PhD Pierre-Yves Boëlle

MD, PhD Dominique Debray

PhD Loic Guillot

MD Harriet Corvol
email: harriet.corvol@aphp.fr

SERPINA1 Z allele is associated with cystic fibrosis liver disease

Keywords: SERPINA1 Z, cystic fibrosis, liver disease, cirrhosis, portal hypertension

Purpose:

The SERPINA1 Z allele is associated with cystic fibrosis (CF)-related liver disease (CFLD), a common manifestation in patients with CF. We estimated CFLD incidence based on the SERPINA1 genotype in 3,328 CF patients with CFLD-phenotype information.

Methods:

The associations of SERPINA1 Z (rs28929474) and S (rs17580) alleles with age at CFLD onset and the development of CFLD-related complications (severe liver disease with cirrhosis, portal hypertension, esophageal varices) were analyzed.

Results: Overall, 3% of patients carried the SERPINA1 Z allele and 13% the S allele. The cumulative incidence of CFLD increased more rapidly in patients carrying the Z allele (HR = 1.6; 95% CI = 1.1-2.4, P = 0.019), reaching 47% by age 25 compared to 30% in non-carriers. Increased risk was similar for patients with severe CFLD (HR = 1.5, 95% CI = 0.7-3.2, P = 0.31) but failed to reach significance due to a limited sample size of Z-allele carriers. No significant effect was found for the S allele.

Conclusion:

CF patients carrying the SERPINA1 Z allele had an increased risk of developing CFLD and related complications compared to non-carriers. Routine SERPINA1 Z genotyping upon CF diagnosis is warranted for identifying patients worthy of closer liver disease monitoring.

INTRODUCTION

Cystic fibrosis (CF) is the most common, severe, autosomal recessive genetic disease in Caucasians.

Pathogenic variants in the gene encoding the chloride channel CF transmembrane conductance regulator (CFTR) 1 induce multi-organ dysfunction, including the lungs, pancreas, intestine, and liver.

A broad spectrum of hepatobiliary abnormalities are covered under the term CF-related liver disease (CFLD). Focal biliary cirrhosis is the most clinically important form of CFLD, since extension of the initially focal fibrogenic process may cause multilobular biliary cirrhosis followed by portal hypertension and associated complications. [START_REF] Debray | Best practice guidance for the diagnosis and management of cystic fibrosis-associated liver disease[END_REF]3 Currently, multilobular cirrhosis ranks as the third leading cause of death in patients with CF after respiratory failure and transplantation-related complications. 4 Although CF is recognized as a monogenic disease, considerable interindividual variability in phenotype exists among patients with the same CFTR pathogenic variants. [5][6][7] In addition to environmental factors, genetic modifiers have also been shown to contribute to this variability. 5,6 We have assembled a large cohort of patients as part of the French CF Modifier Gene Study and recently reported the incidence of CFLD and severe CFLD in these patients. [START_REF] Boelle | Cystic Fibrosis Liver Disease: Outcomes and Risk Factors in a Large Cohort of French Patients[END_REF] We found 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 sex, CFTR F508del homozygosity, and a history of meconium ileus at birth. The SERPINA1 gene has been implicated in the development and progression of CFLD. 9 SERPINA1 encodes the alpha-1 antitrypsin (AAT) protein synthesized by the liver. Several pathogenic variants of SERPINA1 have been determined to cause an AAT deficiency that predisposes an individual to liver disease and early-onset emphysema. The most common variants involve the Z and S alleles, each caused by single nucleotide polymorphisms. [START_REF] Bartels | Real time PCR detection of the PI*Z and PI*S mutations associated with alpha-1 antitrypsin deficiency[END_REF] The Z variant is the allele overwhelmingly associated with liver disease. [START_REF] Zorzetto | SERPINA1 gene variants in individuals from the general population with reduced alpha1-antitrypsin concentrations[END_REF] Indeed, during biogenesis the Z-type AAT protein folds abnormally in the endoplasmic reticulum of hepatocytes and is retained intracellularly instead of being efficiently secreted, resulting in low serum levels of AAT. [START_REF] Stoller | A review of alpha1-antitrypsin deficiency[END_REF] The intracellular accumulation of AAT mutant Z proteins within hepatocytes can lead to liver injury, cirrhosis, or hepatocellular carcinoma. [START_REF] Teckman | Liver disease in alpha-1 antitrypsin deficiency: current understanding and future therapy[END_REF] Individuals heterozygous for AAT that carry one normal protease inhibitor M allele and one pathogenic variant Z allele (PiMZ or MZ) are asymptomatic with regard to liver disease.

Compound heterozygotes for the S and the Z alleles of AAT (PiSZ) may develop liver disease with identical manifestations to those of PiZZ patients, whereas liver disease is absent in PiSS homozygote individuals. [START_REF] Teckman | Liver disease in alpha-1 antitrypsin deficiency: current understanding and future therapy[END_REF] A CF case-control study revealed that carriers of the SERPINA1 Z allele are more common among patients with severe CFLD, with a large odds ratio of 5. [START_REF] Bartlett | Genetic modifiers of liver disease in cystic fibrosis[END_REF] However, the study focused on patients with severe liver disease defined as cirrhosis showing signs of portal hypertension, which prevented the determination of the actual risk associated with the variant for developing CFLD.

Therefore, in the present study, we estimated the cumulative incidence of CFLD based on the SERPINA1 genotype in a cohort of French CF patients of unprecedented size (n = 3,328).

MATERIAL AND METHODS

As previously described, we assembled the French CF Modifier Gene Study cohort that included CF patients treated at French CF centers since 2004 (see also Supplementary Information). [START_REF] Boelle | Cystic Fibrosis Liver Disease: Outcomes and Risk Factors in a Large Cohort of French Patients[END_REF] In brief, 4,798 patients with CF were recruited for the study, corresponding to approximately 80% of all French patients with CF. 14 Among this cohort, 3,328 CF patients with pancreatic insufficiency born after 1985 were available for evaluation of CFLD incidence and risk factors, including patients with severe CFLD. [START_REF] Boelle | Cystic Fibrosis Liver Disease: Outcomes and Risk Factors in a Large Cohort of French Patients[END_REF] CFLD was defined according to the European Best Practice Guidance by Debray et al. [START_REF] Debray | Best practice guidance for the diagnosis and management of cystic fibrosis-associated liver disease[END_REF] Patients with cirrhosis, portal hypertension, and/or esophageal varices were classified as having severe CFLD (see also Supplementary Information). 3,[START_REF] Debray | Cystic Fibrosis-related Liver Disease: Research Challenges and Future Perspectives[END_REF] The study was approved by the French ethical committee (CPP no. 2004/15) and the information collection was approved by the Commission Nationale de L'informatique et des Libertés (no. 04.404). Written informed consent was obtained from each patient and/or guardian.

Genotyping of the SERPINA1 Z (rs28929474) and S (rs17580) alleles was carried out using Kompetitive Allele Specific PCR (KASP) genotyping chemistry (LGC, Teddington, UK). In the dbSNP database (https://www.ncbi.nlm.nih.gov/projects/SNP/), rs28929474 is identified as an A/G variant with G being the ancestral allele, and rs17580 is identified as an A/T variant with A being the ancestral allele.

Descriptive statistics were compiled as the mean ± standard deviation (SD) or percentages as appropriate. All patients were considered to be at risk for CFLD since birth and were censored at the time of the last visit without a CFLD diagnosis before January 2017. For other patients, the age at CFLD diagnosis was determined using the date of the first report in the medical records, allowing for "interval censoring" between birth and the age of the first report when the date of onset was not precisely known. This was the case for 142 of 605 patients with CFLD (24%). Likewise, severe CFLD onset was defined as the first date that cirrhosis, portal hypertension, and/or esophageal varices were reported and uncertainty regarding this date was considered as described above. The date of severe CFLD was interval censored in 19 of 175 patients (11%). We used the log-rank test adapted for interval-censored data for comparisons between the cumulative incidences curves [START_REF] Fay | Rank invariant tests for interval censored data under the grouped continuous model[END_REF] and Cox regression adapted to interval-censored data to determine the association of factors linked to age at CFLD onset. [START_REF] Pan | Extending the Iterative Convex Minorant Algorithm to the Cox Model for Interval-Censored Data[END_REF] Confidence intervals were computed by the bootstrap method. Bonferroni correction was used for multiple comparisons.

RESULTS

Clinical characteristics of the 3,328 CF patients included in this study, along with the distribution of the SERPINA1 Z and S alleles, are shown in Table 1. The minor allele frequencies were similar in our cohort to those reported for Europeans: SERPINA1 Z (A variant): 1.4% vs. 2% respectively, SERPINA1 S (T variant): 6.4% versus 6% respectively. There were no patients homozygous for SERPINA1 Z.

Details on the number of CF patients at risk of developing CFLD and severe CFLD, and the cumulative number of CFLD and severe CFLD events for the entire cohort as well as according to SERPINA1 Z and S genotypes are provided in the Supplementary Information (Table S1, S2 and S3).

Overall, 3% of the CF patients carried the SERPINA1 Z allele and 13% the S allele. The cumulative incidence of CFLD increased more rapidly in patients carrying the SERPINA1 Z allele (HR = 1.6; 95% CI = 1.1-2.4, P = 0.019), reaching 47% by age 25 in the Z allele carriers compared to 30% at age 25 in the others (Table 1 and Figure 1). The increase in risk was similar for patients with severe CFLD (HR = 1.5, 95% CI = 0.7-3.2, P = 0.31), but this did not reach statistical significance. With respect to severe CFLD, there were only seven cases among the Z allele carriers, making the cumulative incidence curve difficult to accurately estimate. Clinical characteristics such as sex, year of birth and of CF diagnosis were not associated with SERPINA1 Z and S alleles (Supplementary Information Table S4).

Adjusting on European origin, CFTR variants and meconium ileus did not change the strength of the association (Supplementary Information Table S5). The effect of carrying one SERPINA1 S allele on CFLD risk was not statistically significant (Table 1 and Figure 1).

DISCUSSION

The French CF Modifier Gene Study provided an unprecedentedly large cohort of 3,328 pancreaticinsufficient patients with CF born after 1985. This gave an opportunity to obtain a more accurate estimate of the incidence of CFLD and severe CFLD with sufficient power to detect associations of clinical relevance. [START_REF] Boelle | Cystic Fibrosis Liver Disease: Outcomes and Risk Factors in a Large Cohort of French Patients[END_REF] We found that the SERPINA1 Z allele was associated with an increased risk of developing CFLD, although the association was weaker than that previously reported. [START_REF] Bartlett | Genetic modifiers of liver disease in cystic fibrosis[END_REF] Nevertheless, 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 non-carrier patients.

