MD Maria-Christina Zennaro
email: maria-christina.zennaro@inserm.fr

Fabio Fernandes-Rosa

Mineralocorticoid Receptor Mutations

Keywords: PHA1, pseudohypoaldosteronism type 1, MR, mineralocorticoid receptor, ENaC, epithelial sodium channel, minor allele frequency (MAF)

Aldosterone and the mineralocorticoid receptor (MR) are key elements for maintaining fluid and electrolyte homeostasis as well as regulation of blood pressure. Loss of function mutations of the MR are responsible for renal pseudohypoaldosteronism type 1 (PHA1), a rare disease of mineralocorticoid resistance presenting in the newborn with weight loss, failure to thrive, vomiting and dehydration, associated with hyperkalemia and metabolic acidosis, despite extremely elevated levels of plasma renin and aldosterone. On the opposite, a MR gain of function mutation has been associated with a familial form of inherited mineralocorticoid hypertension exacerbated by pregnancy. In addition to rare variants, frequent functional single nucleotide polymorphisms of the MR are associated with salt sensitivity, blood pressure, stress response and depression in the general population. This review will summarize our knowledge on MR mutations in PHA1, reporting our experience on the genetic diagnosis in a large number of patients performed in the last 10 years at a national reference center for the disease. We will also discuss the influence of rare MR variants on blood pressure and salt sensitivity as well as on stress and cognitive functions in the general population.

Introduction

Aldosterone and the mineralocorticoid receptor (MR) play a key role in the regulation of electrolyte balance and blood pressure. Abnormalities in aldosterone and MR function lead to salt losing disorders or hypertension. Aldosterone is synthesised in the zona glomerulosa of the adrenal cortex under the control of different stimuli, mainly the renin-angiotensin system and potassium. Aldosterone regulates transepithelial ionic transport by stimulating sodium reabsorption and potassium secretion in the distal tubule of the kidney, the colon, salivary and sweat glands via binding to the MR [START_REF] Pearce | Aldosterone: its receptor, target genes, and actions[END_REF]. The MR is a member of the steroid hormone receptor subgroup of the nuclear receptor superfamily (Nuclear receptor commitee 1999) and acts as a ligand-dependent transcription factor by binding to specific hormone responsive elements located in regulatory regions of target genes. In the distal nephron, the MR induces an aldosterone-dependent dynamic transcriptional program, leading to rapid induction of different signaling pathways and transcriptional cascades modulating the activity of major structural components of ion transport. In the aldosterone-sensitive distal nephron, aldosterone regulates sodium and potassium balance by stimulating the activity of the epithelial sodium channel ENaC and the Na + -K + -ATPase [START_REF] Stockand | New ideas about aldosterone signaling in epithelia[END_REF]. The MR rapidly induces transcription of the serum-and glucocorticoid regulated kinase (sgk) 1, which directly stimulates ENaC activity by phosphorylating Nedd4-2, thus reducing ubiquitylation, retrieval, and degradation of ENaC [START_REF] Bhalla | Disinhibitory pathways for control of sodium transport: regulation of ENaC by SGK1 and GILZ[END_REF]. In parallel, transcriptional regulation of the deubiquitylating enzyme Usp2-45 increases ENaC surface expression and activity [START_REF] Verrey | Early transcriptional control of ENaC (de)ubiquitylation by aldosterone[END_REF]. MR also increases the expression of glucocorticoid-induced leucine zipper protein (GILZ) which acts in parallel with sgk1 to increase ENaC localization at the plasma membrane [START_REF] Soundararajan | A novel role for glucocorticoidinduced leucine zipper protein in epithelial sodium channel-mediated sodium transport[END_REF].

In addition to its effects on the kidney, a large number of studies have highlighted the role of the MR in regulating physiological processes in non-epithelial tissues, including the heart, vessels, adipose tissue and the brain (Jaisser& [START_REF] Jaisser | Emerging Roles of the Mineralocorticoid Receptor in Pathology: Toward New Paradigms in Clinical Pharmacology[END_REF].

It is important to remember that MR possesses the same affinity for aldosterone and for the physiological glucocorticoid cortisol (corticosterone in rats and mice), which has plasma concentrations 100-1000 fold higher than those of aldosterone. In aldosterone target tissues, the enzyme 11-beta-hydroxysteroid dehydrogenase type 2 (11HSD2) converts cortisol (and corticosterone) into the inactive metabolites cortisone and 11-dehydrocorticosterone, thus modulating intracellular glucocorticoid levels and protecting the nonselective MR from occupation by glucocorticoids ( [START_REF] Edwards | Localization of 11b-hydroxysteroid dehydrogenase -tissue specific protector of the mineralocorticoid receptor[END_REF][START_REF] Funder | Mineralocorticoid action: target tissue specificity is enzyme, not receptor, mediated[END_REF]; for a detailed review, see the article by JW Funder in this issue). In addition to local metabolism of the glucocorticoid hormones, intrinsic properties of the MR, in terms of ligand discrimination and coregulator recruitment, contribute to modulate receptor selectivity and transcriptional effects [START_REF] Farman | Multiple aspects of mineralocorticoid selectivity[END_REF]. In unprotected tissues, like adipose tissue and in the brain, MR is a high affinity receptor for glucocorticoids, although depending on the cell type and environmental conditions, glucocorticoids may have agonist or antagonist effects [START_REF] Funder | Mineralocorticoid receptors: distribution and activation[END_REF].

The study of MR variants has contributed enormously to our understanding of structurefunction relationships in the MR. Frequent genetic variants of the MR are associated with salt sensitivity, blood pressure, stress response and depression. In extreme cases, loss of function mutations are responsible for renal pseudohypoaldosteronism type 1 (PHA1), whereas a gain of function mutation has been associated with a familial form of inherited mineralocorticoid hypertension. More than 380 rare coding variants have been described in the major transcript of the NR3C2 gene, coding for the MR, in the Exome Aggregation Consortium (ExAC) [START_REF] Lek | Analysis of protein-coding genetic variation in 60,706 humans[END_REF]) which aggregates exome sequencing data from a wide variety of sequencing projects, with constraint metrics indicating that NR3C2 is intolerant for both missense and nonsense mutations. This suggests that rare variants (MAF less than 0.5%) but also common single nucleotide polymorphism, with a minor allele frequency (MAF) in the population ≥ 5%, may have functional consequences on mineralocorticoid homeostasis in the general population and modulate disease susceptibility whenever they significantly affect protein function or amount.

Pseudohypoaldosteronism type 1

The integrity of the mineralocorticoid axis is particularly relevant in the neonatal period, where renal regulation of water and electrolyte balance is impaired due to immature tubular function [START_REF] Holtback | Molecular determinants of sodium and water balance during early human development[END_REF]. Events such as prematurity or infections may lead to salt loss and dehydration which can be further amplified by physiological partial resistance to aldosterone in the newborn [START_REF] Martinerie | Physiological partial aldosterone resistance in human newborns[END_REF]. PHA1 is a rare disease of mineralocorticoid resistance, first described by Cheek and Perry in the early 50ies of the last century [START_REF] Cheek | A salt wasting syndrome in infancy[END_REF]. The disease presents as a salt wasting syndrome in the neonatal period, with weight loss, failure to thrive, vomiting and dehydration, associated with hyperkalemia and metabolic acidosis, despite extremely elevated levels of plasma renin and aldosterone [START_REF] Zennaro | Aldosterone resistance: structural and functional considerations and new perspectives[END_REF]. Seminal work by Armanini et al and Kuhnle et al has characterized the clinical and pathogenic mechanisms of PHA1 and paved the way to the discovery of the underlying genetic abnormalities, showing abnormalities of aldosterone binding on mononuclear leukocytes from patients with PHA1 and different transmission of the hormonal and binding defects [START_REF] Armanini | Aldosterone-receptor deficiency in pseudohypoaldosteronism[END_REF][START_REF] Kuhnle | Pseudohypoaldosteronism in eight families: different forms of inheritance are evidence for various genetic defects[END_REF]). These results were further confirmed by clinical studies describing the existence of two distinct clinical and genetic entities associated with different severity and evolution: a renal form, in which signs of mineralocorticoid resistance are restricted to the kidney and a generalized form, where systemic mineralocorticoid resistance in the kidney, but also in the colon, salivary and sweat glands and the lungs, leads to severe salt loss from multiple organs [START_REF] Hanukoglu | Type I pseudohypoaldosteronism includes two clinically and genetically distinct entities with either renal or multiple target organ defects[END_REF].

