Alekh Paranjapye

Manon Ruffin

Ann Harris
email: ann.harris@case.edu

Harriet Corvol
email: harriet.corvol@aphp.fr

Genetic variation in CFTR and modifier loci may modulate Cystic Fibrosis disease severity

Keywords: cystic fibrosis, genetics, genomics, CFTR, modifier genes, cis-regulatory elements

In patients with cystic fibrosis (CF), genetic variants within and outside the CFTR locus contribute to the variability of the disease severity. CFTR transcription is tightly regulated by cis-regulatory elements (CREs) that control the three-dimensional structure of the locus, chromatin accessibility and transcription factor recruitment. Variants within these CREs may contribute to the pathophysiology and to the phenotypic heterogeneity by altering CFTR transcript abundance. In addition to the CREs, variants outside the CFTR locus, namely "modifiers genes", may also be associated with the clinical variability. This review addresses variants at the CFTR locus itself and CFTR CREs, together with the outcomes of the latest modifier gene studies with respect to the different CF phenotypes.

Background

Cystic fibrosis (CF) is the most common severe autosomal recessive genetic disease in Caucasians caused by variants in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel expressed in epithelial cells throughout the body [START_REF] Riordan | Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA[END_REF]. To date, over 2,000 variants in the CFTR gene have been identified world-wide. The international CF genetics research community has contributed these data to the CFTR2 database (http://www.cftr2.org), which provides information on different CFTR variants and their disease liability by a phenotype-driven approach [START_REF] Sosnay | Defining the disease liability of variants in the cystic fibrosis transmembrane conductance regulator gene[END_REF]. Available data include genomic and phenotypic analyses, together with molecular studies (CFTR RNA and protein expression) and sweat chloride measurements. Compiling information from more than 88,000 patients with CF, together with in vitro studies, 412 variants were annotated and 346 assigned as "CF causing", 37 as "of varying consequences", 21 as "non-CF causing", and 8 "of unknown significance" [START_REF] Sosnay | Defining the disease liability of variants in the cystic fibrosis transmembrane conductance regulator gene[END_REF].

Missense variants are the most prevalent (40%), compared to frameshift (16%), splicing (12%) and non-sense variants (8%) [START_REF] Castellani | Consensus on the use and interpretation of cystic fibrosis mutation analysis in clinical practice[END_REF][START_REF] Corvol | Translating the genetics of cystic fibrosis to personalized medicine[END_REF]. The most common variant (70% of patients) is a three base deletion (c.1521_1523delCTT) that removes a phenylalanine residue at position 508 (F508del). Annotating these CFTR variants will benefit both clinical diagnosis and personalized therapies [START_REF] Mccague | Correlating CFTR Function with Clinical Features to Inform Precision Treatment of Cystic Fibrosis[END_REF].

CF affects many organs, including the pancreas, the liver, the intestine, and most critically the lungs. Although CF is recognized as a single gene disorder, disease severity varies between mutation types and between patients with the same CFTR variant [START_REF] Cutting | Cystic fibrosis genetics: from molecular understanding to clinical application[END_REF][START_REF] Guillot | Lung disease modifier genes in cystic fibrosis[END_REF][START_REF] Mckone | Effect of genotype on phenotype and mortality in cystic fibrosis: a retrospective cohort study[END_REF][START_REF] O'neal | Cystic Fibrosis Disease Modifiers: Complex Genetics Defines the Phenotypic Diversity in a Monogenic Disease[END_REF]. This variability complicates treatment options and the efficacy of therapeutics. The only strong CFTR genotype-phenotype association is with pancreatic exocrine function, which is either deficient (PI for pancreatic insufficiency), or normal (PS for pancreatic sufficiency). Generally, patients carrying two CF-causing variants have a classical form of CF associated with PI, whereas others have a milder form of disease associated with PS (4). Besides environmental factors, additional genetic variants, both within and outside the CFTR locus, may also contribute to this phenotypic variability [START_REF] Cutting | Cystic fibrosis genetics: from molecular understanding to clinical application[END_REF][START_REF] Guillot | Lung disease modifier genes in cystic fibrosis[END_REF][START_REF] Mckone | Effect of genotype on phenotype and mortality in cystic fibrosis: a retrospective cohort study[END_REF][START_REF] Kerschner | Screening for Regulatory Variants in 460 kb Encompassing the CFTR Locus in Cystic Fibrosis Patients[END_REF]. CFTR transcription is tightly regulated by cisregulatory elements (CREs) that control the three-dimensional structure of the locus, chromatin accessibility and transcription factor recruitment [START_REF] Swahn | Cell-Selective Regulation of CFTR Gene Expression: Relevance to Gene Editing Therapeutics[END_REF]. Variants within these CREs may contribute to pathophysiology by altering CFTR transcript abundance, as shown for those in the gene promoter [START_REF] Kerschner | Screening for Regulatory Variants in 460 kb Encompassing the CFTR Locus in Cystic Fibrosis Patients[END_REF]. Utilization of unique CREs in tissue-specific or cell-type-specific contexts may be involved in the different disease manifestations and severities between affected organ systems. In addition to the CREs, variants outside the CFTR locus, in other "modifiers genes", are also associated with the clinical heterogeneity in CF [START_REF] Cutting | Cystic fibrosis genetics: from molecular understanding to clinical application[END_REF][START_REF] Guillot | Lung disease modifier genes in cystic fibrosis[END_REF][START_REF] Mckone | Effect of genotype on phenotype and mortality in cystic fibrosis: a retrospective cohort study[END_REF][START_REF] O'neal | Cystic Fibrosis Disease Modifiers: Complex Genetics Defines the Phenotypic Diversity in a Monogenic Disease[END_REF]. This review, based in part on a symposium session presented at the 16th ECFS Basic Science Conference, Dubrovnik, Croatia, 27 to 30 March, 2019, describes the results of investigating variants at the CFTR locus itself and CFTR CREs, together with the outcomes of the latest modifier gene studies with respect to the different CF phenotypes.

VARIANTS AT THE CFTR LOCUS AND CFTR REGULATION

Variants at the CFTR locus

Extensive research has focused on mutational diversity of the CFTR gene and the relationship with disease severity. Current therapeutic options are limited to treating patients with relatively few (albeit common) known variants in the coding region, whereas variants elsewhere in the locus (Figure 1) also contribute to the disease-associated alleles. Large deletions and genomic rearrangements constitute a significant proportion of rare CFTR alleles with variable disease phenotype [START_REF] Audrezet | Genomic rearrangements in the CFTR gene: extensive allelic heterogeneity and diverse mutational mechanisms[END_REF][START_REF] Ferec | Gross genomic rearrangements involving deletions in the CFTR gene: characterization of six new events from a large cohort of hitherto unidentified cystic fibrosis chromosomes and metaanalysis of the underlying mechanisms[END_REF]. In a screen for rare CF-causing mutations that are currently unidentified, several variants in 460 kb encompassing the CFTR gene mapped to known CFTR CREs [START_REF] Kerschner | Screening for Regulatory Variants in 460 kb Encompassing the CFTR Locus in Cystic Fibrosis Patients[END_REF]. These include variants at the gene promoter, enhancer elements at the locus, and structural elements that may result in dysregulated CFTR transcription. Many mechanisms could account for how variants in CREs disrupt CFTR transcription, though these have yet to be proven experimentally. Single base changes may introduce transcription factor binding sites such as the c.-8G>C CFTR promoter variant, which creates a binding motif for zinc finger protein 300 (ZNF300), a known transcriptional repressor [START_REF] Gou | Identification and functional analysis of a novel human KRAB/C2H2 zinc finger gene ZNF300[END_REF]. Similarly, variants that destroy the binding site for architectural proteins may change locus organization and hence gene expression.

Regulation of CFTR

CFTR regulatory mechanisms differ between tissues and cell types at organ sites affected by CF [START_REF] Thul | A subcellular map of the human proteome[END_REF]. This diversity is evident from observations on long-range chromatin looping interactions and the location of open chromatin sites, which mark active CREs, at the locus [START_REF] Gheldof | Cell-type-specific long-range looping interactions identify distant regulatory elements of the CFTR gene[END_REF]. Cell-type-selective regulatory elements tightly control CFTR expression by recruiting specific transcription factors and establishing the three-dimensional structure of the expressed locus. CFTR lies within a single topologically associated domain (TAD) maintained by the architectural proteins CCCTC-binding factor (CTCF) and cohesin complex [START_REF] Smith | Invariant TAD Boundaries Constrain Cell-Type-Specific Looping Interactions between Promoters and Distal Elements around the CFTR Locus[END_REF].

