MD PhD FACC Dimitris Tousoulis
email: drtousoulis@hotmail.com

MD Nikolaos Papageorgiou

MD Theodora Psaltopoulou

MD Anastasios Giolis

MD Charalambos Antoniades

MD Eleftherios Tsiamis

MD Antigoni Miliou

MD Kostas Toutouzas

MD Gerasimos Siasos

Christodoulos Stefanadis

M D Facc

MD Ass Prof Dimitris Tousoulis

Divergent anti-inflammatory effects of different oil acute consumption on healthy individuals

Keywords: oil, ICAM-1, VCAM-1, TNF-a, inflammation

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

Evidence suggests that inflammatory process contributes to the development of atherosclerosis [START_REF] Libby | Leducq Transatlantic Network on Atherothrombosis. Inflammation in atherosclerosis: from pathophysiology to practice[END_REF]. The role of several inflammatory biomarkers, such as acute phase proteins, proinflammatory cytokines and adhesion molecules, is nowadays under investigation as regards the relation with cardiovascular disease [START_REF] Biasucci | CDC/AHA Workshop on Markers of Inflammation and Cardiovascular Disease: Application to Clinical and Public Health Practice: clinical use of inflammatory markers in patients with cardiovascular diseases: a background paper[END_REF].

Tumor necrosis factor alpha (TNF-a) is known to be released by macrophages, as well as vascular endothelial and smooth muscle cells, promoting the expression of cell adhesion molecules (CAMs) and the migration of leukocytes into and through the vascular wall [START_REF] Klein | Comparative studies on vascular endothelium in vitro. 3. Effects of cytokines on the expression of E-selectin, ICAM-1 and VCAM-1 by cultured human endothelial cells obtained from different passages[END_REF]. Migration to the arterial wall, rolling and tethering of leukocytes to the endothelium (adhesion) play a crucial role in atherogenesis.

Adhesion is a complex process depending on interactions between molecules such as the vascular adhesion molecule-1 (VCAM-1) and the intercellular molecule-1 (ICAM-1) on the endothelium and the integrins on neutrofils [START_REF] Meydani | Soluble adhesion molecules: surrogate markers of cardiovascular disease?[END_REF]. Interestingly, studies have shown that despite the similar structure and function of VCAM-1 and ICAM-1, these two molecules may participate at different stages of atherogenesis [START_REF] Blann | Cell adhesion molecules in cardiovascular disease and its risk factors--what can soluble levels tell us?[END_REF].

Recently, it is widely reported that dietary habits may modify cardiovascular disease rates in diverse populations. Adhesion molecules and proinflammatory cytokines are implicated as important mediating pathways for cardiovascular diseases, activated though different intakes of food groups, such as alcohol [START_REF] Bau | Alcohol consumption, cardiovascular health, and endothelial function markers[END_REF], as well as obesity [START_REF] Forsythe | Obesity and inflammation: the effects of weight loss[END_REF]. Accordingly, despite the fact that it is not easy to specify substances with anti-atherosclerotic properties comprising diets, studies have shown that components such as olive oil or n-3 fatty acids may play a key role in cardiovascular protection, by especially inhibiting inflammatory process [START_REF] Perez-Martinez | Dietary fat differentially influences regulatory endothelial function during the postprandial state in patients with metabolic syndrome: From the LIPGENE study[END_REF][START_REF] Massaro | Basic mechanisms behind the effects of n-3 fatty acids on cardiovascular disease[END_REF]. However, the impact of acute consumption of different types of oil on proinflammatory cytokines and adhesion molecules remains unknown.

Thus, the aim of the present study was to examine the acute effects of extra virgin olive oil, soy oil, corn oil and cod-liver oil on circulating levels of sVCAM-1, sICAM-1 and TNF-a in young, healthy individuals.

Materials and methods

Study population and protocol

A total number of 66 healthy young subjects were recruited randomly for this parallel study according to their age, gender and body mass index (BMI). Exclusion criteria were the presence of clinical evidence for atherosclerotic peripheral vascular disease or coronary atherosclerosis, the presence of any acute or chronic inflammatory disease (eg. liver disease). Subjects with any classic risk factor for atherosclerosis such as smoking, as well as those receiving any systemic medication, were also excluded from the study. Apart from the questionnaire given asking clinical and medical variables, anthropometric and blood pressure assessments were made.

All the participants were randomly allocated into 4 groups to receive 4 different types of oil, in order to receive an equivalent amount of 50 ml (and almost equal kilocalories) [START_REF] Tousoulis | Acute effects of different types of oil consumption on endothelial function, oxidative stress status and vascular inflammation in healthy volunteers[END_REF][START_REF] Vogel | Table 1. Demographic characteristics of the participants Olive oil Soy oil Corn oil Cod liver oil p[END_REF]. Each type of oil had a known macronutrients composition commercially available. Thus, the composition (provided by the manufacturer) per 100ml of each type of oil was: extra virgin olive oil (824 kcal or 3449.9 kJ, 91.6gr fatty acids, 12.8gr saturated, 70.5gr monounsaturated, 8.3gr polyunsaturated), corn oil (825 kcal or 3454.1 kJ, 92gr fatty acids, 12gr saturated, 25gr monounsaturated, 55gr polyunsaturated), soy oil (825 kcal or 3454.1 kJ, 92gr fatty acids, 14gr saturated, 23gr monounsaturated, 55gr polyunsaturated) and cod liver oil (830 kcal or 3475.0 kJ, 92gr cod oil, 0.00828gr eicosapentaenoic acid, 0.00736gr docosahexaenoic acid, 46gr monounsaturated fatty acids, 30gr polyunsaturated fatty acids, 0.0112gr vitamin A, 0.0001gr vitamin D, 0.068gr vitamin E). It is worth to mention that extra virgin olive oil was administered, as minor components (eg. β-carotene, a-tocopherol, β-sitosterol, tyrosol, oleuropein, hydroxytyrosol) of virgin olive oil are thought to be responsible for much of its beneficial effects. Oils were administered directly from their commercial form (liquid)

and not as part of a meal. Measurements were performed at baseline (0 hours) and 3 hours post oil consumption. None of the participants followed any specific recommendation regarding diet or physical activity prior to the study. All subjects participated normally to all their daily activities without modifications. Briefly, all measurements were performed in the morning in a dark quiet room under controlled temperature (22-24 o C) and after patients were at a rest for at least 10 minutes. All subjects abstained from food, tobacco, alcohol and caffeine-containing drinks or antioxidant supplements such as juices for at least 8 hours before oil ingestion (baseline measurement). In addition all subjects abstained from the aforementioned parameters during the 3 hours (before the second measurement) post oil consumption.

The study protocol was approved by the Institutional Ethics Committee and written informed consent was obtained from all subjects. In addition, the study was carried out in accordance with the Declaration of Helsinki II and European Union guidelines for good clinical practice.

Laboratory measurements

Venous blood samples were obtained at the beginning of each protocol and at 3 hours post intervention. Routine chemical methods were used to determine serum concentrations of total cholesterol, triglycerides, high density lipoprotein and lowdensity lipoprotein cholesterol. After centrifugation at 1150g at 4 o C for 10 minutes, serum was collected and stored at -80 o C until assayed. Quantitative sandwich enzyme immunoassay technique was used to determine serum sVCAM-1 (ng/mL) (Quantikine human sVCAM-1 ELISA kit, R&D Systems), sICAM-1 (ng/mL) (Quantikine human sICAM-1/CD54 ELISA kit, R&D Systems), as well as TNF-a levels (pg/ml) (Quantikine HS, human TNF-a/TNFSF1A ELISA kit, R&D systems).

Statistical analysis

All variables were tested for normal distribution by Kolmogorov-Smirnov test. Normally distributed variables are presented as mean values with standard errors (mean±SD), whereas not normally distributed data are presented as median (25-75th percentiles values). Correlations were assessed by Spearman's correlation coefficient (rho) due to not normally distributed data. Between groups comparisons were performed by X 2 test for categorical variables or one-way ANOVA, followed by Bonferoni post hoc correction for multiple comparisons of normally distributed continuous variables or by Kruskal-Wallis followed by Dunn's post test for not normally distributed variables. Moreover, the effect of oil consumption on each variable was examined by performing paired t-test or Wilcoxon signed-rank test as appropriate. Our study was powered based on the data regarding to sICAM-1: pilot data suggested that the difference in the response of matched pairs is normally distributed with standard deviation 13.0. If the true difference in the mean response of matched pairs is 66.77, we would need to study 4 pairs of subjects to be able to reject the null hypothesis that this response difference is zero with probability (power) 0.99. The Type I error probability associated with this test of this null hypothesis was 0.01. All analyses were carried out by using the SPSS v18.0 (SPSS, Chicago, IL) statistical package.

Results

The demographic characteristics and lipid profile of the participants are presented in Table 1. There were no significant differences between the four groups at baseline.

Effect of oil consumption on sVCAM-1 and sICAM-1

Acute consumption of olive oil, soy oil, cod liver oil as well as corn oil had no significant effect on sVCAM-1 levels (p=NS for all) (Figure 1). Interestingly, cod liver oil (p<0.01), olive oil (p<0.01), as well as soy oil (p<0.01) decreased sICAM-1 levels significantly. Controversially to the neutral/slightly negative effect of corn oil on sVCAM-1, the latter had also a significant effect on sICAM-1 levels (p<0.01) (Figure 2).