Renal PHA1 (MIM#177735, ORPHA#71871, also called autosomal dominant PHA1) is a mild form of primary mineralocorticoid resistance and represents the most frequent form of the disease. It is due to loss of function mutations in the NR3C2 gene [START_REF] Geller | Mutations in the mineralocorticoid receptor gene cause autosomal dominant pseudohypoaldosteronism type I[END_REF][START_REF] Pujo | Mineralocorticoid receptor mutations are the principal cause of renal type 1 pseudohypoaldosteronism[END_REF][START_REF] Sartorato | Different inactivating mutations of the mineralocorticoid receptor in fourteen families affected by type I pseudohypoaldosteronism[END_REF]. The prevalence, as estimated from recruitment through a national reference centre for rare diseases (MC. Zennaro, genetics laboratory, HEGP, see below) is ~1 per 80,000 newborns, but may be underestimated due to phenotypic variability, including asymptomatic cases. Patients show various degrees of failure to thrive, dehydration and vomiting, with hyponatremia, hyperkalemia and inappropriately high urinary sodium excretion. In contrast, urinary potassium excretion is low, with reduced fractional potassium excretion and transtubular potassium gradient [START_REF] Rodriguez-Soriano | Transtubular potassium concentration gradient: a useful test to estimate renal aldosterone bio-activity in infants and children[END_REF]. Diagnosis is confirmed by elevated plasma renin and aldosterone levels. Symptoms of renal PHA1 improve in childhood and, generally, salt supplementation, which is required to correct the sodium losses, can be discontinued around age 18-24 months. Older children are generally clinically asymptomatic. Nevertheless, a recent case-control study investigating 39 adult patients with renal PHA1 carriers of NR3C2 mutations showed that they show life-long increased plasma renin and aldosterone levels as well as increased salt appetite, with normal blood pressure and potassium levels [START_REF] Escoubet | Cardiovascular effects of aldosterone: insight from adult carriers of mineralocorticoid receptor mutations[END_REF], confirming evidence from earlier case reports suggesting persistence of hormonal abnormalities in adulthood [START_REF] Geller | Autosomal dominant pseudohypoaldosteronism type 1: mechanisms, evidence for neonatal lethality, and phenotypic expression in adults[END_REF][START_REF] Kuhnle | Pseudohypoaldosteronism in eight families: different forms of inheritance are evidence for various genetic defects[END_REF][START_REF] Zennaro | Pseudohypoaldosteronism: a case survey and future directions[END_REF]. Remarkably, high aldosterone levels and salt intake in the context of low MR activity are not associated with adverse cardiovascular outcome in these patients, but rather with improved diastolic left ventricular function [START_REF] Escoubet | Cardiovascular effects of aldosterone: insight from adult carriers of mineralocorticoid receptor mutations[END_REF].

In contrast to the renal form, generalized PHA1 (MIM #264350, ORPHA#171876, also called autosomal recessive PHA1) is a severe salt wasting syndrome with profound hyponatremia and hyperkalemia, which may be complicated by cardiac dysrhythmias, collapse, shock or cardiac arrest [START_REF] Speiser | Pseudohypoaldosteronism: a review and report of two new cases[END_REF]. Salivary or sweat tests are positive and a subset of patients show respiratory tract illnesses [START_REF] Kerem | Pulmonary epithelial sodium-channel dysfunction and excess airway liquid in pseudohypoaldosteronism[END_REF] and cutaneous lesions similar to those appearing in miliaria rubra [START_REF] Belot | Pseudohypoaldosteronisms, report on a 10-patient series[END_REF]. Generalized PHA1 is due to loss of function mutations in one of the three genes coding for the subunits of the epithelial sodium channel ENaC (SCNN1A; SCNN1B; SCNN1G) [START_REF] Chang | Mutations in subunits of the epithelial sodium channel cause salt wasting with hyperkalaemic acidosis, pseudohypoaldosteronism type 1[END_REF][START_REF] Strautnieks | A novel splice-site mutation in the gamma subunit of the epithelial sodium channel gene in three pseudohypoaldosteronism type 1 families[END_REF]. Early diagnosis of the disease is critical to survival and prognosis is poor with no remission reported. Patients experience recurrent life-threatening episodes of salt loss, requiring lifelong supplementation with high doses of sodium and ion exchange resins [START_REF] Belot | Pseudohypoaldosteronisms, report on a 10-patient series[END_REF][START_REF] Hanukoglu | Clinical improvement in patients with autosomal recessive pseudohypoaldosteronism and the necessity for salt supplementation[END_REF].

MR mutations in PHA1

Since the studies by Armanini et al, it appeared that PHA1 was due to a tubular insensitivity to aldosterone action. After the cloning of the MR complementary DNA sequence in 1987 [START_REF] Arriza | Cloning of human mineralocorticoid receptor complementary DNA: structural and functional kinship with the glucocorticoid receptor[END_REF], early studies failed however to identify MR mutations in one family with autosomal dominant inheritance [START_REF] Zennaro | No alteration in the primary structure of the mineralocorticoid receptor in a family with pseudohypoaldosteronism[END_REF] and in the index PHA1 case described by Cheek and Perry [START_REF] Komesaroff | Pseudohypoaldosteronism: Molecular characterisation of the mineralocorticoid receptor[END_REF]. No MR abnormalities were also found in one case with generalized PHA, where the authors postulated a defect in a post-MR step of aldosterone action [START_REF] Arai | Physiological and molecular aspects of mineralocorticoid action in pseudohypoaldosteronism: a responsiveness test and therapy[END_REF]. It was only in 1998 that Geller and co-workers confirmed the initial hypothesis of a primary defect in the MR, by identifying two frameshift mutations, two premature termination codons and one splice donor mutation in the MR in four dominant and one sporadic cases of PHA1 [START_REF] Geller | Mutations in the mineralocorticoid receptor gene cause autosomal dominant pseudohypoaldosteronism type I[END_REF]. The main difference between this and previous studies was the technical approach for detecting mutations. Indeed, after the cloning of the NR3C2 gene [START_REF] Zennaro | Human mineralocorticoid receptor genomic structure and identification of expressed isoforms[END_REF], genomic sequencing became available allowing to detect mutations directly on DNA. A p.Arg590X MR nonsense mutation was subsequently detected in affected patients from the kindred studied by Zennaro et al, which was not present on MR mRNA in peripheral blood lymphocytes from the same patient [START_REF] Geller | Autosomal dominant pseudohypoaldosteronism type 1: mechanisms, evidence for neonatal lethality, and phenotypic expression in adults[END_REF]. This is likely a consequence of nonsense mediated mRNA decay, whereby mRNA carrying premature stop codons in nonterminal exons is degraded, and suggests that MR haploinsufficiency is sufficient to cause renal PHA1. Sequencing of the coding exons and intron-exons junction has eventually identified also the causal mutation of the Australian index case of PHA1, who was carrier of a p.Leu938Arg mutation transmitted from his asymptomatic mother [START_REF] Fuller | Pseudohypoaldosteronism type 1: the index case revisited[END_REF].

Subsequently, more than fifty different MR mutations have been reported in the literature [START_REF] Arai | Physiological and molecular aspects of mineralocorticoid action in pseudohypoaldosteronism: a responsiveness test and therapy[END_REF][START_REF] Geller | Mineralocorticoid resistance[END_REF][START_REF] Riepe | Clinical and molecular features of type 1 pseudohypoaldosteronism[END_REF][START_REF] Zennaro | Aldosterone resistance: structural and functional considerations and new perspectives[END_REF]. Disease-causing mutations are located in all exons of the NR3C2 gene and affect all functional domains of the MR. The human MR is a protein of 984 amino acids with a modular structure comprising three separate domains with specific functions [START_REF] Viengchareun | The mineralocorticoid receptor: insights into its molecular and (patho)physiological biology[END_REF][START_REF] Yang | The mineralocorticoid receptor and its coregulators[END_REF]. The NR3C2 gene is composed of 8 coding exons; exon 2 codes for the N-terminal domain (NTD), involved in transcriptional activation and intramolecular interactions, whereas exons 3 and 4 code for the DNA binding domain (DBD). This central domain of the protein folds into two zinc-finger structures that are involved in DNA recognition and receptor dimerization [START_REF] Hudson | Crystal structure of the mineralocorticoid receptor DNA binding domain in complex with DNA[END_REF]. Exons 5-9 code for the ligand binding domain (LBD), which also contains regions involved in interaction with heat shock proteins, transcriptional activation and dimerization (for a detailed review see [START_REF] Fuller | Novel interactions of the mineralocorticoid receptor[END_REF][START_REF] Huyet | Structural determinants of ligand binding to the mineralocorticoid receptor[END_REF][START_REF] Tallec | The mineralocorticoid receptor: a journey exploring its diversity and specificity of action[END_REF]. This region harbours a ligand-dependent activation function AF2. Remarkably, to date only nonsense, frameshift or splice site mutations have been identified in exon 2. Although functional common single nucleotide polymorphisms have been identified in this region [START_REF] Derijk | A common polymorphism in the mineralocorticoid receptor modulates stress responsiveness[END_REF], see below), it appears that changes in MR function supported by the N-terminus are not translating into a sufficiently severe salt losing disorder to be diagnosed as PHA1. This is probably due to the specific conformation of the domain, which is supposed to be unstructured in solution, undergoing induced folding upon DNA and/or protein binding [START_REF] Lavery | Structure and function of steroid receptor AF1 transactivation domains: induction of active conformations[END_REF].

