Complaint Management: Which efforts to satisfy the complainers: the role of relationship quality?

Keywords: Complaint management, theory of justice, customer relationship management Track: Relationship Marketing

The aim of this communication is to study the effectiveness of complaint management according to the quality of the relationship between the firm and the customer. We compare interactional (relational) and distributive (monetary) compensation in terms of preference for loyal and new customers. The results show that loyal customers prefer interactional efforts and purchase vouchers to refunding. New customers prefer distributive compensations and refunding.

Introduction

Facing dissatisfaction, customers have several alternatives (Hirschman, 1995): exit, loyalty and voice. The verbal answer (Voice) can be word-of-mouth communication or a complaint which is a constructive way to express dissatisfaction to obtain a correction or compensation. The management of complaints thus perfectly integrates within the scope of the customer relationship management to increase loyalty since it gives an organization a last chance to retain dissatisfied clients [START_REF] Smith | A Model of Customer Satisfaction with Service Encounters Involving Failure and Recovery[END_REF]Fornell & Wernerfelt, 1987). In addition, complaints are a very rich source of valuable information to improve quality continuously. The investigations on complaint management show that the theory of justice [START_REF] Adams | Inequity in Social Exchange[END_REF] explains the satisfaction of complaining customers [START_REF] Orsingher | A meta-analysis of satisfaction with complaint handling in services[END_REF]. However, the questions about the nature and the valence of the compensations as well as which consumer targets to privilege remain unanswered. The principal contribution of this article is thus to determine the most effective dimensions of the theory of justice in the context of customer complaint management to satisfy and retain customers. We differentiate the effectiveness of the complaint management process according to the relationship quality or strength between the firm and the customer. We first describe the key factors for complaint management and then we explain our conceptual model as well as our hypotheses and methodology. The article afterwards shows the results and finishes with a discussion, managerial implications and research directions.

Key factors in the management of complaints

The management of complaints aims at preserving the quality of a relationship. It is one critical moment during which customers can test the reality of the receiving efforts which the firm is ready to grant to satisfy them.

The role of the relationship quality

The quality of the relationship indicates a psychological connection that customers have with a firm [START_REF] Crosby | Relationship quality in services selling: an interpersonal influence perspective[END_REF]DeWulf et al., 2001). It can be considered as a global judgment of the relationship [START_REF] Garbarino | The different roles of satisfaction, trust, and commitment in customer relationships[END_REF]. In literature a consensus is established about the importance of satisfaction, trust, commitment and identity connection which influence the quality of the relationship [START_REF] Bhattacharya | Understanding the bond of identification: An investigation of its correlates among art museum members[END_REF][START_REF] Sirdeshmukh | Consumer trust, value, and loyalty in relational exchanges[END_REF]. The role of the relationship quality is complex in the complaint management process and service incidents (Gregoire & Fisher, 2006). On one side, a strong relationship quality may have a protective effect [START_REF] Ahluwalia | How Prevalent Is the Negativity Effect in Consumer Environments?[END_REF]: a strong bond between a customer and a company could result in lower expectations, a less important stability and valuation of the problem, more satisfaction with the complaint management [START_REF] Hess | Service Failure and Recovery: The Impact of Relationship Factors on Customer Satisfaction[END_REF]. Furthermore, a bad complaint management might less negatively influence on his/her trust and commitment (Tax et al.,1998). On the other hand, the relationship quality may lead to judgments and behaviors that are relatively more negative for the company [START_REF] Aaker | When Good Brands Do Bad[END_REF]. Indeed, customers with strong relationship quality may have higher expectations in terms of complaint management [START_REF] Kelley | Antecedents to customer expectations to service recovery[END_REF] and they may be more inclined to feel betrayed because their trust they had in the company has been disappointed by an incident of service [START_REF] Robinson | Trust and breach of the psychological contract[END_REF]Grégoire et al., 2009).

The role of the perceived justice

The theory of justice explains how individuals react to situations of conflicts [START_REF] Gilliland | The perceived fairness of selection systems: an organizational justice perspective[END_REF][START_REF] Lind | The social psychology of procedural justice[END_REF]. The perception of justice results from a three-dimensional evaluation (Tax et al. 1998;[START_REF] Smith | A Model of Customer Satisfaction with Service Encounters Involving Failure and Recovery[END_REF]. The meta-analyzes of [START_REF] Orsingher | A meta-analysis of satisfaction with complaint handling in services[END_REF] show that distributive and interactional justice strongly influences the satisfaction and the behavior of complainers while procedural justice plays a very weak role. Thus, when consumers are dissatisfied and when they have a feeling of injustice, they make a complaint to restore the balance of the exchange from an economic and relational point of view. From an economic point of view, they wish to receive a proportional answer to their costs: the utilities of the complaint must therefore be higher than the perceived costs, including those related to the treatment of the complaint [START_REF] Walker | Procedural Justice : A psychological analysis[END_REF]. From a relational point of view, customers wish to be treated with consideration and respect by the company.

Hypotheses

The effectiveness of the complaint efforts of the company must be considered regarding the type of customers [START_REF] Hess | Service Failure and Recovery: The Impact of Relationship Factors on Customer Satisfaction[END_REF]Grégoire et al., 2009). According to Grégoire et al. (2009) the relationship quality with customers plays a fundamental moderating role on the compensations to be granted. If the relationship quality is good (bad) the level of necessary compensation efforts can (must) be lower (higher). A major weakness of their research is that they do not distinguish distributive and interactional efforts. We therefore suggest that the loyal individuals maintaining a strong relationship quality with the firm are searching more for interactional efforts than new customers. This could be justified by the importance of this dimension in the field of complaint management (Tax et al., 1998;[START_REF] Smith | A Model of Customer Satisfaction with Service Encounters Involving Failure and Recovery[END_REF]. In addition, the interactional efforts are more able to restore the status of the customer who might feel betrayed because of the service incident. They would like to be recognized as such [START_REF] Lind | The social psychology of procedural justice[END_REF]): H1: Loyal customers having a strong relationship quality with the firm prefer interactional (not-monetary) compensations to distributive (monetary) ones. On the other hand, new customers, having a low relationship quality with the firm, do not have identity connections with the company. They should therefore be attached to rebalance the exchange in economic terms: H2: New customers having a weak relationship quality with the firm prefer distributive (monetary) compensations to interactional (not-monetary) ones. In this context, we distinguish two types of compensations: money refunding and purchase voucher. The purchase voucher symbolizes the desire of the firm and the customer to continue the relationship. That is why we suggest that the loyal customers having a strong relational quality with the firm are more willing to accept purchase vouchers than refunding. This is co-herent with their complaints' targets to improve a given situation in case of dissatisfaction and to continue the relationship with the firm. H3: Loyal customers having a strong relationship quality with the firm prefer purchase vouchers to money refunding. On the other hand, a new customer does not need to restore trust and inevitably has not committed yet into a new long term relationship. H4: New customers having a weak relationship quality with the firm prefer refunding to purchase vouchers. Finally, the intensity of the effort of compensation (i.e. generosity) must be considered. In H1 we suggest that loyal customers having a strong relationship quality with the firm are more searching for consideration (interactional dimension) than economic benefits (distributive dimension). On the other hand, customers having a relationship quality with the firm are more instrumentally orientated, and consequently, they are probably more sensitive to the intensity of the effort of compensation (i.e. the monetary value; Grégoire et al., 2009). H5: The importance of the intensity of the effort of compensation is lower for loyal customers having a strong relationship quality with the firm than for new customers having a low relationship quality with the firm.

Methodology

To test our assumptions we choose the experimental methodology by scenario. The restaurant sector is selected because the service provider has to cope with many incidents because of the importance and the complexity of the interpersonal relationships (i.e. customers and personnel in contact). We consider two types of distributive compensations to distinguish their relative impact and their valence: 1) the nature: the restaurant offers a) a purchase voucher to be used on a forthcoming consumption or b) money refunding.

2) The intensity (or the monetary value of refunding or the purchase voucher) that corresponds to a) the total amount (100%) or b) a part (66%) of the value of the meal. Concerning interactional compensation, we compare the situation where a) the owner contacts the complainer by telephone in order to apologize (i.e. strong relational value) or b) the restaurant sends an impersonalized email (i.e. low relational value). 8 compensation scenarios with 2x2x2 dimensions (3 attributes of compensations having each 2 levels) were thus generated by an orthogonal design (Orthoplan, SPSS; see table 1).

The investigations have been carried out between 2010 and 2011 with 229 students from 3 French universities. The interviewees, either supposed to be a new consumer (N=86) or a loyal client (N=143), are invited to classify by descending preference the most desired compensation ("1") to the less preferred one ("8"). A random rotation of the scenarios is made before each investigation to avoid systematic bias. To calculate the partial utilities of the attributes, a conjoint analysis is used (Conjoint,SPSS 12.0)

Table 2. Attributes' importance and compensations' partial utilities

For loyal customers, compensations' interactional dimensions (i.e. the quality of the relationship) are much more important (83%) than for new customers (52%). H1 is thus validated. If the quality of the relationship with the firm is good, the compensations' distributive dimensions (nature = 8% and intensity of the effort = 9%) are significantly less important than for new customers. Moreover the purchase voucher, as expected in H3, is preferred to refunding by loyal customers, which confirms the intention to maintain the relationship with the supplier. In order to confirm this result, we carry out a supplementary inter-subject experimentation with another group of students. For this, we create two scenarios: 1) the restaurant offers refunding (N=39) or a purchase voucher (N=31). In both cases, we only consider the case of loyal customers who are contacted by the owner of the restaurant who offers a compensation of a value equivalent to the amount of the meal. The feeling of perceived justice is not significantly different according to the nature of the compensation (m voucher =5,13, m refunding = 5,08, p=0,86)1 . On the other hand, for new customers the nature (28%) and the value (20%) remain important, in spite of the fact that the relational value is the most important dimension. H2 is rejected but it becomes clear that the relational component of the complaint is less important for new customers than loyal ones. Refunding as a compensation which less links to the supplier has more partial utilities (.263) than purchase vouchers (-.263). H4 is validated. Finally in support with H5, the importance of the monetary value of the compensation is lower for loyal customers having a strong relationship quality with the firm. The differences are significant for all the assumptions (Chi-2 test, p< .01 or .05).