The role of SERPINA1 in CFLD was first identified in a two-stage case-control study including CF patients from several countries worldwide. [START_REF] Bartlett | Genetic modifiers of liver disease in cystic fibrosis[END_REF] Both the initial and a replicate studies showed that severe CFLD is associated with the SERPINA1 Z allele (odds ratio of 4.72 and 3.42, respectively). [START_REF] Bartlett | Genetic modifiers of liver disease in cystic fibrosis[END_REF] Analysis of our cohort of patients with CF confirmed this association, but demonstrated a smaller difference in risk than that reported previously. For example, using the cumulative incidence at age 25, the odds ratio for the Z allele was only 2 in our cohort. However, the difference in cumulative risk with age was still clinically relevant since SERPINA1 Z carriers had a 50% greater risk of developing CFLD compared to non-carriers. We did not observe any association of the SERPINA1 S allele with CFLD, which was not surprising since the S allele is recognized to be associated with reduced levels of AAT protein, but not with liver manifestations. [START_REF] Torres-Duran | Alpha-1 antitrypsin deficiency: outstanding questions and future directions[END_REF] Our study had limitations related to its design and the use of medical records as a primary source of information. However, we previously reported that selection bias due to differential mortality was likely to be small. [START_REF] Boelle | Cystic Fibrosis Liver Disease: Outcomes and Risk Factors in a Large Cohort of French Patients[END_REF] A second potential issue was the rarity of the SERPINA1 Z allele in our cohort. This reduced the precision of estimation, despite the large cohort size. Indeed, there were only 90 carriers of the SERPINA1 Z allele (PiMZ) and no homozygous PiZZ patients in the cohort. Furthermore, only seven of these patients experienced severe CFLD, making the cumulative incidence difficult to accurately estimate.

Obtaining a more accurate prediction of the risk of CFLD in CF patients remains an important issue, especially since ursodeoxycholic acid, the treatment commonly prescribed for its prevention, seems to have little to no effect. [START_REF] Boelle | Cystic Fibrosis Liver Disease: Outcomes and Risk Factors in a Large Cohort of French Patients[END_REF][START_REF] Cheng | Ursodeoxycholic acid for cystic fibrosis-related liver disease[END_REF] Given this situation, identifying biomarkers to predict the occurrence of CFLD is fundamental to improving the monitoring of disease progression and for assessing the effects of novel therapies, such as bile-salt analogs, anti-fibrotics, and CFTR correctors and potentiators. SERPINA1 Z genotyping at the time of CF diagnosis may help to single out a population of patients who should be more closely screened for liver disease. Gaining a better understanding of the genetic profiles of patients with CFLD will undoubtedly open new therapeutic avenues and help to develop prospective therapies that focus on high-risk groups.

patients and families who participated in this study and the contributing French CF Centers (see the list of investigators in the Supplementary Information).

Patients and data collection

Since 2001, all patients with cystic fibrosis (CF) in France are evaluated at least once a year by one of 47 French hospital-based CF centers according to the national CF care recommendations (https://www.has-sante.fr/portail/jcms/c_2792719/fr/mucoviscidose). 1 Patients with CF treated in 38 of the participating CF centers from January 2004 to January 2017 were enrolled in the French CF Modifier Gene Study. As of January 1, 2017, 4,798 patients with CF had been included, which corresponded to approximately 80% of all French patients with CF. [START_REF] Debray | Best practice guidance for the diagnosis and management of cystic fibrosis-associated liver disease[END_REF] Longitudinal data were obtained from electronic medical records or abstracted from the patients' paper records; retrospectively before 2004 and prospectively after January 2004. We analyzed patients born after 1985 with pancreatic insufficiency (n = 3,328) since pancreatic sufficient patients are known to have milder disease. Patients born before 1985 were excluded in order to limit selection biases due to an overrepresentation of patients with milder disease contributing to longer survival. 3 Information on liver status was collected at each visit. 4 When no history on CFLD was available in the patient's record, he/she was considered not to be affected. Age at onset of liver disease and severe liver disease were determined as defined below.

CFLD and severe CFLD definitions

CFLD was defined according to the European best practice guidance by Debray et al. 5 when at least two of the following characteristics were present: 1) abnormal physical examination: hepatomegaly and/or splenomegaly; 2) abnormalities of liver function tests defined as an increase of transaminase (alanine aminotransferase and/or aspartate aminotransferase) and/or gammaglutamyl transpeptidase levels above normal upper limits; 3) ultrasonographic (US) evidence of liver involvement (heterogeneous echogenicity, irregular margins, or nodularity), portal hypertension (splenomegaly, increased thickness of the lesser omentum, spontaneous splenorenal anastomosis, large collateral veins, or ascites), or biliary abnormalities (bile duct dilatation). Patients with cirrhosis diagnosed by US, computed tomography (CT), and/or magnetic resonance imaging (MRI), and/or portal hypertension [splenomegaly, hypersplenism (platelets < 150,000 10 9 /L and white blood cells < 3000 10 9 /L), and/or spontaneous portosystemic shunts on US], and/or esophageal varices were classified as having severe CFLD. 6,7

Supplementary tables

Figure 1 .

 1 Figure 1. Cumulative incidence of cystic fibrosis-related liver disease (CFLD) (A and C) and severe

  CFLD and severe CFLD CFLD HR (95%CI), P-value (Adjusted P-value ** ) Severe CFLD HR (95%CI), P-value (Adjusted P-value ** ) SERPINA1 Z (G:A vs G:G) 1.62 (1.08-2.42), P=0.019 (Padj=0.076) 1.48 (0.70-3.14), P=0.31 (Padj=1.0) SERPINA1 S (T:A/T:T vs A/A) 0.87 (0.67-1.12), P=0.27 (Padj=1.0) 0.64 (0.38-1.09), P=0.1 (Padj=0.4) Data are means ± SD or % (numbers) unless otherwise indicated. *Genotyping failed for 41 patients for the Z allele and 48 patients for the S allele. ** Bonferroni adjustment for 4 tests. Abbreviations: CF: cystic fibrosis; CFTR: cystic fibrosis transmembrane conductance regulator, CFLD: cystic fibrosis-related liver disease; HR: hazard ratio; 95% CI: 95% confidence interval.

Abbreviations:

  Abbreviations: CFLD: cystic fibrosis related liver disease

Table 1 :

 1 Patient clinical characteristics, SERPINA1 Z and S alleles distribution and their association with CFLD and severe CFLD.

	Clinical characteristics	Total (n)
	Sex (Male), % (n)	3328	52.0% (1731)
	Current age (years), mean ± SD		15.9 ± 7.7
	Year of birth, % (n)
	1986-1995		35.5% (1183)
	1996-2005		40.1% (1333)
	2006-2017		24.4% (812)
	Age at enrolment, % (n)
	Birth		35.2% (1172)
	1-10 years		35.9% (1195)
	10-18 years		28.9% (961)
	European origin, % (n)		88.9% (2957)
	CF diagnosis <1 year old, % (n)		73.9% (2461)
	CFTR F508del homozygotes, % (n)		49.4% (1645)
	Meconium ileus, % (n)		13.4% (445)
	SERPINA1 Z and S alleles distribution	Total (n)	% (n)
	SERPINA1 Z, % (n)	3287*
	G:G		97.3% (3197)
	G:A		2.7% (90)
	SERPINA1 S, % (n)	3280*
	A:A		87.4% (2868)
	T:A		12.1% (396)
	T:T		0.5% (16)
	Association of SERPINA1 Z and S alleles
	with

Table S1 : Number at risk, cumulative number of events, and cumulative incidence of cystic fibrosis related liver disease (CFLD) and severe CFLD

 S1

			CFLD			Severe CFLD
	Age	At risk	Cumulative	Cumulative	At risk	Cumulative	Cumulative
	(years)	(n)	number of	incidence	(n)	number of	incidence (%)
			events (n)	(%)		events (n)
	0	3287	0	0	3287	0	0
	5	2953	93	3.7	3028	12	0.5
	10	2269	250	9.9	2403	59	2.3
	15	1571	402	16.4	1700	125	5.3
	20	917	513	22.8	1027	151	6.9
	25	391	592	30.5	464	171	9.1

Table S2 : Number at risk, cumulative number of events, and cumulative incidence of CFLD and severe CFLD according to SERPINA1 Z genotypes

 S2

			SERPINA1 Z GG		SERPINA1 Z AG/AA
	Age	At risk	Cumulative	Cumulative	At risk	Cumulative	Cumulative
	(years)	(n)	number of	incidence (%)	(n)	number of	incidence (%)
			events (n)			events (n)
				CFLD
	0	3197	0	0	90	0	0
	5	2875	88	3.6	78	5	8.1
	10	2213	237	9.7	56	13	16.0
	15	1529	386	16.3	42	16	22.4
	20	897	493	22.6	20	20	29.2
	25	380	567	30.0	11	25	46.9
				Severe CFLD
	0	3197	0	0	90	0	0
	5	2946	11	0.5	82	1	1.4
	10	2338	57	2.2	65	2	5.5
	15	1651	120	5.2	49	5	7.2
	20	1002	144	6.8	25	7	11.3
	25	452	164	9.1	12	7	11.3
	Abbreviations: CFLD: cystic fibrosis related liver disease

Table S3 : Number at risk, cumulative number of events, and cumulative incidence of CFLD and severe CFLD according to SERPINA1 S genotypes

 S3

			SERPINA1 S AA		SERPINA1 S AT/TT
	Age	At risk	Cumulative	Cumulative	At risk	Cumulative	Cumulative
	(years)	(n)	number of	incidence (%)	(n)	number of	incidence (%)
			events (n)			events (n)
				CFLD
	0	2868	0	0	412	0	0
	5	2575	76	3.5	373	16	4.6
	10	1971	215	9.9	295	32	8.7
	15	1360	349	16.5	210	48	15.1
	20	787	449	23.2	129	59	19.2
	25	338	519	30.9	53	68	27.0
				Severe CFLD
	0	2868	0	0	412	0	0
	5	2635	11	0.5	387	1	0.3
	10	2084	52	2.4	313	7	2.0
	15	1475	110	5.3	223	13	4.0
	20	887	135	7.2	139	14	4.6
	25	403	154	9.6	60	15	5.3

ACKNOWLEDGEMENTS

The authors would like to thank the clinical research assistants involved in the data collection, namely, Pauline Touche, Tania Kombi, and Julie Mésinèle. We would like to especially thank the

Financial Support

This work was supported by the Institut National de la Santé et de la Recherche Médicale (INSERM),

Conflict of interest notification

The authors certify that they have no potential conflict of interest to declare regarding the subject matter or materials discussed in this manuscript.