Effect of oil consumption on TNF-a

Extra virgin olive (p<0.05), soy oil (p<0.01) and cod liver oil intakes (p<0.01) reduced significantly TNF-a levels, whereas corn oil induced a non-significant decrease in TNF-a levels (p=NS) (Figure 3). Interestingly, the absolute change in TNF-a levels (39 pairs) was significantly correlated with the absolute change in ICAM-1 levels (66 pairs) after oil consumption (rho=0.379, p<0.05) (Figure 4a). However, there was no significant correlation between absolute changes in TNF-a levels and VCAM-1 levels (59 pairs) (rho=0.019, p=NS) (Figure 4b).

Discussion

In the present study we assessed the acute effect of extra virgin olive oil, soy oil, corn oil and cod liver oil consumption on adhesion molecules and proinflammatory cytokines in healthy individuals. We found that all types of oil induced a significant reduction in ICAM-1 levels. In addition, extra virgin olive oil, soy oil and cod liver oil reduced also TNF-a levels. Moreover, we observed a significant correlation between the absolute change of TNF-a and ICAM-1 levels.

These findings indicate that specific types of oil exert significant anti-inflammatory effects by affecting proinflammatory cytokines and specific adhesion molecules.

Effect of oil acute consumption on adhesion molecules

It is a fact that adhesion molecules participate in the development of atherosclerosis. In addition, it has been shown that dietary habits or modifications may prevent atherogenesis by affecting inflammatory molecules such as VCAM-1 and ICAM-1.

Mena et al [START_REF] Mena | Inhibition of circulating immune cell activation: a molecular antiinflammatory effect of the Mediterranean diet[END_REF] while assessing the relationship between Mediterranean diet (supplemented with virgin olive oil) and adhesion molecules, showed a significant inverse correlation of this dietary pattern with ICAM-1 and VCAM serum levels. Interestingly, in the PREDIMED study [START_REF] Salas-Salvadó | Components of the Mediterranean-type food pattern and serum inflammatory markers among patients at high risk for cardiovascular disease[END_REF] among middle aged individuals at high cardiovascular risk , the highest consumption of nuts and virgin olive oil was associated with the lowest concentrations of VCAM-1, ICAM-1, albeit only for ICAM-1 was this difference statistically significant in the case of nuts and for VCAM-1 in the case of virgin olive oil. The results were not in the same direction when analyzing n-3 fatty acids; in a prospective, randomized, double-blind study enrolling patients with myocardial infarction to receive highly concentrated n-3 polyunsaturated fatty acids (PUFAs) or corn oil, inflammatory markers, such as E-selectin and ICAM-1 were unaffected 1 year later [START_REF] Grundt | Clinical outcome and atherothrombogenic risk profile after prolonged wash-out following long-term treatment with high doses of n-3 PUFAs in patients with an acute myocardial infarction[END_REF]. Importantly, Fuentes et al [START_REF] Fuentes | Chronic effects of a highfat diet enriched with virgin olive oil and a low-fat diet enriched with alphalinolenic acid on postprandial endothelial function in healthy men[END_REF] showed that different types of fat composition meals affected differentially (in 4 hours) VCAM-1 levels in healthy men, as monounsaturated fatty acids appeared to be more beneficial than a-linoleic by reducing VCAM-1 levels. Data seem more conflicting, when referring to healthy adults. Studies such as this by Eschen et al [START_REF] Eschen | Soluble adhesion molecules in healthy subjects: a dose-response study using n-3 fatty acids[END_REF] have shown a dose dependent effect as it was observed a significant decrease in ICAM-1 in the PUFA 2.0gr group and a significant increase in VCAM-1 in the PUFA 6.6gr group. However, other studies conducted in young healthy subjects, showed that diets rich in lean fish significantly decreased ICAM-1 levels, but had had no effect on VCAM-1 levels [START_REF] Paulo | Influence of n-3 polyunsaturated fatty acids on soluble cellular adhesion molecules as biomarkers of cardiovascular risk in young healthy subjects[END_REF].

In the present study we have shown that VCAM-1 levels are unaffected after ingestion of all types of oil, a finding which is in line with our previous observation in a smaller cohort [START_REF] Tousoulis | Acute effects of different types of oil consumption on endothelial function, oxidative stress status and vascular inflammation in healthy volunteers[END_REF]. Importantly, we have demonstrated a significant non specific acute effect of olive, soy, corn and cod liver oil on circulating levels of sICAM-1. It is likely that a common ingredient in all types of oil may modify the cleavage rate of sICAM-1 on cell membranes leading to rapid changes of its circulating levels.

Effect of oil acute consumption on TNF-a

Tumor necrosis factor alpha has a crucial role in the inflammatory process implicated in atherogenesis. Thus, dietary modification targeting TNF-a could be beneficial in preventing atherosclerosis, as suggested by studies focusing on olive oil, soy oil and polyunsaturated lipids.

Consumption of olive oil as a single component reduced significantly not only VCAM-1, but also TNF-α serum levels in coronary venous blood, as reported by [START_REF] Serrano-Martinez | A Mediterranean dietary style influences TNF-alpha and VCAM-1 coronary blood levels in unstable angina patients[END_REF]. In addition, not only extra virgin olive oil, but also refined olive oil has been found to downregulate TNF-a [START_REF] Marquez-Martin | Modulation of cytokine secretion by pentacyclic triterpenes from olive pomace oil in human mononuclear cells[END_REF]. Moreover, dietary intervention with n-3 PUFA and plant sterols reduced systemic inflammation in hyperlipidemic individuals, reflected in a decrease of TNF-a levels by 10% [START_REF] Micallef | Anti-inflammatory and cardioprotective effects of n-3 polyunsaturated fatty acids and plant sterols in hyperlipidemic individuals[END_REF]. Interesting results have been reported by studies of soy consumption. Thus, TNF-a levels were significantly lower in the soy nut group compared to the soy protein and control groups as this was demonstrated by Azadbakht et al [START_REF] Azadbakht | Soy protein intake, cardiorenal indices, and C-reactive protein in type 2 diabetes with nephropathy: a longitudinal randomized clinical trial[END_REF] implying a role in the soya lipid fraction. In contrast to these findings, Jenkins et al [START_REF] Jenkins | Effects of high-and low-isoflavone soyfoods on blood lipids, oxidized LDL, homocysteine, and blood pressure in hyperlipidemic men and women[END_REF] showed that there was no significant effect on interleukin-6 or TNF-a in middle aged men and women on the high-isoflavone versus the low-isoflavone soy food diet.

In the present study we have shown that acute consumption of extra virgin olive oil, soy oil and cod liver oil reduced significantly TNF-a levels, while corn oil induced a non significant decrease in TNF-a levels. It is likely that the different types of oils may exert different intracellular "antioxidant" effects, which are then reflected to a variable activation of intracellular redox sensitive pathways (such as nuclear factor-κB and activator protein-1). This different effect on intracellular redox signaling could perfectly explain the differences in the circulating levels of soluble adhesion molecules, which represent a marker of the expression of these molecules on the cellular surface, a process regulated by these redox-sensitive transcriptional pathways. We have also found a significant association between the absolute changes (d) in TNF-a levels and ICAM-1 levels. These findings indicate that the expression of the proinflammatory cytokine TNF-a which was modified by oil consumption may regulate the subsequent expression of ICAM-1. Therefore, a potential pathway linking these molecules may be affected by the anti-inflammatory effects of specific oils.

Although our findings point-out a significant role for oil acute consumption in inflammatory markers, it is difficult to extrapolate our results to clinical practice, as our study was mainly a pathophysiological study designed to assess the potential pathways mediating the effect of different oil acute consumption on inflammatory process.

In conclusion, acute consumption of all types of oil decreased significantly ICAM-1 levels. In addition, olive oil, soy oil and cod liver oil decreased significantly TNF-a levels. Moreover, the degree of change in TNF-a levels was correlated with the degree of change in ICAM-1 levels, indicating that the expression of the proinflammatory cytokine TNF-a after oil consumption, may regulate the subsequent expression of ICAM-1 level. These findings suggest that soy oil, cod liver oil and virgin olive oil exert specific significant anti-inflammatory effects.

Figure 1 .

 1 Figure 1. Effect of different types of oil acute consumption on sVCAM-1 levels.

Figure 2 .

 2 Figure 2. Effect of different types of oil acute consumption on sICAM-1 levels.

Figure 3 .

 3 Figure 3. Effect of different types of oil acute consumption on TNF-a levels.

Acknowledgements:

None

Conflict of interest:

The authors declare that there is no conflict of interest

Figure legends

Figure 1 All types of oil had no significant effect on sVCAM-1 levels (ng/mL) (p=NS for all).Values are expressed as mean±SD.

Figure 2

Cod liver oil, olive oil, soy oil and corn oil decreased sICAM-1 levels (ng/mL) significantly (p<0.01 for all). Values are expressed as mean±SD.

Figure 3

Extra virgin olive (p<0.05), soy oil and cod liver oil consumption (p<0.01 for both) reduced significantly TNF-a levels (pg/ml). No effect was observed for corn oil (p=NS). Values are expressed as mean±SD.

Figure 4 a) Acute oil consumption resulted in a significant correlation between the absolute changes(d) in TNF-a levels (pg/ml) and ICAM-1 levels (ng/mL) b) (rho=0.379, p<0.05). b) There was no significant correlation between the absolute changes(d) in TNF-a levels and VCAM-1 levels (rho=0.147, p=NS).


