 4q35.1-qter deletion in a recombinant of chromosome 4 pericentric inversion[END_REF], with DSD mainly observed in XY patients (hypospadias, hypoplastic scrotum).

P7 had a syndromic XY DSD with micropenis. He carried an association of a 2.2Mb-sized distal 4q35.2 deletion containing 4 coding genes (ZFP42 (OMIM*674572), TRIML1, TRIML2 and FRG1 (OMIM*601278)) and a 46.2Mb-sized distal 7q31.1q36.3 duplication encompassing more than 200 coding genes. Reports of 7q terminal duplications may include DSD (micropenis and cryptorchidism), especially duplications encompassing the 7q31 region [START_REF] Bosfield | Pure Distal 7q Duplication: Describing a Macrocephalic Neurodevelopmental Syndrome, Case Report and Review of the Literature[END_REF]. We suggest FEZF1 (OMIM*613301) and RBM28 (OMIM*612074) as possibly implicated in the DSD phenotypes reported in these patients. The genes contained in the 4q35.2 deletion are not considered as sensitive to haploinsufficiency and similar deletions were reported as VOUS or benign in ClinVar and DECIPHER databases. We thus considered the 7q duplication alone as responsible from the patient's phenotype.

P8 presented with a syndromic XY DSD with micropenis. Cytogenetic studies revealed homogeneous trisomy for the whole chromosome 3 long arm, due to a chromosome 14 derivative of a maternal reciprocal translocation (3;14). Partial or complete 3q trisomy are rare and reported in severe polymalformative phenotypes. Most XY patients present with micropenis, cryptorchidism and J o u r n a l P r e -p r o o f hypospadias [START_REF] Dworschak | Comprehensive review of the duplication 3q syndrome and report of a patient with Currarino syndrome and de novo duplication 3q26.32-q27.2[END_REF]. TBL1XR1 (OMIM*608628) and SOX2 (OMIM*184429) are suggested as the main genes implicated in DSDs in 3q terminal region [START_REF] Dworschak | Comprehensive review of the duplication 3q syndrome and report of a patient with Currarino syndrome and de novo duplication 3q26.32-q27.2[END_REF] and Discussion). P9 had a syndromic XY DSD with hypospadias. This fetus carried both a 8.3Mb-sized 3p26.3p26.1 deletion encompassing 15 coding genes (notably ITPR1 (OMIM*147265)), and a 34.5Mbsized 3q26.1q29 duplication containing more than 150 coding genes, suggesting a derivative chromosome from a pericentric inversion. Parents were not available for testing. Chromosome 3p deletion syndrome is responsible for a syndrome first characterized by neurodevelopmental disorders, cerebellar ataxia and variable facial gestalt [START_REF] Fernandez | Molecular characterization of a patient with 3p deletion J o u r n a l P r e -p r o o f syndrome and a review of the literature[END_REF]. Cryptorchidism was only reported once, in a patient carrying an associated 4q32.2qter duplication [START_REF] Petriczko | Familial distal monosomy 3p26.3-pter with trisomy 4q32.2qter, presenting with progressive ataxia, intellectual disability, and dysmorphic features[END_REF]. Thus, we suggest the 3q trisomy observed in P8 and P9 as more likely to play a role in their DSD phenotype.

P11 had a syndromic XX DSD with unicornuate uterus. CMA revealed a terminal de novo 9.8Mb-sized 11q24.2q25 deletion containing at least 40 coding genes, leading to the diagnosis of Jacobsen syndrome (OMIM#147791). P4 (described above) also presented with a 5.83Mb-sized 11q24.3q25 deletion encompassing 18 genes, leading to partial Jacobsen syndrome, in addition to partial trisomy 9q. Cryptorchidism and hypospadias are reported in Jacobsen syndrome [START_REF] Grossfeld | The 11q terminal deletion disorder: A prospective study of 110 cases[END_REF][START_REF] Penny | Clinical and Molecular Characterization of Patients with Distal 11q Deletions[END_REF], however uterine malformations were not reported to date, and no particular gene was linked to the occurrence of a genital phenotype. Common deleted genes in both P4 and P11 included, among others, BARX2 (OMIM*604823), NTM (OMIM*607938) and OPCML (OMIM*600632), these two last genes predicted to be sensitive to haploinsufficiency. BARX2 seemed the most likely to be linked to DSD, as discussed below, since NTM and OPCML functions seemed distant from genital development (neuronal migration and tumor suppression respectively). sSMC P12 had a syndromic XY DSD with a curved penis and a hypospadias. He carried a supernumerary isochromosome 12p, leading to homogeneous tetrasomy 12p (Pallister-Killian syndrome, (MIM#601803)). DSDs are frequently reported in both XX and XY patients presenting with Pallister-Killian syndrome [START_REF] Izumi | Pallister-Killian syndrome[END_REF][START_REF] Wilkens | Novel clinical manifestations in Pallister-Killian syndrome: Comprehensive evaluation of 59 affected individuals and review of previously reported cases[END_REF]. DSD phenotypes was not linked to a J o u r n a l P r e -p r o o f particular gene on chromosome 12 short arm. However, we suggested CHD4 (OMIM*603277) as a candidate gene for DSD in Pallister-Killian syndrome, as discussed below.

P14 had a syndromic XX DSD with normal-appearing female external and internal genitalia, and histologically normal hypoplastic ovaries, one of them showing a small area of undifferentiated tissue without signs of dysgenesis. She presented with a sSMC derived from chromosome 22, resulting in a partial trisomy 22 (22q11.1q11.21,5Mb) in a mosaic state. This marker contained the cat-eye syndrome (CES, OMIM#115470) region and the LCR-A to LCR-D 22q11.21 region. Genital anomalies are rare in CES: two XX patients are reported with hypoplastic female external genitalia or Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome [START_REF] Alsubaihin | Müllerian Agenesis in Cat Eye Syndrome and 22q11 Chromosome Abnormalities: A Case Report and Literature Review[END_REF]. We suggest CECR2 (OMIM*607576) may be a candidate in explaining the DSD observed in some CES (discussed below).

CNVs <5Mb

P5 had a syndromic XY DSD with micropenis and hypospadias. A 3.8 Mb-sized 4p16.3 deletion and a 6.7 Mb-sized 7p22.3p22.1 duplication due to a paternal reciprocal translocation (4;7) was detected.

The 4p deletion contains the critical region for WHS and includes 45 coding genes, notably NSD2 and NELFA (see above, [START_REF] Battaglia | Wolf-Hirschhorn syndrome: A review and update[END_REF]). The 7p22 duplication contained 54 coding genes, most of them not being considered as triplosensitive. The CNV did not seem to disrupt the frontier of a topologically associated domain (TAD) containing a DSD gene. However, smaller 7p22.1 duplications are reported in the literature, notably in 46,XY patients presenting with syndromic cryptorchidism, the authors suggesting ACTB (OMIM*102630) as the main candidate gene ( [START_REF] Ronzoni | 7p22.1 microduplication syndrome: Refinement of the critical region[END_REF] and Discussion).