Discussion

The results show the importance of the relationship quality with the customer. On the one hand, the interactional efforts are preferred whatever the type of customers (loyal vs. new). It is thus important to establish a direct relationship with the customers; a phone call is preferred to an email. The quality of the relationship thus influences the effectiveness of the complaint management. For loyal customers, the direct contact with the firm is by far the most important compensation element. It might even enable firms to reduce the compensations' amounts. Lastly, loyal customers more easily accept purchase vouchers than refunding, because vouchers symbolize their intention to continue the relationship.

For new customers, even if interactional efforts constitute the most important factor, they try to rebalance the exchange in economic terms. They prefer a refunding because they do not need to restore trust and do not necessarily try to establish a long term relationship. These clients have an instrumental orientation, and are more sensitive to the intensity of the effort of the compensation. It is thus very important that the firm grants them a compensation equal to the totality service's value.

Managerial implications

For the management of complaints, it is important to understand which compensations are most valued according to the type of customers (new vs. loyal). This highlights the need for a differentiated reward management. The firm can offer limited distributive or monetary efforts to loyal customers if managers pay attention to interactional or relational elements. On the other hand, the distributive efforts must be maximized if the objective is to satisfy new customers.

Research directions

Variables such as the sector, the responsibility of the company for the incident and the customer involvement are likely to influence the results and could be integrated. On the theoretical level we study the impact of the compensations via preferences. It would be interesting to consider other variables of process such as the perceived justice or satisfaction. The effectiveness of the complaint management could also be approached by the measure of purchase, word-of-mouth or retaliation intentions. The effects of interactions between the compensations could be studied more thoroughly. Finally, individual financial indicators, such as "Customer Lifetime Value" [START_REF] Bolton | The theoretical underpinnings of customer asset management: a framework and propositions for future research[END_REF], could be integrated in future research, to grant compensations according to the value of the customers. This last point is important as the profitability of a complaint management program must especially be measured by its profit contribution.

Table 1 : Orthogonal scenario compensation design 5. Results The

 1 . relative importance of the compensations varies according to the type of client.

	Scenario 1.	Scenario 2.	Scenario 3.	Scenario 4.
	Call of owner	Call of owner	Call of owner	Call of owner
	Voucher, 66%	Voucher, 100%	Refunding, 66%	Refunding,100%
	Scenario 5.	Scenario 6.	Scenario 7.	Scenario 8.
	E-mail, Voucher	E-mail, Voucher	E-mail, Refunding,	E-mail,	Refunding,
	66%	100%	66%	100%

Perceived justice is measured using an item (with a 7 points Likert scale): "The compensation of the restaurant is just" (inspired by[START_REF] Blodgett | The effects of perceived justice on complainants' negative word-of-mouth behavior and repatronage intentions[END_REF].


PhD Vanessa Cooper
email: vanessa.cooper@pharmacy.ac.uk

Dr Graeme J Moyle

Dr Martin Fisher

Geraldine Reilly

Jacqueline Ewan

PhD Hui C Liu

Professor Rob Horne

Beliefs about antiretroviral therapy,treatment adherence and quality of life in a 48 week randomised study of continuation of zidovudine/lamividine or switch to tenofovir DF/emtricitabine, each with efavirenz

Keywords: HIV, Combivir, Truvada, adherence, beliefs about medicines

Beliefs about antiretroviral therapy

HIV [START_REF] Mocroft | Decline in the AIDS and death rates in the EuroSIDA study: an observational study[END_REF][START_REF] Palella | Declining morbidity and mortality among patients with advanced human immunodeficiency virus infection[END_REF][START_REF] Sterne | Longterm effectiveness of potent antiretroviral therapy in preventing AIDS and death: a prospective cohort study[END_REF], its success is dependent on continuous, long-term treatment [START_REF] El-Sadr | Risk for opportunistic disease and death after reinitiating continuous antiretroviral therapy in patients with HIV previously receiving episodic therapy: a randomized trial[END_REF] The Strategies for Management of Antiretroviral Therapy (SMART) Study Group, 2006) and high level of medication adherence [START_REF] Bangsberg | Less than 95% adherence to nonnucleoside reverse-transcriptase inhibitor therapy can lead to viral supression[END_REF][START_REF] Gross | Boosted PI are more forgiving of suboptimal adherence than non-boosted PI or NNRTI[END_REF][START_REF] Paterson | Adherence to protease inhibitor therapy and outcomes in patients with HIV infection[END_REF]. Rates of persistence with ART and adherence are suboptimal: approximately 28% of patients discontinue treatment over the first year [START_REF] Cooper | The impact of once versus twice daily dosing and baseline beliefs about HAART on adherence to the NRTI component of efavirenz-based HAART over 48 weeks: The NOCTE study[END_REF] and studies using objective measures suggest approximately 30% of patients miss doses each day [START_REF] Gross | Effect of adherence to newly initiated antiretroviral therapy on plasma viral load[END_REF][START_REF] Liu | A comparison study of multiple measures of adherence to HIV protease inhibitors[END_REF][START_REF] Mcnabb | Adherence to highly active antiretroviral therapy predicts virologic outcome at an innercity human immunodeficiency virus clinic[END_REF].

A multitude of barriers to ART adherence have been identified. These can be conceptualised as practical barriers (those that limit the patient's capacity to take their medication, leading to nonintentional non-adherence) and perceptual barriers (beliefs that impact on the individual's intention to take their medication as prescribed) [START_REF] Horne | Treatment perceptions and self-regulation[END_REF]. In order to facilitate adherence, both practical and perceptual barriers to ART need to be addressed [START_REF] Horne | Concordance, Adherence and Compliance in Medicine Taking: A conceptual map and research priorities[END_REF].

In recent years, the complexity of antiretroviral regimens has been greatly reduced and there are now a number of reasonably well tolerated and efficacious once-daily, low pill burden antiretroviral treatment regimens [START_REF] Johnson | A once-daily lopinavir/ritonavirbased regimen provides noninferior antiviral activity compared with a twice-daily regimen[END_REF][START_REF] Maggiolo | Oncea-day therapy for HIV infection: a controlled, randomized study in antiretroviral-naive HIV-1-infected patients[END_REF][START_REF] Molina | Five-year follow-up of once daily HAART with emtricitabine, didanosine and efavirenz (Montana ANRS 091 trial)[END_REF][START_REF] Santos | Simplicity and efficacy of a once-daily antiretroviral regimen with didanosine, lamivudine, and efavirenz in naïve patients: the VESD study[END_REF]. Once daily (QD) dosing is the preference of many patients [START_REF] Moyle | The Assessing Patients' Preferred Treatments (APPT-1) study[END_REF] and increases patient satisfaction with ART [START_REF] Maitland | Switching from twice-daily abacavir and lamivudine to the once-daily fixed-dose combination tablet of abacavir and lamivudine improves patient adherence and satisfaction with therapy[END_REF]. A recent meta-analysis comparing adherence to once daily Vs twice daily ART regimens showed a modest yet significantly greater rate of adherence (+2.9%) to once daily regimens over 11 published studies [START_REF] Parienti | Better adherence with once-daily antiretroviral regimens: a meta-analysis[END_REF]. Despite its benefits, once-daily ART is unlikely to fully address the problem of non-adherence, since perceptual barriers to adherence may remain. A range of perceptual barriers to ART adherence have been identified, [START_REF] Chesney | Self-reported adherence to antiretroviral medications among participants in HIV clinical trials: The AACTG adherence instruments[END_REF][START_REF] Gifford | Predictors of self-reported adherence and plasma HIV concentrations in patients on multidrug antiretroviral regimens[END_REF][START_REF] Molassiotis | Factors associated with adherence to antiretroviral medication in HIV-infected patients[END_REF][START_REF] Spire | Adherence to highly active antiretroviral therapies (HAART) in HIV-infected patients: from a predictive to a dynamic approach[END_REF]. These beliefscan be grouped under two categories: perceptions of necessity for treatment (necessity beliefs) and concerns about adverse effects [START_REF] Horne | Treatment perceptions and self-regulation[END_REF]Horne & Weinman, 1999;[START_REF] Horne | The Beliefs about Medicines Questionnaire: The development and evaluation of a new method for assessing the cognitive representation of medication[END_REF]. This 'necessity-concerns' framework has been found to predict adherence to medication across a variety of long-term illnesses [START_REF] Horne | Self regulation and self management in asthma: Exploring the role of illness perceptions and treatment beliefs in explaining non-adherence to preventer medication[END_REF]Horne et al., 1999), including HIV [START_REF] Cooper | The impact of once versus twice daily dosing and baseline beliefs about HAART on adherence to the NRTI component of efavirenz-based HAART over 48 weeks: The NOCTE study[END_REF][START_REF] Horne | Doubts about necessity and concerns about adverse effects: Identifying the types of beliefs that are associated with non-adherence to HAART[END_REF][START_REF] Horne | Patients' perceptions of Highly Active Antiretroviral Therapy in relation to treatment uptake and adherence: The utility of the Necessity-Concerns Framework[END_REF].