Parents' and children's communication about genetic risk: qualitative

study learning from families' experiences.

Parents' and children's communication about genetic risk: qualitative study learning from families' experiences.

Running title: Genetic risk communication within families

INTRODUCTION

In families affected by genetic conditions, parents not only have the challenge of explaining the illness, its treatment and outcomes but they also have the concomitant stressor of discussing the inherited nature of the condition with their children. Many parents struggle to know when, how and what to tell their children about the genetic risks because they fear causing distress and so feel a need to protect their children from this information [4][5][6][START_REF] Fanos | Family pictures: Growing up with a brother with X-linked severe combined immunodeficiency[END_REF][START_REF] Canam | Coping with feelings: chronically ill children and their families[END_REF] . Many parents also describe a large burden of guilt, which can contribute to their reticence in discussing genetic conditions with their family [START_REF] Fanos | Family pictures: Growing up with a brother with X-linked severe combined immunodeficiency[END_REF] . Previous studies however, have shown that withholding genetic risk information from children until they are adult can affect their coping, self esteem [START_REF] Spiridigliozzi | Genetic Risk Communication: Experiences of Adolescent Girls and Young Women from Families with Fragile X Syndrome[END_REF][START_REF] Fanos | Sib understanding of genetics and attitudes toward carrier testing for X-linked severe combined immunodeficiency[END_REF] reproductive decision-making [START_REF] Fanos | Family pictures: Growing up with a brother with X-linked severe combined immunodeficiency[END_REF][START_REF] Fanos | Sib understanding of genetics and attitudes toward carrier testing for X-linked severe combined immunodeficiency[END_REF] and family cohesion [START_REF] Sobel | Ambigious loss and disenfranchised grief: the impact of DNA predictive testing on the family as a system[END_REF][START_REF] Sobel | Impact of genetic testing for Huntingtons Disease on the family system[END_REF] .

Studies examining families' communication of genetic risk information between children and their parents, which involve all members' perspectives including affected and unaffected children, have to-date been limited. With regard to children and genetic risk, the focus of debate has centred on genetic testing and the best time to carry it out rather than how to help the families cope and come to terms with the condition and its risk. Many health professionals report being asked for advice on talking to children (personal communications). However little is known about children's understanding and experiences of learning about inherited genetic risk within their families. Studies to date are small [START_REF] Metcalfe | Family communication between children and their parents about inherited genetic conditions: a meta-synthesis of the research[END_REF] , limited to a single condition and have rarely included all family members; affected and unaffected individuals and those at risk of carrying the affected gene. Differences related to patterns of inheritance, age of onset, morbidity and mortality, where parents have to discuss the genetic condition with their children at contrasting stages of their development have not been examined.

To address these gaps, this study explored communication about genetic risk and the factors that affect it with all family members. Six different genetic conditions were chosen which encompassed different morbidity and mortality patterns in relation to the more common, mendelian inheritance patterns. The aim was to draw on a breadth of families' experiences to ascertain what information children require at different stages of their childhood, how to provide it and their likely reactions to genetic risk information according to morbidity and mortality outcomes of the genetic condition.

METHOD

Theoretical Framework

Family communication about genetic risk was investigated qualitatively from the perspective of three inter-related models. These focused on the family members' roles and influential factors (family system theory [START_REF] Segrin | Family Communication[END_REF] ), what was communicated and the language used (drawing on symbolic interaction theory [START_REF] Blumer | Symbolic Interactionism: Perspective and Method[END_REF] ) and the behavioural outcomes [START_REF] Bandura | Social Learning Theory[END_REF]

Participant families

Potential participant families were volunteers identified via charity and NHS support groups in England and Wales. Parents and their children (irrespective of their risk status; affected, at risk or unaffected) from families affected by one of six genetic conditions were invited to participate according to the inclusion criteria. It was anticipated that 6-7 families would be recruited for each condition (table 1). The genetic conditions were chosen because not only do they have a distinct pattern of inheritance and vary in the age of onset but also because genetic testing may take place at different ages, some diseases caused are life-limiting and there are different impacts on the individual in terms of illness duration.

Data collection

Semi-structured interviews were conducted with parents, children (8-12years), young people (13-17years) and adult children (18+years). Children were interviewed independently of their parents or siblings. Consent was obtained to audio-record interviews in all cases except one.

Interview schedules were developed from the literature review and discussion with the advisory and steering groups. The interviews used neutral, open ended questions and interviewers were responsive in adapting their questions, language style and question order according to the responses received. The questions focused on family members' roles in discussing the genetic condition and its risk, the language used, and the behavioural outcomes in terms of emotional response, coping and adaptability. For children, art based portfolios were developed for use prior to interviews [START_REF] Coad | Developing art-based techniques for eliciting children and young people's views about their family communication in genetic conditions[END_REF] to provide a sense of control in the activity and assist with establishing a rapport and an environment conducive to the discussion of personal and sensitive information [START_REF] O'kane | The Development of Participatory Techniques Chapter 7[END_REF][START_REF] Mauthner | Methodological Aspects of Collecting Data from Children[END_REF] .

Data Analysis

Interviews were transcribed verbatim and analysed using social constructivist grounded theory [START_REF] Corbin | Basics of Qualitative Research[END_REF] . Due to the large quantity of data collected two analysis frameworks, one for children and the other for adults were developed using interviews from five families affected by different genetic conditions. The two frameworks kept the children's perspective clearly separate from the adults at this stage.

Transcripts and field notes were read for each interview. Two researchers developed each framework, initially working independently to identify phenomena within the interview and develop concepts and a list of categories before agreeing an overall list for the framework with the second researcher. As part of developing the framework the concepts were also considered in terms of family communication models; for example family members' roles in communication, and children and adults' self reporting of behavioural outcomes. Each category had clearly defined properties and dimensions for the concepts that informed its development. Once developed, each framework was rechecked for reliability and applicability against the first five interviews plus an additional two families' interviews chosen randomly.

The framework underwent modification to incorporate new phenomena and concepts as they emerged and interviews previously analysed were revisited to ascertain whether similar phenomena had been overlooked. Data was coded into the framework using the software package NVIVO-7. Each family's transcripts and field notes were grouped together to explore the level of agreement between parent(s) and children including; how children learned about the genetic condition and genetic risk, how much they knew and understood, the level of openness and the shared language used to describe the genetic condition, genetic risk and associated morbidity and mortality. Throughout all stages of the analysis from fieldwork to coding, observations were used to develop a series of grounded theory questions (related to the research questions and to inductive ideas) that were used to examine the data. Each researcher examined data specific to two genetic conditions on the basis of grounded theory questions, and noted any other additional observations including any negative cases. A second researcher randomly reviewed the analyses for consistency and negative cases. All three researchers discussed any disputes until consensus was reached. Finally, the findings from the family groups for each of the six conditions were compared for consensus and difference using a constant comparative analysis [START_REF] Corbin | Basics of Qualitative Research[END_REF] to identify the overall outcomes. The findings are reported in overarching thematic headings, which reflect the grounded theory questions that tested the data [START_REF] Corbin | Basics of Qualitative Research[END_REF] .

1

FINDINGS

A total of 33 families were interviewed. The demographic profile of participants and their families is provided (table 2). Families were recruited from across England and Wales but specific regional and other information that may make families identifiable to each other or to health professionals has been omitted to protect confidentiality. 6 Across all of the genetic conditions, many similarities were observed and exceptions between conditions are indicated in the findings by denoting which conditions the comment is referring to.

When the transcripts of all members of a family were reviewed together there was usually a strong level of consistency between reports about what and when children and young people had been told about a genetic condition and its risks. There was often a shared narrative about the condition and its effects on the family, and parents' understanding and views were often reflected in the explanations and descriptions given by their children. In families where children had better understanding and more insight, parents had usually discussed the condition throughout childhood, often as part of the family story.

Parents, children and most young people thought parents should be the main people to provide genetic risk information because they understood their children best. Young people however thought health professionals should provide guidance and advice to parents. Over half of the young people also wanted information directly from health professionals, preferably ones who understood the contemporary issues faced at school, college and the transitions they faced at this developmental stage.

Roles in communication

Mothers were usually the key person in the family who shared genetic risk information. Some fathers took on this role as key informant or shared it with the mother, but children often described fathers as too angry or too upset to talk. Whilst some parents took shared decisions about talking to the children about the genetic condition, there were also equal numbers who did not discuss it with each other, or if they did, they disagreed on the best course of action.

"well he (younger sibling) doesn't hasn't said that he knows …we haven't spoken to him as such, have we?...he's just aware" Parent -Older child affected by DMD

Making decisions about the sharing of genetic risk information

Across families, parents differed in deciding when and how they talked to their children about a genetic condition. Decisions were not usually related to their child's cognitive development but were more dependent on the genetic condition's morbidity and whether it was life limiting. Only a small number of parents expressed a view that their child(ren) had a moral and ethical right to know about a genetic condition affecting their family.

Delaying or choosing the right time to talk to children about a genetic condition was often very distressing and parents worried they were making the right decision. Most delayed discussions for as long as possible until a particular event either internal or external to the family initiated the need to discuss the condition. In contrast, most children and young people said they had been aware at some level of the condition being in their family from a young age (usually by around 8 years) and wanted to be able to discuss it and understand what was happening in their family from then.