P10 presented with a syndromic XX DSD linked to a 2.14Mb-sized de novo 6q27 terminal deletion. Her DSD was characterized by uterine and fallopian tubes agenesis, associated with a complex cardiopathy and cerebral anomalies. The deletion encompassed 10 coding genes, including ERMARD (OMIM*615532) and DLL1 (OMIM*606582). This patient was previously reported in [START_REF] Lesieur-Sebellin | Terminal 6q deletions cause brain malformations, a phenotype mimicking heterozygous DLL1 pathogenic variants: A multicenter retrospective case series[END_REF]. Terminal deletion 6q are known to be responsible from a recognizable syndrome mainly defined by cerebral anomalies, epilepsy and neurodevelopmental impairment, with DLL1 as the main J o u r n a l P r e -p r o o f candidate gene [START_REF] Lesieur-Sebellin | Terminal 6q deletions cause brain malformations, a phenotype mimicking heterozygous DLL1 pathogenic variants: A multicenter retrospective case series[END_REF]. However, one 46,XY patient with 6q terminal deletion was reported as also having genital hypoplasia [START_REF] Striano | Clinical phenotype and molecular characterization of 6q terminal deletion syndrome: Five new cases[END_REF]. DLL1 is a receptor for NOTCH signaling pathway, which is known to be implicated in genital and gonadal development, albeit not related to DSD in humans to date (see Discussion).

P13 had a syndromic XX DSD with normal-appearing female genitalia and gonads but a bicornuate, bicervix uterus and a vaginal septum. CMA found a 1.7Mb-sized de novo 19q13.42q13.43 deletion.

Similar deletions were only reported in ClinVar without detailed phenotype assertion, but these CNV were considered likely pathogenic (nssv585173). The deletion contained 30 coding genes, including UBE2S (OMIM*610309), the only gene predicted to be impacted by haploinsufficiency, and located on a frontier between 2 TADs. These TADs did not seem to contain another DSD gene. This was the smallest CNV identified in the cohort.

Discussion

Our study focused on the role of chromosomal imbalances/CNVs in DSD. We confirmed the role of some imbalances previously reported (Supplementary Table 1) and reported CNVs containing either known or suspect DSD-causative genes.

Role of CNVs in DSDs

Some chromosomal syndromes are known to include genital features (ex: 9p-syndrome). Apart from recurrent cytogenetic anomalies such as gonosomal aneuploidies and SRY translocations, pathogenic CNVs in DSDs account for 4 to 30% of all cases [START_REF] Ledig | Array-CGH analysis in patients with syndromic and non-syndromic XY gonadal dysgenesis: evaluation of array CGH as diagnostic tool and search for new candidate loci[END_REF][START_REF] Mazen | Advances in genomic diagnosis of a large cohort of Egyptian patients with disorders of sex development[END_REF]. Diagnosing CNVs responsible from DSDs can lead to (1) identification of new genes, through determining the smallest region of overlap (ex: (Scott and Amarillo, 2022)), (2) identification of promoters and other regulating non-coding regions (ex: SOX9, Supplementary Table 1), (3) identify CNV as a new mechanism in a gene previously reported as DSD-causative through point variations (ex: WNT4, Supplementary Table 1).

This study confirmed the prominent role of first tier routine detection of chromosomal imbalances or J o u r n a l P r e -p r o o f CNVs through cytogenetic techniques (karyotype and aCGH). Supplementary Table 1 sums up known DSD-causative CNVs and related genes.

Novel small CNVs Isolated partial 19q13 deletions are rarely described in the literature nor in patients' databases [START_REF] Rose | Phenotypic variability of the cat eye syndrome. Case report and review of the literature[END_REF], DECIPHER 400969, 421866, ClinVar nssv585173). To our knowledge, these patients were not reported as having DSD, contrary to patient P13 (bicornuate bicervix uterus and vaginal septum).

The region deleted in P13 contains genes (UBE2S, NLRP2 and NLRP5) implicated in oocyte maturation in humans (Mu et al., 2019, p. 2) and early embryonic development in animal models [START_REF] Wang | Ube2s regulates Sox2 stability and mouse ES cell maintenance[END_REF], but none of them were previously reported in DSD. UBE2S is an enzyme implicated in the ubiquitin-proteasome protein degradation pathway. Some authors reported UBE2S as a dosagesensitive gene responsible for regulating Sox2 expression in mouse early embryos (Wang et al., 2016, p. 2). SOX2 (SRY-related HMG-box gene 2) is a promising candidate for sex development, since DSDs are reported in patients of both sexes presenting with loss of function variants in this gene or a complete gene deletion [START_REF] Abualsaud | Survey of disorders of sex development in a large cohort of patients with diverse Mendelian phenotypes[END_REF]. Data are currently insufficient to definitely link the 19q13.4 deletion to the occurrence of DSD, but the region appears as an interesting candidate. Further cases are needed to get more insight on the role of UBE2S in sexual development.

Large chromosomal imbalances involve genes known or suspected to intervene in sexual development Partial or complete 3q trisomy are rare and reported in phenotypes close to Cornelia-de Lange syndrome, associated with sacral anomalies, micropenis, cryptorchidism and hypospadias [START_REF] Dworschak | Comprehensive review of the duplication 3q syndrome and report of a patient with Currarino syndrome and de novo duplication 3q26.32-q27.2[END_REF]. These phenotypes are in accordance to what was observed in our patients (P8 and P9, micropenis and hypospadias). The authors suggested TBL1XR1 (3q26.32, OMIM*608628) responsible from Pierpont syndrome, as a candidate gene for DSD. TBL1XR1 encodes a protein capable of interacting with histones and promoting the expression of nuclear receptor's target genes. SOX2 (3q26.33, OMIM*184429) is also a promising candidate, as described above. Partial 4p monosomy, especially 4p16.3 deletion, is responsible for Wolf-Hirschhorn syndrome (WHS), a contiguous genes syndrome. Genital anomalies in WHS include hypospadias with cryptorchidism in J o u r n a l P r e -p r o o f XY patients, and clitoral variations, streak gonads and absence of Müllerian derivatives in XX individuals [START_REF] Battaglia | Wolf-Hirschhorn syndrome: A review and update[END_REF]. A few patients with 4p duplications of various sizes are also reported in the literature: most of the clinical features observed are close to those of WHS syndrome [START_REF] Maurin | Molecular cytogenetic characterization of a 4p15.1-pter duplication and a 4q35.1-qter deletion in a recombinant of chromosome 4 pericentric inversion[END_REF]. These observations are coherent with the phenotype of P5 (4p deletion) and P6 (4p duplication), namely micropenis and/or hypospadias. Two genes, namely NSD2 and NELFA, seem to contribute to the core symptoms of WHS [START_REF] Battaglia | Wolf-Hirschhorn syndrome: A review and update[END_REF]. NSD2 encodes a SET domain-containing transcriptional regulatory protein with histone methyltransferase activity. In mouse embryonic stem cells, Whsc1 (NSD2 homolog) governs cell differentiation through the expression of key transcription factors [START_REF] Nimura | A histone H3 lysine 36 trimethyltransferase links Nkx2-5 to Wolf-Hirschhorn syndrome[END_REF]. NELFA encodes a protein of the negative elongation factor complex (NELF), implicated in the regulation of transcription elongation of RNA polymerase. Studies in mice models suggests that Nelf is required in early embryogenesis, to maintain the pluripotency state of embryonic stem cells. Patients with haploinsufficiency of these genes, especially NSD2, can present with genital variations [START_REF] Zanoni | Loss-offunction and missense variants in NSD2 cause decreased methylation activity and are associated with a distinct developmental phenotype[END_REF].