The functional consequences of certain mutations located in the DBD and the LBD have been studied in vitro for their effects on hormone binding, DNA binding and transcriptional activation. LBD mutations diminish or completely abolish ligand binding, whereas DBD mutations affect basal or dynamic DNA binding and eventually intracellular trafficking. In some cases, those effects could be predicted on the basis of previous experimental evidence and the crystal structure of the MR LBD [START_REF] Fagart | Crystal structure of a mutant mineralocorticoid receptor responsible for hypertension[END_REF][START_REF] Huyet | Structural determinants of ligand binding to the mineralocorticoid receptor[END_REF][START_REF] Pujo | Mineralocorticoid receptor mutations are the principal cause of renal type 1 pseudohypoaldosteronism[END_REF]. Conversely, in some cases, MR mutations identified in PHA1 patients have allowed the identification of crucial amino acids involved in given receptor functions (Sartorato et al. 2004a;[START_REF] Sartorato | Different inactivating mutations of the mineralocorticoid receptor in fourteen families affected by type I pseudohypoaldosteronism[END_REF]. The MR LBD domain contains 11 helices (H1 and H3-H12) and two short β-sheets organized in the three helical sandwich folds common to all members of the nuclear receptor family [START_REF] Bledsoe | A ligand-mediated hydrogen bond network required for the activation of the mineralocorticoid receptor[END_REF][START_REF] Fagart | Crystal structure of a mutant mineralocorticoid receptor responsible for hypertension[END_REF][START_REF] Li | Structural and biochemical mechanisms for the specificity of hormone binding and coactivator assembly by mineralocorticoid receptor[END_REF]. This scaffold delimits a binding pocket at the lower part of the domain. 22 residues of the H3, H5, H7, H11 and H12 helices, the β-strand between the H5 and H6 helices, and the loop between the H11 and H12 helices form the ligand binding cavity of the MR [START_REF] Fagart | Antagonism in the human mineralocorticoid receptor[END_REF]. At one extremity of the ligand-binding cavity, Gln776 (H3 helix) and Arg817 (H5 helix) establish hydrogen bonds with the 3-ketone function, which characterizes all the MR binding steroids. At the other extremity of the ligand-binding pocket, Thr945 (H11 helix) is in contact with the 20-ketone function of steroids and Asn770 with the 21-hydroxyl group of MR agonists. As a consequence, mutations affecting amino acids 770 and 776 have major consequences on aldosterone binding and transcriptional activation by the MR [START_REF] Pujo | Mineralocorticoid receptor mutations are the principal cause of renal type 1 pseudohypoaldosteronism[END_REF]Sartorato et al. 2004a). On the other hand, analysis of a p.Leu979Pro mutation identified in PHA1 has allowed identification of hydrophobic interactions in the extreme Cterminal tail of the MR which are essential for establishing the ligand-binding competent state of the receptor (Sartorato et al. 2004a).

In addition to haploinsufficiency, due to complete loss of function or nonsense mediated mRNA degradation, MR carrying mutations may also exert dominant negative effects on the wild type receptor. This is because MR binds as a dimer on hormone responsive elements in regulatory regions of target genes. In this case the effects are strongly promoter-dependent and functional consequences associated with MR mutations may be distinct on different genes and in different tissues (Sartorato et al. 2004b). Indeed, investigation of four MR mutations affecting residues in the LBD identified in families with PHA1 supports this view. Measure of aldosterone-dependent gene expression of endogenous sgk1, GILZ, NDRG2, and SCNN1A, four bona fide MR target genes with different kinetic responses, in renal cell has shown that different MR mutants with comparable loss of ligand binding differently affect individual gene expression profiles [START_REF] Fernandes-Rosa | Mineralocorticoid receptor mutations differentially affect individual gene expression profiles in pseudohypoaldosteronism type 1[END_REF]. In particular, whereas receptors carrying a p.Leu848Pro and p.Arg947Ter mutation were incapable of inducing sgk1, GILZ, NDRG2, and SCNN1A expression, receptors carrying a p.Ser843Pro mutation retained complete transcriptional activity on sgk1 and GILZ gene expression, and MR carrying a p.Leu877Pro mutation negatively affected the expression of sgk1, NDRG2, and SCNN1A. The gene-dependent differences in transcriptional activity of mutant receptors might be explained by different promoter organizations and by modifications in the three-dimensional structure of the MR LBD, which may differentially affect MR interactions with target promoters and the set of transcriptional coregulators recruited to the receptor. These studies suggest that not only the extent of functional reduction but also the specific qualitative loss of function, in terms of regulated gene expression, modulate the phenotype in PHA1 and may constitute the mechanistic substrate for phenotypic variability of the disease [START_REF] Fernandes-Rosa | Mineralocorticoid receptor mutations differentially affect individual gene expression profiles in pseudohypoaldosteronism type 1[END_REF].

years of genetic diagnosis of PHA1

The genetics laboratory at the Hôpital Européen Georges Pompidou (HEGP) is the French referral center for the genetic diagnosis of PHA1, which is part of the reference network MARHEA for hereditary kidney diseases of the child and adult (www.sfndt.org/sn/marhea/) and is accredited to ISO 15189 standard by the relevant French authority. 236 affected patients have been assessed for genetic diagnosis in our laboratory since 2004. Improved case detection together with genetic analysis has allowed to identify ≈100 NR3C2 mutations or exon deletions in patients with renal PHA1 and more than 30 mutations in SCNN1A, SCNN1B

or SCNN1G in generalized PHA1, and to discover a continuum of phenotypically and/or biologically distinct forms of PHA1 [START_REF] Dirlewanger | A homozygous missense mutation in SCNN1A is responsible for a transient neonatal form of pseudohypoaldosteronism type 1[END_REF][START_REF] Hubert | Mineralocorticoid receptor mutations and a severe recessive pseudohypoaldosteronism type 1[END_REF]. Genetic diagnosis is performed by targeted Sanger sequencing, based on clinical and biochemical characteristics; more recently, development of a targeted NGS kit has allowed simultaneous sequencing of NR3C2, SCNN1A, SCNN1B and SCNN1G in referred patients and better characterisation of cases with diagnostic ambiguity. NR3C2 mutations were found in 62% of patients with renal PHA1 (Table 1). In 61 cases the mutation was inherited from one of the parents, in 24 cases from the father and in 37 cases from the mother. De novo mutations were found in 20 cases, and in 17 cases transmission is unknown because the parents were not investigated. It is important to note that in many familial cases clinical diagnosis was suggestive of a sporadic form with no family history, and genetic diagnosis allowed identification of asymptomatic carriers and subsequent genetic counseling. In particular, genetic diagnosis can be performed on cord blood, allowing for rapid salt supplementation if required and prevention of severe dehydration of newborns in affected families. The exact causes of intrafamilial phenotypic heterogeneity in renal PHA1 are unknown, but intercurrent events, such as infections, vomiting or diarrhea may worsen an otherwise subclinical neonatal salt loss in individuals at risk. Naturally occurring hypomorphic or hyperfunctioning alleles of other genes, coding for proteins involved in distal sodium reabsorption, may also modulate the phenotype.

Twenty mutations were found in exon 2; all of them lead to truncated receptors. Of the 22 mutations identified in exons 3 and 4, coding for the MR DBD, 11 were nonsense or frameshift mutations, the reminder missense mutations. Thirty variants were located in exons 5-9 and affected the LBD; the majority were missense mutations, underscoring the importance of individual amino acids in the ligand binding domain for ensuring an appropriate conformation of the MR ligand binding pocket. Nine splice variants were identified in different introns. In patients with renal PHA1, in whom no NR3C2 mutation was detected, large deletions were searched for by SNP analysis in informative families and by quantitative multiplex PCR of short fluorescent fragments (qMPSF). Large deletions encompassing single or multiple exons and the flanking intronic regions of the NR3C2 gene were identified in 19 of 160 patients with renal PHA1 (12%), confirming previous results suggesting that large deletions of the NR3C2 gene are reasonably frequent in PHA1 patients [START_REF] Pujo | Mineralocorticoid receptor mutations are the principal cause of renal type 1 pseudohypoaldosteronism[END_REF]).

Remarkably, one of our patients was compound heterozygous carrier of two different nonsense mutations of the MR, p.[Ser166Ter];[Trp806Ter], representing the first case of severe recessive renal PHA1 caused by MR mutations [START_REF] Hubert | Mineralocorticoid receptor mutations and a severe recessive pseudohypoaldosteronism type 1[END_REF]. In this family, the index case was diagnosed with PHA1 and genetic screening identified a frameshift mutation in exon 2 (c.497_498delCT) resulting in a premature stop codon at amino acid position 166 (p.Ser166Ter) (Table 1,K096_1). The family history indicated that three others members were possibly affected by PHA1. Familial genetic screening for the Ser166X mutation revealed that the mutation was transmitted from the father to the index case, and was also present in one sibling, patient K096_4 (Table 1), but absent in the other affected sibling. The parents of the family subsequently participated in the clinical trial "PHACARV -Cardiovascular Evaluation of Adult PHA 1 Patients" [START_REF] Escoubet | Cardiovascular effects of aldosterone: insight from adult carriers of mineralocorticoid receptor mutations[END_REF]; both father and mother had a similar phenotype suggesting PHA1 with increased plasma aldosterone levels.