Although the CFTR TAD is invariant in all cells, different CREs within it are recruited in celltype-selective 3D interactions with the promoter. Open chromatin/CRE sites were shown by several protocols to be different in airway and intestinal epithelial cell populations [START_REF] Ott | A complex intronic enhancer regulates expression of the CFTR gene by direct interaction with the promoter[END_REF][START_REF] Smith | A regulatory element in intron 1 of the cystic fibrosis transmembrane conductance regulator gene[END_REF][START_REF] Zhang | Molecular mechanisms controlling CFTR gene expression in the airway[END_REF].

CREs within intron 1 (185 + 10kb) (K) and intron 11 (1811 + 0.8kb) were found to control CFTR expression in the intestine [START_REF] Kerschner | Transcriptional networks driving enhancer function in the CFTR gene[END_REF]. Among activating transcription factors at these two sites are forkhead box protein A1/A2 (FOXA1/A2), hepatocyte nuclear factor 1 (HNF1) and caudal type homeobox 2 (CDX2) [START_REF] Kerschner | Chromatin remodeling mediated by the FOXA1/A2 transcription factors activates CFTR expression in intestinal epithelial cells[END_REF]. In contrast, CREs at open chromatin peaks located -44kb and -35kb upstream of the CFTR transcriptional start site are airway-selective [START_REF] Zhang | Immune mediators regulate CFTR expression through a bifunctional airway-selective enhancer[END_REF][START_REF] Zhang | Oxidative stress regulates CFTR gene expression in human airway epithelial cells through a distal antioxidant response element[END_REF]. The -35 kb site appears to be involved in immune regulation of CFTR, recruiting interferon regulatory factor 1 (IRF1) and nuclear factor Y (NF-Y). The -44 kb site may respond to oxidative stress in airway epithelial cells. In addition to activating factors CFTR expression in airway epithelial cells is regulated by a number of transcription factors acting as repressors such as Ets homologous factor (EHF) and Krüppel-like factor 5 (KLF5) [START_REF] Mutolo | A transcription factor network represses CFTR gene expression in airway epithelial cells[END_REF]. Determining the interactions of CREs at the locus and their associated transcription factors will enhance understanding of the CFTR regulatory landscape in CF-relevant tissues. A number of approaches are being taken to upregulate CFTR transcription for therapeutic benefit, including activation by CRISPRa [START_REF] Villamizar | Targeted Activation of Cystic Fibrosis Transmembrane Conductance Regulator[END_REF] and steric inhibition of 5' UTR regulatory mechanisms by antisense oligonucleotides [START_REF] Sasaki | Steric Inhibition of 5' UTR Regulatory Elements Results in Upregulation of Human CFTR[END_REF].

Cell-Type Specificity of Expression

The abundance of CFTR transcripts varies between tissue types and in different cells within the same tissue [START_REF] Trezise | Expression of the cystic fibrosis gene in human foetal tissues[END_REF]. In the small intestine and the colon, the majority of CFTR mRNA is found in the mucosal epithelium with a decreasing gradient along the crypt to tip axis [START_REF] Strong | Localization of cystic fibrosis transmembrane conductance regulator mRNA in the human gastrointestinal tract by in situ hybridization[END_REF]. Also, a rare epithelial cell type that expresses high levels of CFTR was observed in several studies.

CFTR expression is very high in most ductal epithelial cells in the pancreas [START_REF] Hyde | The cystic fibrosis transmembrane conductance regulator as a marker of human pancreatic duct development[END_REF][START_REF] Reid | Cystic fibrosis of the pancreas: involvement of MUC6 mucin in obstruction of pancreatic ducts[END_REF][START_REF] Wilschanski | The cystic fibrosis of exocrine pancreas[END_REF]. As lung disease is the primary cause of mortality in CF patients, there is an ongoing effort to characterize CFTR-expressing cells in the airway epithelium. CFTR protein is reported in ciliated cells in the epithelium though levels of expression are apparently lower than in intestinal epithelial cells [START_REF] Kreda | Characterization of wild-type and deltaF508 cystic fibrosis transmembrane regulator in human respiratory epithelia[END_REF]. Recent single cell RNA-seq sequencing data from mouse tracheal epithelial cells and primary human bronchial epithelial cells also identified a rare high CFTRexpressing cell type in the lung, named the pulmonary ionocytes, which may have a similar role to the high CFTR-cells in the intestinal epithelium [START_REF] Montoro | A revised airway epithelial hierarchy includes CFTR-expressing ionocytes[END_REF][START_REF] Plasschaert | A singlecell atlas of the airway epithelium reveals the CFTR-rich pulmonary ionocyte[END_REF]. Further work is underway to resolve the cellularity of CFTR expression in the human lung. Identifying the unique combinations of CREs and activating factors utilized by different CFTR expressing epithelial cells will advance understanding of the cell-specific disease mechanisms.

CYSTIC FIBROSIS MODIFIER GENES

For more than a decade, modifier genes in CF were sought using candidate gene approaches, and early studies were hampered by small sample size and poor study design, yielding mixed results [START_REF] Guillot | Lung disease modifier genes in cystic fibrosis[END_REF]. One of the major limitations of the candidate gene approach is that it does not identify genetic locations other than those suspected to influence the disease. That is, it does not detect modifying genes or pathways beyond those involved in our limited understanding of the disease [START_REF] Cutting | Cystic fibrosis genetics: from molecular understanding to clinical application[END_REF]. Thus, it became important to move forward by studying the whole genome. Although whole genome sequencing was still out of range in large part through costs and lack of resources, it became feasible to pursue genome-wide association studies (GWAS). A GWAS can generate information on hundreds of thousands of single nucleotide polymorphisms (SNPs) chosen to capture linkage disequilibrium (LD) structure without bias imposed by pre-existing models. Moreover, the use of imputations made it possible to explore millions of polymorphisms based on the initial GWAS results. Thus, the GWAS data identified novel genes, regulatory loci, and pathways not previously considered. However, the main disadvantage in testing so many variants is that there are statistical penalties that increase as the number of comparisons rises, and, thus, power is a major limitation [START_REF] Cutting | Modifier genes in Mendelian disorders: the example of cystic fibrosis[END_REF]. To overcome this constraint, collaborations were set up in North-America and France to create an international GWAS consortium. Data from these collaborative studied revealed several novel regions of the genome that are associated with numerous CF phenotypes (Figure 2).

Lung function modifiers

Lung disease remains the major cause of morbidity and mortality in CF. CF sibling and twin studies suggested that more than 50% of the variation in lung function could be explained by modifier genes [START_REF] Vanscoy | Heritability of lung disease severity in cystic fibrosis[END_REF]. To evaluate these modifiers, a quantitative lung phenotype is computed for each patient [START_REF] Taylor | A novel lung disease phenotype adjusted for mortality attrition for cystic fibrosis Genetic modifier studies[END_REF]. In addition, this enables direct comparison of the lung function of CF patients irrespective of age and gender, thus reducing the need to stratify individuals and thereby increasing statistical power and avoiding distortions. By applying genome-wide approaches (linkage and association analyses), North-American teams initially showed in 3,444 PI-CF patients that the intergenic region between the Ets Homologous Factor (EHF) and Apaf-1 interacting protein (APIP) loci (chr11p13) was associated with lung disease severity [START_REF] Wright | Genomewide association and linkage identify modifier loci of lung disease severity in cystic fibrosis at 11p13 and 20q13.2[END_REF]. The study was further extended with additional patients from North America and France.

The meta-analysis of 6,365 CF patients confirmed the previous genome-wide association with the EHF/APIP locus, and identified four new regions that contain genes with high biological relevance for the pathophysiology of CF lung disease: regions on chr3q29 (mucins 4 and 20: MUC4/MUC20), chr5p15.3 (solute carrier family 9 member A3: SLC9A3), chr6p21.3 (class II major histocompatibility complex: HLA Class II) and chrXq22-q23 (angiotensin II receptor type 2/solute carrier family 6 member 14: AGTR2/SLC6A14) (Figure 2) [START_REF] Corvol | Genome-wide association meta-analysis identifies five modifier loci of lung disease severity in cystic fibrosis[END_REF]. Of note the mechanisms connecting high P-value SNPs in intergenic regions with the expression or function of nearby genes, has proven to be a major challenge.