To the best of our knowledge, patients with 6q terminal deletion encompassing DLL1 (as observed in P10, Table 1) did not present with DSD. DLL1 is an effector of the NOTCH pathway and, in mouse models, Notch pathway disruption can lead to cryptorchidism and abnormal Sertoli cell function ( [START_REF] Kamińska | Notch signaling regulates nuclear androgen receptor AR and membrane androgen receptor ZIP9 in mouse Sertoli cells[END_REF][START_REF] Yuan | Cryptorchidism in LhrKO animals and the effect of testosterone-replacement therapy[END_REF]. Moreover, some authors performed gene profiling on vaginal samples from MRKH patients and highlighted downregulation of genes implicated in NOTCH pathway, including DLL1. Since P10 presented with a MRKH phenotype, with absent uterus and Fallopian tubes and normal ovaries, it suggests DLL1 as a DSD candidate, especially in MRKH. Partial 7p or 7q terminal duplications are often observed in cases of derivative chromosome from parental reciprocal translocations, as for P5 (presenting with micropenis and hypospadias and a 7p terminal duplication). Some authors suggested ACTB (7p22.1, OMIM*102630) as the critical gene responsible from most of the phenotype observed in 7p duplications [START_REF] Ronzoni | 7p22.1 microduplication syndrome: Refinement of the critical region[END_REF]. ACTB encodes beta-actin, an essential protein of the cytoskeleton, implicated in various functions such as cell shaping, migration and gene expression regulation. ACTB is responsible for Baraitser-Winter J o u r n a l P r e -p r o o f cerebrofrontofacial syndrome, which includes DSD in XY patients, through a gain-of-function mechanism. Large 7q duplications encompassing the 7q31 region [START_REF] Bosfield | Pure Distal 7q Duplication: Describing a Macrocephalic Neurodevelopmental Syndrome, Case Report and Review of the Literature[END_REF] were observed in patients with syndromic micropenis and/or cryptorchidism (as observed in P7). This region contains FEZF1 (7q31.32, OMIM*613301) and RBM28 (7q32.1, OMIM*612074) two genes responsible for recessive hypogonadotropic hypogonadism, a condition that can lead to XY DSD and primary amenorrhea in XX patients, although these genes are not currently known to be dosage-sensitive.

Mosaicism for complete trisomy 9 is a rare condition, with only a few patients reported to date [START_REF] Li | Trisomy 9 mosaic syndrome: Sixteen additional patients with new and/or less commonly reported features, literature review, and suggested clinical guidelines[END_REF]. Clinical findings are quite similar to those observed for partial 9p trisomy [START_REF] Akalin | Partial trisomy 1q41qter and partial trisomy 9pter-9q21.32 in a newborn infant: an array CGH analysis and review[END_REF].

Genitourinary variations are commonly observed in both XX and XY patients, albeit more frequent in XY cases [START_REF] Li | Trisomy 9 mosaic syndrome: Sixteen additional patients with new and/or less commonly reported features, literature review, and suggested clinical guidelines[END_REF][START_REF] Solomon | Trisomy 9 mosaicism and XX sex reversal[END_REF][START_REF] Stoll | Trisomy 9 mosaicism in two girls with multiple congenital malformations and mental retardation[END_REF]. This is in line with the phenotype observed in P1, P2, P3 and P4 (XX sex reversal, micropenis, hypospadias, cryptorchidism). Among the leading genes for DSDs on chromosome 9, DMRT1 (9p24.3, OMIM*602424) plays a critical role in the maintenance of the male phenotype by repressing pro-ovarian genes. XY mice KO for Dmrt1 fully develop as males but present with abnormal Sertoli cells differentiation [START_REF] Raymond | Dmrt1, a gene related to worm and fly sexual regulators, is required for mammalian testis differentiation[END_REF]. In birds, Dmrt1 is responsible for sex determination in a dose-dependent manner, two copies of the gene leading to a male fate whereas a single copy is not sufficient to repress female-determining genes [START_REF] Lambeth | Over-expression of DMRT1 induces the male pathway in embryonic chicken gonads[END_REF]. In humans, disruption of DMRT1 leads to prenatal focal testicular dysgenesis [START_REF] Baetens | Update on the genetics of differences of sex development (DSD)[END_REF] and DMRT1 deletions are associated with XY sex reversal [START_REF] Zarkower | DMRT1: An Ancient Sexual Regulator Required for Human Gonadogenesis[END_REF]. NR5A1 (9q33.3, OMIM*184757) encodes SF1 (Steroidogenic Factor-1), a member of the orphan nuclear receptor family of transcription factors involved in reproduction, steroidogenesis, adrenal and gonadal development in both males and females. This gene is responsible from various phenotypes in humans when disrupted, including adrenocortical insufficiency, 46,XY or XX sex reversal, male or female infertility notably premature ovarian insufficiency (POI) [START_REF] Baetens | Update on the genetics of differences of sex development (DSD)[END_REF]Jaillard et al., 2020, p. 1). Interestingly, overexpression of Nr5a1 in female mice impairs their reproductive functions through hyperandrogenism, disruption of ovarian functions, premature infertility and metabolic disturbance [START_REF] Rotgers | Constitutive expression of Steroidogenic factor-1 (NR5A1) disrupts ovarian functions, fertility, and metabolic homeostasis in female mice[END_REF]. Although isolated DMRT1 or NR5A1 duplications are not reported J o u r n a l P r e -p r o o f to date in DSD patients, we cannot rule out that overexpression of male-determining genes in trisomy 9 plays a role in the occurrence of such phenotypes.

Regarding chromosome 11q anomalies, genital features of Jacobsen syndrome mainly concern XY patients. Cryptorchidism (as observed in P4) is observed in 36-60% of males [START_REF] Mattina | Jacobsen syndrome[END_REF].