Patients' perceptions of their personal necessity for treatment and concerns about adverse effects stem from their perceptions of illness and medicines in general [START_REF] Horne | Treatment perceptions and self-regulation[END_REF] as well as their experiences of symptoms and side effects [START_REF] Cooper | The influence of symptom experiences and attributions on adherence to highly active anti-retroviral therapy (HAART): a six-month prospective, follow-up study[END_REF].., The primary analysis of the SWEET (Simplification With Easier Emtricitabine and Tenofovir) study [START_REF] Fisher | A Randomized Comparative Trial of Continued Zidovudine/Lamivudine or Replacement With Tenofovir Disoproxil Fumarate/Emtricitabine in Efavirenz-Treated HIV-1-Infected Individuals[END_REF]) demonstrated maintenance of virological control, increases in haemoglobin, declines in proatherogenic lipids and limb fat preservation among stable, virologically suppressed efavirenz-recipients who switched from combivir (CBV) (zidovudine plus lamivudine) to truvada (TVD) (emtricitabine and tenofovir).

The aim of this analysis was to assess the effect of switching from twice daily CBV to once daily TVD, each plus efavirenz (EFZ), on adherence, beliefs about ART (perceptions of necessity for ART, concerns about adverse effects and perceived treatment intrusiveness) and quality of life. AZT and 3TC or CBV plus EFV therapy for ≥6 months, with RNA <50 copies/ml on two consecutive visits and <400 copies/ml for ≥3 months prior to screening. Eligible subjects were randomised 1:1 to a) stop CBV and start TVD once-daily, or b) continue CBV twice-daily. Both TVD and CBV were taken as one tablet per dose, with or without food. All subjects continued EFV once-daily. The overall study methods are described elsewhere [START_REF] Fisher | A Randomized Comparative Trial of Continued Zidovudine/Lamivudine or Replacement With Tenofovir Disoproxil Fumarate/Emtricitabine in Efavirenz-Treated HIV-1-Infected Individuals[END_REF].

Methods

These

Measures

Adherence

Adherence was measured using the Medication Adherence Self-Report Inventory (MASRI) [START_REF] Walsh | Responses to a 1 month self-report on adherence to antiretroviral therapy are consistent with electronic data and virological treatment outcome[END_REF] at weeks 4, 12, 24 and 48. Subjects were asked to indicate the percentage of ART medication taken over the previous month on a visual analogue scale (VAS). The VAS has demonstrated good validity against objective measures (electronic monitoring: r = 0.63, P =0.001; pill count: r = 0.75, p= 0.001; and viral load: p= 0.01) [START_REF] Walsh | Responses to a 1 month self-report on adherence to antiretroviral therapy are consistent with electronic data and virological treatment outcome[END_REF]. Low adherence was defined as taking <95% of ART over the past month. Subjects with missing data were defined as having low adherence.

Beliefs about ART were assessed at baseline and weeks 4, 12, 24 and 48 using the Beliefs about Medicines Questionnaire (Horne et al., 1999) which has previously been adapted for antiretroviral therapy (BMQ-ART; [START_REF] Horne | Doubts about necessity and concerns about adverse effects: Identifying the types of beliefs that are associated with non-adherence to HAART[END_REF][START_REF] Horne | Patients' perceptions of Highly Active Antiretroviral Therapy in relation to treatment uptake and adherence: The utility of the Necessity-Concerns Framework[END_REF]). The BMQ-ART comprised 2 scales:

The ART-necessity scale consists of 8 items assessing patients' beliefs about their personal need for ART for controlling their HIV, maintaining their health, preventing illness and keeping them alive, while the ART-concerns scale consisted of 15 items bringing together a range of concerns about the potential adverse effects of ART that have been identified across studies, including fears about short-and long-term side effects, concerns about the timing of pills and the disruptive effects of the ART regimen on daily life, concerns about lacking information about ART and more abstract concerns about becoming dependent on ART, feeling that ART is a ''mystery,'' embarrassment surrounding taking ART and a general feeling of worry about taking the For the specific purposes of this study, an additional four items were included to elicit concerns about unwanted changes to the shape of buttocks, hips, face and arms.

Participants were presented with a series of statements about which they were told, ''These are statements that other people have made about combination therapy.'' They were then asked to rate their level of agreement with each necessity belief or concern on a scale, where responses ranged from strongly agree (scored 5) to strongly disagree (scored 1). Three items (2 concerns and 1 necessity belief) were reverse scored. Scores for the individual items within each scale were summed to give a total scale score. To facilitate comparison of scores between scales, a mean scale score was computed by dividing each scale by the number of items, giving a range of 1 to 5 for necessity and concerns scales with higher scores indicating stronger necessity beliefs or concerns.

Treatment intrusiveness

Perceived intrusiveness of the ART regimen was assessed at baseline and weeks 4, 12, 24 and 48 using the HAART Intrusiveness Scale (HIS) (Newell, Mendes, & Horne, 2002). This scale consisted of 12 items addressing the degree to which ART is perceived to interfere with ten aspects of daily life (e.g. social life, ability to work, relationships). Participants were asked to indicate the degree to which ART interfered with each aspect of their daily life on a scale from 1-5, where 1 indicated low interference and 5 indicated high interference. A total score was computed by adding up responses to each item. For comparison with other scales, an average score was computed by dividing the total score by the number of items. Possible responses ranged from 1-5 with higher scores indicating higher perceived intrusiveness. Quality of life was measured by the SF-12 v2 Health Survey [START_REF] Ware | A 12-Item Short-Form Health Survey: construction of scales and preliminary tests of reliability and validity[END_REF] at baseline and weeks 4, 12, 24 and 48. This scale has previously been used [START_REF] Viswanathan | Nature and correlates of SF-12 physical and mental quality of life components among low-income HIV adults using an HIV service center[END_REF] and validated [START_REF] Delate | The discriminative ability of the 12-item short form health survey (SF-12) in a sample of persons infected with[END_REF] with people with HIV. It contains either one or two items from each the following health concepts: physical functioning, role limitations arising from problems with physical health, bodily pain, general health, vitality (energy/fatigue), social functioning, role limitations arising from emotional problems and mental health (psychological distress and well-being). Two summary scales were generated: the physical component summary (PCS-12) and the mental component summary (MCS-12). Possible scores ranged from 0-100, with higher scores indicating better health. The scale has demonstrated acceptable validity and reliability [START_REF] Ware | A 12-Item Short-Form Health Survey: construction of scales and preliminary tests of reliability and validity[END_REF].

Quality of life

Viral load was recorded at each visit.

Statistical methods

Analyses were conducted using SPSS version 15.0. Significance was evaluated using two-tailed non-parametric tests at alpha=0.05.

The primary outcome of this analysis was adherence at 48 weeks. In an intention to treat analysis, where missing = nonadherent, subjects reporting ≥95% adherence were allocated to a 'high adherence' group and those who reported <95% adherence, discontinued the study or had missing adherence data were allocated to a 'low adherence' group [START_REF] Horne | Patients' perceptions of Highly Active Antiretroviral Therapy in relation to treatment uptake and adherence: The utility of the Necessity-Concerns Framework[END_REF]. A chisquare test was used to test for differences in adherence (high versus low) between randomised treatment groups. Change in the proportion of subjects reporting high (≥95%) adherence over time was assessed within each randomised group between week 4 and week 48 using

McNemar's test.

For all other variables, missing data were treated as missing. Differences in beliefs about ART (ART-Necessity and ART-Concerns), ART-intrusiveness and quality of life were assessed between randomised groups at baseline and week 48 using the Mann Whitney U Associations between continuous variables were assessed using the Spearman's Rho test.

Viral load

In order to compare viral load efficacy, the proportion of subjects with HIV RNA <50 copies/ml at week 48 was compared between randomised groups using the Chi-Square test. Subjects with missing viral load data were considered to have a detectable viral load (≥50 copies/ml).

Results

234 participants (84% male) took at least one dose of study medication, 117 continued CBV (CBV arm) and 117 switched to TVD (TVD arm).

Insert Table 1 about here

Comparisons at baseline

At baseline, there were no significant differences in beliefs about ART (Necessity or Concerns), ART-intrusiveness, or quality of life (PCS-12 or MCS-12) between randomised groups (all p>0.05; Table 3). Subjects randomised to receive TVD reported less concern about unwanted changes to the shape of their buttocks compared to those randomised to continue on CBV (Table 4).

Change over time

Adherence

At week 48, 87/117 (74.4%) subjects in the TVD arm reported ≥95% adherence compared to 73/117 (62.4%) of those randomised to remain on CBV (p=0.049; Table 2). The proportion of subjects reporting ≥95% adherence decreased significantly between weeks 4 and 48 for the group as a whole (McNemar's test, p<0.0001). Within groups analyses showed a significant

Insert Table 2 about here

Beliefs about ART ART-Necessity

Perceptions of necessity for ART did not differ between randomised groups at week 48, and there was no difference between the two groups in the extent to which perceptions of necessity changed over time (Table 3).