Genes associated with meconium ileus susceptibility

Around 15% of the patients with CF are born with meconium ileus (MI), an intestinal obstruction at birth which affects only PI patients. Similarly to the approach applied for lung disease modifiers, the first GWAS in 3,763 North-American patients showed 2 loci near solute carrier genes, SLC6A14 at chXq23-24 and solute carrier family 26 member 9 (SLC26A9) at ch1q32.1, associated with MI susceptibility [START_REF] Sun | Multiple apical plasma membrane constituents are associated with susceptibility to meconium ileus in individuals with cystic fibrosis[END_REF]. Recently, the meta-analysis of >6,500 CF patients from the International CF Gene Modifier Consortium confirmed these associations and identified two new loci comprising the nongastric form of the H + /K + -ATPase (ATP12A) and serine protease 1 (PRSS1) genes (Figure 2) [START_REF] Gong | Genetic association and transcriptome integration identify contributing genes and tissues at cystic fibrosis modifier loci[END_REF]. Further transcriptomics analysis of nasal epithelial cells from CF patients and pancreatic gene expression data from GTEx, showed that lung disease severity is associated with variations in SLC6A14 gene expression in nasal epithelia, and meconium ileus susceptibility with variations in expression of SLC6A14, SLC26A9 and ATP12A in the pancreas.

Modifiers of cystic fibrosis related diabetes (CFRD)

CF-related diabetes (CFRD) is one of the most important complications of CF. While the age at onset varies considerably, CFRD prevalence increases with age, affecting more than 90% by the 5 th decade in PI-CF patients. Onset of CFRD is recognized to be associated with a more rapid decline in lung function and a reduced survival [START_REF] Moran | Cystic fibrosis-related diabetes: current trends in prevalence, incidence, and mortality[END_REF]. A pilot candidate-gene study found that the gene encoding the transcription factor 7-like 2 (TCF7L2), which is known to be associated with type II diabetes prevalence in the general population, was also associated with the risk of developing CFRD [START_REF] Blackman | A susceptibility gene for type 2 diabetes confers substantial risk for diabetes complicating cystic fibrosis[END_REF]. In particular, the variant rs7903146 increased the risk of diabetes threefold, and decreased the age of onset by 7 years. A further GWAS in 3,059 North-American patients including 644 patients with diabetes confirmed the association of CFRD with TCF7L2, and identified a new association with SLC26A9 (Figure 2) [START_REF] Blackman | Genetic modifiers of cystic fibrosis-related diabetes[END_REF]. Interestingly, as the SLC26A9 gene is also associated with the risk of onset of MI at birth (see above) this suggests a pleiotropic action of this gene [START_REF] Sun | Multiple apical plasma membrane constituents are associated with susceptibility to meconium ileus in individuals with cystic fibrosis[END_REF]. Finally, a recent study provides new data suggestive of an impact of SLC26A9 promoter variants on its expression [START_REF] Lam | Increased expression of anion transporter SLC26A9 delays diabetes onset in cystic fibrosis[END_REF].

Cystic fibrosis liver disease (CFLD) modifiers

Focal biliary cirrhosis is the most clinically relevant form of CF liver disease (CFLD) in PI-CF patients, since extension of the initially focal fibrogenic process may lead to multilobular biliary cirrhosis with subsequent portal hypertension and related complications [START_REF] Debray | Best practice guidance for the diagnosis and management of cystic fibrosis-associated liver disease[END_REF][START_REF] Stonebraker | Features of Severe Liver Disease With Portal Hypertension in Patients With Cystic Fibrosis[END_REF].

Currently, multilobular cirrhosis ranks as the third leading cause of death in patients with CF after respiratory failure and transplantation-related complications [START_REF] Rowland | Outcome in patients with cystic fibrosis liver disease[END_REF]. To date, only candidategene approaches have been performed to look for liver modifiers [START_REF] Debray | Modifier genes in cystic fibrosis-related liver disease[END_REF]. Some genes have been shown to be associated to an increased frequency of liver disease, such as ATP binding cassette subfamily A member 4 (ABCA4), glutathione S-transferase (GSTP1), and a mannosebinding lectin (MBL) haplotype [START_REF] Flamant | Glutathione-S-transferase M1, M3, P1 and T1 polymorphisms and severity of lung disease in children with cystic fibrosis[END_REF][START_REF] Tomaiuolo | An MBL2 haplotype and ABCB4 variants modulate the risk of liver disease in cystic fibrosis patients: a multicentre study[END_REF]; and other to a more severe liver disease such as serpin family A member 1 (SERPINA1, previously known as the α1-antitrypsin gene) (Figure 2) [START_REF] Bartlett | Genetic modifiers of liver disease in cystic fibrosis[END_REF]. The French CF Modifier Gene Study recently reported the incidence of CFLD and severe CFLD in patients with CF and showed that the risk of CFLD increases with age, with a frequency up to 32% by age 25, and is associated with several risk factors, including male gender, CFTR F508del homozygosity, and a history of meconium ileus at birth [START_REF] Boelle | Cystic Fibrosis Liver Disease: Outcomes and Risk Factors in a Large Cohort of French Patients[END_REF]. In a cohort of unprecedented size for such study (n = 3,328), we confirmed that the incidence of CFLD increased more rapidly in patients carrying the SERPINA1 Z allele, with up to 47% of the Z allele carriers developing liver disease before the age of 25 compared to only 30% for noncarrier patients [START_REF] Boelle | SERPINA1 Z allele is associated with cystic fibrosis liver disease[END_REF].

Pharmacogenomics

In recent years, considerable efforts have led to the development of therapies that target the CFTR protein. Significant clinical benefits of ivacaftor, such as gain of lung function and reduced number of pulmonary exacerbations, were initially observed in patients older than 12 years and carrying at least one Gly551Asp (G551D) CFTR variant [START_REF] Ramsey | A CFTR potentiator in patients with cystic fibrosis and the G551D mutation[END_REF]. Subsequently, ivacaftor was approved for other CFTR-gating variants and for patients older than 2 years [START_REF] Davies | Efficacy and safety of ivacaftor in patients aged 6 to 11 years with cystic fibrosis with a G551D mutation[END_REF][START_REF] Avella | SLC26A9 stimulates CFTR expression and function in human bronchial cell lines[END_REF][START_REF] Boeck | Efficacy and safety of ivacaftor in patients with cystic fibrosis and a non-G551D gating mutation[END_REF][START_REF] Davies | Safety, pharmacokinetics, and pharmacodynamics of ivacaftor in patients aged 2-5 years with cystic fibrosis and a CFTR gating mutation (KIWI): an open-label, single-arm study[END_REF]. Since the airway response to ivacaftor was variable between individuals, it was hypothesized that modifier genes could play a role. Though genome-wide analyses are as yet incomplete, candidate studies targeted SLC26A9, a gene that may have a pleiotropic role in CF as discussed above [START_REF] Sun | Multiple apical plasma membrane constituents are associated with susceptibility to meconium ileus in individuals with cystic fibrosis[END_REF][START_REF] Gong | Genetic association and transcriptome integration identify contributing genes and tissues at cystic fibrosis modifier loci[END_REF][START_REF] Blackman | Genetic modifiers of cystic fibrosis-related diabetes[END_REF]. A pilot study in Canadians showed that one variant of this gene was indeed associated with the lung-response to ivacaftor, explaining 28% of the variability (Figure 2) [START_REF] Strug | Cystic fibrosis gene modifier SLC26A9 modulates airway response to CFTR-directed therapeutics[END_REF]. We also found that this gene was associated with the variability of the lung response in French [START_REF] Corvol | SLC26A9 Gene Is Associated With Lung Function Response to Ivacaftor in Patients With Cystic Fibrosis[END_REF]. Specifically, focusing on the variant rs7512462 of the SLC26A9 gene in the patients carrying at least one G551D CFTR mutation, we observed that the CC genotype was associated with a lesser response, with a decrease in FEV1pp of -7.7% compared to the patients carrying the ancestral allele T. Pharmacogenetics testing in CF patients is a growing current and future challenge that may be predictive for efficacy and for adverse drugs effects.

FIGURESFigure 1 .

 1 FIGURES

Figure 2 .

 2 Figure 2. Cystic fibrosis modifier genes.

Acknowledgements

CF work in the Harris lab is funded by the National Institutes of Health, USA and the Cystic Fibrosis Foundation; and in the Corvol lab by the Institut National de la Santé et de la Recherche Médicale, the Assistance Publique-Hôpitaux de Paris, Sorbonne Université, the Association Vaincre La Mucoviscidose, the Chancellerie des Universités (Legs Poix), and the Cystic Fibrosis Foundation.