Uterine anomalies, as seen in P11, were not reported to date. Among the genes reported in the 11q telomeric region and included in the overlapping deleted region in our patients, we suggested BARX2 (OMIM*604823) as a candidate gene for DSD. BARX2 is a homeobox transcription factor able to bind regulatory elements of genes encoding neural adhesion molecules. In animal models, Barx2 is known to play a role in neural and craniofacial structures, and hair follicles [START_REF] Olson | Barx2 functions through distinct corepressor classes to regulate hair follicle remodeling[END_REF]. BARX2 was found to interact with estrogen receptor-alpha (ESR1) to regulate its splicing and expression [START_REF] Stevens | BARX2 and estrogen receptor-alpha (ESR1) coordinately regulate the production of alternatively spliced ESR1 isoforms and control breast cancer cell growth and invasion[END_REF]. Variations in genes regulating ESR1 mRNA degradation led to hypospadias (as observed in P4), sex reversal and/or gonadal dysgenesis in XY patients [START_REF] Tsai | Genetic analysis of a Taiwanese family identifies a DMRT3-OAS3 interaction that is involved in human sexual differentiation through the regulation of ESR1 expression[END_REF]. A recent study also suggested ESR1 as a candidate gene for MRKH syndrome [START_REF] Brucker | Sequence variants in ESR1 and OXTR are associated with Mayer-Rokitansky-Küster-Hauser syndrome[END_REF] P14 presented with hypoplastic ovaries and a sSMC leading to mosaicism for proximal trisomy 22. Such anomalies are usually responsible for CES (Rosias et al., 2001). The sSMC observed in P14 region encompassed various genes suggested to play a role in gonadal development and maintenance: CECR2, J o u r n a l P r e -p r o o f COMT (OMIM*116790) and HIRA (OMIM*600237), of which only CECR2 is included in the CES critical region [START_REF] Footz | Analysis of the Cat Eye Syndrome Critical Region in Humans and the Region of Conserved Synteny in Mice: A Search for Candidate Genes at or near the Human Chromosome 22 Pericentromere[END_REF]. In CES, genital anomalies are rare, mainly observed in XY patients. Male mice KD for Cecr2 are infertile with testis histological defects [START_REF] Norton | Subfertility in young male mice mutant for chromatin remodeller CECR2[END_REF]. This can suggest that variations of CECR2 expression may impair cellular maturation and growth in gonads. COMT or HIRA, are suggested to play a role in oocyte maturation and POI [START_REF] Jaillard | 22q11.2 rearrangements found in women with low ovarian reserve and premature ovarian insufficiency[END_REF], and may contribute to the hypoplastic ovaries observed in P14, but such a hypothesis needs to be further explored.

Phenotype complexity of DSD

In our cohort, micropenis and Müllerian anomalies were the most common phenotypes observed in XY and XX patients, respectively. Micropenis is a frequent symptom resulting from androgen insensitivity or ineffective testicular testosterone secretion. Beyond testicular dysfunction or defects, any disruption of the gonadotropic axis may lead to micropenis. Genes implicated in cerebral development or in ubiquitous cellular functions such as cell migration or chromatin remodeling may also cause micropenis (discussed in [START_REF] Gazdagh | Novel Genetic Associations and Range of Phenotypes in Children with Disorders of Sex Development and Neurodevelopment: Insights from the Deciphering Developmental Disorders Study[END_REF]). Similar conclusions can be made for Müllerian anomalies, which also rely on disruption of genes implicated in ubiquitous pathways (i.e. Wnt-β-catenin, HOX genes…(Kyei-Barffour et al., 2021)). Thus, in front of large genomic imbalances in DSD patients, it may be difficult to differentiate genes directly implicated in sex determination from DSD as an indirect consequence of disruption of ubiquitous functions. We carefully reviewed published functional studies and/or animal models for the suggested genes presented in this study, but the size of the imbalances highlighted in most of our patients remains a limitation in such approach. The imbrication of environmental factors and this genetic complexity leads to difficulties in diagnosing the etiology of DSDs, and almost 50% of the patients may be stuck in a "diagnostic odyssey" [START_REF] Globa | Disorders of Sex Development in a Large Ukrainian Cohort: Clinical Diversity and Genetic Findings[END_REF][START_REF] Mazen | Advances in genomic diagnosis of a large cohort of Egyptian patients with disorders of sex development[END_REF]. Phenotypic variability and emerging proofs of oligogenism in DSDs (Camats et al., 2020) complicate the interpretation of variants. High-throughput sequencing techniques such as WES, and more recently, whole genome sequencing (WGS), may be of help in addressing both CNVs and point variations detection [START_REF] Audi | GENETICS IN ENDOCRINOLOGY: Approaches to molecular genetic diagnosis in the management of differences/disorders of sex development (DSD): position paper of EU COST Action BM 1303 'DSDnet[END_REF][START_REF] Délot | Towards improved genetic diagnosis of human differences of sex development[END_REF] in patients with normal J o u r n a l P r e -p r o o f conventional karyotype. Finally, "mild" DSD are also underreported in syndromic patients presenting with severe non-genital symptoms, such as neurodevelopmental impairment [START_REF] Gazdagh | Novel Genetic Associations and Range of Phenotypes in Children with Disorders of Sex Development and Neurodevelopment: Insights from the Deciphering Developmental Disorders Study[END_REF].

Description of recurrent CNVs associated with DSDs still provide new genes and new questions, as demonstrated with the patients reported here.

Conclusion

This study shed the light on the variable phenotypes and chromosomal imbalances/CNV encountered in DSDs and highlights the clinical impact of these genetic variations. Molecular genetic analysis is becoming the gold standard for DSDs diagnosis but cytogenetics studies may still be relevant as (1) most of DSDs are linked to gonosomal aneuploidies or structural chromosomal anomalies, including low-level mosaicism, (2) non-coding genome CNV also probably account for a non-negligible part of DSDs, (3) such routine techniques may provide a good diagnostic yield at a relatively low cost.

Innovative technologies such as long-read WGS and Optical Genome Mapping, may combine the abilities to detect both structural, copy number and point variations, thus increasing the diagnosis yield of DSDs and ending the patient's "odyssey".

All patients reported here were registered in the DECIPHER database. Accession numbers are available in Table 1. 2q25(125,101,271-134,927,114)x1 dn,22q12.3(36,668,889-36,971,301) J o u r n a l P r e -p r o o f

CRediT Author Statement

Laura

  , a DSD linked to abnormal regression of the Müllerian ducts, as partially observed in P11. We can thus postulate that the DSD phenotypes observed in patients P4 and P11 are partially linked to BARX2 haploinsufficiency and ESR1 dysregulation. Supernumerary marker chromosomes are a cause of DSD As observed in P12, cryptorchidism is the most frequent DSD feature in Pallister-Killian patients presenting with DSD. We proposed CHD4 (12p13.31, OMIM*603277) as an interesting candidate, since it encodes a protein from the nucleosome remodeling and histone deacetylase (NURD) repressor complex, which is involved in epigenetic regulation of gene transcription, DNA repair, and cell cycle progression. A recent study in a mouse model demonstrated the role of Chd4 in spermatogonia survival and normal XY gonad development (de Castro et al., 2022).