"

How and what children are told

All children whether affected, at risk or unaffected showed a high level of insight into how they learned. Children said they wanted small amounts of information at a time and to be able to discuss what was happening in their families with their parents. Children and young people reflecting retrospectively on their experience of learning about a genetic condition and its risks said that they came to learn and understand about the condition and its implications quite slowly. It took time for them to become aware of the risks either to themselves or to other family members and most children and young people said they did not spend time repeatedly thinking about it once they had initially learnt about the risks.

Information given in the gradual way children preferred, increased their understanding over time, and gave them insight without the shock of learning about the condition and its implications all at once. Parents and young people said that receiving information as younger children was less shocking, and because they learnt gradually as they grew up, it made new information and its implications easier to cope with. This lack of shock for younger children probably occurred because they had less insight and fewer experiences to draw on to recognise the fuller implications. Unlike the young people who pieced information together very quickly to ask very challenging questions when they were not always prepared for the answers.

It was important to go at the child's pace, as information that was too graphic or went beyond what the child asked about, could stop them wanting to discuss the condition further. However, this was not achieved simply by waiting for children to ask questions, which parents often did. The lack of acknowledgement of the genetic condition by parents inhibited children aged 8-11years onwards from asking questions about it because they thought it would upset their parents. Parents therefore, had to make it clear to their child that they were happy for them to ask questions. Children and young people thought less formal discussions were best, and they liked to discuss the genetic condition in situations where they were also doing other things alongside their parent(s) such as preparing a meal, gardening or riding in the car. In some families there was a 'story' about the condition and parents told their children how they had learnt about the condition and had learned to cope with its effects. Parents thought this taught children about coping with the condition, offered opportunities to talk and allowed different family members insight into each others' experiences and feelings. For some it was also an opportunity for children to look to parents or older siblings as role models. The affected child was usually given more information than siblings (CF, FAP, HbO, NF).

However, if the condition was life-limiting and the child likely to die during their adolescence, affected children were given limited information but their older siblings were told more about the outcomes, which they were expected to keep secret (DMD). Overall parents' and children's discussions of genetic risk intertwined with the communication about the management of the genetic condition. This may explain why many children and young people often struggled to explain what they knew and understood about the genetic risks separately from knowledge and discussion about the genetic condition and its day-today management.

Children's understanding of genetic risk and inheritance patterns

Based on self-reports of children and observations made by parents, patterns emerged in children and young people's understanding and reactions during different stages of their childhood. This included those affected, those at risk and unaffected children (figure 2).

However, some differences in knowledge were identified across conditions and between inheritance patterns.

The dominant pattern of inheritance was easier for children to understand than the recessive or X-linked patterns but it was also accompanied by most misconceptions. Children often thought that 50 % risk meant that one of two children would be affected. Even adult children who knew this was not the case said it was difficult not to think in this way. Both parents and young people looked for phenotypic traits of a genetic condition in family members and sometimes thought that children who were most similar to an affected parent in personality or looks were more likely to inherit the condition.

In families with dominant conditions (HD, FAP, NF) children from the age of 10 -11 years upwards understood that the condition was 'passed down' through their families with some using the word 'inherited'. Most knew it had something to do with genes and sometimes talked about chromosomes and DNA. Where the condition did not manifest until adulthood or adolescence (HD and FAP) it could be more difficult for children to conceptualise risk in terms of how the condition was going to affect their future lives.

" born with it, something to do with my DNA when I was being made" Child 8-11y HbO

Young people usually understood the risks for themselves during teenage years (around 12-15years), but were usually 15-17 years old before they realised the reproductive implications. At this age some were also vaguely aware of possible interventions, such as in vitro fertilisation that would be available to them to prevent their own future children being affected. Across the age range children and young people talked about '50:50' chance of inheriting the affected gene but only a few young people aged 12-15 years and those aged 16 or older used the word 'dominant' to describe the genetic risk appropriately in the relevant conditions. With FAP, affected young people had a more detailed knowledge of dominant inheritance than unaffected siblings did, usually because parents chose not to discuss risk information until after children had received a positive gene test.

"Erm I think the FAP comes from like other people's DNA…and it goes, and goes through the family sometimes." Young person 12 -14 years FAP -affected

Very few young people could explain recessive inheritance patterns accurately. Children and young people affected by recessive conditions (CF or HbO) usually knew there was a risk to their potential children, although they did not understand the details of this. Some affected (CF or HbO) girls knew that pregnancy would present some risks for their own health, and this was more meaningful to them than concerns about the genetic risk for their future children. Siblings at risk of carrier status who were younger than the affected child had been tested at birth but did not necessarily understand what this meant. Siblings who had not been tested did not understand risk until they were about 16 years old and had been told prior to testing or when parents thought they might begin sexual relationships. When young people knew they were or could be carriers of a recessive condition they had little idea of population risk, and thus the likelihood of them having a child with another carrier.

In families affected by DMD, girls up to the age of 16 did not understand the risks to their potential children. Children affected by DMD generally knew little about the condition, and heredity and genetic risk had not been discussed with them.

Children's emotional response to genetic risk

In families where parents treated communication about the genetic condition and its risks as an ongoing process throughout childhood, these children and young people had greater insight and appeared to cope well with the condition and their increasing knowledge of associated risk. By contrast, families who had initially tried to keep risk information secret later viewed a lack of knowledge as having lead to increased stress and negative emotional experiences for children, as well as for parents.

"It wasn't ooh boo hoo Mummy's poorly, it was a relief cause she understood why Mummy was the way, she had been treating her the way she was…" Parent of child< 8years old -Partner had HD Where communication was or had been less open, children struggled to understand what was happening and parents said they had observed signs of stress, such as children and young people becoming withdrawn and easily upset. Many young people said they had felt better when they knew about and understood the implications of the condition and its risks.

Young people often said they had felt scared about the risk of inheriting something they did not fully understand and they had experienced problems with self-esteem. Some individuals thought this may have been a contributory factor to them being bullied or self harming.

In some families, parents encouraged and supported young people to make their own decisions about treatments, genetic testing and care provision. This appeared to encourage young people to discuss information and their thoughts about the genetic condition with their parents and usually to follow their advice. In contrast, we observed that young people did not discuss the genetic condition and their concerns with parents who made decisions and took a more authoritative role.

DISCUSSION

This is the largest study to date that explores the sensitive issues surrounding communication about genetic risk between parents, children and siblings. It has taken into consideration the views and experiences of families from six different genetic conditions that vary in inheritance pattern, age of onset, morbidity and life expectancy. Due to differences in these factors, the detailed content of family discussions varied slightly between the genetic conditions but how children learnt, their level of insight and understanding and the emotional impact of the knowledge gained was similar across each age range for each of the six conditions.

All children and young people valued being able to discuss genetic conditions affecting their family. Gradually learning about the condition as they grew up helped them cope and come to terms with the risk to either themselves or other family members, including their own future offspring, irrespective of the disease outcomes. Demonstrating that assimilation of genetic risk information is a process rather than a single act [START_REF] Gaff | Process and outcome in communication of genetic information within families: a systematic review[END_REF] .

Where children had not always received explanations from their parents about a genetic condition, this had caused considerable strain on the individuals, parents and children, and on their relationships with each other but also their wider social networks. Once family communication became more open, this helped re-establish understanding and a shared reality that enabled parents and children to support each other and cope better.

Most parents found deciding how and when to talk to their children about the genetic condition, a distressing and major issue in family life. Despite being encouraged to talk to their children about the genetic condition by health professionals, most parents or children were unable to access appropriate advice and support at different developmental stages.

However this reflected the limited support available generally for these families, who often experienced other problems and issues related to their experiences of the genetic condition.

A future manuscript will explore this in more detail.

Children and young people, irrespective of their risk status also highlighted their need for opportunities to meet with a health or social care professional with whom they could discuss the implications of genetic risk. Many young people were only just beginning to realise and understand the implications of genetic risk and the possibility of a carrier status when they were having genetic tests carried out. This raises serious ethical questions about how much the young people involved really understand when and after they have consented to the tests, and may indicate a need for further advice and guidance on the genetic testing of children [START_REF] Borry | on behalf of the Public and Professional Policy Committee (PPPC) of the European Society of Human Genetics (ESHG)[END_REF] .

Young people often had difficulty understanding genetic risk information, in terms of risk statistics and probability, and often relied on subjective judgments such as shared family phenotype features. Often this reflected parents' views but also characterises how lay people often view hereditary [START_REF] Walter | Coming down the line.' Patients' understanding of their family history of common chronic diseases[END_REF] and the well-documented difficulties many people have with understanding risk probabilities [START_REF] Etchegary | Information processing in the context of genetic risk: implications for genetic risk communication[END_REF] . All age groups of children and young people have difficulty in understanding probability terms, and younger adolescents (10 to 15 years) particularly have difficulty distinguishing between 'probably' and 'possibly' 25 . Probability information or risk percentages presented as a pie chart 26 are the most effective method to assist children's understanding. Therefore, based on this evidence and the findings presented, more effective genetic risk communication tools are required, to aid parents and health professionals in providing better explanations.