SUPPLEMENTARY INFORMATION

Contents


P Didier

B Piotrowski

M Fischer

P Laheurte

Mechanical stability of custom-made implants: Numerical study of anatomical device and low elastic Young's modulus alloy

The advent of new manufacturing technologies such as additive manufacturing deeply impacts the approach for the design of medical devices. It is now possible to design custom-made implants based on medical imaging, with complex anatomic shape, and to manufacture them. In this study, two geometrical configurations of implant devices are studied, standard and anatomical. The comparison highlights the drawbacks of the standard configuration, which requires specific forming by plastic strain in order to be adapted to the patient's morphology and induces stress field in bones without mechanical load in the implant. The influence of low elastic modulus of the materials on stress distribution is investigated. Two biocompatible alloys having the ability to be used with SLM additive manufacturing are considered, commercial Ti-6Al-4V and Ti-26Nb. It is shown that beyond the geometrical aspect, mechanical compatibility between implants and bones can be significantly improved with the modulus of Ti-26Nb implants compared with the Ti-6Al-4V.

Introduction

Titanium-based alloys are widely used in the medical field for standardized implants manufacturing [START_REF] Guillaume | Dental implants: a review[END_REF][START_REF] Williams | Custom-made titanium cranioplasty: early and late complications of 151 cranioplasties and review of the literature[END_REF]. They exhibit several advantages: pure titanium cp-Ti and titanium alloy Ti-6Al-4V have remarkable osseointegration properties [START_REF] Shah | Commercially pure titanium (cp-Ti) versus titanium alloy (Ti6Al4V) materials as bone anchored implants-is one truly better than the other ?[END_REF], excellent corrosion resistance and biocompatibility [START_REF] Gepreel | Biocompatibility of Ti-alloys for long-term implantation[END_REF]. Their availability allows their production and sale on a large scale. Titanium devices are used for many type of implants, such as hip endoprosthesis for structural support to replace deficient bones [START_REF] Kaegi | The thrust plate hip prosthesis: a follow-up of 15-20 years with 102 implants[END_REF], tibia and femur titanium nails [START_REF] Ali | Management of compound fracture tibia in children with titanium elastic nails[END_REF][START_REF] Bhuyan | Titanium elastic nailing in pediatric femoral diaphyseal fractures in the age group of 5-16 years-a short term study[END_REF].

However, standard titanium implants have limitations. One of the most important constraints and reasons for failure is that they are not adapted to the morphology of every patient. In many cases, custommade (or anatomical) implants have shown excellent fit to the defect, reduction of the rate of implant failure, and high patient satisfaction [START_REF] Williams | Custom-made titanium cranioplasty: early and late complications of 151 cranioplasties and review of the literature[END_REF][START_REF] Wehmöller | CAD/CAM-prefabricated titanium implants for large skull defects-clinical experience with 166 patients from 1994 to 2000[END_REF][START_REF] Xu | Custommade locked plating for acetabular fracture: a pilot study in 24 consecutive cases[END_REF]. Therefore, the recent developments of additive manufacturing with titanium-based alloys have significantly facilitated the fabrication of these kinds of implants [START_REF] Leordean | Customized implants with specific properties, made by selective laser melting[END_REF][START_REF] Cox | Adding functionality with additive manufacturing: fabrication of titanium-based antibiotic eluting implants[END_REF]. This type of customized solution can be applied to a large field of traumas, as cranio-facial for bone augmentation devices [START_REF] Otawa | Custom-made titanium devices as membranes for bone augmentation in implant treatment: modeling accuracy of titanium products constructed with selective laser melting[END_REF], or mandible prosthesis [START_REF] Li | Evaluation of biological properties of electron beam melted Ti6Al4V implant with biomimetic coating in vitro and in vivo[END_REF]. Another example of limitation of standard implants (non anatomical) is the plates used to attach aesthetic epitheses on facial bones [START_REF] Treatment | Cranial and maxillofacial epithesis treatment on osseointegrated implants: concept and principles[END_REF]. This specific case is particularly widespread with traumas resulting from face cancers, where the removal of an organ such as an eye, a nose or an ear is necessary [START_REF] Greig | Reconstruction of the exenterated orbit with osseointegrated implants[END_REF][START_REF] Malard | Pertes de substance étendues de la pyramide nasale: rôle de l'épithèse[END_REF]. The facial reconstruction is composed of a metallic part fixed on the bone with a screw, on which is attached an aesthetic epithesis. The standardized metallic devices have to be adapted to the morphology of the patient by the surgeon during surgery. These pieces are shaped gradually by successive plastic deformations in order to embrace as best as possible the bone shape on which they will be fixed [START_REF] Lee | Standardization of surgical techniques used in facial bone contouring[END_REF]. This step, named contouring, extends the duration of the operation and the cumulative plasticity weakens the piece; it is requested to avoid it [START_REF] Xu | Custommade locked plating for acetabular fracture: a pilot study in 24 consecutive cases[END_REF]. Moreover, even after classical contouring on standard plates, the shape of the implant cannot perfectly match the physiological topology of the bone. This induces that the surgeon has to force to adapt the piece on the bone before screwing the two parts together. Thus, without any load condition on the implant, it is, once screwed, subject to stresses.

One other cause of implant failure is the mismatch in modulus of elasticity between bone and implant. That induces stress-shielding effect [START_REF] Gepreel | Biocompatibility of Ti-alloys for long-term implantation[END_REF][START_REF] Santis | Biomechanical effects of titanium implants with full arch bridge rehabilitation on a synthetic model of the human jaw[END_REF]. It has already been demonstrated that the application of a load on metallic rigid device implanted in a bone induces stress shielding, resulting in unloading of some areas and overloading others [START_REF] Goshulak | The biomechanical effect of anteversion and modular neck offset on stress shielding for short-stem versus conventional long-stem hip implants[END_REF][START_REF] Joshi | Analysis of a femoral hip prosthesis designed to reduce stress shielding[END_REF]. This phenomenon is all the more significant when the implant is rigid, and titanium-based alloys such as Ti-6Al-4V exhibit a relatively high Young's modulus (110 GPa) [START_REF] Li | New Developments of Ti-Based Alloys for Biomedical Applications[END_REF]. In the last few years, new beta-titanium alloys have been developed with the particularity to present an elastic modulus notably inferior to that of Ti-6Al-4V. Indeed, after a specific thermo-mechanical treatment, Ti-XNb alloys reach a low young modulus up to 30 GPa [START_REF] Elmay | Effects of thermomechanical process on the microstructure and mechanical properties of a fully martensitic titanium-based biomedical alloy[END_REF]. Implants made with this type of material have better mechanical stability, on the one hand by reducing the stress shielding in the bone compared to a physiological situation, and on the other hand by minimizing the stress gap between the implant and the bone responsible for micro movements [START_REF] Piotrowski | Interaction of bone-dental implant with new ultra low modulus alloy using a numerical approach[END_REF][START_REF] Handan | Evaluation of stress distributions occurring on zirconia and titanium implant-supported prostheses: a three-dimensional fi nite element analysis[END_REF][START_REF] Eduardo | The role of prosthetic abutment material on the stress distribution in a maxillary single implant-supported fi xed prosthesis[END_REF]. Moreover, a lot of studies highlight the excellent biocompatibility of this type of materials, beyond cytotoxicity and corrosion tests [START_REF] Niinomi | Development of new metallic alloys for biomedical applications[END_REF][START_REF] Cremasco | Effects of alloying elements on the cytotoxic response of titanium alloys[END_REF][START_REF] Metikoš-Huković | The influence of niobium and vanadium on passivity of titanium-based implants in physiological solution[END_REF][START_REF] Bai | Electrochemical corrosion behavior of Ti-24Nb-4Zr-8Sn alloy in a simulated physiological environment[END_REF][START_REF] Mcmahon | A comparative study of the cytotoxicity and corrosion resistance of nickel-titanium and titanium-niobium shape memory alloys[END_REF][START_REF] Eisenbarth | Biocompatibility of beta-stabilizing elements of titanium alloys[END_REF]. In addition to all these advantages, a key point is that Ti-XNb alloys are suitable to additive manufacturing technology [START_REF] Fischer | In situ elaboration of a binary Ti-26Nb alloy by selective laser melting of elemental titanium and niobium mixed powders[END_REF].

To investigate the implant-bone mechanical behavior, several approaches can be employed. In this work, finite elements method is adopted. Many finite element studies have already been proposed to explore bone-implant interface, revealing the benefits of the adapted structure of implants [START_REF] Leordean | Customized implants with specific properties, made by selective laser melting[END_REF], or the advantages of low Young's modulus alloy [START_REF] Piotrowski | Interaction of bone-dental implant with new ultra low modulus alloy using a numerical approach[END_REF]. The specific scope of dental field is particularly investigated [START_REF] Favot | Numerical study of the influence of material parameters on the mechanical behaviour of a rehabilitated edentulous mandible[END_REF][START_REF] Verri | Effect of crown-to-implant ratio on peri-implant stress: a finite element analysis[END_REF][START_REF] Santiago | Finite element analysis on influence of implant surface treatments, connection and bone types[END_REF]. Some studies take into account some surgical aspects of implants fixing by finite element methods [START_REF] Darwich | Assessment of the biomechanical performance of 5 plating techniques in fixation of mandibular subcondylar fracture using finite element analysis[END_REF][START_REF] Huang | Biomechanical optimization of a custom-made positioning and fixing bone plate for Le Fort I osteotomy by finite element analysis[END_REF]. However, few papers take into considerations both effects of structural and material optimization.

The aim of this study is to highlight the combined influence of low elastic modulus (material) and anatomical shape (structure) on the mechanical stability of an implant. In the first part of this work, we propose to describe the setting up of the finite element models composed with three simplified parts (screw, bone and implant) used to respond to the presented problem. At the same time, we will present the mechanical properties of the two materials Ti-6Al-4V and Ti-26Nb. In the second part, results for quantifying the advantages of custom-made or anatomical implants in comparison to the use of a standardized implant are presented and commentated.

Materials and methods

Anatomical and standard implant finite element models

The study appreciates the benefits of an anatomical implant in Ti-26Nbin observing what happened (i) when firstly a surgeon establishes and screws an implant on the skull; (ii) when secondly a displacement is applied on this implant. Thus two parameters are studied. The first one is the global shape of the implant, considering an anatomical or a standard implant. The second one is the stiffness of the material, depending on whether the implant is made ofTi-6Al-4V or Ti-26Nb biocompatible alloy. This induced two comparisons: anatomical Ti-6Al-4V versus standard Ti-6Al-4V, and anatomical Ti-6Al-4V versus anatomical Ti-26Nb. Thus two main finite element models are considered (standard and anatomical), with a possible changing material. A synthetic diagram of the study strategy is presented in Fig. 1. The different mechanical characteristics of the materials (Ti-6Al-4V, Ti-26Nb, cortical bone) used in the models are presented in Section 2.2 Identification of material properties.