  Mary: Investigation, Writing -original draft; Mélanie Fradin, Laurent Pasquier, Chloé Quelin, Philippe Loget, Maela Le Lous, Gwenaëlle Le Bouar, Sylvie Nivot-Adamiak, Anna Lokchine, Christèle J o u r n a l P r e -p r o o f Dubourg, Vincent Jauffret, Bénédicte Nouyou, Catherine Henry, Erika Launay, Sylvie Odent: Investigation, Resources; Marc-Antoine Belaud-Rotureau: Supervision, Writing -Review & Editing, Sylvie Jaillard: Conceptualization, Supervision, Writing -Review &Editing.

Figure 1 :

 1 Figure 1: Inclusion and cohort flowcharts

Figure 2 :

 2 Figure 2: Histological aspect of patient P1's gonad.

Table 1 : Phenotype and genotype description of the patients reported in this study.

 1 TOP: termination of pregnancy, exam: examination, NA: not assessed, NIPD: non-invasive prenatal diagnosis, IUGR: intrauterine growth retardation, SUA: single umbilical artery.

J o u r n a l P r e -p r o o f J o u r n a l P r e -p r o o f

Funding source

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Data sharing and accessibility

Conflicts of interest

The authors declare no conflict of interest.


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COVID-19 infection in patients on long-term home parenteral nutrition for chronic intestinal failure

Keywords: COVID -19, SARS-CoV-2, pandemic, Home Parenteral Nutrition, Intestinal Failure, epidemiology

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

COVID-19 INFECTION IN PATIENTS ON LONG-TERM HOME PARENTERAL NUTRITION FOR CHRONIC INTESTINAL FAILURE

Abstract

Background and aims

To investigate the incidence and the severity of COVID-19 infection in patients enrolled in the database for home parenteral nutrition (HPN) for chronic intestinal failure (CIF) of the European Society for Clinical Nutrition and Metabolism (ESPEN).

Methods

Period of observation: March 1 st , 2020 March 1 st , 2021. Inclusion criteria: patients included in the database since 2015 and still receiving HPN on March 1 st , 2020 as well as new patients included in the database during the period of observation. Data related to the previous 12 months and recorded on March 1 st 2021: 1) occurrence of COVID-19 infection since the beginning of the pandemic (yes, no, unknown); 2) infection severity (asymptomatic; mild, no-hospitalization; moderate, hospitalization no-ICU; severe, hospitalization in ICU); 3) vaccinated against COVID-19 (yes, no, unknown); 4) patient outcome on March 1st 2021: still on HPN, weaned off HPN, deceased, lost to follow up.

Results

Sixty-eight centres from 23 countries included 4,680 patients. Data on COVID-19 were available for 55.1% of patients. The cumulative incidence of infection was 9.6% in the total group and ranged from 0% to 21.9% in the cohorts of individual countries. Infection severity was reported as: asymptomatic 26.7%, mild 32.0%, moderate 36.0%, severe 5.3%. Vaccination status was unknown in 62.0% of patients, non-vaccinated 25.2%, vaccinated 12.8%. Patient outcome was reported as: still on HPN 78.6%, weaned off HPN 10.6%, deceased 9.7%, lost to follow up 1.1%.. A higher incidence of infection (p=0.04), greater severity of infection (p<0.001) and a lower vaccination J o u r n a l P r e -p r o o f

Introduction

The coronavirus SARS-CoV-2 (COVID-19) has been a challenging worldwide pandemic in recent years [START_REF] Zhu | A novel coronavirus from patients with pneumonia in China, 2019[END_REF]. The clinical spectrum of COVID-19 ranges from an asymptomatic infection or mild upper respiratory tract symptoms to severe pneumonia with acute respiratory distress syndrome (ARDS) [START_REF] Zhu | A novel coronavirus from patients with pneumonia in China, 2019[END_REF][START_REF] Huang | Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China[END_REF]. Older age and the presence of comorbidities, such as diabetes, cardiovascular diseases and obesity, were reported to be risk factors for progression of pulmonary disease as well as for death [START_REF] Wu | Risk Factors Associated With Acute Respiratory Distress Syndrome and Death in Patients With Coronavirus Disease 2019 Pneumonia in Wuhan, China[END_REF][START_REF] Hu | Risk Factors Associated with Clinical Outcomes in 323 COVID-19 Patients in Wuhan, China[END_REF]. Patients affected by chronic organ failure are particularly vulnerable subgroups requiring epidemiological investigation to better understand their associated morbidity and mortality risk and to devise prevention and treatment strategies.

In May 2020, a few months after the beginning of the pandemic, the Home Artificial Nutrition and Chronic Intestinal Failure (HAN&CIF) special interest group of the European Society for Clinical Nutrition and Metabolism (ESPEN) carried out a survey of centres looking after patients on home parenteral nutrition (HPN) for Chronic Intestinal Failure (CIF) [START_REF] Pironi | Home Artificial Nutrition & Chronic Intestinal Failure; Acute Intestinal Failure Special Interest Groups of ESPEN. ESPEN endorsed recommendations. Definition and classification of intestinal failure in adults[END_REF] to assess the impact of the COVID-19 pandemic on patient care [START_REF] Allan | Home Artificial Nutrition & Chronic Intestinal Failure special interest group of ESPEN. An International Survey of Clinicians' Experience Caring for Patients Receiving Home Parenteral Nutrition for Chronic Intestinal Failure During the COVID-19 Pandemic[END_REF]. A total of 78 centers from around the world contributed to the survey, representing ≥3500 patient experiences from both adult and pediatric centers; at that time, 53 centres (67.95%) reported to have no known COVID-19 infected patients, 7 (8.97%) centres were aware that some patients had been infected but were unsure of the exact number of cases, while 18 (23.08%) centres reported that a total of 37 patients had been infected with COVID-19. [START_REF] Allan | Home Artificial Nutrition & Chronic Intestinal Failure special interest group of ESPEN. An International Survey of Clinicians' Experience Caring for Patients Receiving Home Parenteral Nutrition for Chronic Intestinal Failure During the COVID-19 Pandemic[END_REF].

In 2015, the HAN&CIF group developed an international multicenter research project to develop consensus criteria for the severity of CIF; the study protocol involved annual prospective data collection of HPN-dependent patients using a structured database [START_REF] Pironi | Clinical classification of adult patients with chronic intestinal failure due to benign disease: An international multicenter cross-sectional survey[END_REF][START_REF] Pironi | Intravenous supplementation type and volume are associated with 1-year outcome and major complications in patients with chronic intestinal failure[END_REF]. As part of the project, data collection in 2021 was used to perform an international multicenter survey to investigate the incidence and severity of COVID-19 infection in patients on long-term HPN for CIF.