ART-Concerns

Subjects who switched to TVD reported fewer concerns about ART at the 48 week follow-up than those remaining on CBV (p=0.038). Furthermore, there was a greater decline in ART-concerns between baseline and 48 weeks among those who switched to TVD (p=0.007) (Table 3).

Insert Table 3 about here

Specific Concerns about unwanted changes to body shape

Switching to TVD resulted in a reduction of concerns about unwanted changes to the shape of their hips (p=0.032), buttocks (p=0.002), face (p=0.004) and arms (p=0.011) between baseline and week 48 (Table 4). These concerns decreased between baseline and 48 weeks among those who switched to TVD and increased among those who remained on CBV.

Insert Table 4 Subjects who switched to TVD reported less treatment intrusiveness than those who stayed on CBV at week 48 (p=0.004), however, the difference between groups in the change in intrusiveness from baseline to week 48 did not reach significance (p=0.057) (Table 3).

Quality of life

There were no significant differences between randomised groups in physical component summary (PCS) or mental component summary (MCS) scores at week 48, and no difference in the extent to which these variables changed over time (Table 3).

Associations between adherence, beliefs about ART and quality of life

Lower adherence at week 48 was associated with doubts about necessity for ART (p=0.020), stronger concerns about adverse effects (p=0.010), greater treatment-intrusiveness (p=0.010) and poorer mental health-related quality of life at week 48 (p=0.008) (Table 5).

Insert Table 5 about here

Viral load 99/117 (84.6%) of those randomised to remain on CBV and 102/117 (87.2%) of those who switched to TVD recorded an undetectable viral load (<50 copies/ml) at 48 weeks (p=0.573).

Discussion

Long-term adherence to antiretroviral therapy is pivotal to treatment success [START_REF] Bangsberg | Less than 95% adherence to nonnucleoside reverse-transcriptase inhibitor therapy can lead to viral supression[END_REF][START_REF] Gross | Boosted PI are more forgiving of suboptimal adherence than non-boosted PI or NNRTI[END_REF][START_REF] Paterson | Adherence to protease inhibitor therapy and outcomes in patients with HIV infection[END_REF]. In this study, switching from twice daily CBV to once daily TVD in people established on EFV resulted in higher levels of adherence and reduced both patients' concerns about adverse effects and the degree to which ART was perceived to intrude into daily life. We found no significant effect of switching from CBV to TVD on viral load or quality of life.

The results of this study concur with those of previous studies showing a benefit of once-daily ART for adherence [START_REF] Parienti | Better adherence with once-daily antiretroviral regimens: a meta-analysis[END_REF]. Reducing the frequency of ART to once-daily may reduce practical barriers to adherence by minimizing the opportunity to miss doses and facilitating the assimilation of treatment into daily routines. The effect of the change in regimen frequency on practical barriers to adherence is reflected in less reported intrusiveness at week 48 and a reduction of intrusiveness between baseline and week 48 among those who switched to TVD (although this result was of borderline significance).

Switching from CBV to TVD also reduced perceptual barriers to adherence. Several previous studies [START_REF] Cooper | The impact of once versus twice daily dosing and baseline beliefs about HAART on adherence to the NRTI component of efavirenz-based HAART over 48 weeks: The NOCTE study[END_REF][START_REF] Horne | Doubts about necessity and concerns about adverse effects: Identifying the types of beliefs that are associated with non-adherence to HAART[END_REF][START_REF] Horne | Patients' perceptions of Highly Active Antiretroviral Therapy in relation to treatment uptake and adherence: The utility of the Necessity-Concerns Framework[END_REF] have shown that patients who have strong concerns about adverse effects of ART are less likely to be highly adherent. In this study, switching from CBV to TVD reduced patients' concerns about their treatment. The concerns measure encompassed patients' concerns about side effects and potential long term effects, as well as concerns about the disruption of treatment to daily life and embarrassment about taking ART. The reduction in the overall score may therefore reflect both the reduction in the number of daily dosing and an improvement in side effects and longer-term effects including lipodystrophy [START_REF] Fisher | A Randomized Comparative Trial of Continued Zidovudine/Lamivudine or Replacement With Tenofovir Disoproxil Fumarate/Emtricitabine in Efavirenz-Treated HIV-1-Infected Individuals[END_REF]. In this case, switching from CBV to TVD also reduced specific concerns about changes to the shape of hips, buttocks, face and arms. This finding reflects the findings of the primary analysis of the SWEET study [START_REF] Fisher | A Randomized Comparative Trial of Continued Zidovudine/Lamivudine or Replacement With Tenofovir Disoproxil Fumarate/Emtricitabine in Efavirenz-Treated HIV-1-Infected Individuals[END_REF] [START_REF] Rajagopalan | Impact of lipoatrophy on quality of life in HIV patients receiving anti-retroviral therapy[END_REF] and a reduction in quality of life [START_REF] Kavouni | The face of HIV and AIDS: can we erase the stigma?[END_REF]. These types of side effects have also previously been linked to non-adherence [START_REF] Ammassari | Relationship between HAART adherence and adipose tissue alterations[END_REF].

Both individuals randomised to switch to TVD and those who remained on CBV remained convinced of their necessity for ART over the 48 weeks. In contrast to previous data showing an inverse relationship between persistent symptoms and perceptions of necessity for ART [START_REF] Cooper | The influence of symptom experiences and attributions on adherence to highly active anti-retroviral therapy (HAART): a six-month prospective, follow-up study[END_REF], the higher prevalence of side effects among those who remained on CBV (Fisher et al., 2009) did not impact on perceptions of necessity. This may be due to relatively strong perceptions of the need for ART among this highly adherent sample.

Despite the benefit of switching to TVD on adherence, we found no significant effect of switching from CBV to TVD on viral load at 48 weeks. The lack of a corresponding effect of regimen on viral load may be a result of the relatively high adherence threshold used. In setting our cut-off point for high adherence, we adopted the convention of 95% [START_REF] Paterson | Adherence to protease inhibitor therapy and outcomes in patients with HIV infection[END_REF]. Viral suppression may be achieved at lower rates of adherence nonnucleoside reverse transcriptase inhibitor (NNRTI)-based regimens [START_REF] Bangsberg | Less than 95% adherence to nonnucleoside reverse-transcriptase inhibitor therapy can lead to viral supression[END_REF]. Despite a greater level of adherence, fewer concerns about side-effects and less perceived treatment intrusiveness among those who switched to TVD, we did not find a difference in quality of life. It could be that the SF-12, a generic (rather than HIV specific) measure of quality of life lacked sensitivity to detect differences in physical and mental wellbeing resulting from the different regimens, since it does not include some of the relevant aspects of quality of life such as somatic symptoms and body image [START_REF] Grossman | Quality of Life and HIV: Current Assessment Tools and Future Directions for Clinical Practice AIDS Read[END_REF]. It is also worth noting that quality of life was good at baseline for the subjects in this study, with very little room for improvement.

Consistent with other studies, lower adherence was associated with patients' doubts about their necessity for ART and strong concerns about adverse effects [START_REF] Cooper | The impact of once versus twice daily dosing and baseline beliefs about HAART on adherence to the NRTI component of efavirenz-based HAART over 48 weeks: The NOCTE study[END_REF][START_REF] Horne | Doubts about necessity and concerns about adverse effects: Identifying the types of beliefs that are associated with non-adherence to HAART[END_REF][START_REF] Horne | Patients' perceptions of Highly Active Antiretroviral Therapy in relation to treatment uptake and adherence: The utility of the Necessity-Concerns Framework[END_REF], a higher level of perceived treatment intrusiveness [START_REF] Newell | Assessing the psychological and therapyrelated barriers to optimal adherence: an observational study[END_REF] and reduced mental health-related quality of life (Mannheimer et al., . We found no significant relationship between adherence and physical health-related quality of life. Again, this may be explained by the use of a generic rather than HIV specific quality of life scale, or to the fact that the study only included people who were already doing well on ART. A previous study found increases in quality of life over the first 12 months of ART among individuals who consistently reported higher adherence and a decrease in quality of life among patients reporting <80% adherence [START_REF] Mannheimer | Quality of life in HIV-infected individuals receiving antiretroviral therapy is related to adherence[END_REF].

The results of this study should be interpreted in light of its limitations. Adherence was not measured at baseline, therefore it was not possible to determine whether adherence differed between randomised groups at baseline, or to examine change from baseline within or between the two groups. The fact that this was a clinical trial with inclusion criteria of undetectable viral load on two consecutive visits means that the sample were biased in terms of being highly adherent. Although further studies with different populations would be useful, we would speculate that the benefits of TVD over CBV in terms of once daily dosing [START_REF] Parienti | Better adherence with once-daily antiretroviral regimens: a meta-analysis[END_REF] and fewer side effects [START_REF] Fisher | A Randomized Comparative Trial of Continued Zidovudine/Lamivudine or Replacement With Tenofovir Disoproxil Fumarate/Emtricitabine in Efavirenz-Treated HIV-1-Infected Individuals[END_REF] would also reduce perceptual and practical barriers to ART and improve adherence among broader clinic populations. Additional bias may have resulted from the fact that the trial was open-labelled, however, we are confident that our findings are not solely due to more positive expectations of the newer drug (TVD) because beliefs about ART became increasingly positive over the 48 weeks the participants were receiving TVD. The differences between randomised groups were modest. A further limitation was our reliance on self-report questionnaires. To minimize the possibility that individuals would give socially desirable responses, emphasis was placed on the fact that the individuals' responses were confidential and questionnaires were carefully worded in a nonjudgmental manner. Self-report measures have advantages over other methods of measuring adherence, including high face validity and high specificity for nonadherence; however, they are subject to self-presentation and recall bias. Our rating of adherence may underestimate the true extent of nonadherence (some people may be giving socially desirable responses, reporting higher adherence rates than they actually attain in practice), however, this bias was likely to be similar between randomised groups. The findings of this study present a clear rationale for the implementation of interventions to maintain higher levels of adherence by addressing perceptual and practical barriers to ART.