Summary and Future directions

Recent advances in research into the genetics of CF provide some insights on the phenotypic heterogeneity of the disease, potential targets to predict disease severity and personalize therapeutic care, and new therapeutic avenues for CF patients. Studying larger cohorts and using cutting-edge protocols of functional genomics may allow the identification of new CFTR variants and modifier genes that could better explain the heterogeneity of the disease. In addition, exploring the mechanisms of CFTR transcriptional regulation and evaluating the impact of CFTR regulatory variants and modifier genes in CF is crucial to better understand the disease and improve patient care. Current challenges are to develop clinical diagnostic tools based on identified CFTR variants and modifier genes as well as new specific and personalized therapies to improve survival and quality of life of all the patients with CF.

Conflict of interest

The authors have no conflicts of interest to declare.


Anne Villot

Anne-Cécile Pizzoferrato

Annie Longie

Bernard-Jean Paniel

Arnaud Fauconnier

M.D Pizzoferrato Anne-Cécile

Technical considerations and mid-term follow-up after vaginal hysterocolpectomy with colpocleisis for pelvic organ prolapse

Keywords: hysterocolpectomy, colpocleisis, pelvic organ prolapse, symptoms, satisfaction

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

Introduction

Cumulative incidence of POP surgery affects 11% of women after 70 years old [START_REF] Lousquy | Update on the epidemiology of genital prolapse[END_REF]. Three surgical techniques can be used to repair prolapse: the first is prosthetic surgery by the vaginal or abdominal route (sacrocolpopexy) the main complication of which is mesh erosion [START_REF] Maher | Surgical management of pelvic organ prolapse in women[END_REF]; the second is the use of native tissue (autologous repair) which results in prolapse recurrence of the anterior compartment in up to 55% of women [START_REF] Maher | Surgery for women with anterior compartment prolapse[END_REF]; and the third is obliterative surgery (colpocleisis) which is an interesting option for the more elderly patient who does not wish to preserve sexual function. One of the main difficulties of evaluating the satisfaction rates and effectiveness of obliterative surgery is the heterogeneity of the surgical approaches (e.g., with or without hysterectomy, and with or without perineorraphy).

We describe in this study the technique of vaginal obliteration associating with vaginal hysterocolpectomy (as described by Rouhier) and vaginal vault repair (as described by Campbell) and present mid-term results including pelvic floor symptoms and quality of life, patient satisfaction and surgical complications.

Materials and methods

This was a retrospective single-center study of women who underwent hysterocolpectomy and vaginal vault repair and colpocleisis at the Gynecology Unit of a University Teaching Hospital between June 2006 and June 2016. Since 2006, we have systematically collected patient data including evaluation by a standardized questionnaire. All women who underwent the procedure were examined according to the Pelvic Organ Prolapse Quantification (POP-Q) [START_REF] Bump | The standardization of terminology of female pelvic organ prolapse and pelvic floor dysfunction[END_REF] before the surgery. Women completed validated questionnaires including the French validated short version of the Pelvic Floor Distress Inventory 20 (PDFI-20) which evaluates the severity of POP-related symptoms. The mean for the total PFDI-20 score and the mean score for each sub-questionnaire of the PFID-20 (Urinary Distress Inventory (UDI-6), Pelvic Organ Prolapse Distress Inventory (POPDI-6) and Colorectal-Anal Distress Inventory (CRADI-8)) were reported [START_REF] De Tayrac | The use of quality of life scales in women with pelvic organ prolapse: gadget or real progress?[END_REF]. Most of the patients underwent urodynamics before surgery. This exam was carried out according to good practice [START_REF] Rosier | International Continence Society Good Urodynamic Practices and Terms 2016: Urodynamics, uroflowmetry, cystometry, and pressure-flow study[END_REF]. The severity of stress urinary incontinence (SUI) was assessed by the French validated Urinary Handicap Measurement (MHU) questionnaire [START_REF] Amarenco | Echelle d'évaluation du handicap pelvien : mesure du handicap urinaire (MHU)[END_REF]. It is a graduated scale, completed by the physician, including seven subscales (urgency severity, urge urinary incontinence, day-time and night-time frequency, SUI and hesitancy).

Surgical technique

Following induction of general anesthesia, the patient is positioned in the dorsolithotomy position. A sterile Foley catheter is placed into the urethra.

I-Anterior dissection. The anterior wall is infiltrated with diluted lidocaine injection. An anterior quadrangular colpectomy is performed so the anterior colpocele can be resected from point Aa to the anterior insertion of the cervix, corresponding to the bladder aspect of the vagina [START_REF] Bump | The standardization of terminology of female pelvic organ prolapse and pelvic floor dysfunction[END_REF] (Figure 1a). Halban's fascia is preserved if possible in order to allow subsequent suburethral plication with 2-0 Vicryl sutures. IV-Sub-urethrovesical plication. Suburethral-vesical plication is performed with 3-0 Vicryl in one or two parallel planes to correct the distal part of the anterior colpocele. The important point here is to take the plication beyond the neck of the bladder, under the urethra to avoid de novo urinary incontinence V-Lateral and apical fixations (Figure 1c). The two round ligaments are ligated and fastened well forward under the bladder (Crossen technique) with 2-0 Vicryl [START_REF] Crossen | [END_REF].

Then both uterosacral ligaments are shortened, crossed and fastened to the vaginal wall by two sutures of 2-0 Vicryl [START_REF] Campbell | Vaginal Hysterectomy[END_REF]

Follow-up

The women had post-operative examinations at 1 and 6 months and then yearly. They were all contacted by a letter between June 2016 and October 2016 to evaluate their symptoms and postoperative satisfaction. Both the pre-and postoperative questionnaires included the PFDI-20, POPDI-6, CRADI-8 and UDI-6 scores as well as the Pelvic Floor Impact Questionnaire (PFIQ-7) about the impact of the POP-related symptoms [START_REF] De Tayrac | Development of a linguistically validated French version of two short-form, condition-specific quality of life questionnaires for women with pelvic floor disorders (PFDI-20 and PFIQ-7)[END_REF]. The International Consultation on Incontinence Questionnaire Short Form (ICIQ-SF) questionnaire was added to explore symptoms of incontinence [START_REF] Avery | ICIQ: a brief and robust measure for evaluating the symptoms and impact of urinary incontinence[END_REF]. Patient satisfaction was assessed using the validated Patient Global Impression of Improvement (PGI-I) questionnaire [START_REF] Srikrishna | Validation of the Patient Global Impression of Improvement (PGI-I) for urogenital prolapse[END_REF].

As this type of surgery is usually performed on elderly and frail women, we used the ADL (Activities of Daily Living) and IADL (Instrumental Activities of Daily Living) scales. These scales were developed to assess capabilities relating to self-care and lifestyle, in order to quantify an approximate level of dependency and frailty [START_REF] Katz | Studies of illness in the aged. The index of ADL: a standardized measure of biological and psychosocial function[END_REF][START_REF] Graf | The Lawton Instrumental Activities of Daily Living (IADL) Scale[END_REF]. Postoperative complications were described according to the Clavien-Dindo classification [START_REF] Dindo | Classification of surgical complications: a new proposal with evaluation in a cohort of 6336 patients and results of a survey[END_REF].

Statistical analysis

We used Chi-2 and Fisher exact tests to compare qualitative variables and Student and Mann-Whitney tests for quantitative variables, with a significance level of 5%. To define risk factors for postoperative satisfaction a binary logistic regression model was constructed comparing very satisfied patients (PGI-I = 1) to others.

Analyses were conducted using Stata 13.0 (Stata Corp., College Station, Texas).

Ethics committee approval was obtained from the French "Comité d'éthique de la recherche en obstétrique et gynécologie" (CEROG 2018-GYN-0601).

Results

Population characteristics (Table 1)

During the study period, 37 surgical procedures were performed. Eight patients (21.6%) had a history of pelvic surgery including five (13.5%) hysterectomies, two (5.4%) Burch colposuspensions, and three (8.1%) abdominal surgeries for POP (Kapandji and sacrocolpopexy). Thirty-two patients (91.4%) had a grade III cystocele, 25 (71.4%) a grade III uterine prolapse, and 23 (67.7%) a grade II rectocele. Twenty-five patients (67.6%) had SUI before surgery.

Surgery characteristics (Table 2)

All the procedures were performed by two experienced surgeons in pelvic surgery (AF and a Hospital Service Practitioner, GB). The mean operative mean time was 112 mins. Eleven (29.7%) patients were treated for SUI by suburethral sling at the same time. All these patients complained of SUI and had urodynamics to study the intrinsic urethral sphincteric competence and bladder contractility. One intraoperative complication occurred (2.7%): a rectal wound that required suturing. Three (8.1%) new surgical interventions were required within 15 days for serious adverse events. One patient needed her suburethral sling lowered because of urinary retention 5 days after the procedure. She resumed spontaneous urination on postoperative Day 8. On Day 14 another woman developed a pararectal abscess which required surgical drainage. On Day 15 one woman required unilateral section of a tension-free vaginal tape (TVT) for acute urinary retention. Two days after the section, urinary retention was considered fully resolved : postvoid residual volume was < 100 ml with a peak flow rate greater than 20 ml/s. The mean duration of hospitalization was 5.5 +/-4.2 days.