Geometrical characteristics

Three entities are necessary to build the models: a screw, a piece of a skull and an implant. The screw and the bone parts are exactly the same in the two finite element models.

Concerning the bone part, a simplified model is considered in order to avoid parasite effects. Indeed, the important feature is the curvature and the no-plate topography of the skull bone, so the bone is modeled by a semi-cylinder. The dimensions are shown in Fig. 2.

The screw is composed of three areas, on the one hand the head, on the other hand a threaded part in contact with the bone and a smooth part in contact with the implant (the shank). The geometry characteristics are presented in Fig. 2.

The difference between these two models is due to the geometry of the perforated extremity of the implant. It is assumed that this portion is deformed by the surgeon in the standard case when he sets the screw, but not deformed in an anatomical case, because of the adapted shape of the anatomical implant. To approach the real case, the central part of the implant is fitted to the bone in both cases. It simulates the fact that a surgeon firstly deforms the implant with pliers for maximal adjustment of the implant shape to the skull shape.

So in the anatomical model, the implant has exactly the same curvature as the bone. And in the standard model, the extremity with the hole is linear and does not bond to the bone. In the two simulations, the implant is 4 mm wide and 1 mm thick, as showed in Fig. 2.

Mesh

All parts were meshed with C3D10 tetrahedron elements with quadratic interpolation, as illustrated in Fig. 3.

In order to define the size of the elements, a preliminary convergence analysis was performed. The implant was meshed with a maximum size of 0.5 mm with a progressive decrease around the hole down to 0.1 mm. The bone was also meshed with progressive discretization from 1 mm at the borders to 0.1 mm around the threaded hole of the screw. The screw was entirely meshed with 0.1 mm sized elements. The number of elements were respectively 44,711, 52,575, 62,365 and 70,763 for the standard implant, the anatomical implant, the bone and the screw.

Interactions between components

The bone and the screw were merged together to simplify the contact between them, because of the complexity of the threaded geometry. The merge operation has the advantage of still allowing the assignation of different materials to different partitions and the contact is assumed to be perfect.

A simple contact without friction for the other interactions was applied, namely between the inferior surface of the implant and the bone, the internal surface of the implant's hole and the screw's shank, and the superior surface of the implant and the screw's head. Previous tests with various friction penalties (0.1 and 0.5) were carried out to justify the frictionless behavior, and it appears that the results are uniform and identical regardless of the friction penalty. The different interaction properties are summarized in Table 1.

Successive steps of the numerical models

Different geometries induce different model constructions, but it is necessary to compare their performance. This justifies a similar loading condition applied to each model from a geometrically similar situation. Consequently, preliminary calculation steps are needed in the standard case in order to implement this similar initial condition.

In the anatomical case, the surgeon just has to place the implant at the surface of the bone and screw it without any deformation of the implant. This is the main interest of an anatomic implant. The only loading ever applied on the metallic implant is the daily positioning and removal of the epithesis. The epithesis not being considered in the models, these actions are modeled with a displacement imposed on the implant, referred to as epithesis loading. Thus the simulation is only composed of the final loading of the implant, the screw being placed at the beginning of this study.

In the standard case, contrary to the anatomical case, it is not possible to place and directly screw the implant on the bone, because it simply does not fit. Therefore the surgeon deforms the implant when he screws it, and forces it to adapt its shape to the bone, as explained previously. To simplify this stage, this first simulation is separated into several steps:

-Application of a pressure at the top of the holed extremity of the implant in order to deform it, shown in Fig. 4; -Placing of the screw; -Unloading of the pressure, which induces by elastic return a contact between implant and head of screw; hence the device is considered in the initial state for the application of the epithesis loading; -Application of the epithesis loading.

The load is applied at the extremity of the implant and is equivalent to a force of 195.2 N, which is the minimum force for a necessary deformation of the implant for the placing of the screw.

Load and boundary conditions

Once the preliminary steps are achieved, similar loads and boundary conditions can be applied. Boundary conditions are the same in the two models. However the epithesis loading is slightly different, modeled with a displacement applied to the implant.

First of all, the bone is always fixed through 3 surfaces.

The other condition, the epithesis loading, is applied on the inferior extremity of the implant. The locations of these two conditions are presented in Fig. 5. It can be composed of 2 stages: firstly, the implant is moved in the x axis direction until contact between the implant and the screw's shank, in order to reduce the slack between the screw and the implant. This first displacement is not exactly the same in the two cases because of the previous plastic deformation of the implant in the standard model. Indeed, the implant hole's border is not at the same distance from the screw shank in these models. When the contact is initiated between the screw and the implant, another displacement which is identical in each case, in x and y axis, is applied to pull on the screw. The adopted displacement imposed for epithesis loading was selected arbitrarily. Thus, the resulting values are not necessarily cal, but allow comparison between cases. They are recapitulated in Table 2.

All the models were made with Abaqus 6.13 (Dassault), and all the simulations were performed with nodes of 16 CPU. The calculation times for a standard case and an anatomical case were respectively 5 h and 3 h.

Identification of material properties

In the present study, for the metallic parts (screw and implant), two materials have been considered: Ti-6Al-4V and Ti-26Nb both processed by Selective Laser Melting (SLM). Material properties were obtained experimentally with uniaxial compression tests on parallelepiped samples elaborated with a SLM280HL machine (SLM Solutions). These characterization tests are required because there are no compression curves of these alloys elaborated by SLM in literature. The Fig. 6 shows that the Young's Modulus measured for Ti-6Al-4V and for Ti-26Nb were respectively 110 GPa and 60 GPa. Very close plastic behaviors can be observed between the two alloys. Hence, for both materials, the same isotropic plastic behavior has been adopted and only the Young's modulus was changed, in order to focus on the stiffness influence. Thus a typical Ti-6Al-4V plastic behavior is adopted for both alloys [START_REF] Tuninetti | Anisotropy and tension-compression asymmetry modeling of the room temperature plastic response of Ti-6Al-4V[END_REF].

For the modeling of the bone, cortical bone, an elastic modulus of 15 GPa was considered [START_REF] Bernard | Accurate measurement of cortical bone elasticity tensor with resonant ultrasound spectroscopy[END_REF][START_REF] Turner | The elastic properties of trabecular and cortical bone tissues are similar: results from two microscopic measurement techniques[END_REF], neglecting cancellous bone. This choice of definition was retained principally because in practice the screw mainly penetrates the cortical bone. Moreover, it is well known that the cortical bone stiffness is around 10 times greater than that of cancellous bone [START_REF] Prot | The behavior of cancellous bone from quasistatic to dynamic strain rates with emphasis on the intermediate regime[END_REF]. The materials elastic properties are summed up in Table 3.

Table 3 Mechanical elastic properties of the different materials used for the finite element models.

Materials

Young's modulus (GPa) Ti-6Al-4V 110 Ti-26Nb 60 Cortical bone 15 Fig. 7. Von Mises stress distribution in the standard case, after forming the implant, before the application of the displacement.

Fig. 8. Plastic equivalent strain in the implant, after its deformation, before the application of the displacement.

Results and discussion

Four kinds of results have been retained to compare the different cases:

-The residual stresses in the bone in the standard case due to the deformation of the implant before the application of the solicitation; -The general stress fields in different parts in the two models, especially the stress shielding in the bone; -The shear stress field along the screw; -The stress gap between the bone and the screw along the interface of the hole.

The symmetry plane, normal to the z axis, was chosen to observe the different results, in order to have a global view in all parts of the field outputs.

As mentioned in the previous section, the load applied to the systems has been chosen arbitrarily. Hence, the relative data highlighting advantages of configurations compared to others are investigated.

Residual stresses in the bone in the standard model

In this section, the residual stresses in the bone due to contact pressure of the forming of the implant are observed. Once the standard implant is screwed on the bone, even before the solicitation is applied to the model, an important stress field can be observed mostly in the bone as illustrated in Fig. 7. In the anatomical framework, the implant is already shaped to correspond to the bone's surface. Therefore this type of implant does not need any preliminary deformation and the bone is not solicited.

The Von Mises stress field in the bone and the plastic equivalent strain in the implant are investigated, and presented in Figs.s 7 and 8. After the deformation of this implant and the placing of the screw, which retains its elastic return despite a plastic deformation, a high level of Von Mises stresses can be observed in the skull part. Two main areas are identified, where the stresses are concentrated and exceed 150 MPa. Firstly the bone is compressed by the implant on the right side of the threaded hole. On the other hand, the relaxation of the pressure on the extremity of the implant after the establishment of the screw creates another resulting pressure on the inferior left side of the screw's head. This resulting pressure involves a concentration of stresses on the left side of the top of the hole.

These residual stresses have two misgivings. Firstly, they could induce bone densification in unsuitable areas, and also deeply impact the physiological state. Under loading of these bones, stress shielding can induce unloaded areas, which can be responsible for osteoporosis. The second point is the admissible additional stress. It can be seen that the stress field is around 150 MPa in bone. With a plastic yield stress between 150 and 200 MPa, this means that this bone cannot accept additional stress.

General Von Mises stress fields

Before analyzing the results in specific areas, it is important to have an overview of the whole frameworks. The Von Mises stress field in the same cutting plane as previously is also studied as illustrated in Figs.s 9 and 10.

Concerning the first comparison, which highlights the importance of an adapted geometry, Fig. 8 shows that a higher stress concentration can be observed in each part of the standard model despite a similar displacement initially applied, especially in the screw. It can be seen that in the standard case, the screw part in contact with implant (area A) reaches around 550 MPa, whereas it does not exceed 200 MPa in the anatomical case.

In the second study, where the influence of the material is tested, Fig. 9 reveals that the difference in stress repartition is less important. Nonetheless, the use of Ti-26Nb implant reduces the Von Mises stresses in every part. For instance, the Von Mises stress in the internal surface of the implant's hole (area B) is around 350 MPa for the Ti-6Al-4V alloy and less than 200 MPa for the Ti-26Nb alloy.

The Von Mises stresses in the bone where the concentration is the highest, at the interface with the screw, is particularly investigated. The solicitations in the bone are the most important because the bone should be the latest to be damaged. To do so, two paths on each side of the screw following nodes along the hole were used, presented in Fig. 11. For each node, the Von Mises stresses were picked for comparisons between models. The distribution being very similar on each side with the same values, it has been decided to just focus on side 1.

Firstly, concerning the influence of geometry, Fig. 12 highlights the fact that there are less solicitations in the bone all along the areas of interest in the anatomical case. On average, the diminution reaches 72% (Table 4). This is mainly explained by initial stress state in the bone due to geometric incompatibilities (before loading) and by a better stress repartition. Thus, replacing a standard implant by a custom one is less traumatic for the bone.