Open family communication underpins the cohesion of families, and improves family members' commitment to long term care of a parent or sibling [START_REF] Canam | Coping with feelings: chronically ill children and their families[END_REF][START_REF] Spiridigliozzi | Genetic Risk Communication: Experiences of Adolescent Girls and Young Women from Families with Fragile X Syndrome[END_REF] . Strong and supportive families have the ability to transform both children's and parents' lives, and help them cope with illness, the risks involved and the care provision that may be needed. Therefore, the care of the family needs to go beyond preparing for genetic testing or carrying out treatments for the individual affected by the genetic condition. There is a need for greater family centred care to support parents in advising and helping them to manage the condition whilst maintaining pivotal family relationships.

Supporting better family communication is likely to lead to better health outcomes for all family members. Greater family-centred care from health professionals will help family members cope and adjust to genetic risk information. This may be a role for a family support worker within existing professional groups 27 . Such a worker could provide advice and guidance on family communication about genetic risk communication to parents, give young people the further information they require and generally support the family dynamic in managing and coping with the genetic condition.

The study does not allow us to know the findings' generalisability to the wider population of families affected by these and other genetic conditions. Future survey studies are required to find out the generalisability and test our theories further. In addition, the 3 recruitment of these families via support groups is a potential bias. However, others have suggested the majority of parents in families affected by genetic conditions, share the experiences we have outlined 28 . Further studies are therefore required to establish the prevalence of the problems the parents and children identified, and to develop and evaluate interventions and resources to assist parents in talking to their children about genetic risk information. x Cystic fibrosis (CF) (autosomal recessive -onset from birth)

REFERENCE

x Neurofibromatosis (NF) (autosomal dominant -onset from birth, wide variation in signs and symptoms)

x Duchenne Muscular Dystrophy (DMD) (X-linked recessive -onset of symptoms in early childhood, progressive deterioration until death in late adolescence early adulthood)

x Haemoglobinopathies (HbO) (autosomal recessive -acute episodes throughout child and adulthood, vary in frequency)

x Familial Adenomatous Polyposis(FAP) (autosomal dominant -adolescence onset)

x Huntingtons Disease (HD) (autosomal dominant -usually adult onset, often middle age)

x Participant families had been diagnosed with or at risk from the genetic condition for a minimum of 12 months, and at least one member (either child or adult) was affected or at risk from the condition, including the possibility of being a carrier.

x The family had access to a peer support group or a health professional following the interview so that any personal or family issues unintentionally triggered by the interviewers could be discussed with experienced people capable of giving appropriate support and advice.

x The research ethics committee directed that the children to be interviewed should be aged 8 years and above, except for families affected by HD were only young people 16 years and above could be interviewed.

x Children were only included in the interviews where parents confirmed they had personally explained to the child about the genetic condition and its risks, and they were willing to answer any questions the interview may raise for the child.

x Definition of a family member according to Metcalfe et al., (2008).

Emotional reaction

The most difficult time emotionally. Young adults were coming to terms with the implications of the condition at a time when so many other important life decisions and choices are being made.

Young people reported upset and shock as they better understood the mortality, responded to test results or faced surgery. However they looked for positives and did not let the condition monopolise their life.

A period of rebellion for some children who became angry and questioned 'why me?' Some children challenged treatments and routines.

Children continued to accept information at a relatively superficial level and looked for positives and did not let the condition become the central focus of their lives.

Children seemed to accept information at a relatively superficial level. There were some behavioural problems or signs of stress when they were not given information in difficult family situations.

years plus 15 -17 years 12 -14 years 8 -11 years Up to 7 years

Young adults began to understand the wider implications of the condition and relate it more to themselves. They realised how it might influence their career choices and personal relationships and how their decisions relating to genetic testing had impact on other family members.

Young people understood that they may carry a gene that could or would affect their health. They had a clearer understanding of mortality although still not always the stages of progression. They were more able to describe hereditary patterns and began to realise the implications for their own future children.

Children began to understand more about the condition in terms of expected future symptoms and about mortality. Most understood hereditary in terms of their relationship to their parent(s) but not to their future children. Most use genetic language but without an underlying understanding, and they could not describe hereditary patterns accurately or quantify risk.

Children understood the condition in terms of

what they could see and how it currently affects their daily lives. Most understood the notion of hereditary in terms of the condition being passed down through the family but had no idea of hereditary patterns.

Children began to notice and question visible difference and some asked if they were likely to be affected in the same way as what they observed. Some understood that the condition was a result of the biological relationship between parents and child in the same way as eye colour.

Understanding of genetic condition and risks

  of children and parents. Each model informed the design of the interview schedule and was used as part of the grounded theory development for the data analysis. Prior to commencing the study a psycho-social definition of family 13 was established. An advisory group of six parents and five young people from diverse cultural and social backgrounds were consulted about the conduct of the study and later, the credibility of the findings. The group discussed the findings in context and contributed to the development of subsequent recommendations. The study was approved by the Liverpool Children's Research Ethics Committee (REC No:07/Q1502/16).

"

  …absolutely not. My dad went "La la la under the sea." Anything to do with HD that was it, switched off…he couldn't cope with it I don't think…" Young person 15-17 years at risk from HD Siblings rarely discussed with each other, the genetic condition or its risk. Most communication took place between an individual child and their parent(s), whom they trusted above any other information source until they reached 15-17 years. Parents often recalled talking to their older children for the first time but many overlooked talking to their younger, unaffected children. Parents assumed that because the younger children witnessed the effects of the condition in the family everyday, they automatically knew what was happening. Children did not talk to parents' new partners about the condition but saw this person as source of support for their parent (Figure 1).

"

  …me and my mum would erm, in the mornings me and my mum we're sort of people who get up first and so we would sort of just sit down by the sofa, like one of us would be on the computer and just sort of talking and, you know, it (HD) would come into the conversation …." Young person 15-17 years at risk from HD Many parents said they were completely open to discussion with their children but further discussion revealed that this often meant answering questions as they arose. Many also talked at a different time in the interview about protecting children from certain aspects of information about the condition or risk until they needed to know. Most children and young people recognised these limitations and when talking about the level of their parents' openness qualified this with adjectives such as 'quite' or 'fairly'.

Figure 1 :

 1 Figure 1: Family roles in the communication of genetic risk information

Figure 2 .

 2 Figure 2. Children's developing understanding and response to genetic risk.

  I might be a bit angry if they didn't tell me because I would just want to know, so I don't think there is anything to hide really when you have got a disease, you have got a disease really, so but no I don't think there should be any secrets" Young person 15 -17 years CF -affected

A number of factors often compromised parents' capability of discussing genetic risk.

These were related to one or more of the following, depending on the genetic condition and stage of disease progression; shock at diagnosis, which prevented parental discussion; guilt and fears pre and post genetic testing; increased emotional and physical care giving; and experiences of grief and bereavement. A second article, currently in preparation, will expand on these factors in detail.

Table 1 : Explanation of the genetic condition and inclusion criteria used to recruit participants GENETIC CONDITION and summary of inheritance pattern INCLUSION CRITERIA

 1

	1 Beale EA, Baile WF, Aaron J (2005) Silence is not golden: communicating with children
	dying from cancer. J Clin Oncol 23;15:3629-3631.
	2 Kreicbergs U, Valdimarsdottir U, Onelov E, Henter JI, Steineck G (2004) Talking about
	death with children who have severe malignant disease. N Engl J Med 351; 12:1175-1186
	3 Ranmal R Prictor M Scott JT (2008) Interventions for improving family communication
	with children and adolescents about their cancer. Cochrane Database of Systematic
	Reviews Issue 4 Art. No. CD002969 DOI:10.1002/14651858.CD002969.pub2
	4 Holt K. What Do We Tell the Children? Contrasting the Disclosure Choices of Two HD
	Families Regarding Risk Status and Predictive Genetic Testing. J Genet Counseling
	2006;15:4; 253-265.
	5 Gallo AM, Angst D, Knafl KA, Hadley E, & Smith C (2005) Parents sharing information
	with their children about genetic conditions. Journal of Pediatric Health Care 2005;19: 5:
	267-275.
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	among children of hereditary breast cancer gene (BRCA1/2) testing participants.
	Psychooncology 2002;10;4: 336-346.

Table 2 -Demographic Profile of Participants Individuals interviewed Family backgrounds

 2 The information in the rows bear no relation to each other, the findings are to be read in their columns only to give an indication of the different types of family that took part in the study.

			Age Range of Parents Years (N and Gender)	Age range of Children Years (Gender)	Ethnicity	Educational background of Parents	Genetic condition affecting families (N)	Status of children in relation to genetic condition
	52 Parents = 34 Women and 18 Men	Children living with parents in 29 families	30-34 (4Ƃ0ƃ)	8-11 (5Ƃ4ƃ)	5 Afro-Caribbean or Black British	21 left school at 16 years	Cystic Fibrosis (CF) (4)	21 affected or positively tested for genetic condition
						15 left school
	33 Children (aged <18years)	In 21 of these, children lived with both parents	35-39 (6Ƃ2ƃ) 12-14 (3Ƃ7ƃ)	3 Asian or British Asian or Middle East origins	at 18 years following advanced level vocational qualifications or	Duchenne Muscular Dystrophy (DMD) (6)	11 were at risk of being affected or carriers
						course
	11 Adult children (aged 18+ years)	In five, children lived with single mother	40-44 (9Ƃ9ƃ) 15-17 (7Ƃ7ƃ)	25 White and from the UK	16 had University level education	Familial Adenomatous (6) Polyposis (FAP)	12 were unaffected siblings
	16 Parents	In three, children					Haemoglobinopathies
	interviewed as a	lived with mother	45-49 (8Ƃ5ƃ) 18+ (9Ƃ2ƃ)			(HbO)
	couples	and new partner					(6)
	17 Women interviewed alone including 6 single mothers		50-54 (2Ƃ1ƃ)				Huntington's Disease (HD) (7)
	4 Men interviewed						Neurofibromatosis
	alone (2 separated		55-59 (3Ƃ0ƃ)				(NF)
	fathers)						(4)
			65+ (4Ƃ0ƃ)
	The families represented a wide spectrum of religious beliefs and included: Atheism, Christianity, Church of England, Hindu, Humanist, Methodist, Muslim,
	None, Pentecostal, Roman Catholic and Sikh.
	Note:

We also wish to acknowledge the invaluable contribution of advisory and steering groups.