Functional outcomes and satisfaction

Twenty-nine women returned the survey (78.4% response rate). No difference was noted between responders and non-responders concerning pre-and postoperative characteristics (Table 3). The mean time between the surgery and the last follow-up and the medical survey were respectively: 16.9 months +/-22.09 [range 1.03-105.53] and 44.09 months +/-30.15 [range 3.5-120.8]. All validated questionnaires concerning symptoms and quality of life (POPDI-20, UDI-6, CRADI-8, POP-DI 6) decreased significantly after the surgery (Table 4).

The mean PGI-I was 1.55 +/-0.8 [START_REF] Lousquy | Update on the epidemiology of genital prolapse[END_REF][START_REF] Maher | Surgical management of pelvic organ prolapse in women[END_REF][START_REF] Maher | Surgery for women with anterior compartment prolapse[END_REF][START_REF] Bump | The standardization of terminology of female pelvic organ prolapse and pelvic floor dysfunction[END_REF]. The mean postoperative ICIQ-SF score was 2.15 +/-

4.4 [0-17].
The mean PGI-I score was 1.55 +/-0.8 corresponding to patients "much better" or "better": PGI-I = 1 ("much better") for 58.6% (17/29) of responders ; PGI-I = 2 ("better") for 31.0% (9/29) of responders. The only criterion associated with high satisfaction (PGI-I = 1) was the grade of prolapse [3.7 (0.1) versus 3.1 (0.2) p=0.01] (Table 5).

Urinary incontinence.

Among the 37 women, 25 (67.6%) presented UI before the surgery. Among these, 11 (44%) underwent concomitant UI surgery. Of these 11 women, two second surgeries (18%) were required for urinary retention: one to lower the sling, and the other to section one arm of the sling. Two women (18%) needed prolonged intermittent urinary catheters for dysuria.

After the colpocleisis, 13 women (35%) described UI but only one (7%) underwent a second procedure. This woman's preoperative urodynamic testing showed a major sphincter deficiency. The postoperative severe urinary incontinence (ICIQ score = 17) was due to the suburethral plication. A ProACT ® sphincter device was implanted but later explanted because of urethral erosion. Retropubic TVT was then inserted and the patient described a 60% improvement in incontinence afterwards.

Complications

One case of rectal injury occurred which was sutured without postoperative complication (no fever, no rectovaginal fistula) suites and one pararectal abscess that required surgical drainage on day 14. One patient had a prolonged hospitalization due to hypokalemia that was secondary to postoperative equilibration of diuretic therapy for high blood pressure. None of the patients experienced prolapse recurrence, although one presented a rectal prolapse requiring surgical management.

Discussion

According to this single-center retrospective study in 37 women, we confirmed that colpocleisis is an effective procedure for the treatment of severe POP in women who do not wish to retain coital function.

In our study, 58.6% (17/29) of responders declared to be much better (PGI-I=1) and 31.0% (9/29) of them declared to be better (PGI-I = 2).

In a prospective cohort study including 40 patients, Hullfish et al. found that 95% of their patients were either "very satisfied" or "satisfied" with their surgical outcome which is in agreement with the 89.6% satisfaction rate (PGI-I ≤ 2) we found [START_REF] Hullfish | Colpocleisis for pelvic organ prolapse: patient goals, quality of life, and satisfaction[END_REF]. In a French cohort of 25 women having undergone the same procedure, 56% (14/25) of the patients reported being very satisfied and 32% (8/25) as being satisfied [START_REF] Deval | Hysterocolpectomy with colpocleisis for massive genital prolapse in women aged over 70 years[END_REF].

All the scores used in this study showed significant improvement (POPDI-6, CRADI -8, UDI-6, PFDI-20) after surgery. In a cohort of women having undergone colpocleisis without hysterectomy the PFDI-20 decreased from 121.3 (IQR 80.3-167.2) to 33.8 (IQR 12.5-72.9) at 6 weeks of follow up (p<0.01) confirming our results [START_REF] Fitzgerald | Colpocleisis and urinary incontinence[END_REF]. In Hullfish's cohort, 35% of the women had concomitant surgery for SUI which was similar to our cohort where 44% of the patients had a midurethral sling implanted for preoperative incontinence (11/25) [START_REF] Hullfish | Colpocleisis for pelvic organ prolapse: patient goals, quality of life, and satisfaction[END_REF].

Eighteen percent (2/11) of the women in our study required sling revision surgery for urinary retention and 18% (2/11) required intermittent urinary catheterization. Similarly, Fitzgerald and Brubaker reported that 14% of patients who underwent colpocleisis together with fascial sling placement required sling revision surgery [START_REF] Fitzgerald | Colpocleisis and urinary incontinence[END_REF]. Oliphant et al. performed a decision analysis based on staged versus concomitant midurethral sling (MUS) surgery to treat women with occult SUI planning to undergo colpocleisis. In their study, fewer than a quarter of the women in the staged group underwent sling placement within 1 year [START_REF] Oliphant | Midurethral Sling for Treatment of Occult Stress Urinary Incontinence at the Time of Colpocleisis: A Decision Analysis[END_REF]. In our study, among the 13 patients (13/37, 35%) who presented SUI before the POP surgery, only one woman (1/13, 7%) required sling placement later. Van der Ploeg et al. showed that only 52% of women with a diagnosis of occult SUI experienced urinary incontinence after the surgery [START_REF] Van Der Ploeg | Vaginal prolapse repair with or without a midurethral sling in women with genital prolapse and occult stress urinary incontinence: a randomized trial[END_REF]. However, of the women with occult SUI undergoing POP-only surgery, 13 % needed additional MUS surgery. In Deval's retrospective study of 30 hysterocolpectomy with colpocleisis procedures, no concomitant SUI surgery was performed, only suburethral plication. In this study symptoms of SUI persisted in four cases (13%) and de novo SUI occurred in two (6.6%) [START_REF] Deval | Hysterocolpectomy with colpocleisis for massive genital prolapse in women aged over 70 years[END_REF].

The risk of a de novo UI is an important element to discuss with the patient as some authors found an association between postoperative UI and women's dissatisfaction. Indeed, Hullfish and al. showed a statistical correlation between the postoperative UDI score and mean personal satisfaction goal attainment [START_REF] Hullfish | Colpocleisis for pelvic organ prolapse: patient goals, quality of life, and satisfaction[END_REF]. Crips et al. used a different satisfaction score: the Decision Regret Scale and the Satisfaction with Decision scale. These authors showed that 12 of their 87 subjects (13.8%) described regret or dissatisfaction with eight regrets due to de novo urinary complaints [START_REF] Crisp | Body image, regret, and satisfaction following colpocleisis[END_REF]. We found that the presence of postoperative urinary incontinence tended to limit patient satisfaction, although the study lacked the strength to demonstrate this. In the group of "very satisfied patients" (PGI-I = 1) the proportion of postoperative ICIQ-SF score >1 was 13.3% versus 50% in the PGI-I > 1 group (p=0.06).

None of the preoperative scores of symptoms and quality of life influenced patient satisfaction (Table 5).

As described in the surgical technique, the creation of a deep ligamentous floor by the Campbell-Crossen technique is essential. It is very important to pull the plication beyond the neck of the bladder, under the urethra to avoid de novo incontinence. The suspension of the bladder neck by this natural sling, and the final insertion of the bladder in the abdominal enclosure has the double effect of contributing greatly to the treatment and prevention of SUI.

The main complications in our cohort were postoperative dysuria and rectal injury. For the patient with rectal injury sutured at the time of surgery and for the patient requiring postoperative abscess drainage, the PGI-I index was 1. In recent French recommendations, Deffieux et al. estimated the rate of dysuria and the need for intermittent urinary catheterization between 0 and 10% [START_REF] Deffieux | Colpocleisis: guidelines for clinical practice[END_REF]. Von Pechmann et al. observed that concomitant hysterectomy was associated with more blood loss and blood transfusion (35% versus 12%, p=0.02) without increasing the rate of other surgery complications [START_REF] Pechmann | Total colpocleisis with high levator plication for the treatment of advanced pelvic organ prolapse[END_REF]. In the cohort conducted by Katsara et al. in 44 women undergoing colpocleisis, five (11.4%) had major complications (four bladder injuries and one bowel perforation) which were sutured during surgery [START_REF] Katsara | Long-term quality of life, satisfaction, pelvic floor symptoms and regret after colpocleisis[END_REF].