Then, the anatomical case is compared to the same model with low Young modulus Ti-26Nb instead of Ti-6Al-4V. Again, the distribution of Von Mises stresses along the path shows an average decrease by 34%, so it can be concluded that the influence of material is significant. Finally, the Von Mises stress field in the bone is reduced by 81% with the use of a Ti-26Nb custom implant.

Stress gap between the bone and the screw

The observation of solicitations in the bone is a key to highlight the influence of studied parameters. However, the appreciation of the stress gap between the screw and the bone, responsible for micro-movements inducing a loss of implant stability, can be more effective. Along the contact surface between the screw and the bone, the same paths of nodes presented in Fig. 10 have been defined in the post-treatment files. The stress gap is calculated on each common node between the screw and the bone, thanks to the merge operation between the two components during the design of the models. The evolution of the stress gap along the path is represented in Fig. 13. The evolution being very similar on each side with the same aspect, results on side 1 are presented.

The screw threads can be recognized because of the successive rough decreases on the chart Fig. 14; therefore the most solicited areas are between the threads. When the geometry parameter is changed in the anatomical case, the stress gap of Von Mises stress is significantly reduced, especially between the threads. For example on the second threads, the gap exceeding 300 MPa in the standard case decreases less than 100 MPa in the custom case. Table 5 shows that the results of the Von Mises stress gap on both sides, in terms of average, are almost the same. The decrease reaches 73% on side 1 and 71% on side 2. The value is slightly different but the case is not symmetric. Indeed, the direction of the applied load is more favorable for side 1.

Secondly, when the material influence is investigated by comparison between Ti-6Al-4V and Ti-26Nb, a significant diminution can also be observed all along the sides. In fact, in the custom low modulus model, the stress jump does not even exceed 25 MPa along the entire interface, except for the first point. Respective average diminutions of 61% and 60% are noted for side 1 and 2 with this second comparison, which are almost as high as the first comparison. These results are explained by the screw and the implant's low Young's modulus, which comes close to the bone Young's modulus. This is an important result because it demonstrates that the stress-shielding in bone due to a load in the implant is minimized with a Ti-26Nb custom device, which ensures a better stability of implant.

Finally the stress jump is reduced by 90% and 89% when the two changing parameters are considered. This emphasizes the interest of these two possible improvements.

Shear stresses in the screw

Interest was set in the shear stress field (σ xy ) in the same symmetry plane as previously, to consider the stability of the screw. The shear XY component stress is investigated because it is the most important in the global Cauchy stress tensor.

The general shear stress field in the screw was observed with the first comparison concerning the adaptive geometry, in order to have an overview of the repartition of shear stresses in the screw. Even if the maximum values reach 600 MPa in absolute value in the two cases, the shear stresses are less important and extended in the anatomical case, as showed in Fig. 15.

To quantify our expectations, paths have been defined on the borders of the screw, showed in Fig. 16.

Figs. 17 and 18 represent shear stress evolution along these two different paths on the screw. On side 1, the screw is impacted by the implant which shears it because of the displacement imposed. It is different on side 2, where the screw is in contact with the bone. This explains the different distribution of shear stresses along both sides.

Despite different distributions, the same tendency can be observed: when the implant geometry is changed, the stresses significantly decrease, mostly on the first half of the paths. On average, the diminution is 69% on the first side and 74% on the second side, as can be seen on Table 6.

The replacement of Ti-6Al-4V with Ti-26Nb induces a less significant decrease in shear stress, which is however not negligible. All along the paths, Ti-26Nb stresses are below Ti-6Al-4V stresses and are weaker by 44% on side 1 and 49% on side 2. With the two cumulated improvements, a benefit of 83% and 87% are obtained on side 1 and 2, respectively. Thus, the principal solicitation of the screw being a shear solicitation, it can be concluded that an anatomical implant in Ti-26Nb widely contributes to the sustainability of the screw.

Conclusion

Custom-made Ti-26Nb implants, including the combined effects of low modulus and anatomical shape, provide better mechanical stability compared with Ti-6Al-4V plate usually implanted, especially with surrounding bones. a) In the placing and tightening of the screw, in the standard model, the bone is stressed by the plastic deformation of the implant, when it is being screwed on the bone surface. b) When the implant is loaded, preservation of the bone is better when using a custom-made implant the manufacturing of which is made feasible thanks to SLM technology: the general Von Mises stress field, the shear stresses and the stress gap between the screw and the bone are all considerably minimized in the anatomical model compared to the standard case. Concerning the influence of the material, it has been seen that the use of an ultra low Young's modulus alloy also reduces the different stress fields in the bone and in the screw as well as the stress gap between the screw and the bone, thus minimizing stress shielding.

Fig. 1 .

 1 Fig. 1. Schematic representation of the study strategy.

Fig. 2 .

 2 Fig. 2. Geometrical characteristics of the considered entities: the screw (A), the bone part (B), the entire standard model (C) and the entire anatomical model (D).

Fig. 3 .

 3 Fig. 3. Mesh representation of the considered parts of the two models: the screw (A), the bone part (B), the anatomical implant (C) and the standard implant (D).

Table 1 4 .

 14 Interaction strategy between the different contact pairs of the finite element models, namely bone-screw, implant-screw and implant-bone. Interaction Bone-screw Implant-screw Implant-bone Type Merge Contact without friction Contact without friction Fig. Location of the pressure load in the standard finite element model, to induce forming of the implant.

Fig. 5 .Fig. 6 .

 56 Fig. 5. Load applied and boundary conditions locations.

Fig. 9 .

 9 Fig. 9. Comparison of the general stress fields of the standard model (left) and the anatomical model (right).

Fig. 10 .

 10 Fig. 10. Comparison of the general stress fields of the anatomical model with Ti-6Al-4 V (left) and Ti-26Nb (right).

Fig. 11 .Fig. 12 .

 1112 Fig. 11. Location of the two paths used along the screw-bone interface.

Fig. 13 .Fig. 14 .

 1314 Fig. 13. Representation of the Von Mises stresses along the side 1 comparing the anatomical model in Ti-6Al-4V and Ti-26Nb.

Table 5 Fig. 16 .

 516 Fig.[START_REF] Malard | Pertes de substance étendues de la pyramide nasale: rôle de l'épithèse[END_REF]. Location of the two paths used to capture the XY shear stresses along the screw.

Fig. 15 .

 15 Fig. 15. Comparison of the general shear XY stress fields in the standard model (left) and in the anatomical model (right) in Ti-6Al-4V.

Fig. 17 .

 17 Fig. 17. Representation of the shear stresses in the screw, along the side 1, according to different model configurations.

Fig. 18 .

 18 Fig.[START_REF] Santis | Biomechanical effects of titanium implants with full arch bridge rehabilitation on a synthetic model of the human jaw[END_REF]. Representation of the shear stresses in the screw, along the side 2, according to different model configurations.

Table 4

 4 Results of the average decrease of the Von Mises stresses on the two sides of the screw-bone interface, according the different comparisons.

	Average decrease of the Von Mises stress in the bone	Standard Ti-6Al-4V vs. anatomical	Anatomical Ti-6Al-4V vs. anatomical	Total: standard Ti-6Al-4V vs. anatomical
		Ti-6Al-4V	Ti-26Nb	Ti-26Nb
	Side 1	72%	34%	81%
	Side 2	64%	34%	76%

Table 6

 6 Results of the average decrease of the shear stresses in the screw according to different comparisons.

	Average diminution of the shear stress in the	Standard Ti-6Al-4V vs. anatomical	Anatomical Ti-6Al-4V vs. anatomical	Total: standard Ti-6Al-4V vs. anatomical
	screw	Ti-6Al-4V	Ti-26Nb	Ti-26Nb
	Side 1	69%	44%	83%
	Side 2	74%	49%	87%


MSc Maguelonne Roux

Claire Perret Msc

MD Eva Feigerlova

Badreddine Mohand

Oumoussa Bsc

MD Pierre-Jean Saulnier

Carole Proust Msc

PhD David-Alexandre Trégouët

MD Samy Hadjadj

Plasma levels of miR-152-3p are associated with diabetic nephropathy in patients with type 2 diabetes

Keywords: Circulating microRNA, microRNA-Sequencing, Type 2 diabetes, Diabetic nephropathy, biomarkers

Background

MiRNAs are small non-coding RNAs participating in genes post-transcriptional regulation. Their key role in modulating the susceptibility to human diseases is now widely recognized, in particular in the context of cardiometabolic disorders. The aim of the present study was to identify miRNAs associated with diabetic nephropathy (DN) in patients with type 2 diabetes (T2D).

Methods

A next generation sequencing-based miRNA profiling was performed in a case-control study for DN in plasma samples of 23 T2D patients with DN (cases) and 23 T2D without (controls). The main associations were confirmed using qRT-PCR and tested for replication in an independent case-control collection of 100 T2D patients, 50 with DN and 50 without.

Results

From the 381 known mature miRNAs that were found highly expressed in the discovery samples, we observed and replicated an association between increased plasma levels of hsa-miR-152-3p and DN (p = 4.03 10 -4 in the combined samples). Hsa-miR-152-3p plasma levels were further found to be positively correlated (p = 0.003) to plasma osmolarity, a surrogate marker for solute carrier net activity, whose regulation is controlled by several genes including SLC5A3, one of the predicted targets of hsa-miR-152-3p.

Conclusions

We observed strong evidence for the association of hsa-miR-152-3p plasma levels and DN in patients with T2D confirming an association previously observed in patients with T1D.

INTRODUCTION

Diabetes is considered as the leading cause of end-stage renal failure, contributing to more than onethird of all end-stage renal disease based on the 2016 report of United States Renal Data System 1 .

Diabetic nephropathy (DN) remains incompletely understood and occurs in 30 to 40% of diabetic patients [START_REF] Rossing | Prediction, progression and prevention of diabetic nephropathy. The Minkowski Lecture[END_REF] , and even in patients with good glycemic control [START_REF] Zhang | Factors predictive of nephropathy in DCCT Type 1 diabetic patients with good or poor metabolic control[END_REF] . Abnormalities in several signaling pathways and cytokines have been demonstrated to contribute to DN development, including the renin-angiotensin system, reactive species of oxygen, endoplasmic reticulum stress, pro-inflammatory cytokines and formation of advanced glycation end-products [START_REF] Forbes | Mechanisms of diabetic complications[END_REF][START_REF] Brownlee | The pathobiology of diabetic complications: a unifying mechanism[END_REF] .