The Department of Health, England funded the study and the views expressed do not necessarily reflect those of this Authority.

CONFLICTS OF INTEREST:

All authors have declared that they have no conflict of interests in relation to carrying out or reporting this study.


Ph.D Elisabet Einarsdottir
email: elisabet.einarsdottir@helsinki.fi

Marianna R Bevova

Alexandra Zhernakova

Lotta L E Koskinen

Ruben Vant Slot

Chris J J Mulder

M Luisa Mearin

Ilma R Korponay-Szabo

Katri Kaukinen

Alienke Monsuur

Ruben Van't Slot

Kalle Kurppa

Juha Kere

Markku Mäki

Cisca Wijmenga

Päivi Saavalainen

Multiple independent variants in 6q21-22 associated with susceptibility to celiac disease in the Dutch, Finnish and Hungarian populations

Keywords: Celiac, linkage, association, genome-wide, 6q21-22

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

Introduction

Celiac disease is a chronic autoimmune disorder of the small intestine with autoimmune features. It is triggered in genetically susceptible individuals by dietary exposure to gluten, a protein present in wheat, barley and rye. Ingestion of gluten by celiac disease patients leads to flattening of the duodenal mucosa, a gradual process classified according to Marsh [START_REF] Marsh | Gluten, major histocompatibility complex, and the small intestine. A molecular and immunobiologic approach to the spectrum of gluten sensitivity ('celiac sprue')[END_REF] . With a prevalence above 1% [START_REF] Lohi | Increasing prevalence of coeliac disease over time[END_REF] , celiac disease is the most common food intolerance in general western populations. Long regarded as a gastrointestinal disorder of childhood, the disease is now considered to be a chronic systemic autoimmune disease and is more and more often diagnosed in adults [START_REF] Ravikumara | The changing clinical presentation of coeliac disease[END_REF] .

Celiac disease is a multifactorial disorder and both environmental factors, such as gluten, and a multigenic predisposition are needed to explain the aetiology of the disease. Concordance rates for celiac disease have been shown to be higher in monozygous twins (86%) than in dizygous twins (20%), indicating a strong genetic component in the development of celiac disease [START_REF] Greco | The first large population based twin study of coeliac disease[END_REF] . Celiac disease aggregates in families with an approximate 10% recurrence risk for siblings of celiac disease patients. There is, however, no clear Mendelian inheritance of the disease in families, suggesting that the genetic predisposition is derived from the presence and interaction of more than one susceptibility gene.

Over 90% of celiac disease patients express the HLA-DQ2 heterodimer, encoded by DQA1*0501 and DQB1*02. A causal role for HLA-DQ2 has been demonstrated as this molecule can present the gluten-derived peptides to T-cells [START_REF] Molberg | Tissue transglutaminase selectively modifies gliadin peptides that are recognized by gut-derived T cells in celiac disease[END_REF] . A further 5% of celiac disease patients carry HLA-DQ8. Although HLA-DQ2 or DQ8 seem to be necessary for celiac disease development, they are not sufficient, as HLA-DQ2 or DQ8 alone can only explain some 40% of the genetic variation underlying celiac disease. This implies that other, non-HLA genes, are also important determinants of disease aetiology.

A recent whole-genome association scan identified a number of genetic variants involved in celiac disease susceptibility [START_REF] Van Heel | A genome-wide association study for celiac disease identifies risk variants in the region harboring IL2 and IL21[END_REF] . However, all the known risk variants explain not more than 50% of the genetic variance of celiac disease, indicating that novel genes and novel risk variants remain to be identified. This was supported by the results of the second whole-genome association study in celiac disease [START_REF] Dubois | Multiple common variants for celiac disease influencing immune gene expression[END_REF] . This study confirmed a number of the previously identified risk variants, as well as identifying numerous novel variants.

A study in the Dutch population obtained evidence for linkage of celiac disease to the 6q21-22 region, approximately 70 cM downstream of the HLA region [START_REF] Van Belzen | A major non-HLA locus in celiac disease maps to chromosome 19[END_REF] . The region of interest spans approximately 102 to 122 Mb and is clearly distinct from the HLA region as well as from the previously identified TNFAIP3 and TAGAP celiac susceptibility genes [START_REF] Hunt | Newly identified genetic risk variants for celiac disease related to the immune response[END_REF][START_REF] Trynka | Coeliac disease associated risk variants in TNFAIP3 and REL implicate altered NF-{kappa}B signalling[END_REF] . An independent study by Einarsdottir et al. (unpublished) of susceptibility genes for celiac disease in Finnish and Hungarian families also showed linkage to 6q16-22, to a region overlapping with the linkage found in the Dutch population. Taken together, these results indicate the possibility of a genetic factor within the chromosome 6q21-22 region that is involved in susceptibility to celiac disease in multiple populations. We therefore performed a fine-mapping scan in Dutch unrelated individuals with celiac disease in order to identify the possible candidate gene. This study was further followed up in family datasets from the Finnish and Hungarian populations.

Material and methods

Patients and controls

Dutch cohort:

DNA from whole blood was available from a cohort of Dutch individuals with celiac disease. All the affected individuals were diagnosed in accordance with the revised ESPGHAN criteria [START_REF]Anonymous Revised criteria for diagnosis of coeliac disease. Report of Working Group of European Society of Paediatric Gastroenterology and Nutrition[END_REF] . The control cohort comprising random blood bank donors and has been described previously [START_REF] Van Heel | A genome-wide association study for celiac disease identifies risk variants in the region harboring IL2 and IL21[END_REF] . All 446 Dutch cases and 641 controls were from the Netherlands and of European descent, and at least three of their four grandparents were also born in The Netherlands. The sample set has been described previously [START_REF] Monsuur | Myosin IXB variant increases the risk of celiac disease and points toward a primary intestinal barrier defect[END_REF] . This study was approved by the Medical Ethical Committee of the University Medical Centre Utrecht.

Finnish and Hungarian cohorts:

The Finnish families and cases were collected at the University of Tampere and have been described previously [START_REF] Koskinen | Association study of the IL18RAP locus in three European populations with coeliac disease[END_REF] . The collection of the Hungarian samples has also been described previously [START_REF] Koskinen | Myosin IXB gene region and gluten intolerance: linkage to coeliac disease and a putative dermatitis herpetiformis association[END_REF] . A total of 1001 individuals from 284 Finnish families (19 unknown phenotype, 359 healthy, 623 affected) and 1070 individuals from 357 Hungarian families (7 unknown phenotype, 471 healthy, 592 affected) were included for genotyping. All sample collections were approved by the ethical committees of the Tampere and Helsinki University Hospitals, Finnish National Public Health Institute, Heim Pal Children's Hospital, Budapest, and the University of Debrecen. All participants were informed about the study according to the study protocol and gave their written informed consent.

Marker selection and genotyping

For a comprehensive follow-up screen in an independent Dutch dataset, we took the lod-1 region (95% confidence interval; ranging from rs9390679 (101,261,700 bp) to rs11154152 (123,694,693 bp) (National Center for Biotechnology Information NCBI34/hg16) of the previously identified linkage peak on chromosome 6 [START_REF] Van Belzen | A genomewide screen in a four-generation Dutch family with celiac disease: evidence for linkage to chromosomes 6 and 9[END_REF] . SNPs for fine-mapping were selected by downloading all the SNPs typed in the CEPH (Centre d'Etude du Polymorphisme Humain) population (Utah residents with ancestry from northern and western Europe) that were located in the genomic sequence of the region of interest from the HapMap database (November 2004, Phase I, http://www.hapmap.org/). The program Tagger (available at http://www.broad.mit.edu/mpg/tagger/) was subsequently used to select tag SNPs to cover the region. We used r 2 ≥0.7 to perform pairwise tagging. Additionally we set up a minor allele frequency (MAF) cut off ≥5% and took Illumina quality design scores into account for preferential picking of SNPs that are more likely to work on the Illumina platform. The obtained tag SNPs were genotyped using the GoldenGate assay on an Illumina BeadStation 500 GX (Illumina Inc., San Diego, USA). After genotyping all tag SNPs from the initial scan in the Dutch cohort were examined for their resulting quality; all those that were not polymorphic, had a low signal or had clusters that were too wide were excluded (data not shown). In addition, tag SNP that deviated from Hardy-Weinberg equilibrium (HWE) in the controls (p <0.001) were excluded from further analysis, yielding a final set of 872 tag SNPs obtained for genotype analysis.