Switching to a once daily, lower toxicity regimen is one way of addressing these barriers. Given that our classification of low adherence included both taking less than 95% of doses, and stopping treatment completely, and the link between taking less than prescribed and discontinuation of treatment [START_REF] Vrijens | Adherence to prescribed antihypertensive drug treatments: longitudinal study of electronically compiled dosing histories[END_REF], these findings are particularly important in light of the current recommendation of continuous ART (Gazzard et al., 2008).

In summary, patients on long-term, twice daily CBV may benefit from switching to once daily TVD in order to reduce concerns about ART, facilitate integration of treatment into daily life and optimise adherence over the long-term.

  (ART) has vastly improved morbidity and mortality associated with

  data were collected as part of phase IV, open-label, UK-based, multi-centre, randomised, 48-week trial, which compared the continuation of CBV with substitution with TVD in patients treated with EFV-based ART. Eligibility criteria included being aged ≥18 years, being stable on

  in these variables between baseline and week 48 (48 week scores minus baseline scores) were compared between randomised groups using the Mann Whitney U test.

  who remained on CBV (McNemar's test p<0.0001) but not among those who switched to TVD (McNemar's test p=0.137).

  in which switching from CBV to TVD preserved limb fat and led to limb fat recovery. Previous studies have shown distribution to cause psychological distress

  about here

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	Treatment Intrusiveness
	10

Table 1 :

 1 Demographics and Baseline Characteristics (Treated Analysis Set)

		FTC/TDF + EFV	AZT+3TC + EFV	Total
	Characteristic	(N = 117)	(N = 117)	(N = 234)
	Sex (n, %)
	Male	101	86%	96	82%	197	84%
	Female	16	14%	21	18%	37	16%
	Age (years)
	Mean (SD)	42 (8.8)		42 (9.1)	42 (8.9)
	Min, Max	20, 69		24, 72		20, 72
	Race (n, %)
	White	77	66%	68	58%	145	62%
	Black	36	31%	44	38%	80	34%
	Asian	3	3%	4	3%	7	3%
	Other (Black Caribbean, Mixed	1	< 1%	1	< 1%	2	< 1%
	heritage)

Table 2 :

 2 Proportion of subjects reporting ≥95% adherence at each assessment during study

		FTC/TDF + EFV	AZT/3TC + EFV	Pearson's	p-value
		(N = 117)	(N = 117)	chi-square
	4 weeks (n,%)	96 (82.1)	95 (81.2)	0.028	0.866
	12 weeks (n,%)	93 (79.5)	86 (73.5)	1.165	0.281
	24 weeks (n,%)	92 (78.6)	81 (69.2)	2.683	0.101
	36 weeks (n,%)	87 (74.4)	74 (63.2)	3.365	0.067
	48 weeks (n,%)	87 (74.4)	73 (62.4)	3.874	0.049

Table 3 :

 3 Median scores for beliefs about ART, ART-intrusiveness and quality of life at baseline and week 48 and changes between baseline and week 48 in each treatment group, and p values for differences between groups (Mann Whitney U Test).

		FTC/TDF + EFV (N = 117)	AZT/3TC + EFV (N = 117)	p-value
	Measure	n	Median (IQR)	n	Median (IQR)
	Baseline
	ART-Necessity	107	4.37 (3.88, 4.63)	111	4.38 (3.75, 4.75)	0.935
	ART-Concerns	107	2.33 (2.00, 2.67)	111	2.47 (2.00, 2.87)	0.329
	ART-intrusiveness	109	1.25 (1.00, 1.58)	111	1.33 (1.00, 1.83)	0.376
	SF-12 Physical component	101	55.1 (48.9, 57.5)	108	53.7 (46.3, 57.5)	0.405
	SF-12 Mental component	101	49.1 (38.1, 54.7)	108	48.6 (40.5, 55.1)	0.548
	Week 48
	ART-Necessity	104	4.37 (3.75, 4.75)	97	4.50 (3.88, 4.88)	0.276
	ART-Concerns	104	2.31 (1.93, 2.31)	97	2.53 (2.13, 2.93)	0.038
	ART-intrusiveness	104	1.17 (1.00, 1.42)	97	1.33 (1.00, 1.88)	0.004
	SF-12 Physical component	97	53.2 (46.7, 56.7)	92	52.3 (42.6, 56.4)	0.191
	SF-12 Mental component	97	47.4 (40.9, 54.5)	92	47.6 (40.9, 54.3)	0.694
	Change to Week 48
	ART-Necessity	96	-0.00 (-0.25, 0.15)	93	0.13 (-0.13, 0.25)	0.159
	ART-Concerns	97	-0.05 (-0.50, 0.25)	93	0.07 (-0.13, 0.40)	0.007
	ART-intrusiveness	97	0.00 (-0.25, 0.00)	93	0.00 (-0.25, 0.21)	0.057
	SF-12 Physical component	85	0.00 (-2.79, 3.26)	86	-1.29 (-5.83, 2.84)	0.211
	SF-12 Mental component	85	0.46 (-4.92, 6.03)	86	-0.04 (-6.32, 5.43)	0.318

Table 4 :

 4 Median scores for concerns about unwanted changes in body shape at baseline and week 48 and changes between baseline and week 48 in each treatment group, and p values for differences between groups (Mann Whitney U Test)

		FTC/TDF + EFV (N = 117)	AZT/3TC + EFV (N = 117)
	Type of concern	n	Median (IQR)	n	Median (IQR)	p-value
	Baseline
	Shape of hips	107	2.00 (1.00, 3.00)	111	2.00 (2.00, 3.00)	0.171
	Shape of buttocks	107	2.00 (1.00, 3.00)	110	2.50 (2.00, 3.00)	0.039
	Shape of face	107	2.00 (2.00, 3.00)	111	2.00 (2.00, 3.00)	0.177
	Shape of Arms	106	2.00 (2.00, 3.00)	111	2.00 (2.00, 3.00)	0.114
	Week 48
	Shape of hips	104	2.00 (1.00, 3.00)	97	2.00 (2.00, 3.00)	0.061
	Shape of buttocks	102	2.00 (1.00, 3.00)	97	2.00 (2.00, 4.00)	0.016
	Shape of face	104	2.00 (1.00, 3.00)	97	2.00 (2.00, 3.00)	0.007
	Shape of Arms	104	2.00 (1.00, 3.00)	97	2.00 (2.00, 3.00)	0.063
	Change from baseline to
	Week 48
	Shape of hips	97	0.00 (-1.00, 0.00)	93	0.00 (0.00, 1.00)	0.032
	Shape of buttocks	95	0.00 (-1.00, 0.00)	92	0.00 (0.00, 1.00)	0.002
	Shape of face	97	0.00 (-1.00, 0.00)	93	0.00 (0.00, 1.00)	0.004
	Shape of Arms	96	0.00 (-1.00, 0.00)	93	0.00 (0.00, 1.00)	0.011

Table 5 :

 5 Associations between adherence, beliefs about ART and quality of life

	Self-reported adherence (MASRI-VAS)
	Spearman's rho	p-value

http://mc.manuscriptcentral.com/ac-phm-vcy Health Sciences

Acknowledgments

The SWEET Study Group, were: J Ainsworth (North Middlesex, London), J Anderson (Homerton University, London), G Brook (Central Middlesex, London), A de Burgh Thomas (Gloucester Royal), S Das (Coventry & Warwickshire), J Dhar (Leicester Royal Infirmary), M Fisher (Brighton & Sussex University), R Fox (Gartnavel General), V Harindra (St. Mary's, Portsmouth), P Hay (St. Georges, London), M Johnson (Royal Free, London), M Kapembwa (Northwick Park, London), M Kingston (Manchester Royal Infirmary), N Larbalestier (St. Thomas's, London), C Leen (Western General, Edinburgh), R Maw (Royal Victoria, Belfast), G Moyle (Chelsea & Westminster, London), F Mulcahy (St. James's, Dublin), E Ong (Newcastle General), C Orkin (Barts & Royal London), M Shahmanesh (Selly Oak, Birmingham), D White (Birmingham Heartlands), E Wilkins (North Manchester General), I Williams (University College London), Gilead Sciences Limited, Cambridge. The research was funded by Gilead Sciences.