This unexpected result prompted us to sequence the entire NR3C2 coding sequence and intron-exon junctions in the asymptomatic mother. A missense mutation was found in exon 6 (c.2418G>A) replacing tryptophan at amino acid position 806 with a stop codon. Genetic screening for this second mutation in the pedigree revealed that the mutation had been transmitted to the asymptomatic patient K096_2 (Table 1). Patient K096_4 was compound heterozygous for both familial mutations. Analysis of MR expression and residual MR function showed that both alleles were expressed in lymphocytes from affected carriers, but ex vivo experiments indicated that MR carrying the p.Ser166Ter mutation were degraded. MR carrying the p.Trp806Ter mutation completely lost aldosterone binding, but showed minimal constitutive ligand-independent transcriptional activity. Altogether, these results suggested that the PHA1 phenotype resulted from combined haploinsufficiency and partial loss of function. This exceptional case demonstrates that minimal residual activity of MR is compatible with life. It also suggests that rare hypomorphic NR3C2 alleles may be more common than expected from the prevalence of detected PHA1 cases and may affect renal salt handling and blood pressure in the general population.

An activating MR mutation in a rare Mendelian form of arterial hypertension

By screening for NR3C2 in 75 patients with early onset severe hypertension, Geller et al identified a heterozygous p.Ser810Leu mutation in a 15-ys old boy with severe hypertension associated with suppressed plasma renin and low aldosterone levels [START_REF] Geller | Activating mineralocorticoid receptor mutation in hypertension exacerbated by pregnancy [see comments[END_REF].

Screening of the family of the index case identified 11 relatives that had been diagnosed with severe hypertension before age 20, thus defining a new Mendelian form of arterial hypertension. Remarkably, two female carriers of the mutation had all their pregnancies complicated by marked exacerbation of hypertension, accompanied by low serum potassium levels and undetectable aldosterone levels, but without signs of preeclampsia.

The MR p.Ser810Leu mutation lies in the MR ligand binding pocket. It induces a major change in the LBD conformation, leading to antagonist ligands switching to become agonists.

Functional studies in cells have shown that although the transcriptional activity of wild type and mutant MR where indistinguishable in response to aldosterone, progesterone and its derivatives all activated transcription via MR carrying the p.Ser810Leu mutation.

Spironolactone, which is clinically used as MR antagonist, was also a potent agonist on the mutant receptor. Structural analysis of the MR ligand binding pocket via 3D homology models and later through established X-ray crystal structures has explained the structural determinants of these changes in ligand properties. Under normal circumstances agonist ligands, such as aldosterone, stabilize the active conformation of the LBD via anchoring of their 21-hydroxyl function on Asn770 of helix 3 of the MR (see above). Antagonist ligands such as progesterone and RU26752 are unable to establish this contact [START_REF] Fagart | Antagonism in the human mineralocorticoid receptor[END_REF].

The antagonist behavior of spironolactones has been explained by a clash between the Ala773 residue and substitutions at the 11β position in spironolactones, which are expected to hinder their accommodation in the ligand binding pocket. Structural and biochemical studies indicated that the p.Ser810Leu mutation results in the gain of a van der Waals interaction between helix 5 and helix 3 that substitutes for interaction of the steroid 21-hydroxyl group with helix 3 in the wild-type receptor, thus stabilizing the receptor in an active conformation.

The first MR LBD X-ray crystal structure to be reported was that of the mutant MR carrying the p.Ser810Leu mutation [START_REF] Fagart | Crystal structure of a mutant mineralocorticoid receptor responsible for hypertension[END_REF] complexed with deoxycorticosterone (DOC) and progesterone, which both act as agonists on the mutated receptor. The most striking observation was that the Leu810 residue (in helix 5) establishes hydrophobic contacts with the Gln776 residue (helix H3) and with the 19-methyl group of both DOC and progesterone In presence of these contacts, contact between the ligand and the Asn770 residue is no longer required, and antagonist ligands that do not contact the Asn770 residue in the wild-type MR are able to stabilize MRS810L in its active conformation [START_REF] Fagart | Crystal structure of a mutant mineralocorticoid receptor responsible for hypertension[END_REF].

In subsequent studies, Rafestin-Oblin et al have identified cortisone and 11dehydrocorticosterone as being the endogenous ligands responsible for constitutive MR activation and hypertension in men and non-pregnant women carrying the MR p.Ser810Leu mutation [START_REF] Rafestin-Oblin | The severe form of hypertension caused by the activating S810L mutation in the mineralocorticoid receptor is cortisone related[END_REF]. Those steroids are the main metabolites of cortisol and corticosterone produced by the action of the 11HSD2. In contrast with their low affinity for the wild type MR, cortisone and 11-dehydrocorticosterone bind MRS810L with high affinity, leading to its activation and induction of MR-dependent transcriptional activation. Since plasma concentration of cortisol is about 30-fold higher than that of corticosterone, it is likely that cortisone triggers most of the phenotype in affected carriers.

Despite intensive search of the MR p.Ser810Leu mutation in patients with early-onset hypertension and/or pregnancy-induced hypertension (unpublished data), no further family has been reported in the literature. However, the variant is not described in exome data from the ExAC (http://exac.broadinstitute.org/), which has collected data from >60,000 unrelated individuals sequenced as part of various disease-specific and population genetic studies [START_REF] Lek | Analysis of protein-coding genetic variation in 60,706 humans[END_REF]. It therefore remains to be established whether this remains a textbook case from which we have learned enormously in terms of structure-function relationships of the MR, or whether it might be relevant for hypertensive pregnant women in a larger context. Given the functional characteristics of MR carrying the p.Ser810Leu mutation, spironolactone, as well as eplerenone, cannot be used to treat hypertension in these patients [START_REF] Hultman | The liganddependent interaction of mineralocorticoid receptor with coactivator and corepressor peptides suggests multiple activation mechanisms[END_REF]. In contrast, both BR-4628 and finerenone, two MR antagonists derived from the chemical class of dihydropyridines, are potent MR antagonists that retain their antagonist character at the MRS810L mutant [START_REF] Amazit | Finerenone Impedes Aldosterone-dependent Nuclear Import of the Mineralocorticoid Receptor and Prevents Genomic Recruitment of Steroid Receptor Coactivator-1[END_REF][START_REF] Fagart | A new mode of mineralocorticoid receptor antagonism by a potent and selective nonsteroidal molecule[END_REF]. As finerenone is currently developed for clinical use and has been studied in different clinical trials, it might be a therapeutic option to treat patients carrying the MR p.Ser810Leu mutation.

Influence of MR gene variants on blood pressure, stress and the HPA axis

In addition to rare MR variants, frequent NR3C2 polymorphisms have been shown to exert quantitative effects on MR function and to modulate salt balance, blood pressure, stress and the hypothalamic-pituitary-adrenal (HPA) axis. The NR3C2 polymorphism c.-2G>C (rs2070951, MAF ≈0.45) is a frequent single nucleotide polymorphism located in the 5'untranslated region of the NR3C2 gene, 2 nucleotides upstream of the first translation start site. In vitro characterization has revealed that the G allele is associated with decreased MR protein levels and reduced transcriptional activation compared to the C allele both in the presence of aldosterone and cortisol. In different groups of patients, the G allele of the c.-2G>C polymorphism is associated with increased activation of the renin-angiotensinaldosterone axis and with increased blood pressure, probably related to decreased MR expression. Subjects with the GG genotype had significant higher plasma renin levels both in a mild hypertensive group subjected to a salt sensitivity test and in a healthy normotensive group included in a crossover study to receive both a high and low Na/K diet compared to homozygous C carriers; the GG genotype was also correlated with higher plasma aldosterone levels in healthy subjects. In mild hypertensives and in a large cohort for Depression and Anxiety the genotype GG was associated with higher systolic blood pressure in males. These studies provide evidence that frequent polymorphisms of the MR may exert quantitative effects on the activity of the renin-angiotensin-aldosterone axis and blood pressure in the general population, modulating vulnerability for hypertension [START_REF] Van Leeuwen | The functional c.-2G>C variant of the mineralocorticoid receptor modulates blood pressure, renin, and aldosterone levels[END_REF].

In addition to its function in maintaining salt homeostasis and regulating blood pressure, the MR also plays a role in the brain, where it regulates salt appetite, blood pressure, stress response and cognitive processes [START_REF] De Kloet | Brain mineralocorticoid receptors and centrally regulated functions[END_REF][START_REF] Gomez-Sanchez | ICV infusion of corticosterone antagonizes ICV-aldosterone hypertension[END_REF]; for a detailed review, see the article by M Joëls and ER de Kloet in this issue). In the brain, MR is expressed in limbic regions, particularly in hippocampal neurons; in the absence of 11HSD2, the brain MR is essentially occupied and activated by cortisol. Coordinately with the glucocorticoid receptor the MR regulates the onset and termination of the stress response.