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Materials and methods

Study protocol, patient population and data collection

The ESPEN international multicenter survey for CIF is based on the retrospective analysis of data prospectively recorded during 12-month follow-up periods. The study started on March 1 st , 2015, and data collection was performed on March 1 st of each subsequent year, analysing data recorded during the previous 12 months for those patients already included in the database and for new patients who started HPN for CIF during the preceding 12 months. Center invitation to participate in the study occurred via representatives of the national Parenteral and Enteral Nutrition (PEN) societies of the ESPEN Council, who were asked to send the study protocol to members of their PEN societies. Both pediatric (≤18-year-old) and adult patients who were dependent on HPN for either benign-CIF or malignant-CIF were included. The term 'malignant-CIF' indicates the presence of active malignant disease [START_REF] Wu | Risk Factors Associated With Acute Respiratory Distress Syndrome and Death in Patients With Coronavirus Disease 2019 Pneumonia in Wuhan, China[END_REF]. Data were collected in a structured questionnaire embedded in an Excel (Microsoft Co., 2013) database, termed "the CIF Action day" [START_REF] Wu | Risk Factors Associated With Acute Respiratory Distress Syndrome and Death in Patients With Coronavirus Disease 2019 Pneumonia in Wuhan, China[END_REF][START_REF] Alfano | The frail world of haemodialysis patients in the COVID-19 pandemic era: a systematic scoping review[END_REF].

For each patient, the following data were collected at first inclusion in the database (baseline): age and gender; body weight and height; underlying disease and its benign or malignant nature; pathophysiological mechanism of CIF; HPN requirements (duration, number of days of infusion per week, type of parenteral nutrition admixture, intravenous supplementation (IVS) volume and energy for each day of infusion). The pathophysiological mechanisms of IF were classified as short bowel syndrome with end-jejunostomy (SBS-J), with jejuno-colic anastomosis (SBS-JC) or with jejuno-ileal anastomosis and total colon in continuity (SBS-JIC), intestinal dysmotility (dysmotility), intestinal fistulas (fistulas), mechanical obstruction (obstruction) and extensive small bowel mucosal disease (mucosal disease). The severity of CIF was divided into eight categories, based on the type (Fluid and electrolyte alone, FE; parenteral nutrition including macronutrients, PN) and volume of J o u r n a l P r e -p r o o f IVS, calculated as daily mean of the total volume infused per week (volume per day of infusion × number of infusions per week/7 (mL/day)): FE1 or PN1, ≤1000mL; FE2 or PN2, 1001-2000mL; FE3 or PN3, 2001-3000mL; FE4 or PN4, >3000mL [START_REF] Pironi | Intravenous supplementation type and volume are associated with 1-year outcome and major complications in patients with chronic intestinal failure[END_REF]. At the end of the 12-month follow-up period, patient outcome was classified as still on HPN, weaned off HPN or deceased. Weaning from HPN equated to stopping IVS. The causes of death were grouped as HPN/IF-related, underlying diseaserelated and other causes (neither HPN/IF nor underlying disease-related).

For the present study, the period of observation was March 1 st , 2020 (baseline) to March 1 st 2021 (end of follow-up) and the date of data collection was March 1 st 2021. Patients included at baseline were those already in the database still requiring HPN on March 1 st , 2020 and new patients who started HPN between March 1 st ,2020 and March 1 st ,2021. In order to evaluate occurrence and outcomes associated with COVID-19 infection, a section was added to the ESPEN CIF database including three questions:1) had the patient suffered from COVID-19 infection? (answers: yes; no; unknown); 2) if yes, what was the severity of infection? (answers: asymptomatic; mild, nohospitalization; moderate, with hospitalization not in the intensive care unit; severe, hospitalization in the intensive care unit); 3) has the patient been vaccinated against COVID-19 (answers: yes, no, unknown).

Ethical statement

The research was based on anonymized information taken from patient records at the time of data collection. The study was conducted with full regard to confidentiality of the individual patient.

Ethical committee approval was obtained by the individual HPN centers according to local regulations.

Statistical analysis

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Data are reported as absolute and relative frequencies. The frequency of COVID-19 infection was reported as "cumulative annual incidence". Pearson chi-square test and Fisher's exact test were used to analyze frequencies where appropriate. All analyses were carried out using Stata v.15.1, and the significance level was set to p<0.05

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Results

Patient cohort

Sixty-eight HPN centers from 23 countries included 4,860 patients (Tables 1 and2). J o u r n a l P r e -p r o o f J o u r n a l P r e -p r o o f

COVID-19 infection severity

The clinical severity of COVID-19 infection was reported for 172 of the 241 infected patients.

Infection was asymptomatic in 26.7%, mild in 32.0%, moderate in 36.0% and severe in 5.3% (Figure 2). The severity of infection did not differ between sex (p=0.864), age (p=0.720), underlying disease (0.564), mechanism of CIF (p=0.603) and IVS categories (p=0.378). infection by the end of 2020 was 15.8% [11]. The 2022 USRDS report went on to show that patients with CKD had consistently higher COVID-19 testing rates than those without CKD, and rates were higher still for patients undergoing dialysis, suggesting that patients with CKD were consistently more likely to be diagnosed with COVID-19 than those without CKD [12]. We also compared our data with the epidemiological update published by the World Health Organization (WHO) on February 28 th , 2021, which reported cumulative cases per 100 thousand population since the beginning of the pandemic [13]. In many countries, the calculated incidence of infection in HPNdependent patients appeared at least two-fold higher than that in the general population (Figure 3), suggesting either greater susceptibility and/or risk of exposure to COVID-19 infection and/or a greater rate of testing in patients on HPN for CIF. The incidence of infection was significantly higher in adults than in children and in patients with benign rather than malignant disease. The reasons for these differences are not evident from our data. However, as expected, vaccination rates were higher in patients with cancer and lower in children; it is, of course, possible that both adults and J o u r n a l P r e -p r o o f 20 patients with benign disease were at greater risk of exposure to the infection as they spent more time in contact with others outside of their homes. Overall, our data confirm that patients on HPN are a fragile population to be timely protected by repeated vaccination against COVID-19.

COVID-19 infection was asymptomatic or led to only mild symptoms without the need for hospitalization in the majority of cases; notably, these data are comparable to the impact of the disease on the general population observed at the beginning of the pandemic and suggest that HPNdependence for CIF was not a risk factor for developing more severe clinical features following infection [START_REF] Wu | Characteristics of and Important Lessons From the Coronavirus Disease 2019 (COVID-19) Outbreak in China: Summary of a Report of 72 314 Cases From the Chinese Center for Disease Control andPrevention[END_REF][START_REF] Singh | COVID-19: Current knowledge in clinical features, immunological responses, and vaccine development[END_REF].