Dr Eric Pasmant
email: eric.pasmant@gmail.com

Philippe Goussard

Laetitia Baranes

Ingrid Laurendeau

Samuel Quentin

Philippe Ponsot

Yann Consigny

Olivier Farges

Bertrand Condat

Dominique Vidaud

Michel Vidaud

Jian-Min Chen

Béatrice Parfait

First description of ABCB4 gene deletions in familial low phospholipid-associated cholelithiasis

Keywords: ABCB4, Deletion, Biliary disease, LPAC, MDR3

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

INTRODUCTION

ABCB4 (ATP-binding cassette subfamily B member 4) membrane transporter translocates phosphatidylcholine from the inner to the outer leaflet of the canalicular membrane of the hepatocyte. This floppase activity makes phosphatidylcholine available for extraction into the canalicular lumen by bile salts. The ABCB4 gene (MIM 171060) has shown to play a crucial role as evidenced by cholestatic liver diseases caused by its deficiency. [START_REF] Elferink | Hepatocanalicular transport defects: pathophysiologic mechanisms of rare diseases[END_REF] ABCB4 is also known as multidrug resistance 3 gene (MDR3), a member of the MDR/TAP subfamily involved in multidrug resistance as well as antigen presentation. The human ABCB4 gene is located on chromosome 7q21.1, contains 27 coding exons, and spans approximately 74 kb. [START_REF] Lincke | Structure of the human MDR3 gene and physical mapping of the human MDR locus[END_REF] The pathophysiology of ABCB4 alterations resides in lack of phospholipid protection in the bile against the detergent effect of bile salts, resulting in damage to the biliary epithelium, bile ductular proliferation, and potential progressive portal fibrosis. As biliary cholesterol solubilisation depends not only on the concentration of the sterol itself but also on the bile salt and phospholipid concentration, a decreased rate of phospholipid excretion can also be a cause of gallstone formation.

The wide clinical spectrum of the ABCB4 deficiency syndromes in humans covers cholestatic disorders presenting from the neonatal period of life to late adulthood. [START_REF] Elferink | Function and pathophysiological importance of ABCB4 (MDR3 P-glycoprotein)[END_REF][START_REF] Lucena | A multidrug resistance 3 gene mutation causing cholelithiasis, cholestasis of pregnancy, and adulthood biliary cirrhosis[END_REF][START_REF] Kano | Mutations identified in the human multidrug resistance P-glycoprotein 3 (ABCB4) gene in patients with primary hepatolithiasis[END_REF] At least three distinct syndromes with variable severity have been clearly identified: progressive familial intrahepatic cholestasis type 3 (PFIC3; MIM 602347), low phospholipid-associated cholelithiasis (LPAC alias gallbladder disease 1, GBD1; MIM 60080), and familial intrahepatic cholestasis of pregnancy (ICP; MIM 147480). Evidences of ABCB4 mutations have also been found in transient neonatal cholestasis, [START_REF] Jung | Prenatal molecular diagnosis of inherited cholestatic diseases[END_REF] or adult idiopathic biliary fibrosis or cirrhosis. [START_REF] Lucena | A multidrug resistance 3 gene mutation causing cholelithiasis, cholestasis of pregnancy, and adulthood biliary cirrhosis[END_REF][START_REF] Jacquemin | The wide spectrum of multidrug resistance 3 deficiency: from neonatal cholestasis to cirrhosis of adulthood[END_REF][START_REF] Rosmorduc | ABCB4 gene mutations and primary sclerosing cholangitis[END_REF][START_REF] Ziol | ABCB4 heterozygous gene mutations associated with fibrosing cholestatic liver disease in adults[END_REF][START_REF] Denk | ABCB4 deficiency: A family saga of early onset cholelithiasis, sclerosing cholangitis and cirrhosis and a novel mutation in the ABCB4 gene[END_REF][START_REF] Poupon | The cholangiographic features of severe forms of ABCB4/MDR3 deficiency-associated cholangiopathy in adults[END_REF] A recessive inheritance pattern of PFIC3 has been observed. [START_REF] Jacquemin | The wide spectrum of multidrug resistance 3 deficiency: from neonatal cholestasis to cirrhosis of adulthood[END_REF][START_REF] Vree | Mutations in the MDR3 gene cause progressive familial intrahepatic cholestasis[END_REF] Most ABCB4 mutations in patients with PFIC3 have been reported to be homozygous or compound heterozygous. 3,7,9,12- 4 [START_REF] Degiorgio | Molecular characterization and structural implications of 25 new ABCB4 mutations in progressive familial intrahepatic cholestasis type 3 (PFIC3)[END_REF] These mutations include missense and nonsense mutations and short frameshift deletions or insertions. ABCB4 mutations are associated with an absence or with a weak level of canalicular ABCB4 protein and with a low level of biliary phospholipids. [START_REF] Jacquemin | The wide spectrum of multidrug resistance 3 deficiency: from neonatal cholestasis to cirrhosis of adulthood[END_REF][START_REF] Vree | Mutations in the MDR3 gene cause progressive familial intrahepatic cholestasis[END_REF][START_REF] Deleuze | Defect of multidrug-resistance 3 gene expression in a subtype of progressive familial intrahepatic cholestasis[END_REF] Patients with PFIC3 usually present at a few years of age and suffer from severe chronic and progressive cholestasis. Liver histology often reveals fibrosis with portal inflammation and strong bile duct proliferation in an early stage. [START_REF] Maggiore | Diagnostic value of serum gamma-glutamyl transpeptidase activity in liver diseases in children[END_REF] A characteristic high serum gamma-glutamyltransferase activity is found in PFIC3. As a consequence of the cirrhosis, the PFIC3 patients are prone to gastrointestinal bleeding. About 50% of the patients need a liver transplantation. Interestingly, the other half may benefit from treatment with ursodeoxycholic acid (UDCA). [START_REF] Jacquemin | The wide spectrum of multidrug resistance 3 deficiency: from neonatal cholestasis to cirrhosis of adulthood[END_REF] Mutations in the ABCB4 gene that may reduce but not eliminate or drastically decrease the protein (leaving residual activity of the transporter) have been shown to cause a variety of milder cholestatic phenotypes, including LPAC and ICP. LPAC is a peculiar form of biliary gallstone disease characterized by intrahepatic sludge and/or symptomatic cholesterol cholelithiasis in young adults (usually before 40 years). LPAC is typically associated with mild chronic cholestasis, recurrence of symptoms after cholecystectomy, and prevention of recurrence by UDCA. About one third to half of patients have missense, frameshift, or nonsense mutations -mostly heterozygous -in the ABCB4 gene. [START_REF] Rosmorduc | MDR3 gene defect in adults with symptomatic intrahepatic and gallbladder cholesterol cholelithiasis[END_REF][START_REF] Rosmorduc | ABCB4 gene mutation-associated cholelithiasis in adults[END_REF][START_REF] Davit-Spraul | The spectrum of liver diseases related to ABCB4 gene mutations: pathophysiology and clinical aspects[END_REF] One of the hallmarks of LPAC is the response and remission induced by UDCA therapy. Heterozygous ABCB4 mutations were also identified in up to 15% of women suffering from ICP. [START_REF] Vree | Mutations in the MDR3 gene cause progressive familial intrahepatic cholestasis[END_REF][START_REF] Jacquemin | Heterozygous nonsense mutation of the MDR3 gene in familial intrahepatic cholestasis of pregnancy[END_REF][START_REF] Dixon | Heterozygous MDR3 missense mutation associated with intrahepatic cholestasis of pregnancy: evidence for a defect in protein trafficking[END_REF][START_REF] Gendrot | A second heterozygous MDR3 nonsense mutation associated with intrahepatic cholestasis of pregnancy[END_REF][START_REF] Müllenbach | ABCB4 gene sequence variation in women with intrahepatic cholestasis of pregnancy[END_REF][START_REF] Pauli-Magnus | BSEP and MDR3 haplotype structure in healthy Caucasians, primary biliary cirrhosis and primary sclerosing cholangitis[END_REF][START_REF] Keitel | Combined mutations of canalicular transporter proteins cause severe intrahepatic cholestasis of pregnancy[END_REF][START_REF] Wasmuth | Intrahepatic cholestasis of pregnancy: the severe form is associated with common variants of the hepatobiliary phospholipid transporter ABCB4 gene[END_REF][START_REF] Hay | Liver disease in pregnancy[END_REF][START_REF] Schneider | Linkage between a new splicing site mutation in the MDR3 alias ABCB4 gene and intrahepatic cholestasis of pregnancy[END_REF][START_REF] Floreani | Intrahepatic cholestasis of pregnancy; three novel MDR3 gene mutations[END_REF][START_REF] Floreani | Hepatobiliary phospholipid transporter ABCB4, MDR3 gene variants in a large cohort of Italian women with intrahepatic cholestasis of pregnancy[END_REF][START_REF] Bacq | ABCB4 gene mutations and single-nucleotide polymorphisms in women with intrahepatic cholestasis of pregnancy[END_REF] ICP is a reversible form of cholestasis that may develop in the third trimester of pregnancy, usually rapidly resolves after delivery and recurs in 45% to 70% of subsequent pregnancies. [START_REF] Hay | Liver disease in pregnancy[END_REF] The main symptoms are pruritus and, to a lesser extent, jaundice. Serum bile salt and aminotransferases levels are elevated. Increased incidence of foetal complications (including placental insufficiency, premature labor, and sudden fetal death) were reported in association with ICP. UDCA is the treatment of choice for ICP and produces relief of pruritus with improvement in liver tests with no adverse maternal or fetal effects. [START_REF] Hay | Liver disease in pregnancy[END_REF][START_REF] Mazzella | Ursodeoxycholic acid administration in patients with cholestasis of pregnancy: effects on primary bile acids in babies and mothers[END_REF][START_REF] Paus | Diagnosis and therapy of intrahepatic cholestasis of pregnancy[END_REF] It is generally accepted that women who have suffered from intrahepatic cholestasis of pregnancy are also susceptible to development of cholestasis on the use of oral contraceptives (oral contraceptives-induced cholestasis: CIC). [START_REF] Pauli-Magnus | Hepatobiliary transporters and drug-induced cholestasis[END_REF][START_REF] Ganne-Carrié | Progressive familial intrahepatic cholestasis type 3 revealed by oral contraceptive pills[END_REF] Evidences of ABCB4 heterozygous mutations have also been found in patients with drug-induced cholestasis or liver injury. [START_REF] Rosmorduc | MDR3 gene defect in adults with symptomatic intrahepatic and gallbladder cholesterol cholelithiasis[END_REF][START_REF] Rosmorduc | ABCB4 gene mutation-associated cholelithiasis in adults[END_REF][START_REF] Ganne-Carrié | Progressive familial intrahepatic cholestasis type 3 revealed by oral contraceptive pills[END_REF][START_REF] Lang | Mutations and polymorphisms in the bile salt export pump and the multidrug resistance protein 3 associated with drug-induced liver injury[END_REF] Loss of function ABCB4 mutations can present a large spectrum of cholestasis phenotypes and genetic analysis of ABCB4 can now be performed to confirm the diagnosis. [START_REF] Poupon | Combined features of low phospholipid-associated cholelithiasis and progressive familial intrahepatic cholestasis 3[END_REF] However, no ABCB4 mutations are found in a significant proportion of patients. The lack of ABCB4 mutation detection in remaining cases might be attributed to phenotyping errors, genetic heterogeneity and inadequacy of genetic screening methods. In the present study, we have tested the last hypothesis by using recent high resolution gene dosage methodologies in a large series of 102 adult patients with symptomatic intrahepatic cholelithiasis/cholestasis.