Central MR is also involved in the control of autonomic outflow as demonstrated in rats by modulation of stress-induced heart rate and blood pressure responses through central application of MR antagonists [START_REF] De Kloet | Brain mineralocorticoid receptors and centrally regulated functions[END_REF].

Several studies performed by the group of Roel de Rijk and co-workers have explored the effects of common MR variants on stress response and depression. By studying the association between the common NR3C2 single nucleotide polymorphism c.538A>G/p.Ile180Val (rs5522, MAF 0.12) and outcome variables in a healthy cohort subjected to psychosocial challenge (Trier Social Stress Test, TSST), it was shown that carriers of the MR180Val allele had higher plasma and saliva cortisol levels and higher heart rate responses to the TSST than non-carriers [START_REF] Derijk | A common polymorphism in the mineralocorticoid receptor modulates stress responsiveness[END_REF]. In contrast, there was no difference in salt sensitivity according to genotypes in a group of mild hypertensives undergoing a controlled sodium diet followed by a salt sensitivity test in terms of blood pressure, cardiac frequency, and renal sodium handling. The difference in central and renal MR effects were explained by the functional consequences of the p.Ile180Val variant on receptor function. Whereas aldosterone-mediated transactivation of different reporter genes was undistinguishable whatever the amino acid at position 180, in the presence of cortisol the transcriptional activity of MR180Val was significantly lower than that of the MR180Ile. This suggests that p.Ile180Val polymorphism, which is located in the NTD, may affect intramolecular interactions within the MR or binding of specific coactivators and modulate specifically cortisol mediated MR effects.

In addition to p.Ile180Val and c.-2G>C, exonic sequencing of NR3C2 in fifty individuals from the Dutch population resulted in the identification of two major haplotype blocks, one in the 5'-region (based on p.Ile180Val and c.-2G>C) and one in the 3 '-region (ter Heegde et al. 2015). One haplotype in particular, associating c.-2C and p.Ile180 with SNPs in the promoter region of the NR3C2 gene has a frequency of 0.38 in the general population. In addition to increased MR protein expression and differences in transcriptional responses related to c.-2C and p.Ile180, the SNPs in the promoter region are associated with higher promoter activity, resulting in enhanced MR activity [START_REF] Klok | A common and functional mineralocorticoid receptor haplotype enhances optimism and protects against depression in females[END_REF]. Haplotype 2 was associated, in women, with heightened dispositional optimism in a cohort of elderly subjects, with less hopelessness and rumination in a cohort of 150 university students and with a lower risk of depression in a large genome-wide association study. These and other studies suggest that common functional MR haplotypes are important determinants of inter-individual variability in basal and stressinduced HPA-axis activity and stress-related appraisal and learning, by differentially mediating cortisol effects on different systems [START_REF] Ter Heegde | The brain mineralocorticoid receptor and stress resilience[END_REF]. Remarkably, in adult PHA1 patients carrying MR loss-of-function mutations, personal history revealed an increase in depression compared to paired non carriers [START_REF] Escoubet | Cardiovascular effects of aldosterone: insight from adult carriers of mineralocorticoid receptor mutations[END_REF].

Study of adult PHA1 patients carriers of MR mutations has also allowed definition of the contribution of MR to negative feedback of the HPA axis in human [START_REF] Walker | Activation of the hypothalamic-pituitaryadrenal axis in adults with mineralocorticoid receptor haploinsufficiency[END_REF]. In a study performed on a subset of patients from the cohort described in [START_REF] Escoubet | Cardiovascular effects of aldosterone: insight from adult carriers of mineralocorticoid receptor mutations[END_REF], patients with MR mutations had higher morning plasma cortisol and increased 24-hour urinary excretion of cortisol, independent of gender. Higher plasma cortisol was associated with higher plasma renin, lower serum high-density lipoprotein-cholesterol, and higher waist circumference but not with blood pressure, carotid intima-media thickness, or echocardiographic parameters. These data suggest that hypercortisolemia is related to the severity of MR deficiency and has features of glucocorticoid excess mediated by glucocorticoid receptors on liver lipid metabolism and adipose tissue distribution, without adversely affecting cardiac and vascular remodeling in the absence of normal signaling through the MR [START_REF] Walker | Activation of the hypothalamic-pituitaryadrenal axis in adults with mineralocorticoid receptor haploinsufficiency[END_REF].

Concluding remarks

The MR cDNA was cloned by [START_REF] Arriza | Cloning of human mineralocorticoid receptor complementary DNA: structural and functional kinship with the glucocorticoid receptor[END_REF][START_REF] Arriza | Cloning of human mineralocorticoid receptor complementary DNA: structural and functional kinship with the glucocorticoid receptor[END_REF], followed by the determination of the human mineralocorticoid receptor gene structure and identification of expressed isoforms [START_REF] Zennaro | Human mineralocorticoid receptor genomic structure and identification of expressed isoforms[END_REF], the characterization of two different promoters in the human mineralocorticoid receptor gene 5'-regulatory region [START_REF] Zennaro | Characterization of the human mineralocorticoid receptor gene 5'-regulatory region: evidence for differential hormonal regulation of two alternative promoters via nonclassical mechanisms[END_REF] and the identification of different human MR splice variants, some with particular functional properties possibly modulating corticosteroid effects in target tissues [START_REF] Zennaro | A new human MR splice variant is a ligand-independent transactivator modulating corticosteroid action[END_REF] [START_REF] Fuller | Pseudohypoaldosteronism type 1: the index case revisited[END_REF]

  . It appeared from these and other data in rats and mice that the NR3C2 gene undergoes different levels of regulation, leading to tissue and developmentally regulated MR protein expression of different receptor isoforms. Additional mechanisms of hormone selectivity and transcriptional coregulator recruitment contribute to specific and multiple sets of physiological responses. In addition, recent genomewide mapping of MR binding on regulatory sites of target genes has further highlighted the complexity of transcriptional regulation exerted by the MR (Le Billan et al. 2015). As a result, MR mutations identified in PHA1 not only differentially affect gene expression profiles in a promoter-dependent manner (Fernandes-Rosa et al. 2011), but may also have different consequences depending on target tissues, developmental stage and MR ligand, which may explain the heterogeneous phenotypic expression of the disease, even within the same family, and the absence of signs of mineralocorticoid resistance outside the kidney in renal PHA1. Remarkably, in the context of reduced MR function, high salt and high

Figure legends Figure 1 :

 legends1 Figure legends

Table 1 .

 1

	Kindred	Mutation	Protein	Exon	MR domain	Transmission	References
	K158	c.415C>T	p.Gln139Ter	2	NTD	de novo
	K074	c.497_498delCT	p.Ser166Ter	2	NTD	father	(Hubert et al. 2011)
	K096_1 c.497-498delCT	p.Ser166Ter	2	NTD	father	(Hubert et al. 2011)
	K096_4 c.[497-498delCT] ;[2418G>A]	p.[Ser166Ter] ;[Trp806Ter]	2/6	NTD/LBD	mother/father (Hubert et al. 2011)
	K161	c.531dupG	p.Arg178Alafs*5	2	NTD	de novo
	K306	c.556_557del	p.Met186Valfs*3	2	NTD	mother
	K066	c.887-888delCT	Ser296Cysfs*3	2	NTD	father
	K390	c.981delC	p.Ser328Valfs*10	2	NTD	de novo	(Pujo et al. 2007)
	K440	c.1029 C>A	p.Tyr343Ter	2	NTD	mother	(Pujo et al, 2007)
	K115	c.1108C>T	p.Gln370Ter	2	NTD	father
	K261	c.1108C>T	p.Gln370Ter	2	NTD	de novo
	K155	c.1169delG	p.Gly390Alafs*9	2	NTD	de novo
	K128	c.1213delG	p.Ala405Leufs*7	2	NTD	father

Sources of Funding

This work was funded through institutional support from INSERM and by the Fondation pour la Recherche Médicale (DEQ20140329556).

aldosterone do not exert deleterious effects on the cardiovascular system [START_REF] Escoubet | Cardiovascular effects of aldosterone: insight from adult carriers of mineralocorticoid receptor mutations[END_REF], pointing to a central role of the receptor in aldosterone-or glucocorticoid-mediated end organ damage. It is particularly good news, 30 years after its cloning, that the MR is not only responsible for maintaining blood pressure and salt homeostasis, but that a good MR function may contribute to enhance optimism and protect against depression (although in females only!).

Disclosures

The authors have nothing to disclose.

K043

c.