For the replication in Finnish and Hungarian cohorts, we chose the 18 markers showing the highest allelic association in the Dutch cohort (p< 0.01). 14 of the SNPs showed the highest association in the unadjusted model and 4 additional SNP were chosen from the top markers in a sex adjusted model (p<0.01). As the differences in the analysis were not very large we have not proceeded further with the sex adjusted model (data not shown). The markers were genotyped by MALDI-TOF analysis on the Massarray Analyzer platform (Sequenom, San Diego, USA). Genotyping was performed at the MAF core facility in Karolinska Institute, Stockholm, Sweden.

Genotyping results and sample information were collected in a BC|GENE LIMS database (Biocomputing Platforms, Espoo, Finland). As five markers in the replication study failed (assay design failed or call rate < 90%, data not shown), genotype information for twelve SNP markers was available for analysis.

Deviations from Mendelian inheritance in the family materials were determined with PEDCHECK 1.1 [START_REF] O'connell | PedCheck: a program for identification of genotype incompatibilities in linkage analysis[END_REF] . Mendelian errors were detected in 0.21% of genotypes in the Finnish samples, and in 0.17% of the genotypes in the Hungarian samples. All genotypes of a marker were discarded in a family if inheritance errors were detected.

To ensure that the annotation of the rs9391227 SNP on both the Illumina and Sequenom platforms was performed on the same strand, we performed an additional genotyping of rs9391227 on the Taqman platform using 12 controls from Finland (previously genotyped by Sequenom) and 54 Dutch celiac disease cases and controls (previously genotyped by Illumina). All the samples showed 100% concordance of the genotypes obtained on different platforms (data not shown).

Association analysis

For the initial association analysis in the Dutch sample set, we performed a single SNP association test using the Haploview program [START_REF] Barrett | Haploview: analysis and visualization of LD and haplotype maps[END_REF] .

Unphased (version 3.0) [START_REF] Dudbridge | Pedigree disequilibrium tests for multilocus haplotypes[END_REF] was used to look for association in families by calculating allele counts in affected offspring and comparing these with counts from untransmitted alleles from parents and unaffected offspring. All affected individuals in a family were used in the association analysis. As each affected individual within a family is not independent, this constitutes in effect, a test of linkage in the presence of possible association.

Unphased was also used to perform a meta-analysis of the Finnish, Hungarian and Dutch sample sets. Population differences were assessed by a global Wald test. All reported p-values two-tailed and uncorrected unless noted. Only single-marker association analysis was performed as the genetic markers in the follow-up study were too distant from each other. Permutation testing (1000 permutations) was used to estimate empirical p-values, allowing for multiple testing corrections over all tests performed in a run. PLINK (version 1.06) was used to perform quantitative trait analysis in order to explore if the associated markers were possible eQTLs for the genes within a 200 kb window around them.

Power analysis of follow-up study

The Genetic Power Calculator program (http://pngu.mgh.harvard.edu/~purcell/gpc/) [START_REF] Purcell | Genetic Power Calculator: design of linkage and association genetic mapping studies of complex traits[END_REF] was used to estimate the power of the Finnish and Hungarian family-based association analysis to replicate the association in the Dutch case-control samples.

Parameters for power estimation in the Finnish and Hungarian follow-up materials were estimated from the initial Dutch results and marker rs1998166 as it showed the highest association in the discovery phase. We estimated the power to replicate the association to rs1998166 (protective allele frequency 0.123, odds-ratio 0.616, and celiac disease prevalence 0.01) in the 284 Finnish families to be 64%, and in the 357 Hungarian families to be 74%. When combining the Finish and Hungarian samples we had an estimated power of 93%.

Results

Dutch association

In this study, we performed finemapping of the 6q linkage region by genotyping 872 SNP markers in 446 cases and 641 controls. The association of each SNP to celiac disease is shown in figure 1 and supplementary table 1. From 872 SNPs, 18 showed association at p<0.01 and were included for replication in CD families from Finland and Hungary.

Because population sub-structure is a possible cause for false positive results, this issue has been investigated previously for all the Dutch cases and a majority of the controls as a part of another study (Carolien de Kovel, unpublished data). No significant population structure was detected using the STRUCTURE software (http://pritch.bsd.uchicago.edu/structure.html). The likelihood of increasing numbers of subpopulations decreased continuously, indicating that it is more likely that the individuals were drawn from a single population than from two or more. Also, the assignment of the individuals to the different sub-populations, when the software was forced to create more than one, was never significantly different between cases and controls (Caroline de Kovel, unpublished data). In view of these results, it is unlikely that the results of the association analysis in the Dutch dataset were due to population sub-structure.

Follow-up association in Finnish and Hungarian families

Table 1 shows the markers studied in the follow-up association study. The empirical 5% quantile of the best p-values for the Finnish dataset was 0.0029, 0.0033 for the Hungarian dataset, and 0.0027 for the combined Finnish, Hungarian and Dutch dataset (the markers with p-values lower than these are denoted by * in table 1). Three SNPs showed consistent replication in either the Finnish or Hungarian families, to the same allele as observed in the Dutch population. Association analysis revealed that the T-allele of rs9391227 was associated with protection from celiac disease in Finnish families (p 0.0032, OR 0.66), as well as allele T of rs4946111 (p 0.006479, OR 0.66). Allele T of rs9391227 also showed a trend of association with celiac disease in Hungarian families. No other markers were associated with celiac disease in the Hungarian family material.

Association to SNP rs9391227 located downstream of HACE1 and within a region of strong LD surrounding the HACE1 gene was confirmed in a meta-analysis of the Finnish and Hungarian family datasets and the Dutch case-control dataset (p 3.625*10-5, protective allele T OR 0.76). Even if we correct for multiple testing of the 872 SNPs originally tested for, this signal remains significant (Pc = 0.032). This represents the strongest association in the combined materials, and it is stronger than in the Dutch case-control dataset alone. Two additional markers showed a strong association in the meta-analysis, these were rs1998166 (p 0.0002, OR 1.56), located in the RWDD1 gene and rs4946578 (p 0.0018, OR 1.25), located upstream from the GJA1 gene. The association signal of these two markers to celiac disease appears to be contributed to by all three populations, as the meta-p value is lower than that in the Dutch dataset only, and the allele frequency differences in patients and controls show the same trend in each of the populations.

Tests of heterogeneity between the datasets yielded significant differences (p < 0.05) between populations in the markers rs2216084, rs1146229, rs9487060 and rs4946111.

It is expected that there will be significant differences between allele frequencies even in well sampled datasets from different populations. The markers rs2216084, rs1146229, and rs9487060, while showing some association in the Dutch dataset, show very little indication of association in the Finnish and Hungarian datasets. This is not likely to be only due to different allele frequencies as the Dutch and Hungarian datasets show similar frequencies in these markers. Rather, it may be due to different haplotype backgrounds in the different populations. Marker rs4946111 shows some association in the Finnish dataset, but less in the Dutch populations. Furthermore, the effect is even reversed in the Hungarian population. This marker may thus constitute a locus on a highly heterogeneous haplotype background between the studied populations. The association in the Finnish samples may be due to an otherwise rare mutation enriched in the Finnish population through genetic drift.

An analysis (data not shown) in 40 Finnish control samples (Dukes et al. 2010, unpublished) identified no evidence of eQTLs regarding markers rs9391227 and HACE1, rs1998166 and RWDD1, rs4946578 and GJA1, nor of rs9391227 and PREP expression. PREP is not in LD with rs9391227 (data not shown), further arguing against genetic factors within PREP itself explaining the association with rs9391227. Variation at rs9391227 may still, however, be in LD with and have effects on PREP regulatory regions outside of the gene itself.

DISCUSSION

In this study we followed up previous findings of linkage to 6q21-22 in celiac disease by performing a fine-mapping study in the Dutch population, and a replication study in the Finnish and Hungarian populations.

HACE1 & rs9391227

In the combined analysis we identified association of rs9391227 to celiac disease in three populations using both case-control and family datasets. Rs9391227 is an intergenic SNP downstream of and within a region of strong LD surrounding the HACE1 gene (suppl fig 1). HACE1 (HECT domain and ankyrin repeat containing, E3 ubiquitin protein ligase 1) is an ubiquitination-protein ligase. As celiac disease is believed to be due, at least to some extent, to dysregulation of the immune system and antigen presentation, the importance of ubiquitination in celiac disease is plausible.

Whether rs9391227 is involved in this mechanism is unknown. .

Rs1998166 and rs4946578

The marker that is second most strongly independently associated with celiac disease in the meta-analysis is rs1998166. This marker is within an intron of the RWD domain containing 1 (RWDD1) gene. Little is known about this gene, but the RWD domain is found in RING finger and WD repeat containing proteins and in the DEXDc-like helicase subfamily, related to the ubiquitin-conjugating enzymes domain.

The third, independent, association found in the meta-analysis was to rs4946578. This marker is approx. 124kb away from, but contained within an LD block that contains the Gap junction protein, alpha 1, 43kDa (GJA1, CX43) gene. This gene is a member of the connexin gene family and encodes for a component of gap junctions. Connexin-43 is the major protein of gap junctions in the heart, and gap junctions are thought to have a crucial role in the synchronized contraction of the heart and in embryonic development. Mutations in this gene have also been found in deafness [START_REF] Paznekas | GJA1 mutations, variants, and connexin 43 dysfunction as it relates to the oculodentodigital dysplasia phenotype[END_REF] and in palmoplantar keratoderma [START_REF] Van Steensel | A 2-bp deletion in the GJA1 gene is associated with oculo-dento-digital dysplasia with palmoplantar keratoderma[END_REF] .