Only 12.8% of patients were reported to be vaccinated at the time of data collection, although this was more frequent in adults, in those with active cancer and for some underlying CIF pathophysiological mechanisms. That said, this low rate of vaccination would, of course, fit with the study's time frame since the first vaccine was approved in December 2020 and therefore only available during the latter two months of the survey, and not in all countries. Furthermore, differences in country policies to prevent COVID-19 infection could have impacted on the incidence rates of both infection and vaccination status. Differences between age categories were due to the initial approval of the vaccine for adults only, while difference between diseases and mechanisms of CIF may have related to vaccine prioritization for those with frailty in some countries.

Both the frequency and severity of COVID-19 infection were greater in the deceased and in those lost to follow up, with corresponding low vaccination rates in both latter groups. Moreover, around half of all deaths were due to COVID-19 infection. Again, these data are in keeping with observations in the general population and, of course, serve to highlight the importance of preventative measures, such as mask-wearing and handwashing, physical distancing, and timely vaccination to mitigate infection and its severity [START_REF] Singh | COVID-19: Current knowledge in clinical features, immunological responses, and vaccine development[END_REF].

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The strength of this study is based on the large patient cohort and its international and multicenter design. However, limitations may include missing data, which likely relate to difficulties encountered in monitoring outpatients, resultant from lockdown restrictions brought into force in virtually all the countries during the follow-up period of the survey. Furthermore, our study design did not investigate the country policies to prevent COVID-19 infection during the period of the data collection, a factor that could have contributed to the differences observed among countries.

Nevertheless, the results of this unique study on COVID-19 infection in HPN-dependent patients are in keeping with the impact of the infection on the general population, as well as on patients requiring HD for CKD, which is a useful proxy model of disease.

In conclusion, in patients on HPN for CIF, the incidence of COVID-19 infection differed greatly among countries and was asymptomatic or led to only mild symptoms in the majority of cases but was the cause of death in a consistent number of infected patients. COVID-19 infection, infection severity and lack of vaccination were associated with a higher risk of death. The results highlight the importance of measures to prevent infection.

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Figure 1 .

 1 Figure 1. Annual cumulative incidence of COVID-19 infection in the total cohort of 2,503 patients on

Figure 2 .

 2 Figure 2. Percentages of clinical severity of COVID-19 infection in 172 patients on home parenteral

Figure 3 .

 3 Figure 3. Cumulative cases of COVID-19 infection in patients on home parenteral nutrition (HPN), from March 1 st , 2020 to March 1 st , 2021 and

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  n a l P r e -p r o o f FP: none LM: none LCE: Consulting fees, Payment or honoraria for lecture and Support for attending meetings for Takeda UA: Payment or honoraria for lectures for Takeda, Baxter; Support for attending meetings and Participation on a Data Safety Monitoring for Takeda or contracts from any entity and for BBraun, FreseniusKabi, Nestle; Payment or honoraria for lectures for BBraun, OlimpLabs, FreseniusKabi, Baxter, Nestle; Support for attending meetings for or honoraria for lectures, for Abbott, Abela Farm, Danone/Nutricia, Fresenius, GM Pharma, Nestle, Nestle Nutrition Institute, Oktal Pharma, Shire/Takeda; Non-restricted grant delivered to the hospital from BioGaia BC: none GM: none MHMdC: Grants or contracts, Consulting fees, Payment or honoraria, Support for attending meetings, Participation on a Data Safety Monitoring for lectures for Takeda Pharmaceutical Brazil J o u r n a l P r e -p r o o f MK: Payment or honoraria for manuscript writing and educational events for Nutricia, FreseniusKabi ASS: none GB: none J o u r n a l P r e -p r o o f

Table 1 .

 1 Contributing home parenteral nutrition (HPN) centers and patients with chronic intestinal failure enrolled in the study, grouped by country of origin

		HPN Centers	Patients
	Country France Poland Italy USA UK Belgium Israel Denmark Spain Australia Argentina Sweden Germany Croatia Finland Thailand Brazil	n. J o u r n a l P r e -p r o o f n. % 887 18.25 806 16.58 675 13.89 639 13.15 626 12.88 304 6.26 239 4.92 145 2.98 128 2.63 82 1.69 68 1.40 57 1.17 46 0.95 32 0.66 32 0.66 28 0.58 17 0.35
	Bulgaria	16	0.33
	Hungary	15	0.31
	Mexico	9	0.19
	Latvia	5	0.10
	Malaysia	3	0.06
	Turkey	1	0.02
	Total	4,860	100

Table 2 .

 2 Patient demographic and clinical characteristics at baseline enrollment on March 1 st , 2020. COVID-19 infection was reported as known (answer: yes or no) in 2,503 (51.5%) patients, unknown in 2,039 (42.0%) and was not reported in 318 (6.5%). COVID-19 infection occurred in 241 patients, corresponding to an annual cumulative incidence rate of 9.6% of the 2503 patients for whom the item was known. Among contributing countries, the calculated annual cumulative incidence ranged from 0 (Australia) to 21.9% (Israel) (Supplemental table1). Figure1shows the incidence of infections in countries that included in the study at least 10 patients with known infection status. The infection

	Patient categories

Abbreviations: CIF, chronic intestinal failure; SBS, short bowel syndrome; CIPO, chronic intestinal pseudo-obstruction; IVS, intravenous supplementation; FE, fluids and electrolytes; PN, parenteral nutrition J o u r n a l P r e -p r o o f 13 COVID-19 infection rate was 6.1% in children and 10.2% in adults (p=0.036), 10.3% in benign disease and 7.0% in active cancer (p=0.032) and did not differ between sex (p=0.891), mechanisms of CIF (p=0.743) or IVS (p=0.170) categories.

Table 3 .

 3 COVID-19 infection incidence, severity of infection, and COVID-19 vaccination in the 12-month outcome categories of patients on home

	parenteral nutrition (HPN) for chronic intestinal failure
		COVID-19 infection		COVID-19 infection severity			COVID-19 vaccination
		n.	Infected n. (%)	p	n.*	Asymptomatic n. (%)	Mild n. (%)	Moderate n. (%)	Severe n. (%)	p	n. **	Vaccinated n. (%)	p
	Outcome			0.042						<0.001			0.017
	Still on HPN	1930 183 (9.5)		129	38 (29.5)	47 (36.4)	42 (32.6)	2 (1.6)		1376 480 (34.9)
	Weaned off HPN	258	19 (7.4)		15	5 (33.3)	5 (33.3)	5 (33.3)	0		45	10 (22.2)
	Deceased	239	29 (12.1)		20	0	1 (5.0)	12 (60.0) 7 (35.0)		43	8 (18.6)
	Lost to follow-up	27	6 (22.2)		4	2 (50.0)	0	2 (50.0)	0		14	2 (14.3)

*number of infected patients for whom the severity of infection was reported **number of patients for whom the vaccination status (yes or no) was known J o u r n a l P r e -p r o o f

Table 4 .