Here, we describe for the first time ABCB4 partial or complete deletions in patients with LPAC and CIC.

SUBJECTS AND METHODS

Patients

A panel of 102 clinically diagnosed index cases was phenotypically selected from routine genetic diagnosis. All patients were examined by reference hepatologists or gastroenterologists. The full clinical and radiological available information were recorded.

The written informed consent from each patient included in the study was obtained. In total, 59 unrelated adult women with ICP and/or CIC and 43 unrelated adult patients with a clinical presentation compatible with LPAC syndrome were included in this present study. For the latter group, we have considered patients presenting with intrahepatic cholelithiasis despite cholecystectomy before 40 years associated with at least one of the following criteria: presence of complications (cholecystitis, cholangitis or acute pancreatitis), intrahepatic hyperechoic foci with or without sludge or microlithiasis, response to UDCA therapy, and family history.

ABCB4 mutation screening

Total DNA was extracted from whole-blood leukocytes using the Nucleon BACC2 genomic DNA extraction kit (GE Health Care Europe, Amersham, UK). Genomic DNA was amplified with primers specific for the 27 ABCB4 (NM_018849.2) coding exons and their intron boundaries. The primer sequences and PCR conditions are available upon request. Mutation was identified using bidirectional DNA sequencing of purified PCR products. Sequences were aligned with Seqscape ® v5.1 analysis software (Applied Biosystems). ABCB4 molecular analysis was performed in the Biochemistry and Molecular Genetics Laboratory, Beaujon hospital, Clichy, France.

ABCB4 multiplex ligation-dependent probe amplification (MLPA) analysis

ABCB4 deletion screening was performed by MLPA analysis using the SALSA MLPA kit P109 ABCB4, as recommended in the manufacturer's protocol (MRC-Holland, Amsterdam, the Netherlands). This SALSA MLPA kit is designed to detect deletions/duplications of one or more exons of the ABCB4 gene. It contains 22 probes covering ABCB4 coding exons, three probes for ABCB4 promoter and three probes for the centromeric adjacent ABCB1 gene. For reference, several probes for other human genes located on different chromosomes are also included. Briefly, four control samples and patient samples (each containing 100 ng of genomic DNA) were used for overnight hybridization with the probe mixes. After ligation and amplification performed with FAM-labelled primers, PCR products were analysed on an ABI Prism 3130 automatic DNA sequencer (Applied Biosystems). Peak areas for each separated fragment were measured by using Genemapper software v4.0 (Applied Biosystems).

Normalized ratios of <0.6 and >1.3 were considered as deletions and duplications, respectively. Ratios profile between 0.6 and 0.85 were considered as doubtful. .DNA samples with ABCB4 copy number variation were further analyzed by real-time PCR-based gene dosage for confirmation.

Real-time PCR-based gene dosage

We quantified ABCB4 exons 10 and 11 by determining the threshold cycle (Ct) number at which the increase in the signal associated with exponential growth of PCR products begins.

We also quantified the ALB gene (encoding albumin) as an endogenous DNA control, and each sample was normalized on the basis of its ALB content, as previously described. [START_REF] Pasmant | Characterization of a 7.6-Mb germline deletion encompassing the NF1 locus and about a hundred genes in an NF1 contiguous gene syndrome patient[END_REF] ALB was selected as endogenous control because it maps to chromosome 4q11-q13, while ABCB4 is at chromosome 7q21.1. The relative copy number of the ABCB4 exon targets was also normalized to a calibrator, consisting of genomic DNA from a normal subject. Final results, expressed as N-fold differences in the ABCB4 exonic targets copy number relative to the ALB gene and the calibrator were determined as follows: N-fold = 2 (ΔCtsample -ΔCtcalibrator) , where ΔCt values of the sample and calibrator are determined by subtracting the average Ct value of the ABCB4 exon target from the average Ct value of the ALB gene. An N-fold value of <0.6 was considered deleted. All PCRs were performed on a LightCycler® 480 with the LightCycler® 480 SYBR Green I Master kit (Roche). PCR conditions and primer sequences are available on request. Experiments were done with triplicates for each data point.

Microarray characterization of ABCB4 complete deletions

The practical aspects of array-Comparative Genomic Hybridization (CGH) are described in detail elsewhere. [START_REF] Pasmant | Detection and characterization of NF1 microdeletions by custom high resolution array CGH[END_REF]

RESULTS

102 index cases with LPAC and ICP/CIC were screened on the basis of their phenotype for mutations in the ABCB4 gene. 32 ABCB4 heterozygous loss of function point or short insertion/deletion mutations (including nonsense, frameshift, splice, or previously reported/predicted to be deleterious missense mutations) were identified among the 102 tested index cases: in 37% (16/43) of LPAC patients and in 27% (16/59) of ICP/CIC patients.

The 70 adult patients with no ABCB4 mutation found (27 with LPAC and 43 with ICP/CIC) were consequently screened for large rearrangements using MLPA. Four heterozygous deletions were identified with this approach in 7% (3/43) of LPAC patients (BOC propositus of family 1, BEA propositus of family 2, and TIL) and in ~2% (1/59) of ICP/CIC patients (ROC) (Figure 1). The four deletions were confirmed using real-time PCR-based gene dosage.

Clinical features of individuals with ABCB4 gene deletions (family 1: patients BOC, ARE, and BOJ, family 2: patient BEA, and patients ROC and TIL) are summarized in table 1.

Figure 2 shows families 1 and 2 pedigrees including 12 and 5 patients with LPAC syndrome, respectively.

The four ABCB4 gross deletions were then accurately characterized. They included two whole gene deletions (TIL and ROC), and two partial intragenic deletions (BEA: exon 10 deletion and BOC: exons 11 to 19 deletion). Figure 3 The breakpoints of the two intragenic deletions (patients BEA and BOC) were precisely determined by sequencing the junction long-range PCR products. Only DNA sample from BEA yielded a 3055-bp fragment with primers JONCTIONBEA-U (5'-TGTGACTCGGACTATGGATTGTT-3') and JONCTIONBEA-L (5'-CCAAAACTGGATTCACACGCA-3'). Subsequent sequencing revealed that this deletion is in fact a complex rearrangement (Figure 4). A healthy control sample and BEA wild-type allele yielded a 4053-bp non deleted fragment using the same primers. Only DNA sample from BOC (family 1 propositus) yielded a 2,680-bp fragment with primers JONCTIONBOC-U (5'-AAAATAGACCCACTCAGGCAA-3') and JONCTIONBOC-L (5'-

ATGCTACATGCTTATCTAAAACCAT-3'). A healthy control sample yielded no PCR

product, the wild-type allele being too large (~27 kb) to be amplified. Sequencing the PCR product revealed a simple deletion of 24,720 bp (Supplementary Figure 1). This deletion was also detected in three other affected individuals with available DNA samples (Figure 2:

individuals II:4, II:7, and II:10 of family 1).

DISCUSSION

In this study, we showed for the first time that a significant subset of patients with symptomatic cholestasis/cholelithiasis has underlying ABCB4 deletions. Partial or complete heterozygous ABCB4 deletions were found in 7% of patients with LPAC and in ~2% patient with CIC. A large family of 12 affected patients with severe LPAC and cholecystectomy (Family 1) was notably reported (Table 1; Figure 2).

Recent gene dosage methodologies have allowed identification of these ABCB4 deletions. Our observations urge to systematically test patients with LPAC for the presence of ABCB4 deletions. MLPA, a sensitive, cost effective and rapid approach seems particularly adapted to routine diagnosis in molecular genetics. We developed a molecular algorithm tailored to ABCB4 routine analysis that includes ABCB4 gene dosage by MLPA in case of ABCB4 negative sequencing in patients with suggestive phenotype. MLPA allows a fast and inexpensive first-line screening for both partial and complete ABCB4 deletions, complementary to a high-resolution technique such as array-CGH that can be used to characterize larger deletions. Real-time PCR-based gene dosage is useful for deletions confirmation, particularly for samples with ratios profile considered as doubtful, i.e. between 0.6 and 0.85 (patient ROC; Figure 1).