Urinary albumin excretion is widely used as a non-invasive marker for DN although it does not fully reflect the disease process [START_REF] Caramori | The need for early predictors of diabetic nephropathy risk: is albumin excretion rate sufficient?[END_REF] . Histological abnormalities may be present before the detection of microalbuminuria. Similarly, some patients have decreased glomerular filtration rate (GFR) despite normoalbuminuria [START_REF] Bangstad | Early glomerulopathy is present in young, type 1 (insulin-dependent) diabetic patients with microalbuminuria[END_REF][START_REF] Fioretto | Glomerular structure in nonproteinuric IDDM patients with various levels of albuminuria[END_REF] . This phenotype seems to be rather common in type 2 diabetes (T2DM) [START_REF] Macisaac | Nonalbuminuric renal insufficiency in type 2 diabetes[END_REF] , challenging traditional classification of DN [START_REF] Mogensen | The Stages in Diabetic Renal Disease: With Emphasis on the Stage of Incipient Diabetic Nephropathy[END_REF] . An important effort is currently undertaken to identify innovative biomarkers of diabetic nephropathy, as illustrated by the recent grant from the UE on biomarkers of DN (https://ec.europa.eu/research/participants/portal/doc/call/h2020/imi2-2015-05-06/1662621-c5_imi2_c5_text_9_july_2015_en.pdf).

Recently, post-transcriptional regulation of mRNA has emerged as a major mechanism modulating gene expression. Non-coding RNAs such as microRNAs (miRNAs) participate in regulating mRNA stability and aberrant miRNA expression has been shown to account for renal fibrosis and podocyte injury [START_REF] Chung | miR-192 mediates TGF-beta/Smad3-driven renal fibrosis[END_REF][START_REF] Lai | MicroRNA-21 in glomerular injury[END_REF] . In response to various pathophysiological conditions, miRNAs can be released by cells into different extracellular fluids, including blood and could serve as biomarkers of diverse diseases including diabetes and its complications. Circulating miRNAs are more stable due to protection from endogenous RNase activity through their incorporation into lipoprotein complexes or microvesicles such as exosomes [START_REF] Arroyo | Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma[END_REF][START_REF] Vickers | MicroRNAs are transported in plasma and delivered to recipient cells by high-density lipoproteins[END_REF][START_REF] Valadi | Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells[END_REF] . Recent reports have demonstrated distinct profiles of miRNAs and exosomal miRNAs in plasma and urine samples across various stages of diabetic nephropathy [START_REF] Argyropoulos | Urinary microRNA profiling predicts the development of microalbuminuria in patients with type 1 diabetes[END_REF][START_REF] Barutta | Urinary exosomal microRNAs in incipient diabetic nephropathy[END_REF][START_REF] Delić | Urinary exosomal miRNA signature in type II diabetic nephropathy patients[END_REF][START_REF] Glowacki | Increased circulating miR-21 levels are associated with kidney fibrosis[END_REF][START_REF] Feigerlová | Role of post-transcriptional regulation of mRNA stability in renal pathophysiology: focus on chronic kidney disease[END_REF] .

Widely used techniques for identification of differentially expressed miRNAs, such as miRNA microarrays, suffer from a low accuracy due to low specificity of probe hybridization and variable sensitivity to detect less abundant miRNAs. The next-generation sequencing has a high-resolution potential, even though limited data is available in the relevant literature on its use as a discoverybased approach to identify enriched or dysregulated miRNAs in DN. The aim of the present study was to perform a comprehensive sequencing of plasma miRNAs in subjects with T2DM to identify candidate miRNAs for DN.

MATERIAL & METHODS

Study populations

Studied participants were patients with T2DM selected from the cross-sectional DIAB2NEPHROGENE study, which aimed to compare genetic and non-genetic determinants of renal complications in T2D patients. This study design was previously reported [START_REF] Hadjadj | Prognostic value of the insertion/deletion polymorphism of the ACE gene in type 2 diabetic subjects: results from the Non-insulin-dependent Diabetes, Hypertension, Microalbuminuria or Proteinuria, Cardiovascular Events, and Ramipril (DIABHYCAR), Diabete de type 2, Nephropathie et Genetique (DIAB2NEPHROGENE), and Survie, Diabete de type 2 et Genetique (SURDIAGENE) studies[END_REF][START_REF] Leveziel | Association between diabetic macular edema and cardiovascular events in type 2 diabetes patients: a multicenter observational study[END_REF] . The DIAB2NEPHROGENE study was approved by the Poitiers University Ethics Committee and was in accordance with the Helsinki Declaration of 1975, as revised in 2000. All participants gave written informed consent.

Cases were patients with T2DM and DN defined as increased urinary albumin (micro or macroalbuminuria) with or without altered renal function (defined as estimated glomerular filtration rate eGFR below 60 ml/min per 1.73m 2 , according to CKD-EPI equation [START_REF] Levey | A new equation to estimate glomerular filtration rate[END_REF] ) and no clinical and/or biological suspicion of non-diabetic kidney disease. Micro and macroalbuminuria were diagnosed according to urinary albumin determined on two out of three sterile urine collections: microalbuminuria range > 20 -199 mg/l or 30-299 mg/24 hrs, and macroalbuminuria range > 200 mg/l or 300 mg/24 hrs. Controls were patients with T2DM with normal urinary albumin (range < 20 mg/l or 30 mg/24 hrs) and eGFR above 60 ml/min/1.73m 2 .

DN patients and controls were matched for the following criteria: recruiting center, age (+/-5 years), sex. Although not strictly considered as a matching criterion, a special effort was made to have cases and controls not largely differing for diabetic retinopathy (absent, simple, severe non-proliferative or proliferative retinopathy) in order to focus on DN rather than on microvascular risk in general.

Biological samples were stored at -80°c at the CHU Poitiers biobanking facility (CRB Poitiers BB0033-00068) until use (first freeze-thawing cycle).

Biological determinations

Biological determinations of classical biological variables are briefly reported here : HbA1c by HPLC (Adams A1c HA-8160 analyzer; Menarini, Florence, Italy); creatinine by colorimetry on an automated analyzer (Kone Optima; Thermo Clinical Labsystems, Vantaa, Finland) for plasma determinations and on a Hitachi 911 automatic analyzer (Roche Diagnostics, Meylan, France) for urinary determinations; urinary albumin by nephelometry on a Modular System P (Roche Diagnostics GmbH) and osmolarity by the measure of the point of congelation of aqueous solutions with an automatic micro-osmometer (Fisher Bioblock Scientific, Illkirch, France).

Experimental protocol for miRNA sequencing

Plasma miRNA profiling was conducted in a sample of 46 T2D patients, half of which being patients with DN and the remaining half being free of DN. Total RNA containing miRNA was extracted from EDTA plasma sample (400 µl) with Qiagen miRNeasy Serum/Plasma kit (Qiagen, Hilden, Germany). Plasma miRNA libraries were then prepared from 6µl of total RNA using the NEBNext Multiplex Small RNA Library Prep Set for Illumina (New England Biolabs France, Evry) according to manufacturer's instructions with a specific dilution of adaptors to 1/10 and 15 cycles for PCR amplification. Specific small RNAs bands were then selected by a 3-steps optimized AMPure XP Beads (Agencourt Bioscience Corporation, Beverly, Massachusetts, USA) protocol in 96-well plate (200 µl), without QIAQuick PCR purification: 1-a 1.5X AMPure XP beads purification step was first performed on 100 µl of amplified library to remove smaller fragments and the bead product was resuspended with H2O (42 µl); 2-1.1X AMPure XP beads were added to the first purification products (42 µl); 3-After discarding beads with the smallest fragments, the resulting supernatant was kept and purified with 1.8X AMPure XP beads. The beads product, resuspended in H2O (15 µl), served as the final purified library. Two plasma samples were analyzed in duplicate. A pool of equal quantity of 24 purified libraries was constructed, each library being tagged with a different index. Two pools of 24 libraries were constituted in order to analyze the 46 plasma samples, each pool being loaded on 2 lanes of a rapid flowcell. A 55bp single read sequencing of miRNAs pool libraries was performed on an Illumina Hiseq 1500 instrument (Illumina, San Diego, California, USA) at the Post-Genomic Platform of the Pitié-Salpêtrière Hospital.

miRNA sequencing data analysis

After demultiplexing, reads that did not pass Illumina filters were removed with fastq_illumina_filter [START_REF]Illumina CASAVA-1.8 FASTQ Filter[END_REF] . Cutadapt 25 was used to trim 3' and 5' adapters and remove low-quality bases (quality <28) from the 3' and 5' ends. Reads with a length between 15 and 30 bases, i.e. reads that could correspond to miRNAs, were selected for further alignment. Bowtie v1.1.1 [START_REF] Langmead | Ultrafast and memory-efficient alignment of short DNA sequences to the human genome[END_REF] was used to align reads against the Homo Sapiens mature miRNA sequences from the miRBase 21 database [START_REF] Kozomara | miRBase: annotating high confidence microRNAs using deep sequencing data[END_REF] . Only reads that uniquely aligned without any mismatch were kept for the present study. miRNA abundance was expressed as the number of reads (i.e counts) that aligned to a given miRNA. To avoid any background noise due to very low expressed miRNAs, only miRNAs with counts higher than 10 in more than 5 subjects were kept for analysis. To normalize abundances, we first applied the size factor correction as proposed in the Deseq method [START_REF] Anders | Differential expression analysis for sequence count data[END_REF] followed by the application of the variance stabilizing transformation [START_REF] Huber | Variance stabilization applied to microarray data calibration and to the quantification of differential expression[END_REF] . Impact of the normalization procedure can be evaluated by inspecting the miRNA expression profiles that are more comparable across samples after normalization (Supplementary Figure S1), without impacting on the reproducibility of the technical replicates (Supplementary Figure S2). For the subsequent differential analysis, replicates with the lower count variability were used.

Association between detected miRNAs and DN was performed using linear regression analysis adjusted for age, sex, and counts of 3 let7 controls (let7d/7g/7i) miRNAs in order to avoid any uncontrolled bias in libraries preparation [START_REF] Chen | A Combination of Let-7d, Let-7g and Let-7i Serves as a Stable Reference for Normalization of Serum microRNAs[END_REF] . Beforehand, miRNAs were normalized using the normal inverse quantile transformation [START_REF] Peng | Normalizing a large number of quantitative traits using empirical normal quantile transformation[END_REF] .

qRT-PCR validation

Candidate miRNAs showing suggestive statistical evidence for association with DN in the miRNA sequencing analysis were re-quantified for technical validation by qRT-PCR after a new extraction. miRNA qRT-PCR was performed by specific TaqMan miRNA assays using the TaqMan microRNA Reverse Transcription kit and TaqMan Universal PCR Master Mix no UNG (Thermo Fisher Scientific, Waltham, USA) according to the manufacturer's instructions (Supplementary Methods).

MiRNA levels were normalized to the miRNA content of a mix of 3 miRNAs -let7d, let7g and let7iand the relative miRNA levels were calculated using the 2 -ΔCT method. The same methodologies were applied to the candidate miRNAs further measured in an independent sample of 100 T2D patients composed of 50 patients with DN and 50 without. Linear regression analysis, adjusting for age and sex, were employed to test for the association between the qRT-PCR miRNA levels and DN status, in the discovery and replication samples, separately.