Chen Zhang

Caroline Lafond

Anaïs Barateau

Julie Leseur

Bastien Rigaud

Diane-Barbara Chan

Sock Line

Guanyu Yang

Huazhong Shu

Diane Barbara

Chan Sock Line

Jean-Louis Dillenseger

Renaud De Crevoisier

Antoine Simon

Automatic segmentation for plan-of-the-day selection in CBCT

come

Introduction

The standard treatment for locally advanced cervical cancer (LACC) is external beam radiotherapy (EBRT) with chemotherapy, followed by brachytherapy. Although Intensity-Modulated Radiation Therapy (IMRT) is used to reduce normal tissue toxicity [START_REF] Naik | Comparison of dosimetric parameters and acute toxicity of intensity-modulated and three-dimensional radiotherapy in patients with cervix carcinoma: A randomized prospective study[END_REF][START_REF] Gandhi | Early clinical outcomes and toxicity of intensity modulated versus conventional pelvic radiation therapy for locally advanced cervix carcinoma: a prospective randomized study[END_REF], it is limited by large and complex intrapelvic anatomical variations occurring between the treatment fractions [START_REF] Buchali | Impact of the filling status of the bladder and rectum on their integral dose distribution and the movement of the uterus in the treatment planning of gynaecological cancer[END_REF][START_REF] Han | Interfractional dose variation during intensity-modulated radiation therapy for cervical cancer assessed by weekly CT evaluation[END_REF]. The position and shape of the clinical target volume (CTV, including the cervix, uterus, upper-vagina, and parametrium) are highly dependent on bladder and rectum filling, and on tumor regression during treatment [START_REF] Jadon | A systematic review of organ motion and image-guided strategies in external beam radiotherapy for cervical cancer[END_REF][START_REF] Chan | Inter-and intrafractional tumor and organ movement in patients with cervical cancer undergoing radiotherapy: a cinematic-MRI point-of-interest study[END_REF][START_REF] Beadle | Cervix regression and motion during the course of external beam chemoradiation for cervical cancer[END_REF]. In the context of adaptive radiation therapy (ART), plan-of-the-day (PoD) strategies have been proposed based on the generation of a treatment plan library, including several treatment plans optimized according to multiple planning CTs (pCT) acquired with various bladder fillings [START_REF] Bondar | Individualized nonadaptive and online-adaptive intensity-modulated radiotherapy treatment strategies for cervical cancer patients based on pretreatment acquired variable bladder filling computed tomography scans[END_REF], Heijkoop et al 2014[START_REF] Van De Schoot | Dosimetric advantages of a clinical daily adaptive plan selection strategy compared with a non-adaptive strategy in cervical cancer radiation therapy[END_REF]. At each treatment fraction, the treatment plan is then selected among those of the library ("plan-of-the-day") based on an in-room image (e.g., CBCT image, see Figure 1). Although this strategy appears to be adequate to compensate for uterine motions [START_REF] Heijkoop | Clinical implementation of an online adaptive plan-of-the-day protocol for nonrigid motion management in locally advanced cervical cancer IMRT[END_REF][START_REF] Van De Schoot | Dosimetric advantages of a clinical daily adaptive plan selection strategy compared with a non-adaptive strategy in cervical cancer radiation therapy[END_REF][START_REF] Gobeli | Benefit of a pretreatment planning library-based adaptive radiotherapy for cervix carcinoma? Cancer Radiotherapie[END_REF], it remains complex in a clinical workflow.

Different factors limit the rapid advancement of PoD ART. The PoD selection is actually a difficult process, mainly due to the poor contrast of CBCT images and large anatomical deformations. Thus, the expert needs to visualize the full 3D volume to assess the coverage of the whole target by the available treatment plans. The PoD selection is therefore a time-consuming process which is submitted to interobserver variability, as demonstrated in [START_REF] Gobeli | CBCT guided adaptive radiotherapy for cervix cancer: uncertainty of the choice of the plan of the day[END_REF] where 26 operators manually selected PoD on 24 CBCT images. This study showed a high inter-observer variablility since the optimal PoD was chosen on average by 60% of users.

In order to automatize PoD selection, the automatic segmentation of CBCT images has been proposed to measure, at each treatment fraction, the ability of the treatment plans to treat the target [START_REF] Bondar | Intra-patient semi-automated segmentation of the cervix-uterus in CT-images for adaptive radiotherapy of cervical cancer Physics in[END_REF]. Langerak et al. [START_REF] Langerak | Towards automatic plan selection for radiotherapy of cervical cancer by fast automatic segmentation of cone beam CT scans International Conference on Medical Image Computing and Computer-Assisted Intervention[END_REF] proposed to use a multi-atlas-based segmentation method. On a total of 224 CBCT, the CBCT images corresponding to low confidence levels were firstly removed, resulting to 187 images on which the Dice values were 0.85, 0.81, and 0.80 for the uterus, bladder, and rectum, respectively. However, the CBCT image quality is limited by noise, artifacts, and low soft-tissue CTs (pCTs). For this second step, the three pCTs in the planning library were registered to the daily CBCT image to simulate patient repositioning. Then, the coverage of the segmented daily CTV by the CTVs of pCTs was computed. Finally, the best treatment plan was selected as the one corresponding to the highest daily CTV coverage.

Materials and methods

Data acquisition and experimental settings

In this study, we collected 272 CBCT scans and 63 CT scans from 23 patients. All patients were treated with a combination of external beam radiation therapy (EBRT) and pulse-dose-rate brachytherapy (PDR-BT). EBRT delivered a total dose of 45 Gy to the pelvis (supine position), at 1.8 Gy per fraction, using IMRT along with concomitant weekly cisplatin (40 mg/ m 2 ). PDR-BT was delivered following the GEC-ESTRO recommendations. All patients provided signed informed consent.

Each patient underwent two or three planning CTs with different bladder volumes: empty bladder (EB), intermediate bladder (IB), and full bladder (FB). One hour before the first IB CT, the patient had to drink 250 mL of water. Then another 500 mL of water should be consumed to obtain FB CT after 20 minutes. For EB CT, the patient emptied her bladder. All CTs were scanned (Big Bore, Philips) with voxel spacing ranging from 0.87×0.87×3.00 mm 3 to 1.21×1.21×3.00 mm 3 . The dimensions range of CT volumes was from 75×512×512 to 168×512×512.

During the radiation therapy treatment, 5 to 16 CBCT images were acquired (XVI mounted on a Synergy linac, Elekta) for each patient with voxel spacing of 1.00×1.00×2.00 mm 3 and dimensions of 132×410×410.

All images (i.e., planning CTs and CBCTs) were manually contoured slice-by-slice by one radiation oncologist. These contours were the primary CTV, including the cervix, uterus, and upper-vagina, and the rectum, bladder, and bowel bag (including the sigmoid). These delineations were considered as the reference in this study.

CBCT segmentation using deep-learning

The segmentation model was trained using the deep learning-based method nnU-Net, which has been demonstrated to be efficient in multiple medical image segmentation tasks [START_REF] Isensee | nnU-Net: a self-configuring method for deep learning-based biomedical image segmentation[END_REF]. nnU-Net's automatic configuration runs without human intervention when it is applied to a new dataset. The nnU-Net focuses on pre-processing, training, inference strategies, and post-processing. The 3D full resolution U-Net was used since it has been shown to be one of the best performing models in many medical image segmentation tasks [START_REF] Malimban | Deep learning-based segmentation of the thorax in mouse micro-CT scans[END_REF][START_REF] Isensee | nnU-Net: a self-configuring method for deep learning-based biomedical image segmentation[END_REF].

All images were cropped to the region of nonzero values with a cropping size of 84×410×410 voxels. The resampling voxel size was the median voxel spacing of the dataset, i.e., 1.00×1.00×2.00 mm 3 . A z-score normalization was applied to each image.

In the post-processing procedure, a connected component analysis was performed to eliminate the detection of spurious false positives. The model was trained using Pytorch and stochastic gradient descent (SGD) optimizer. The initial learning rate was set to 0.01 and decreased according to the "Poly" scheme [START_REF] Chen | Deeplab: Semantic image segmentation with deep convolutional nets, atrous convolution, and fully connected crfs[END_REF]. The loss function was the sum of Dice similarity coefficient and cross-entropy with the same weight.

We randomly split all 23 patients into four folds using a cross-validation scheme.

Plan-of-the-day selection

After obtaining the segmentation results for each CBCT image, the following three steps were performed: (1) Bone-based rigid registration between each pCT with different bladder fillings (FB, IB, EB) and the daily CBCT to simulate patient repositioning;

(2) computation of the coverage between the CTV of CBCT ( ) and CTV of the CTs ; 1,3,3],[3,3,3],[3,3,3],[3,3,3],[3,3,3],[3,3,3]] Table 2. Network configurations generated by nnU-Net.

Rigid registration

For each patient, a bone-based rigid registration was performed between each CBCT and each pCT, using the ROI of the CBCT which was limited to the treated region. The goal of rigid registration was, in this study, to simulate patient repositioning. In the clinical setup, the rigid transform would result from the applied couch shift/rotation. The library Elastix was used [START_REF] Klein | Elastix: a toolbox for intensity-based medical image registration[END_REF] to estimate the rigid transform (translation and rotation) with normalized correlation as the metric, applied to thresholded images. The resulting rigid transform was visually validated by checking bone alignment and was applied to the pCT's corresponding delineations.