Strengths and limitations:

One of the strengths of our study was the use of validated instruments designed to assess the impact of surgery. Furthermore, the follow-up period was the second longest period (44 months), after the cohort by Hanson et al in 1969, for this type of elderly population with a responders' rate of 78.4% (29/37) [START_REF] Hanson | The Neugebauer-Le Fort operation. A review of 288 colpocleises[END_REF].

Nevertheless, the retrospective nature and the low number of included patients raises some limitations.

Conclusions

Hysterocolpectomy with colpocleisis is an effective treatment for POP in a population of elderly women. Better assessment of patient expectations before surgery could improve the satisfaction score. The severity of prolapse appears to be the main satisfaction criterion. The occurrence of urinary complications (incontinence or dysuria) should be discussed with the patients preoperatively as well as concomitant or staged treatment options.

Legend of Figures

  The dissection is pushed caudally by cutting the edge of the vagina medially to free the suburethral part of the vagina. The vesico-uterine septum is identified and opened and the bladder pushed back with compresses. The bladder is then mobilized by disinserting the bladder pillars. An anterior valve is placed in this space. II-Posterior dissection. The posterior colpocele is infiltrated and a rectangular colpectomy performed of the posterior vaginal wall to allow removal of the upper, Douglas portion of the posterior vaginal wall (Figure 1b). The rectum is carefully pushed down, and the Douglas pouch opened. A posterior valve and a wick is placed in this space. III-Hysterectomy. The uterosacral ligaments on each side are then sectioned individually and retained by wire to preserve them for the apical fixation of the vaginal vault. The cervicovaginal pedicles and then the uterine pedicles are sectioned and coagulated on each side with a thermofusion coagulation instrument. The round ligaments are individualized on each side, sectioned and retained on wire. The utero-ovarian ligaments and Fallopian tubes are sectioned and coagulated. The peritoneum is closed by two continuous sutures of 2-0 Vicryl and extraperitonization of the uterosacral and round ligaments is performed to leave them free for the following steps.

  . The vagina is closed along the sagittal plane by continuous suture with 2-0 Vicryl. VI-Posterior colpoperineorraphy with rectovaginal fascial plication. The tissues are infiltrated and a horizontal perineal incision performed. The anal cap and tendinous center of the perineum are then dissected. The rectovaginal septum is cleaved and the anus identified before performing a quadrangular resection of the caudal part of the posterior vaginal wall. Plication of the prerecti fascia is performed with 3-0 Vicryl. The vaginal angle is reconstituted by myorraphy with one or two sutures of 0 Vicryl and superficial perineorraphy with one to three 2-0 Vicryl sutures, and the natural obliquity of the vagina thus restored. The Foley catheter is removed on postoperative day 1.

Figure 1 .

 1 Figure 1. Operative technic

Figure 1 .

 1 Figure 1. Operative technic a. Anterior quadrangular colpectomy of the anterior colpocele between Aa and anterior vaginal fornix b. Rectangular colpectomy of the upper part of the posterior vaginal wall c. Anterior and apical fixation by suburethral vesical plication, Crossen-Campbell technique (round ligaments plications are not visible because they were performed before the uterosacral ligaments were plicated)

Table 1 .

 1 Characteristics of our population

Table 2 .

 2 Surgical characteristics

Table 3 .

 3 Comparison of pre-and postoperative characteristics between responders and nonresponders

Table 4 .

 4 Results of symptom and quality of life questionnaires before and after the surgery

Table 5 .

 5 Satisfaction criteria based on patient characteristics (bivariate analysis)

Table 1 .

 1 Characteristics of our population

	Total (n= 37)
	Mean (±SD) / n (%)

Table 2 .

 2 Surgical characteristics

	Total (n=37)
	Mean (±SD) / n (%)

Table 3 .

 3 Comparison of pre-and postoperative characteristics between responders and non-

	responders
		Non-responders	Responders	p value
		(n= 8)	(n= 29)
	Age at surgery (years, mean, SD)
		81.7 (1.3)	81.1 (1.2)	0.79
	BMI (Kg/m², mean, SD)	23.6 (1.9)	25.7 (0.7)	0.20
	Preoperative prolapse grade
	(POP-Q)
	2	0	2 (7.1)
	3	5 (71.4)	12 (42.9)
	4	2 (28.6)	14 (50.0)	0.50
	History of pelvic surgery
	No	8 (100.0)	21 (72.4)	0.16
	Yes	0	8 (27.6)
	Postoperative events
	No	7 (87.5)	22 (75.9)	0.65
	Yes	1 (12.5)	7 (24.1)
	BMI Body Mass Index

Table 4 .

 4 Results of symptom and quality of life questionnaires before and after the surgery 20, Pelvic Floor Disorder Inventory; POPDI-6, Pelvic Organ Prolapse-Distress Inventory; CRADI-8, Colo-Rectal-Anal Distress Inventory; UDI-6, Urinary Distress Inventory

		Pre-operative (m+/-SD)	Post-operative (m+/-SD)	p
		[min-max]	[min-max]
	POP-DI 6	39.8 +/-17.2 [16.7-66.7]	2.43 +/-6.6 [0-29.2]	<0.0001
	CRADI-8	15.7 +/-14.2 [0-46.9]	7.55 +/-13.6 [0-43.7]	0.047
	UDI-6	35.9+/-19.5 [0-75]	8.42 +/-9.2 [0-31.2]	<0.0001
	PFDI-20	93.3 +/-46.3 [25-188]	18.4 +/-24.0 [0-77.1]	<0.0001
	ICIQ-SF	.	2.15 +/-4.4 [0-17]	.

PFDI

Table 5 .

 5 Satisfaction criteria based on patient characteristics (bivariate analysis) Pelvic Organ Prolapse-Distress Inventory; CRADI-8, Colo-Rectal-Anal Distress Inventory; UDI-6, Urinary Distress Inventory

		Group PGI =1	Group PGI >1	p
		(n=17)	(n=12)
		Mean (±SD) / n (%)	Mean (±SD) / n (%)
	Age	82.3 (2.0)	85.2 (1.6)	0.30
	BMI* (Kg/m 2 )	26.5 (1.0)	23.9 (1.2)	0.11
	Grade of prolapse	3.7 (0.1)	3.1 (0.2)	0.01
	Duration of hospitalisation	5.6 (0.8)	4 (0.3)	0.12
	Hysterectomy
	No	8 (47.1)	3 (25.0)
	Yes	9 (52.9)	9 (75.0)	0.23
	Postoperative urinary incontinence
	No	11 (64.7)	6 (50.0)
	Yes	6 (35.3)	6 (50.0)	0.42
	Postoperative dysuria
	No	15 (88.2)	11 (91.7)
	Yes	2 (11.8)	1 (8.3)	0.76
	IADL**	23.6 (0.2)	24 (0)	0.40
	ADL**	6.9 (0.5)	7.7 (0.2)	0.36
	Sub urethral sling during the surgery
	No	15 (88.2)	9 (75.0)
	Yes	2 (11.8)	3 (25.0)	0.62
	pre-operative EVA	6.8 (0.4)	6.0 (0.7)	0.28
	pre-operative MHU (SUI) †	1.6 (0.4)	0.6 (0.4)	0.09
	Preoperative PFDI-20 (median) †
	< 100	7 (53.8)	3 (37.5)
	≥ 100	6 (46.2)	5 (62.5)	0.60
	Preoperative POPDI-6 (median) †
	< 37.5	6 (46.1)	3 (37.5)
	≥ 37.5	7 (53.9)	5 (62.5)	0.90
	Preoperative UDI-6 (median) †
	< 37.5	6 (46.1)	4 (50.0)
	≥ 37.5	7 (53.9)	4 (50.0)	0.90
	Preoperative CRADI-8 (median) †
	< 14.06	8 (61.5)	3 (37.5)
	≥ 14.06	5 (38.5)	5 (62.5)	0.30

* BMI: Body Mass Index, **IADL: Instrumental Activities of Daily Living / ADL: Activities of Daily Living †MHU, Urinary Handicap Measurement; PFDI 20, Pelvic Floor Disorder Inventory; POPDI-6,

Conflict of Interest:

The authors declare that they have no conflict of interest.