RESULTS

miRNA identification results

Before pre-processing steps, the average number of reads per sample was 10.5 10 6 , ranging from 7.5 10 6 to 13.8 10 6 . After quality controls, a per-sample average of 64.67% of reads (min = 46.55%, max = 86.59%) was kept for alignment analysis. The percentage of reads that uniquely matched to human mature miRNAs ranged from 2.4% to 16.44%, with a mean of 6.47%, which led to the detection in the 46 studied plasma samples of 1,424 known mature miRNAs. From these miRNAs, 384 miRNAs (including the 3 housekeeping miRNAs serving as positive controls -see Methods) were considered as expressed and entered into the differential expression analysis with respect to DN.

Discovery step

Clinical and biological characteristics of cases and controls in both the discovery and replication cohorts are presented in Table 1. Differential expression analysis between cases and controls, with the full list of results given in Supplementary Table S3, did not reveal any significant association that satisfied the statistical threshold of 1.3 10 -4 corresponding to the Bonferroni correction for the number of tested miRNAs (n = 381). The lowest p-value was observed at p = 1.57 10 -3 for hsa-miR-362-5p.

Nevertheless, 4 miRNAs exhibited a marginal association with DN at p < 0.01. Boxplot representation of their expression levels in cases and controls are shown in Figure 1. Among these 4 miRNAs, three were over-expressed in cases compared to controls, hsa-miR-362-5p (p = 1.57 10 -3 ), hsa-miR-152-3p (p = 4.34 10 -3 ) and hsa-miR-196b-5p (p = 4.47 10 -3 ) while the remaining hsa-miR-140-3p (p = 6.28 10 -3 ) demonstrated opposite pattern of association. These 4 miRNAs were selected for further technical validation.

Technical validation

We checked the consistency between miRNA levels measurements obtained by sequencing and qRT-PCR for the 4 miRNAs with p-values <0.01, selected at the discovery step. The correlation between qRT-PCR levels and sequencing data was satisfactory for hsa-miR-152-3p (r = 0.64), hsa-miR-196b-5p (r = 0.53) and hsa-miR-362-5p (r = 0.42) but not for hsa-miR-140.3p (r = 0.19).

Nevertheless, using the qRT-PCR measurements, only the association of hsa-miR-152-3p with DN was still significant (p = 0.002) with increased expression in cases compared to controls (Table 2), leading us to select hsa-miR-152-3p for replication in an independent sample.

Replication step

Following these observations, the association of miRNA hsa-miR-152-3p with DN was tested in another sample of 100 T2D patients made of 50 cases and 50 controls, whose characteristics are detailed in Table 1. Consistently with the results observed in the discovery cohort, the hsa-miR-152-3p showed increased plasma levels in DN patients compared to non-DN patients (0.061 vs 0.055) with association reaching borderline significance (p = 0.053) (Table 2).

In the combined discovery and replication samples, the statistical evidence between hsa-miR-152-3p plasma levels and DN was p = 4.03 10 -4 .

Intermediate phenotype

Interestingly, the identified hsa-miR-152-3p is computationally predicted [START_REF] Agarwal | Predicting effective microRNA target sites in mammalian mRNAs[END_REF] to target the SLC5A3 gene previously suspected to be involved in acute renal failure [START_REF] Kitamura | Inhibition of myo-inositol transport causes acute renal failure with selective medullary injury in the rat[END_REF] of which DN is an important contributing factor [START_REF] Chawla | Acute kidney injury and chronic kidney disease as interconnected syndromes[END_REF] . As the SLC5A3 protein is involved in the regulation of osmolarity, we examined the relationship between hsa-miR-152-3p plasma levels and plasma osmolarity, which is a surrogate marker for solute carrier net activity. Plasma osmolarity did not differ between cases and controls (Table 1). In the total samples, we observed a positive correlation (r = 0.26, p = 0.003) between hsa-miR-152-3p plasma levels and plasma osmolarity (Supplementary Figure S4). This correlation was observed both in the discovery (r = 0.20) and the replication (r = 0.28) samples, and in cases (r = 0.16) and controls (r = 0.24).

Of note, we did not evidence any correlation between hsa-miR-152-3p plasma levels and urinary albumin/creatinine ratio nor eGFR (data not shown).

DISCUSSION

To our knowledge, the pilot work reported here is the first to apply an agnostic next-generation sequencing technology to plasma samples of T2D patients in order to identify candidate miRNAs for DN. In addition, our study included a technical validation step of the main results observed in a discovery phase as well as a statistical replication in an independent sample. This work revealed that hsa-miR-152-3p plasma levels were increased in DN cases compared to controls, both in the discovery and replication cohorts. Interestingly, this miRNA had been previously found associated with DN in type 1 diabetes patients by a candidate miRNA approach [START_REF] Bijkerk | Circulating microRNAs associate with diabetic nephropathy and systemic microvascular damage and normalize after simultaneous pancreaskidney transplantation[END_REF] . These results strongly suggest that the hsa-miR-152-3p is involved in the DN pathophysiology in the population of patients with diabetes. Computational prediction includes SLC5A3 gene as one of the hsa-miR-152-3p's targets.

SLC5A3 encodes the SMIT1 (Sodium/myo-inositol transporter) protein, an inositol transported contributing to maintaining the osmotic balance in different tissues/organs, including the kidney [START_REF] Schneider | Inositol transport proteins[END_REF] , whose evidence for its role in diabetes-associated metabolism is accumulating [START_REF] Kitamura | Inhibition of myo-inositol transport causes acute renal failure with selective medullary injury in the rat[END_REF][START_REF] Li | Identification and Functional Implications of Sodium Myo-Inositol Cotransporter 1 in Pancreatic β-Cells and Type 2 Diabetes[END_REF][START_REF] Van Steenbergen | Sodium-myoinositol cotransporter-1, SMIT1, mediates the production of reactive oxygen species induced by hyperglycemia in the heart[END_REF] . Besides, we evidenced an association of hsa-miR-152-3p plasma levels with plasma osmolarity, an intermediate phenotype for the activity of solute carrier transporters genes, including SLC5A3. Of note, SLC5A3 has been reported to associate with acute renal failure [START_REF] Kitamura | Inhibition of myo-inositol transport causes acute renal failure with selective medullary injury in the rat[END_REF] . Unfortunately, we were not able to detect SLC5A3 mRNA in our T2D plasma samples (Supplementary Methods) which prevented us from testing any association between DN, SLC5A3 mRNA and hsa-miR-152-3p levels. We did not observe any correlation between hsa-miR-152-3p plasma levels and markers of DN (eg eGFR, urinary ACR)

suggesting the identification of a potential pathway not involved in glomerular pathway but more likely related to tubular ion transfer. A recent study [START_REF] Wang | MicroRNA 152 regulates hepatic glycogenesis by targeting PTEN[END_REF] has highlighted the role of hsa-miR-152-3p in the hepatic regulation of glucose metabolism through a mechanism involving the PTEN pathway. Of note, Kato et al [START_REF] Kato | An endoplasmic reticulum stress-regulated lncRNA hosting a microRNA megacluster induces early features of diabetic nephropathy[END_REF] have demonstrated in mice that the PTEN pathway is also a key target for several glomerular miRNAs associated with DN. In the same mice model, glomerular levels of hsa-miR-152-3p were found elevated in diabetic animals compared to controls [START_REF] Kato | An endoplasmic reticulum stress-regulated lncRNA hosting a microRNA megacluster induces early features of diabetic nephropathy[END_REF] . Of note the main glomerular DNassociated miRNA identified in their work, miR-379, was not among those showing strong association with DN in our plasma samples (not shown data). Finally, obesity, a risk factor for chronic kidney disease [START_REF] Vd | Obesity-related glomerulopathy: clinical and pathologic characteristics and pathogenesis[END_REF] has been reported to associate with increased serum levels of hsa-miR-152-3p in non-diabetic patients [START_REF] Wu | Profiling peripheral microRNAs in obesity and type 2 diabetes mellitus[END_REF] . We thus questioned if obesity could be a confounding factor between hsa-miR-152-3p and DN. In our study population with T2DM, we did not observe any association between hsa-miR-152-3p and body mass index (data not shown).

Several limitations must be acknowledged. We were not able to find any significant association between miRNA and DN that passed the study-wise statistical threshold in our discovery cohort. This is likely to be a power issue due to the small size of the T2D samples processed for miRNA sequencing. As an illustration, the power of our discovery cohort to detect, at the p = 1. 3 10 -4 threshold, any association observed with the hsa-miR-152-3p was only 50%. We were also limited by the available quantity of mRNA material we could have access to in order to validate all suggestive associations observed in the miRNA sequencing phase. As a consequence, we cannot exclude we have missed some additional miRNAs that could associate with DN. In addition, a very stringent bioinformatics pipeline was applied to miRNA sequenced data where mismatch and multiple alignments were not taken into account in this study. This could have limited our chance to discover additional relevant miRNAs. Finally, even though cases and controls were well-matched for important determinants of DN such as age, gender [START_REF] De Hauteclocque | The influence of sex on renal function decline in people with Type 2 diabetes[END_REF] but also diabetic retinopathy [START_REF] Hammes | Risk Factors for Retinopathy and DME in Type 2 Diabetes-Results from the German/Austrian DPV Database[END_REF] , the phenotype between discovery and replication steps was slightly different for cases, with only proteinuric patients in the discovery and a combination of microalbuminuric and proteinuric patients in the replication population. In that way, the replication step could be considered as a replication and an extension step rather than a mere replication with strictly concordant inclusion criteria. These phenotypic differences might explain why the hsa-miR-152-3p plasma levels tend to be higher in cases from the discovery group than in those from the replication phase. However, we found no confounding factors for the association between hsa-miR-152-3p levels and patients' characteristics (see Table 1). The lower association observed in the replication compared to the discovery step could also be due to the wellknown winner curse effect [START_REF] Göring | Large upward bias in estimation of locus-specific effects from genomewide scans[END_REF][START_REF] Zhong | Correcting "winner's curse" in odds ratios from genomewide association findings for major complex human diseases[END_REF] .

Although our research strategy was not aiming to validate candidate miRNAs, we were able to find 23 miRNAs with nominal p-values <0.05 (Supplementary Table S3) and some of them including hsa-miR-132p, hsa-miR-192-5p and hsa-miR-326 were already described in previous work [START_REF] Bijkerk | Circulating microRNAs associate with diabetic nephropathy and systemic microvascular damage and normalize after simultaneous pancreaskidney transplantation[END_REF] . The association observed with hsa-miR-132p was however in the opposite direction as that observed in type 1 patients. Finally, our research design was cross-sectional and led to the identification of an association between DN and plasma levels of hsa-miR-152-3p. In this context, an important caveat must be kept in mind as hsa-miR-152-3p dysregulation could be a consequence of DN rather than causally implicated. The positive correlation between plasma osmolarity and hsa-miR-152-3p plasma levels adds support to the predicted relation between hsa-miR-152-3p and SLC5A3 regulation. But the lack of association of plasma osmolarity and DN tends to exclude that the association of hsa-miR-152-3p plasma levels with DN could be mediated via a direct influence on plasma osmolarity.