Selection of the PoD

To evaluate the ability of each treatment plan to treat the CTV in its daily position, a coverage index was computed between the segmented by the deep learning model and the different CTV of the patients's library ( 𝑪𝑻𝑽 𝑪𝑩𝑪𝑻 𝑪𝑻𝑽 𝑪𝑻 ∈ {𝑪𝑻𝑽 𝑬𝑩 , 𝑪𝑻𝑽 𝑰𝑩 ,

): 𝑪𝑻𝑽 𝑭𝑩 } (1) 𝒄𝒐𝒗 = |𝑪𝑻𝑽 𝑪𝑩𝑪𝑻 ∩ 𝑪𝑻𝑽 𝑪𝑻 | |𝑪𝑻𝑽 𝑪𝑻 |
where | . | is the cardinality of the set. In this way, each CBCT had a coverage value associated with each of the three pCTs (IB, EB, FB) of the patient.

All the three pCTs of the considered patient were ranked according to the corresponding coverage. The pCT corresponding to the highest coverage value was selected as the PoD of the considered treatment fraction.

Evaluation

The data of the 23 patients were used to evaluate the proposed PoD selection process.

Segmentation evaluation

The segmentation of CBCT images was evaluated using four-fold cross-validation, considering the manual expert delineations as the reference. The following geometric metrics were computed for CTV, bowel bag, rectum, and bladder.

i) Volume-based metric

The Dice similarity coefficient (DSC) allows for comparing the automatic segmentation results (𝑆𝑝) with the reference delineation (𝑆r) where a perfect overlap returns 1, and no overlap returns 0:

(2)

𝐷𝑆𝐶 = 2 |𝑆 𝑟 ∩ 𝑆 𝑝 | |𝑆 𝑟 | + |𝑆 𝑝 | ii) Distance-based metrics
Distance-based metrics allow for evaluating the segmentation in terms of the location and shape accuracies of the segmented region boundaries. They use two point sets, A and B, from 𝑆𝑝 and 𝑆r. N is the number of points in A.

Mean absolute distance (MAD) measures the average distance of one boundary pixel to the closest boundary pixels in the other segmentation:

(3) 𝑀𝐴𝐷 = 1 𝑁 ∑ 𝑎 ∈ 𝐴 𝑚𝑖𝑛 𝑏 ∈ 𝐵

‖𝑎 -𝑏‖

Hausdorff distance (HD) measures the similarity between two boundaries and can be expressed as:

(4) 𝐻𝐷 = 𝑚𝑎𝑥 (ℎ(𝐴,𝐵),ℎ(𝐵,𝐴))

where

We reported HD95, i.e., the 95 th percentile of HD, to limit the influence of small outliers.

h(A,B) = max 𝑎 ∈ 𝐴 min 𝑏 ∈ 𝐵 ‖𝑎 -𝑏‖.

PoD selection evaluation

For each treatment fraction, the PoD resulting from the proposed automatic process was compared to the one obtained using the reference delineation of the CTV in the CBCT image. Thus, this reference PoD corresponded to the best coverage between the manual delineation of the and .

𝑪𝑻𝑽 𝑪𝑩𝑪𝑻 𝑪𝑻𝑽 𝑪𝑻 ∈ {𝑪𝑻𝑽 𝑬𝑩 , 𝑪𝑻𝑽 𝑰𝑩 , 𝑪𝑻𝑽 𝑭𝑩 }
To consider that multiple treatment plans may provide a satisfying coverage of the target, a 5% tolerance was applied to the maximum coverage, and the corresponding treatment plan(s) were also selected.

The accuracy of the PoD selection was determined by calculating the number of automatically selected PoD that were identical to the reference PoD or included in the selected treatment plans.

Results

Performance of segmentation

Table 3 reports the mean (range) values of the geometric metrics for the automatic segmentation. Figure 3 summarizes the quantitative results in boxplots. The median DSC of primary CTV, bowel bag, rectum, and bladder were 0.79, 0.81, 0.75, and 0.84, respectively. Figure 4 shows the results of automatic contouring for four patients in axial and sagittal views.

Organ

Performance of treatment plan selections

Considering the accuracy of automatic PoD selection without the 5% tolerance, an agreement between the reference PoD and the automatically identified PoD was observed for 91.5% of CBCTs. This resulted in 23 out of 272 tested CBCTs having a suboptimal selected PoD, compared to the reference.

Journal XX (XXXX) XXXXXX

Author et al 10

Using the 5% tolerance value for PoD selection, multiple (up to three) pCTs could be selected as optimal PoD. This resulted in an increased PoD selection agreement to 99.6%, with one CBCT having suboptimal PoD selection. Figure 5 shows the PoD selection on four different patients corresponding to an agreement between the proposed and selected PoDs. Figure 6 illustrates the only case with suboptimal PoD selection. For this case, the automatic segmentation results were poor (Dice of CTV segmentation was 0.52), which resulted in the wrong proposed PoD.

Discussion

This study aimed at improving the PoD selection for LACC ART on CBCT images. Simulating the proposed process on 272 treatment fractions, it resulted in an agreement between the reference PoD and the automatically identified PoD for all fractions except one.

The first step of the process was based on the automatic segmentation of the daily images using a deep learning network.

The resulting average DSC on the 272 CBCT images were higher than 0.75 for the primary CTV and the three main OARs (bowel bag, rectum, and bladder).

Only two works in the literature, to the best of our knowledge, have proposed cervix CBCT automatic segmentation

Conclusion

This work proposed and evaluated an automatic workflow to select PoD for LACC ART. Based on CBCT image segmentation using a deep-learning method, it selects the optimal treatment plan based on daily CTV geometrical coverage.

The evaluation of 272 treatment fractions showed a high agreement with the reference resulting from expert delineation. The proposed workflow should be further evaluated in a clinical workflow and on a larger number of patients.

  Figure 1. Flowchart of plan-of-the-day ART for cervical cancer. The process includes three steps (1) Planning: acquisition of multiple planning CT scans with variable bladder volumes. (2) Acquisition of the CBCT image of the day. (3) Selection of the most appropriate treatment plan to maximize the target coverage. The CTV, bowel bag, bladder, and rectum are represented as red, green, yellow, and blue-filled contours. For plan-of-the-day selection, empty bladder (EB), intermediate bladder (IB), full bladder (FB), and daily CTV are represented on the daily CBCT as red, blue, green, and yellow contours, respectively.

Figure 2

 2 Figure 2 describes the flowchart of the study. Based on three planning CT scans, corresponding to empty bladder, intermediate bladder and full bladder, and on daily CBCT scans, it contains two parts applied to each daily CBCT image: (1) segmentation of the CBCT image using a deep learning network; (2) selection of the most appropriate PoD among the available planning

Figure 2 .

 2 Figure 2. Flowchart of the study. The steps are: (1) CBCT segmentation using deep learning and (2) plan-of-the-day (PoD) selection using clinical target volume (CTV) contours. The PoD selection relies on: (a) Bone-based rigid registration of the planning CTs (pCTs) with the daily CBCT to simulate patient repositioning; (b) computation of the coverage between the daily CTV ( ) and CTV of the pCTs ( ); (c) selection of the best treatment plan based on target 𝑪𝑻𝑽 𝑪𝑩𝑪𝑻 𝑪𝑻𝑽 𝑬𝑩 , 𝑪𝑻𝑽 𝑰𝑩 , 𝑪𝑻𝑽 𝑭𝑩 coverage: the pCT corresponding to the highest coverage was selected. (EB: empty bladder; IB: intermediate bladder; FB: Full bladder; cov: coverage value).

Figure 3 .Figure 4 .

 34 Figure 3. Quantative segmention results of CTV (uterus), bowel bag, rectum and bladder presented as boxplots. (a) DSC = Dice similarity coefficient ; (b) MAD = Mean absolute distance ; (c) HD95 = 95 th percentile Hausdorff Distance

Figure 5 .

 5 Figure 5. Examples of automatic PoD selection for four patients. (Left) Result of segmentation and clinical target volumes (CTVs) corresponding to the planning library for PoD selection; (Right) Automatic and reference CTV segmentations. Illustrated patients were selected based on the following criteria: single selected PoD (Patients 1 and 2); multiple PoD selections due to the tolerance value of 5% (Patient 3 and 4).