Cécilia Enault

Fabien Aujoulat

Alix Pantel

Nicolas Cellier

Catherine Lechiche

Bernard Mégy

Jean-Philippe Lavigne

Hélène Marchandin
email: helene.marchandin@umontpellier.fr

Nemausus' Sp

Nov

Cécilia Enault

Prof Hélène Marchandin

Surgical site infection after hip replacement due to a novel Peptoniphilus species, provisionally named 'Peptoniphilus nemausus' sp. nov

Keywords: Peptoniphilus, infection, anaerobe, resistance, surgical site infection, prosthetic joint infection

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

Text

Gram-positive anaerobic cocci (GPAC) are important members of the human microbiota, that can also act as opportunistic pathogens in humans. GPAC were shown to be the more frequently isolated anaerobes in microbiological laboratories (24-31% of the total number of isolated anaerobes) [START_REF] Murdoch | Gram-positive anaerobic cocci[END_REF][START_REF] Murphy | Gram-positive anaerobic cocci--commensals and opportunistic pathogens[END_REF]. While Finegoldia magna and Parvimonas micra represent about half of the isolated GPAC [START_REF] Jean-Pierre | Les cocci à Gram positif anaérobies, identification et sensibilité aux antibiotiques : à propos de 170 souches isolées au CHRU de Montpellier. 30ème Réunion Interdisciplinaire de Chimiothérapie Anti -Infectieuse[END_REF][START_REF] Veloo | Antimicrobial susceptibility of clinically relevant Gram-positive anaerobic cocci collected over a three-year period in the Netherlands[END_REF] and are the most studied, several less known genera of GPAC like Anaerococcus and Peptoniphilus, are involved in various opportunistic human infections, mainly as part of polymicrobial infections [START_REF] Murphy | Gram-positive anaerobic cocci--commensals and opportunistic pathogens[END_REF].

A 66-year old woman was admitted to the rehabilitation unit of the University Hospital of Nîmes on March 8, 2018, after revision of her total hip prosthesis on March 1.

The patient presented with morbid obesity (body mass index 52 defining grade 3 obesity). She had no hormone replacement treatment since menopause that occurred at the age of 55. Her history includes arterial hypertension, breast cancer in remission after surgery and radiotherapy, and under current hormonal therapy by letrozole, an aromatase inhibitor with bone loss side effects. Initial arthroplasty was performed on February 8, for painful hip and functional impotence revealing extensive osteolysis of the femoral head with previously undiagnosed osteoporosis. At the same time, a Vitamin D deficiency of 10 nmol/L (normal range: 30-100 nmol/L) was found requiring supplementation. Early periprosthetic fracture occurred at weightbearing initiation and hip prosthesis replacement, including removal of the failed implant, lavage and implantation of a femoral component that has a long stem, was performed on March 1. Microbiological investigations showed an early prosthetic joint infection (PJI), as the 3 samples taken during hip prosthesis revision were positive for Proteus mirabilis. PJI was treated by intravenous ofloxacin (600 mg per day) and the introduction of bisphophonates to correct the osteoporosis and the vitamin D deficiency.

In the rehabilitation unit, a surgical site infection was suspected at the beginning of April and confirmed by CT-scan on April 10. Surgical lavage and debridement were performed on April, 12th as part of the management of the infection with Debridement, Antibiotics, Irrigation and Retention (DAIR). Eight surgical samples were obtained (1 periprosthetic fluid, 4 periprosthetic tissue and 3 bone samples). Direct examination showed either rare or rather numerous polymorphonuclear depending on the sample and Gram-positive cocci were visualized after Gram stain of a periprosthetic tissue sample leading to the instauration of an intravenous antimicrobial therapy by cefotaxime plus vancomycin. Samples were analyzed according to national recommendations [START_REF]Infections osseuses et articulaires[END_REF]. Anaerobic cultures were positive after 7 days of incubation of the periprosthetic fluid and the 4 tissue specimens and grew a strictly anaerobic Gram-positive coccus. The three bone samples remained negative. Identification by MALDI-TOF mass spectrometry (Vitek® MS, bioMérieux, Marcy-l'Etoile, France) was unsuccessful.

Antimicrobial susceptibility testing was performed using Etest strips (bioMérieux) according to the recommendations of the Antibiogram committee of the French Society for Microbiology for anaerobes [START_REF]Bactéries anaérobies à Gram positif[END_REF]. The isolate was susceptible to all antibiotics tested (MICs of imipenem and rifampin < 0.02 mg/L, MICs of amoxicillin and coamoxiclav. < 0.016 mg/L, MIC of metronidazole 0.016 mg/L, MIC of linezolid 0.125 mg/L and MIC of clindamycin 0.75 mg/L) except ofloxacin (MIC > 32 mg/L). The multidisciplinary team for the management of PJI of our hospital decided an antimicrobial treatment switch to clindamycin (2400 mg per day) and rifampin (1200 mg per day) for 3 months, on April 23th. A favourable outcome was noted after the end of the treatment and a one-year period of clinical follow-up after a novel total hip prosthesis has been implanted in July 2018.

For the identification of the GPAC isolated in pure culture from a deep-tissue infection, we tested the isolate with another commercially available MALDI-TOF MS system, (Maldi Biotyper Microflex®, Bruker Daltonics, Bremen, Germany), as differences in identification performances between MALDI-TOF systems have been previously reported for identification of anaerobes [START_REF] Veloo | Comparison of two matrix-assisted laser desorption ionisation-time of flight mass spectrometry methods for the identification of clinically relevant anaerobic bacteria[END_REF]; however, no identification was obtained for the clinical isolate using this alternative MALDI-TOF MS system. We also performed 16S rRNA gene sequencing as previously described [START_REF] Carlier | Anaeroglobus geminatus gen. nov., sp. nov., a novel member of the family Veillonellaceae[END_REF]. Sequence analysis (1388 nt) showed the isolate, belonged to the genus Peptoniphilus, but to an as yet unknown species. Indeed, a similarity table constructed using utilities implemented in Biological sequence alignment editor (BioEdit) software (http://www.mbio.ncsu.edu/bioedit/bioedit.html) revealed that the type strains of Peptoniphilus coxii (97.9% of 16S rRNA gene sequence identity) and Peptoniphilus ivorii (94.6%), as well as the type strains of the two non validated species 'Peptoniphilus urinimassiliensis' (96.6%) and 'Peptoniphilus pacaensis' (96.1%) were the most closely related species of the clinical isolate [START_REF] Citron | Peptoniphilus coxii sp. nov. and Peptoniphilus tyrrelliae sp. nov. isolated from human clinical infections[END_REF][START_REF] Diop | Description of three new Peptoniphilus species cultured in the vaginal fluid of a woman diagnosed with bacterial vaginosis: Peptoniphilus pacaensis sp. nov., Peptoniphilus raoultii sp. nov., and Peptoniphilus vaginalis sp[END_REF][START_REF] Brahimi | Peptoniphilus urinimassiliensis' sp. nov., a new bacterial species isolated from a human urine sample after de novo kidney transplantation[END_REF][START_REF] Song | Peptoniphilus gorbachii sp. nov., Peptoniphilus olsenii sp. nov., and Anaerococcus murdochii sp. nov. isolated from clinical specimens of human origin[END_REF]. However, the highest 16S rRNA gene sequence identity observed between the clinical isolate (strain 1804121828, GenBank accession number: MK945758) and the type strain of Peptoniphilus coxii was below the threshold for species identification, i.e., less than 98.7% of 16S rRNA gene identity [START_REF] Stackebrandt | Taxonomic parameters revisited: tarnished gold standards[END_REF], suggesting the clinical isolate to belong to a novel species in the genus Peptoniphilus [START_REF] Dereeper | Phylogeny.fr: robust phylogenetic analysis for the nonspecialist[END_REF]. The 16S rRNA gene sequence of the clinical isolate was also compared with those of the type strains of species of the genus Peptoniphilus through phylogenetic analysis. Evolutionary distances were analysed using the neighbour-joining (NJ) method (Kimura two-parameter substitution model) using phylogenetic analyses available at http://www.phylogeny.fr [START_REF] Dereeper | Phylogeny.fr: robust phylogenetic analysis for the nonspecialist[END_REF]. Phylogenetic analysis supported the inclusion of the isolate in a new species based on a clearly individualized branching within the genus Peptoniphilus and the cluster P. coxii / 'P.

pacaensis' / 'P.urinimassiliensis' / P. ivorii (Figure 1). A formal characterization of the novel species is ongoing and the name 'Peptoniphilus nemausus' sp. nov. is proposed for this novel species pertaining to the Nîmes town in the south of France, where the strain supporting the description of the species was isolated.