 4 Causes of death in patients on home parenteral nutrition infected with COVID-19 during the 12-month follow up periodDiscussionThis study reports the incidence and severity of COVID-19 infection as well as the vaccination status of a large cohort of patients requiring HPN for CIF during the early phase of the pandemic (March

	Death in patients on HPN infected with COVID-19, n.	29
	Cause of death, n. (%)
	Not reported	1 (3.4)
	Underlying disease-related	14 (48.3)
		• Cancer	7
	Other	• Cancer+COVID-19 • Not reported • COVID-19 • Intracerebral hemorrage • Renal failure J o u r n a l P r e -p r o o f 1 6 14 (48.3) 11 2 1

The United States Renal Data System (USRDS) 2021 annual data report showed that, among Medicare beneficiaries in February 2020 undergoing HD, the cumulative incidence of COVID-19

table 1 .

 1 Patient cohort included in the survey by country, categorized according to the Covid-19 infection status and the infection rate, calculated in those with known infection status. Grants or contracts from any entity for Fresenius Kabi, Nestle, Realfood Blends, VectivBio, Rockfield, Zealand; Consulting fees, Northsea; Participation on a Data Safety Monitoring Board for EndoBarrier M D-S: none TV: Grants or contracts from any entity for Vectiv Bio, Takeda; Consulting fees for Vectiv Bio, Zealand Pharma, Takeda, Baxter, Hamni, NorthSea Therapeutics; Payment or honoraria for lectures for Vectiv Bio, Takeda, Baxter; Support for attending meetings for Takeda, Vectiv Bio, Zealand Pharma, Fresenius Kabi; Receipt of equipment, materials, drugs for VectivBio ZK: Support for attending meetings for Abbott, Fresenius, Nutricia, Nestle, Takeda; Leadership for Croatian Medical Association-The President

	Supplementary Covid-19 infection status		Infection in patients with known infection status
		Patients	Not reported	Unknown	Known	No	Yes	Incidence of infection
	Country	n.	n.	n.	n.	n.	n.	%
	France	887	57	472	358	311	47	13.1
	Poland	806	64	448	294	267	27	9.2
	Italy United States United Kingdom Belgium Israel Denmark Spain Australia Argentina Sweeden Germany Finland	675 639 626 304 239 145 128 82 68 57 46 32	98 23 14 0 45 0 1 1 2 4 0 0 J o u r n a l P r e -p r o o f 101 476 432 298 318 298 333 279 232 10 294 280 162 32 25 145 0 0 127 119 1 80 80 9 57 46 7 46 44 46 0 0 32 30	44 20 47 14 7 8 0 11 2 2	9.2 6.3 16.8 4.8 21.9 6.3 0.0 19.3 4.3 6.3
	Croatia	32	1	0	31	27	4	12.9
	Thailand	28	0	0	28	28	0	0.0
	Brazil	17	1	0	16	14	2	12.5
	Bulgaria	16	0	0	16	13	3	18.8
	Hungary	15	7	7	1	0	1	100.0
	Mexico	9	0	0	9	9	0	0.0
	Latvia	5	0	0	5	3	2	40.0
	Malaysia	3	0	0	3	3	0	0.0
	Turkey	1	0	0	1	1	0	0.0
	Total	4860	318	2039	2503	2262	241	9.6

NVC: Payment or honoraria for lectures for Takeda, Nutricia; Payment for expert testimony, Support for attending meetings and Participation on a Data Safety Monitoring Board for Takeda AL: Consulting fees, Support for attending meetings, Participation on a Data Safety Monitoring Board or Advisory Board for Nestlè; Participation on a Data Safety Monitoring Board or Advisory Board for Takeda; Payment or honoraria for lectures for baxter

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Acknowledgements

Contributing coordinators and centers by country

Argentina

Adriana N. Crivelli, Hector Solar Muñiz; Unidad de Soporte Nutricional, Rehabilitación y Trasplante de Intestino, Hospital Universitario Fundacion Favaloro, Buenos Aires, Argentina Australia Brooke R. Chapman; Austin Health, Melbourne Looi C. Ee; Queensland Children's Hospital, Brisbane, Australia Margie O'Callaghan; Flinders Medical Centre, Adelaide Emma Osland; Royal Brisbane and Women's Hospital, Herston Belgium Marianna Arvanitakis; Erasme University Hospital, ULB, Brussels Tim Vanuytsel, Nathalie Lauwers, Karlien Geboers, Marleen Pijpops; University Hospital Leuven; Leuven Intestinal Failure and Transplantation (LIFT), Leuven; Andre Van Gossum; Medico-Surgical Department of Gastroenterology, Hôpital Erasme, Free University of Brussels, Belgium Brazil Mariana Hollanda Martins da Rocha; Hospital das Clinicas da Faculdade de Medicina da Universidade de São Paulo, São Paulo Bulgaria Maryana Doitchinova-Simeonova; Bulgarian Association of Patients with Malnutrition, Sofia Croatia Zeljko Krznaric, Dina Ljubas Kelecic; University Hospital Centre Zagreb, Zagreb Sanja Kolaček; Children's Hospital Zagreb, Zagreb Denmark Henrik Højgaard Rasmussen; Center for Nutrition and Bowel Disease, Aalborg University Hospital, Aalborg Finland Laura Merras-Salmio, Mikko Pakarinen;Helsinki University Hospital, Children's Hospital, Helsinki France Cecile Chambrier; Hospices Civils de Lyon, Centre Hospitalier Lyon Sud, Lyon J o u r n a l P r e -p r o o f

Vaccination against COVID-19

COVID-19 vaccination status was reported for 3,962 patients as follows: vaccinated 12.8%, not vaccinated 25.2% and unknown 62.0%. The vaccination rate was 2.6% in children and 39.7% in adults (p<0.001), 31.7% in benign disease and 44.9% in malignant disease (p<0.001), 38.3% in SBS-J, 29.7% in SBS-JC, 18.6% in SBS-JIC, 42.4% in fistulas, 21.2% in dysmotility, 14.8% in mechanical occlusion and 25.0% in mucosal disease (p<0.001), and did not differ between sex (p=0.248), mechanisms of CIF (p=0.743) and IVS categories (p=0.800).

Association between COVID-19 incidence, severity and vaccination status and patient outcome

At the end of the 12-month follow up period, the outcomes of 2,454 patients were reported as follows: still on HPN 78.6%, weaned off HPN 10.6%, deceased 9.7%, lost to follow up 1.1%. The infection rate was significantly higher in the deceased and in those lost to follow up (p=0.042), while infection severity was higher in the deceased group (p<0.001) and those still requiring HPN p=0.017) (Table 3).

Twenty-nine patients infected with COVID-19 died, with causes of deaths shown in Table 4. No death was HPN-related. Deaths due to COVID-19 infection accounted for the 42.8% of the 29 deaths occurred in patients who had the infection.

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