(

  [START_REF] Langerak | Towards automatic plan selection for radiotherapy of cervical cancer by fast automatic segmentation of cone beam CT scans International Conference on Medical Image Computing and Computer-Assisted Intervention[END_REF][START_REF] Beekman | Improving predictive CTV segmentation on CT and CBCT for cervical cancer by diffeomorphic registration of a prior[END_REF]. Langerak et al.[START_REF] Langerak | Towards automatic plan selection for radiotherapy of cervical cancer by fast automatic segmentation of cone beam CT scans International Conference on Medical Image Computing and Computer-Assisted Intervention[END_REF] used a multi-atlas-based segmentation method.On a total of 224 CBCT, the images corresponding to low confidence levels were firstly removed, resulting to 187 images on which the Dice values were 0.85, 0.81, and 0.80 for the uterus, bladder, and rectum, respectively. Without discarding any CBCT image, the DSC values obtained in our study were close to these ones, with lower values for CTV and rectum, and higher values for bladder. More recently, Beekman et al.[START_REF] Beekman | Improving predictive CTV segmentation on CT and CBCT for cervical cancer by diffeomorphic registration of a prior[END_REF] compared different deep learning approaches and showed that the best performances were obtained by a deformation-based registration network with a mean DSC of 0.80 on the uterus, computed on a test set of 20 CBCT images. In our study, we obtained similar DSC on 272 CBCT images. This study and[START_REF] Beekman | Improving predictive CTV segmentation on CT and CBCT for cervical cancer by diffeomorphic registration of a prior[END_REF] are the only ones using deep learning for cervix CBCT segmentation. Although 2D and 3D U-Net (or V-Net) networks have been used on planning CT images[START_REF] Liu | Segmentation of organs-at-risk in cervical cancer CT images with a convolutional neural network[END_REF][START_REF] Rhee | Automatic contouring system for cervical cancer using convolutional neural networks[END_REF][START_REF] Rigaud | Automatic segmentation using deep learning to enable online dose optimization during adaptive radiation therapy of cervical cancer[END_REF] resulting to larger DSC for CTV (0.86 ± 0.08), lower contrast, higher noise, but also limited availability of reference segmentation, challenge the exploitation of these methods on CBCT. To our knowledge, this is the first study exploiting nnU-Net on CBCT images and on the uterine region. It confirms its good performances which have been shown on other imaging modalities[START_REF] Isensee | nnU-Net: a self-configuring method for deep learning-based biomedical image segmentation[END_REF].

Figure 6 .

 6 Figure 6. One case with suboptimal PoD selection. (Left) Result of segmentation and clinical target volumes (CTVs) corresponding to the planning library for PoD selection; (Right) Automatic and reference CTV segmentations. The poor segmentation of CTV results in the suboptimal PoD selection.

  Table 1 presents the partition of the dataset. Multiple images (i.e., planning and daily images) from individual patients were not distributed among datasets. The test data was only used to evaluate the performance of the model in this fold and was not involved in training. nnU-Net further divided the training data into training and validation sets and performed a five-fold cross-validation to automatically select the

best network configuration. In the end, the model (or ensemble) which got the best performance was chosen to perform the inference on the test sets of this fold. The number of epochs during training was 1000 for every fold. The evaluation of the segmented volumes is described in part 2.4.1. Table

2

reports the network configurations generated by nnU-Net for the considered dataset.

)

  𝑪𝑻𝑽 𝑪𝑩𝑪𝑻 (𝑪𝑻𝑽 𝑬𝑩 , 𝑪𝑻𝑽 𝑰𝑩 , 𝑪𝑻𝑽 𝑭𝑩 ) selection of the pCT corresponding to the best coverage.

	Model	#Patient Train	Test	#CBCT volume Train Test
	1	18	5	216	56
	2	17	6	197	75
	3	17	6	206	66
	4	17	6	199	73

Table 1 . Partition of the dataset for the deep learning network training and testing. The

 1

	population was randomly

Table 3 .

 3 Quantitative evaluation results of CBCT segmentation.

		DSC	MAD (mm)	HD95 (mm)
	CTV	0.79 (0.42 -0.94) 2.90 (0.65 -11.48) 10.49 (3.02 -46.43)
	Bowel bag 0.81 (0.53 -0.92) 5.22 (2.12 -12.15) 18.40 (6.12 -60.43)
	Rectum	0.75 (0.30 -0.91) 3.14 (0.65 -16.04) 14.19 (2.24 -78.79)
	Bladder	0.84 (0.13 -0.96) 2.91 (0.61 -17.9)	9.45 (2.03 -58.22)

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Acknowledgments

This work was partly funded by the French National Research Association (ANR) as part of the Investissement d'Avenir programme (Labex CAMI) under reference ANR-11-LABX-0004, by the DELPEL project with financial support from ITMO Cancer AVIESAN (National Alliance for Life Sciences & Health) within the framework of the Cancer Plan, by the French organizations "Ligue contre le Cancer" and "La Vannetaise", and by the Embassy of France in China.

Author et al

Author et al 12

In this study, the deep learning model was trained to segment not only the CTV (which is important for PoD selection), but also the main OARs. Intuitively, a binary segmentation task (segmenting only CTVs) could be easier than a multi-class segmentation task (segmenting OARs and CTVs) and thus could achieve better segmentation results. However, in terms of geometric considerations, multi-class segmentation provides a positional prior (e.g. the uterus should be between the bladder and rectum). Such a positional prior helps the convolutional neural network to constrain the position of the uterus during segmentation. Preliminary experiments have validated this assumption, with better results when including OARs in the segmentation task.

The evaluation of the proposed process, not only in terms of segmentation accuracy (e.g., with Dice score) but in terms of PoD selection, is of clinical interest since this PoD selection is, in this adaptive strategy, the most important and difficult step.

In the current clinical practice, the treatment plan is selected visually, resulting in potentially high inter-observer variations [START_REF] Gobeli | CBCT guided adaptive radiotherapy for cervix cancer: uncertainty of the choice of the plan of the day[END_REF]. The identification of the optimal treatment plan appears particularly difficult in the context of complex deformations and limited image quality. Langerak et al. [START_REF] Langerak | Towards automatic plan selection for radiotherapy of cervical cancer by fast automatic segmentation of cone beam CT scans International Conference on Medical Image Computing and Computer-Assisted Intervention[END_REF] proposed a PoD selection, after CBCT segmentation, by comparing the segmented bladder volume to the preoperative planning library (empty and full bladder).

However, the CTV shape may be influenced by other factors than bladder filling, including rectum filling, tumor shrinkage, or non-moving cervix. The PoD selection should thus ideally be primarily based on the shape of the target.

In this paper, the criteria used to select the PoD was the coverage of the daily CTV by the CTVs corresponding to the different planning CTs. It enabled to consider directly the treatment of the target instead of a surrogate of the target position.

Moreover, selecting the best plan as the one providing the best CTV coverage is the approach considered in the literature [START_REF] Gobeli | CBCT guided adaptive radiotherapy for cervix cancer: uncertainty of the choice of the plan of the day[END_REF], Heijkoop et al 2014). On the other hand, since the OARs were also segmented in the proposed process, considering the OARs in the PoD selection is technically possible. However, it would require the definition of a decision process, or a metric, considering and weighting the different criteria (CTV and OARs coverage).

In our study, only 1 case (out of 272) resulted in wrong PoD selection (Figure 6). This case with a relatively small uterus, was caused by poor segmentation result. In a clinical context, this kind of result would be easily visually detected. Then, the treatment plan could be manually selected in the library or a backup plan could be used as proposed in [START_REF] Heijkoop | Clinical implementation of an online adaptive plan-of-the-day protocol for nonrigid motion management in locally advanced cervical cancer IMRT[END_REF].

Online adaptive radiotherapy has recently seen important improvements, especially with the development of MRI-linac and CBCT-based online optimization. For both of them, some challenges remain and hamper their clinical deployment to treat cervical cancer, including the limited quality of CBCT images and long treatment times of MRI-Linac. Very few studies have thus considered these kinds of systems to treat cervical cancer patients [START_REF] Shelley | Adaptive Radiotherapy in the Management of Cervical Cancer: Review of Strategies and Clinical Implementation[END_REF][START_REF] Yock | Initial analysis of the dosimetric benefit and clinical resource cost of CBCT-based online adaptive radiotherapy for patients with cancers of the cervix or rectum[END_REF]. Moreover, the combination of online adaptation with PoD strategies may be of interest to optimize the clinical workflow and limit treatment times.

There are some limitations to our study. The PoD selection criterion was only based on geometrical coverage. It may be of interest to consider dosimetric criteria, which is challenging since it would require computing the dose distribution on the CBCT images. Another limitation is related to the limited number of patients in this study. The proposed workflow will have to be evaluated on a larger cohort. Especially, the evaluation of the segmentation network will have to be deepened, potentially resulting in the need to improve the robustness of the model by training it with new data. Finally, the PoD ART strategy based on multiple planning CTs has shown some limitations, and some more complex strategies have been proposed to improve the libraries. For example, an "evolutive library" strategy has been proposed, enriching the library by including some CBCT anatomies into the library when the daily clinical target volume (CTV) shape differed from the ones in the library (Rigaud et al 2018). Including modeled anatomies resulting from a population analysis has also been proposed [START_REF] Rigaud | Statistical Shape Model to Generate a Planning Library for Cervical Adaptive Radiotherapy[END_REF]. All

Ethical Statement

This research was conducted in accordance with the principles embodied in the Declaration of Helsinki and in accordance with local statutory requirements. It has been approved by the regional research ethics committee (Comité de Protection des Personnes, Ouest V, Rennes, AR-COL 2015-02-45-01). All participants gave written informed consent to participate in the study.