The genus Peptoniphilus was individualized in 2001 to accommodate strictly anaerobic Gram-positive cocci previously classified in the genus Peptostreptococcus that were butyrate-producers, non-saccharolytic and that used peptone and amino acids as major energy sources [START_REF] Ezaki | Proposal of the genera Anaerococcus gen. nov., Peptoniphilus gen. nov. and Gallicola gen. nov. for members of the genus Peptostreptococcus[END_REF]. Since then, a growing number of species has been described and currently 17 species are validly published (http://www.bacterio.net/peptoniphilus.html) [START_REF] Parte | LPSN -List of Prokaryotic names with Standing in Nomenclature (bacterio.net), 20 years on[END_REF] and 9 others have been proposed without current valid publication (May 20, 2019) (Figure 1).

Among the genus, species can be distinguished by phenotypic assays (allowing the determination of a metabolic profile) that are not routinely performed in clinical microbiology laboratories, particularly since the development of MALDI-TOF MS; therefore, species identification is currently based on mass spectrometry and, when unsuccessful, on molecular tools [START_REF] Nagy | ESCMID Study Group of Anaerobic Infections. How to isolate, identify and determine antimicrobial susceptibility of anaerobic bacteria in routine laboratories[END_REF]. MALDI-TOF MS is a powerful and rapid identification tool; however, databases are currently incomplete and optimization of current databases for anaerobes is ongoing [START_REF] Veloo | Identification of Grampositive anaerobic cocci by MALDI-TOF mass spectrometry[END_REF][START_REF] Veloo | The optimization and validation of the Biotyper MALDI-TOF MS database for the identification of Gram-positive anaerobic cocci[END_REF][START_REF] Veloo | Validation of MALDI-TOF MS Biotyper database optimized for anaerobic bacteria: The ENRIA project[END_REF]. MS was unable to identify our clinical isolate and 16S rRNA gene sequence analysis was required revealing the clinical isolate to belong to an unknown species and showing a still underestimated diversity in this genus. A formal description of this new species based on a polyphasic taxonomy approach has been undertaken. Members of the different human microbiota, Peptoniphilus spp. have been reported in a large variety of human endogeneous polymicrobial infections due to the pathogenesis process of such infections, i.e., polymicrobial infections involving members of the contiguous microbiota through contamination of initially sterile anatomical sites. Clinical relevance of Peptoniphilus spp. has been mainly demonstrated after isolation from skin and soft tissues, chronic wounds (pressure ulcer, diabetic foot wounds), osteoarticular samples, genitourinary (vaginal infections) and respiratory tract (pleural empyema, chronic rhinosinusitis) [START_REF] Murphy | Gram-positive anaerobic cocci--commensals and opportunistic pathogens[END_REF].

Anaerobic infections remain rare in patients with prosthetic joints and mostly involved species originating from the cutaneous microbiota like Cutibacterium (formerly Propionibacterium) acnes and Finegoldia magna [START_REF] Walter | Bone and joint infections due to anaerobic bacteria: an analysis of 61 cases and review of the literature[END_REF][START_REF] Shah | Anaerobic prosthetic joint infection[END_REF][START_REF] Lebowitz | Clinical features of anaerobic orthopaedic infections[END_REF][START_REF] Rieber | Periprosthetic joint infection caused by anaerobes. Retrospective analysis reveals no need for prolonged cultivation time if sensitive supplemented growth media are used[END_REF]. Despite Peptoniphilus spp. have been previously identified during osteoarticular [START_REF] Walter | Bone and joint infections due to anaerobic bacteria: an analysis of 61 cases and review of the literature[END_REF][START_REF] Scola | Burden of emerging anaerobes in the MALDI-TOF and 16S rRNA gene sequencing era[END_REF][START_REF] Verma | Peptoniphilus asaccharolyticus-associated septic arthritis and osteomyelitis in a woman with osteoarthritis and diabetes mellitus[END_REF] and soft tissue infections [START_REF] Brazier | ESCMID Study Group on Antimicrobial Resistance in Anaerobic Bacteria. European surveillance study on antimicrobial susceptibility of Gram-positive anaerobic cocci[END_REF][START_REF] Thabit | Antibiotic penetration into bone and joints: An updated review[END_REF],

we were unable to find a case similar to that described herein, i.e., surgical site infection following PJI, among the 122 publications retrieved in the PubMed database using the "Peptoniphilus" search term (July 8, 2019). In the present case, despite anaerobes were not reported during initial infection and the portal of entry or origin of the Peptoniphilus isolate remained unidentified, it is likely that it has been selected by ofloxacin therapy towards initial P. mirabilis infection, as the isolate displayed high level resistance to ofloxacin.

Fluoroquinolones are one of the therapeutic options in the management of osteoarticular infections in case of susceptibility of the causative microorganism, as they displayed good penetration profiles into bone tissues and synovial fluid [START_REF] Jeverica | Evaluation of the routine antimicrobial susceptibility testing results of clinically significant anaerobic bacteria in a Slovenian tertiary-care hospital in 2015[END_REF]. If the antimicrobial susceptibility patterns of the main encountered anaerobic pathogens in bone and joint infections, C. acnes and F. magna, is documented, antimicrobial resistance patterns of the overall GPAC have received less interest being for long considered as microorganisms susceptible to antibiotics with anti-anaerobic activity. However, studies including or focused on GPAC revealed high rate of resistance towards some antibiotics used in the management of osteoarticular anaerobic infections, 25% of GPAC displayed resistance to clindamycin in most recent studies for example [START_REF] Shah | Anaerobic prosthetic joint infection[END_REF][START_REF] Jeverica | Evaluation of the routine antimicrobial susceptibility testing results of clinically significant anaerobic bacteria in a Slovenian tertiary-care hospital in 2015[END_REF]; reported some multidrug resistant clinical isolates [START_REF] Shilnikova | Evaluation of antibiotic susceptibility of Gram-positive anaerobic cocci isolated from cancer patients of the N.N.Blokhin Russian cancer research center[END_REF] while revealing heterogeneity in antibiotic susceptibility patterns between species [START_REF] Veloo | Antimicrobial susceptibility of clinically relevant Gram-positive anaerobic cocci collected over a three-year period in the Netherlands[END_REF][START_REF] Jeverica | Evaluation of the routine antimicrobial susceptibility testing results of clinically significant anaerobic bacteria in a Slovenian tertiary-care hospital in 2015[END_REF][START_REF] Brazier | Antibiotic susceptibilities of Gram-positive anaerobic cocci: results of a sentinel study in England and Wales[END_REF][START_REF] Veloo | Antibiotic susceptibility profiles of anaerobic pathogens in The Netherlands[END_REF]. Regarding ofloxacin, a large study conducted in France, i.e., 170 GPAC isolated from diverse anatomical sites including 16.5% of Peptoniphilus spp. all identified by 16S rRNA gene sequencing, showed a global rate of resistance of 63% but revealed that all Peptoniphilus -but also all Anaerococcus -clinical isolates studied displayed resistance to ofloxacin [START_REF] Jean-Pierre | Les cocci à Gram positif anaérobies, identification et sensibilité aux antibiotiques : à propos de 170 souches isolées au CHRU de Montpellier. 30ème Réunion Interdisciplinaire de Chimiothérapie Anti -Infectieuse[END_REF]. In case of deep monomicrobial infection of a normally sterile body site as in the present case, antimicrobial susceptibility testing is recommended to guide the treatment [START_REF] Gajdács | Identification and antimicrobial susceptibility testing of anaerobic bacteria: Rubik's cube of clinical microbiology?[END_REF]; however, in case of polymicrobial infection involving both aerobes and anaerobes or several anaerobes, antimicrobial susceptibility testing is usually less systematically performed on all isolated anaerobes and one should then consider the presence of potentially resistant microorganisms, not only members of the Bacteroides fragilis group but also some GPAC, among the cultivable microbiota in the choice of the best therapeutic option.

Legend to figure

Fig. 1 .'

 1 Fig. 1. Neighbor-joining phylogenetic tree showing the relationship between the 16S rRNA gene sequences of Peptoniphilus strain 1804121828 T (type strain of the proposed novel

Acknowledgments

The authors thank Nathalie Jeanney for excellent technical assistance.

Authors belong to the Centre Regional des Infections Ostéo-Articulaires complexes (CRIOAc) Sud Méditerranée (AP, NC, CL, BM, and JPL), to the Clinique du Pied Diabétique Gard Occitanie (NC, CL, BM, and JPL) and/or to the FHU INCh (Fédération Hospitalo-Universitaire Infections Chroniques, Aviesan) (all authors). We thank the Nîmes University Hospital for its structural, human and financial support through the award obtained by our team during the internal call for tenders « Thématiques phares ».