The two intragenic deletion breakpoints were cloned at the nucleotide level and no recurrent breakpoints were found. That no significant sequence similarity was found between the centromeric and telomeric breakpoints of both deletions effectively excluded homologous recombination as the underlying mutational mechanisms in both cases. However, the presence of microhomology in each of the aberrant junctions is consistent with both the microhomology-dependent replication-based recombination (MMRDR) and non-homologous end joining (NHEJ) mechanisms. [START_REF] Chen | Genomic rearrangements in inherited disease and cancer[END_REF] Thus, the complex rearrangement detected in patient BEA can be perfectly explained by either serial replication slippage (SRS, a sub-pathway of MMRDR) or NHEJ repair of simultaneously generated double-strand breaks (Figure 4) whilst the simple 24 kb deletion (Supplementary Figure 1) is consistent with a single step of replication slippage (a sub-pathway of MMRDR), microhomology-mediated break-induced replication (MMBIR, a sub-pathway of MMRDR) or NHEJ. [START_REF] Chen | Genomic rearrangements in inherited disease and cancer[END_REF] In this study, adult patients with LPAC were screened for ABCB4 deletions. We assume that ABCB4 deletions may also be found in children with PFIC3. In some patients with PFIC3, only one ABCB4 mutation or no mutation have been previously reported while no ABCB4 protein was detected by immunohistochemistry analysis. [START_REF] Davit-Spraul | The spectrum of liver diseases related to ABCB4 gene mutations: pathophysiology and clinical aspects[END_REF] Some of these genotype-phenotype discrepancies could be explained by presence of ABCB4 deletions in PFIC3 patients.

However, in PFIC3 patient with no mutation found, a heterozygous ABCB4 deletion would not be sufficient to explain the phenotype because PFIC3 is an autosomal recessive disease.

Our observations urge to reassess ABCB4 molecular analysis in these patients using molecular tools allowing detection of rearrangements. PFIC3 patients who do respond to UDCA therapy generally have a partial ABCB4 defect (missense mutations) and the residual phospholipid concentration in bile combined with UDCA replacement, may be sufficient to reduce bile salt toxicity below a critical threshold. [START_REF] Davit-Spraul | The spectrum of liver diseases related to ABCB4 gene mutations: pathophysiology and clinical aspects[END_REF] ABCB4 genotyping (including deletions detection) should help to select those PFIC3 patients who could benefit from UDCA therapy.

In clinical practice, establishment of a comprehensive ABCB4 alteration screening algorithm will permit the use of ABCB4 genotyping to confirm the diagnosis of LPAC syndrome in 3 young adults who present with a symptomatic cholelithiasis and allow familial testing. One argument in support of molecular testing for ABCB4 deletion is benefit of UDCA therapy of both symptomatic and asymptomatic cholelithiasis in patients with ABCB4 deficiency. [START_REF] Rosmorduc | ABCB4 gene mutation-associated cholelithiasis in adults[END_REF] Depending on the results, long-term curative or prophylactic UDCA therapy could be initiated early to prevent the occurrence or recurrence of syndromes and their potential severe complications. Patient ROC who suffered from CIC may also be susceptible to development of ICP that carries a risk of premature delivery and sudden foetal death. Identification of ABCB4 deletions may also benefit patients with CIC and ICP, as UDCA is recommended to reduce pruritus, and probably prematurity without adverse side effects. [START_REF] Hardikar | Intrahepatic cholestasis of pregnancy: when should you look further?[END_REF] Comorbidity factors, environment influences, or unknown genetic modifiers may modulate 3 these phenotypes. [START_REF] Colombo | Clinical features and genotype-phenotype correlations in children with progressive familial intrahepatic cholestasis type 3 related to ABCB4 mutations[END_REF][START_REF] Davit-Spraul | The spectrum of liver diseases related to ABCB4 gene mutations: pathophysiology and clinical aspects[END_REF] Our observations reinforce the potential existence of these genetic modifiers.

Gene dosage technologies have allowed identification of ABCB4 deletions in a significant subset (7%) of patient with LPAC syndrome. An early diagnosis of this biliary disease would be beneficial because of the potential preventive effect of UDCA on biliary complications.

These data must now be taken into account in patient diagnosis and follow-up.

TITLES AND LEGENDS TO FIGURES

  Briefly, array-CGH labelling and hybridization were performed on Agilent whole human genome 400K microarrays as recommended in the Agilent manual (Protocol v6.3, October 2010, Agilent technologies). Patients' genomic DNA and six pooled normal control DNAs (reference) were labelled with Cy5-dUTP and Cy3-dUTP, respectively. Arrays were scanned with an Agilent DNA Microarray Scanner (G2565BA). Log2 ratios were determined with Agilent Feature Extraction software (v 9.1.3.1). Results were visualized and analyzed with Agilent's Genomic Workbench 5.0 software. DNA sequence information referred to the public UCSC (University of California Santa Cruz) database [Human Genome Browser, February 2009 assembly: hg 19, National Center for Biotechnology Information (NCBI) Build 37]. Fine characterization of ABCB4 intragenic deletion breakpoints Long-range PCR was performed with the Expand 20 kb Plus PCR kit as recommended by the manufacturer (Roche Applied Science). Primer pairs and PCR conditions used to characterize the gross deletions are available upon request. PCR products were sequenced with the ABI BigDye terminator sequencing kit (Applied Biosystems) on an ABI Prism 3130 automatic DNA sequencer (Applied Biosystems).

Figure 1 .

 1 Figure 1. ABCB4 SALSA MLPA kit P109 peak areas normalized ratio profiles of patients BEA, BOC, TIL, and ROC and one normal control DNA. Two partial intragenic deletions (patients BEA and BOC) and two whole gene deletions (patients TIL and ROC)were identified. The mean of normalized ratios in patients without deletion was 1.02.

Figure 2 . Pedigrees of families 1 and 2 .

 22 Figure 2. Pedigrees of families 1 and 2. Squares and circles indicate males and females, respectively. Clear symbols indicate unaffected individuals. Arrows indicate propositus. Individuals II:4, II:7, II:10, and IV:1 in family 1 and individual III:1 in family 2 were molecularly tested. Individual II:2 in family 1 was diagnosed with cholangiocarcinoma at 71 years of age.

Figure 3 .

 3 Figure 3. Array CGH profiles of the two partially characterized ABCB4 complete deletions: patients TIL and ROC. Zoom on ABCB4 region at chromosome 7q easily identified deletions. Horizontal blue line shows ABCB4 position. Human Genome Browser, February 2009 assembly: hg 19, National Center for Biotechnology Information (NCBI) Build 37.

Figure 4 .

 4 Figure 4. Illustration of two possible mechanisms underlying the complex rearrangement detected in patient BEA. (A) In the serial replication slippage (SRS) model, 42-43 the newly synthesized leading strand (C) misaligned with the lagging template strand (B) through inverted short repeats (the first step of slippage); having synthesised a short DNA sequence tract, the C strand dissociated from B and re-annealed to its original template strand (A) via short repeats (the second step of slippage); continued DNA synthesis (right-handed dotted arrow). Then replication against the synthesized strand C resulted in the observed complex rearrangement. (B) In the non-homologous end joining (NHEJ) model, at least two double-strand breaks (DSB) occurred within the sequences deleted. One short internal sequence was re-captured in an inverted orientation during the NHEJ repair. Numbers

  Management of ICP also includes close monitoring and early delivery for the foetus. All patients with ABCB4 defect in this study have benefited from UDCA treatment. These observations are remarkable examples of the interrelationship between molecular biology and clinical medicine.

	The extreme variability and the wide spectrum of ABCB4 alteration-related phenotypes make
	genotype-phenotype correlations difficult, although they are of crucial importance for patients
	and their families. Further comparative studies of patients with well-characterized genotypes
	(including deletions) and phenotypes will help determine whether ABCB4 mutation types
	influence clinical outcomes. Three of the four patients with deletions (BEA, BOC, and TIL)
	developed LPAC that recurred despite cholecystectomy. Patient ROC presented a very large
	de novo ~5 Mb deletion encompassing the ABCB4 locus and 20 additional genes including
	ABCB1 gene (Supplementary Table 1). Surprisingly, this patient presented a less severe
	phenotype only consisting in CIC, while being haploinsufficient for 21 genes. Unlike the
	ABCB4 gene, none of these 20 genes is known to be associated with an inherited human
	disease according to the OMIM database (http://www.ncbi.nlm.nih.gov/omim). Variable

expressions of liver diseases caused by ABCB4 mutations have previously been reported.

ACKNOWLEDGEMENTS

We thank the patients for their participation. We thank all the clinicians from France, who provided the samples for this study.

CONFLICT OF INTEREST

The authors declare no conflicts of interest. Abbreviations: AST, aspartate aminotransferase; ALT, alanine aminotransferase; GGT, gamma-glutamyl transpeptidase; U/L: units per liter. The usual ranges for AST, ALT and GGT are around 10 to 35 U/L, 10 to 45 U/L, and 10 to 55 U/L, respectively.