In conclusion, our pilot study identified the hsa-miR-152-3p as a miRNA that associate with the risk of DN in patients affected with T2D and that can be measured in plasma material. This miRNA has been previously shown to associate with DN in a cross-sectional study in patients with T1D. Large prospective studies are now required to validate plasma hsa-miR-152-3p as a biomarker for DN and to establish its prognostic value beyond urinary albumin. In addition, functional experiments are also mandatory to deeply characterize the underlying pathophysiological mechanisms.

CRB Poitiers (BB0033-00068) staff for their help with data collection, biobanking and sample handling.

CONFLICT OF INTEREST STATEMENT None declared.

The results presented in this paper have not been published previously in whole or part, except in abstract format.

AUTHORS' CONTRIBUTION

MR was in charge of all bioinformatic and statistical analyses of the biological data under the supervision of DAT. MR drafted the manuscript that was further reviewed by EF, DAT and SH. Boxplot representation of top miRNAs plasma levels in T2D patients with (cases) and without (controls) DN. Association testing was performed on normalized plasma levels adjusted for age, sex, and counts of the 3 controls (let7d, let7g and let7i) miRNAs.

  miRNA extraction and libraries preparation for sequencing were conducted by CPe. EF, PJS and CPr participated to clinical and biological data collection and measurements. Sequencing of the miRNA data was performed by BMO. The study was designed by DAT and SH. All authors approved the submitted version.

Figure 1

 1 Figure 1 Association of top 4 miRNAs with DN status.Boxplot representation of top miRNAs plasma levels in T2D patients with (cases) and without (controls) DN. Association testing was performed on normalized plasma levels adjusted for age, sex, and counts of the 3 controls (let7d, let7g and let7i) miRNAs.

Table 1 Characteristics of the study population

 1 : estimated Glomerular Filtration Rate determined according to the CKD EPI formula. uACR: Urinary albumin:creatinine ratio Data shown represent mean (standard deviation) except for smoking (% of smokers); sex, albuminuria and diabetic retinopathy (counts); uACR (median [1 st quartile -3 rd quartile ]).

		Discovery Sample	Replication Sample
		Cases	Controls	Cases	Controls
		N = 23	N = 23	N = 50	N = 50
	Age (years)	64.56 (8.35) 64.74 (7.29) 65.50 (8.11)	64.70 (7.65)
	Sex (Male/Female)	17/6	17/6	37/13	37/13
	eGFR (ml/min/1.73m 2 )	50.74 (19.11)	82.35	64.64	80.04
			(13.24)	(22.89)	(12.93)
		132.0	0.67	12.8	0.99
	uACR (mgl/mmol)
		[26.8 -189.0]	[0.47 -1.13]	[3.84 -40.98]	[0.64 -1.55]
	Normo/micro/macroalbuminuria	0/0/23	23/0/0	0/27/23	50/0/0
	Diabetic Retinopathy	0/17/2/4	2/15/4/2	0/31/16/3	5/32/8/5
	(absent/simple/severe non
	proliferative/proliferative)
	Systolic Blood Pressure (mmHg)	164.2	140.6	143.6	136.2
		(22.83)	(18.69)	(18.98)	(18.03)
	Diastolic Blood Pressure (mmHg)	83.9	78.8	76.1	75.2
		(11.73)	(8.83)	(8.29)	(10.74)
	BMI (kg.m -2 )	30.88 (5.69) 29.75 (5.24) 30.67 (5.34)	30.53 (6.06)
	HbA1c (%)	7.77 (1.20)	7.70 (1.00)	8.11 (1.74)	7.78 (1.18)
	Known Diabetes duration (years)	22.56 (5.83) 22.26 (5.16) 18.52 (9.06) 19.52 (10.04)
	Current Smoker (%)	0%	13%	17%	6%
	Plasma Osmolarity	308.5 (8.29) 301.5 (5.07) 303.8 (9.12)	300.5 (7.52)
	(mOsm)

eGFR

Table 2 Association of hsa-miR-152-3p with DN

 2 Shown values correspond to mean (standard deviation) of miRNA levels measured by qRT-PCTR and normalized to let7 controls miRNAs using the 2 -ΔCT method. a Adjusted for age and sex.

			Discovery			Replication
		Cases	Controls	P value a	Cases	Controls	P value a
		(n = 23)	(n = 23)		(n = 50)	(n = 50)
	hsa-miR-152-3p	0.072	0.052	p = 0.002	0.061	0.055	p = 0.053
		(0.026)	(0.018)		(0.017)	(0.018)

ACKNOWLEDGEMENTS

We wish to thank all of the patients and physicians who took part in the DIAB2NEPHROGENE study

(see Supplementary Note).

We also wish to thank E Migault, M Grosdenier and S Brishoual (Inserm CIC1402, Poitiers) and the

FUNDING M.R was financially supported by the Fondation pour la Recherche Médicale and the GENMED Laboratory of Excellence on Medical Genomics (ANR-10-LABX-0013). Data analysis was performed using the C2BIG computing cluster, funded by the Région Ile de France, Pierre and Marie Curie University, and the ICAN Institute for Cardiometabolism and Nutrition (ANR-10-IAHU-05). The DIAB2NEPHROGENE study was supported by grants from PHRC-Poitiers 2004, PHRC-IR 2008, and AFD (Research Grant 2003). The Groupe d'Etude des Maladies Métaboliques et Systémiques (Poitiers, France) also supported the study.


A Clodic

R Alami

E Pacherie

V Fernández Castro

Commitments in Human-Robot Interaction

INTRODUCTION

The notion of commitments plays a pivotal role in understanding joint action (Cohen and Levesque 1991;Bratman, 1992). However, the study of commitments in the context of joint action for human-robot interaction is quite new. We discuss two specific proposals to commitments: the functional approach, which argues that the central function of commitments is to reduce different forms of uncertainty; and the normative approach, which argues that commitments create obligations towards one's co-agents who are entitled to demand that these obligations be satisfied. For demonstration purpose, we consider 2 use-cases. In the first one, a robot guide indicates a direction to a visitor by pointing/indicating a destination (e.g. Pepper in a Mall). In the second one, a robot guide brings a visitor toward its destination (e.g. Rackham at the Space City Museum).

We conclude that both approaches capture fundamental aspects to handle commitments in the context of human-robot interaction.

The Functional Approach (Michael & Pacherie, 2014)

Commitments are psychological mechanism for reducing different types of uncertainties.

Different type of uncertainties in Joint Action:

• Motivational uncertainty: participants do not know whether or not the other is motivated to engage in the joint action. • Instrumental Uncertainty: participants do not know whether or not they agree about how to proceed (e.g. means, roles). • Common Ground Uncertainty: it may happen that the instrumental beliefs and motivations are not mutually manifested. By stablishing commitments, participants can stabilize and provide expectations that facilitate the prediction of the other co-actors.

Key aspects of the functional approach is to explore how commitments are established: Commitments create obligations towards one's co-agents who are entitled to demand that these obligations be satisfied, giving rise to expectations that the agent will act as committed or that, if not, co-agents will demand that he/she/it does.

Although commitments serve to provide reliable expectations, these expectations are of a special kind. In the normative view, commitments serves to attribute to oneself or the other normative expectations, so we can ensure that everyone behaves as expected.

The key aspect of the normative approach is how we display different communicative and behavioral strategies to make explicit of our duties and make the co-partner responsible for their own: Blame, signaling expectation or apologizing.

Robot expects the human to look at the direction of its indications

The robot looks at the human to make sure he is looking at the right direction

The human doesn't look in the appropriate direction Robot: Searching for eye contact Repeat

The human expects the robot not to pursue the interaction before the human has understand the indicated place -

The robot continues the interaction by asking the human if he needs something else

The human asks to come back to the explanation, -'Wait, where?"

Apologizing and coming back to the explanation

Robots expects the human to follow it

The robot says "I accompany you to your destiny, please follow me"" During its movement, the robot has some back and forth movement to check if the human is still here

The human doesn't follow -Stop and Wait -Signal he is waiting -"Please, Follow me" -Approaching the human and ask "it is everything alright?"

Human expects the Robot to adapt Speed

The human can enforce the certainty that everything is alright by saying "keep going, I follow you"

The robot is going too fast or too slow

The human does not follow anymore or on the opposite bump the robot because it is too slow

Apologizing and slowing down/speed up CONCLUSIONS 1. There are two approaches to commitment in joint action which emphasizes different behavioural and cognitive aspects of the role of commitment in joint action. However, the two approaches are complementary, 2. Commitments management gives roboticists a way to give meaning to monitoring, however, it comes with duties. We must give robot abilities to signal its expectations on one side and to monitor human's expectations/reactions/signals on the other side. We must give human means to understand the robot expectations/reactions/signals on one side and means to deliver its expectations to the robot on the other side.

=> we need to develop these monitoring and signaling abilities, it is not part of the action by itself but it is needed for the interaction => perhaps we should design a kind of code to help the management of these aspects either on the robot and on the human side(e.g. a kind of common interface that is understandable by every robot) 3. When there is a violation of expectation, we have to consider where this violation comes from. => perhaps it could be a way to link both functional and normative approach since a violation could come from motivational uncertainty, instrumental uncertainty or common ground uncertainty?

Commitments for Human-Robot Interaction: A Scenario.

We consider a scenario where a Robot must operate autonomously in a Mall and its central function is to guide a person to a destination (see Foster et al 2016). The central task requires the robot to represent different locations and areas, be able to self-localize, possess verbal and non-verbal skills for indicating (e.g. giving instructions; pointing), recognize the human partners and her idiosyncrasies (e.g. is the person too old to walk stairs up?) or have perspective-taking (e.g. to see if the human is seeing the indicated way or target).

Importantly, the robot must be able to handle human's commitment with the joint goal (arriving to a particular location) but also making the human aware that he is committed to perform the task. Do the functional or the normative approaches help us to design a robot capable of performing these type of actions?

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  Descriptive Expectations: expectations whose violation or frustration does not necessary triggers reactive attitudes. These expectations are tied to predictions. For example, you can expect your friend to have a beer because this is what she always does but if she doesn't, this may surprise you but not bother you.• Normative expectations: expectations whose violation or frustration triggers reactive attitudes: blame, petitions of justification or sanctions. It is an expectation connected to the notion of putting someone on demand and when it is frustrated the reaction is more emotionally loaded and directed to regulate the other's action.