PREP

The genetic region we studied on 6q also harbours the gene encoding prolyl endopeptidase (PREP), an enzyme able to cleave proline-rich gluten peptides. An altered PREP activity in the intestinal mucosa could be responsible for the inefficient breakdown of gluten peptides, thus contributing to the onset of celiac disease. PREP has previously been identified as a potential celiac disease candidate; however, a previous study indicated no association of this gene with celiac disease in the Dutch population [START_REF] Diosdado | No genetic association of the human prolyl endopeptidase gene in the Dutch celiac disease population[END_REF] . Although this gene is located within the region studied in the current Dutch, Finnish and Hungarian samples, no indication was found that this gene could explain the linkage and/or association to 6q.

Multiple associations and implications

Linkage to 6q21-22 was found in two independent whole-genome linkage studies, and following up the results, we found association to multiple independent SNPs in the Dutch, Finnish, and Hungarian populations. This replication in several independent datasets makes the association signals in the region credible and likely to be true celiac disease risk factors. We did not find one single gene with a major effect on celiac disease susceptibility, suggesting that there may be several susceptibility genes at this locus, each with small effects. The previously conducted GWAS on celiac disease failed to identify strong associations to celiac disease in the studied region on chromosome 6. There are 8 SNPs among the top 1000 signals from the GWAS of Dubois et al. [START_REF] Dubois | Multiple common variants for celiac disease influencing immune gene expression[END_REF] , which are located in this region, These association signals, however, are in distinct LD blocks (D' ≤ 0.2) from the two most highly associated markers in our study (data not shown). Furthermore, the current study is estimated to adequately tag the eight GWAS signals (data not shown). Of the markers in our study, only three (rs1146229, rs2282854, and rs4946578) were also included in the Dubois GWAS study, allowing for a direct comparison of association signals. These three markers were significantly associated with celiac disease neither in the meta-analysis within the Dubois GWAS study nor in the Finnish or Dutch populations (data not shown), these markers were not part of the follow-up study. In addition to this, the arrays used in the GWAS do not contain perfect proxies for the most highly associated markers in our study (data not shown), arguing that the association we see in the current study may have been missed in the GWAS. We thus conclude that our results likely indicate true association that is independent of the GWAS signals, and that it is unlikely that we are missing major associations at this locus due to insufficient marker coverage.

The current study cannot fully explain the underlying cause of the multiple independent association signals in the region. The multiple association signals show the complexity of disease susceptibility in the region, but the results are consistent in the different study populations and the observed risk effect of the associated variants is relatively high. This argues that each of the identified variants (or nearby untyped variants in LD with the associated variants) is a true, novel, susceptibility factor for celiac disease. 1. SNP markers in the study and their association to celiac disease in Dutch cases and controls (A) as well as Finnish (B) and Hungarian (C) families, and a metaanalysis of all three populations (D). Case frequencies, in the family materials, refer to the allele frequencies of the affected offspring ("cases") and the control frequencies refer to both unaffected offspring ("healthy") and the untransmitted alleles in parents. P-values lower than 0.01 are considered significant (marked in bold) and odds ratios (OR) and 95% OR confidence intervals for significant markers are given. Emp permutation p-values are based on 10000 permutations. Supplementary table 1. Allelic and genotypic association of 860 SNP markers to celiac disease in Dutch cases and controls. The 12 markers chosen for follow-up analysis are shown in bold and underlined. Genotype counts (11,12,22), Hardy-Weinberg (HW) p-values and allelic and genotypic p-values are shown.

TITLES AND LEGENDS TO FIGURES

Figure 1 .

 1 Figure 1. Association of SNPs in the chromosome 6 region to celiac disease in the Dutch cohort. SNP positions are based on NCBI Build 35 and are given in Mb. Diamonds represent individual SNPs. Table1. SNP markers in the study and their association to celiac disease in Dutch cases and controls (A) as well as Finnish (B) and Hungarian (C) families, and a metaanalysis of all three populations (D). Case frequencies, in the family materials, refer to the allele frequencies of the affected offspring ("cases") and the control frequencies refer to both unaffected offspring ("healthy") and the untransmitted alleles in parents. P-values lower than 0.01 are considered significant (marked in bold) and odds ratios (OR) and 95% OR confidence intervals for significant markers are given. Emp permutation p-values are based on 10000 permutations. Supplementary table 1. Allelic and genotypic association of 860 SNP markers to celiac disease in Dutch cases and controls. The 12 markers chosen for follow-up analysis are shown in bold and underlined. Genotype counts(11,12,22), Hardy-Weinberg (HW) p-values and allelic and genotypic p-values are shown.

  Figure 1

Table 1 -SNP markers and association in follow-up study A DUTCH (446 cases, 641 controls) B FINNISH (623 cases, 359 healthy) C HUNGARIAN (592 cases, 471 healthy) D FINNISH, HUNGARIAN, DUTCH (1661 cases, 1471 controls)

 1

	Markers	Position (bp) Alleles case,contr freq	p value emp p-value OR (95% CI)	case,contr freq	p value OR (95% CI)	case,contr freq	p value OR (95% CI)	case,contr freq	p value	OR (95% CI)
	rs1648957	CT	0.498, 0.433	0.003	0.005 1.30 (1.09-1.55) 0.449, 0.481	0.351		0.545, 0.519	0.440	0.498, 0.460	0.085
	rs9391227	AT	0.497, 0.559	0.005	0.003 0.78 (0.66-0.93) 0.541, 0.651	0,003*	0.66 (0.49-0.88) 0.495, 0.555	0.054	0.507, 0.577	3.07*10-5 * 0.76 (0.66-0.86)
	rs2895615	CG	0.690, 0.642	0.020	0.018 1.25 (1.03-1.50) 0.668, 0.641	0.398		0.679, 0.678	0.871	0.683, 0.649	0.061
	rs2216084	CT	0.646, 0.688	0.045	0.058 0.83 (0.69-0.99) 0.809, 0.757	0.048	1.41 (0.99-1.99) 0.706, 0.676	0.603	0.700, 0.699	0.836
	rs1146229	CT	0.085, 0.052	0.002	0.003 1.72 (1.22-2.43) 0.111, 0.118	0.673		0.070, 0.095	0.082	0.087, 0.075	0.259
	rs9487060	CG	0.079, 0.048	0.003	0.002 1.73 (1.21-2.47) 0.067, 0.072	0.724		0.060, 0.062	0.763	0.072, 0.054	0.032	1.36 (1.03-1.79)
	rs2503770	AT	0.233, 0.274	0.031	0.028 0.80 (0.66-0.98) 0.324, 0.313	0.884		0.237, 0.217	0.376	0.255, 0.269	0.536
	rs571060	CT	0.970, 0.987	0.007	0.010 0.44 (0.24-0.81) 0.992, 0.993	1.000		0.982, 0.978	0.841	0.978, 0.986	0.085
	rs2282854	CT	0.933, 0.962	0.003	0.003 0.55 (0.37-0.82) 0.968, 0.965	0.796		0.948, 0.938	0.452	0.946, 0.958	0.170
	rs4946111	GT	0.445, 0.502	0.011	0.008 0.80 (0.67-0.95) 0.385, 0.482	0.007	0.66 (0.49-0.90) 0.434, 0.390	0.280	0.429, 0.474	0.020	0.86 (0.76-0.98)
	rs1998166	CT	0.921, 0.877	0.001	0.003 1.62 (1.20-2.19) 0.940, 0.917	0.105		0.956, 0.928	0.087	0.934, 0.896	2.17*10-4 * 1.56 (1.22-1.98)
	rs4946578	AG	0.584, 0.534	0.023	0.023 1.23 (1.03-1.46) 0.732, 0.695	0.323		0.564, 0.536	0.421	0.606, 0.558	0.002	1.25 (1.09-1.44)

ACKNOWLEDGEMENTS

All the study subjects are thanked for participating in the study. We thank the Dutch Celiac Disease Foundation for participating in this study, H. van Someren and F.

Mulder for clinical database management, and Ruben van't Slot at UMC Utrecht for technical assistance. We thank Wieke HM Verbeek for help in sample collection and Jackie Senior for critical reading of the manuscript. We thank Hanne Ahola and Anne Heimonen for excellent technical assistance and Erzsébet Szathmári, Judit B.Kovács, Margit Lörincz and Anikó Nagy for their work with the Hungarian families. We thank Carolien de Kovel at UMC Utrecht for providing unpublished data on the population structure of the Dutch cohort. We acknowledge funding from the Coeliac Disease Consortium (an innovative cluster approved by the Netherlands Genomics Initiative and partly funded by the Dutch Government, grant BSIK03009 to CW); the Netherlands Organization for Scientific Research (VICI grant 918.66.620 to CW) and the Dutch Digestive Disease Foundation. This work has been funded by the EU Commission through a Marie Curie Excellence Grant (FP6 contract MEXT-CT-2005-025270 to PS), by the Academy of Finland, the Hungarian Scientific Research Fund (contract OTKA 61868), the University of Helsinki Funds, Biocentrum Helsinki, the Competitive Research Funding of the Tampere University Hospital, the Foundation of Pediatric Research and the Sigrid Juselius Foundation.

CONFLICT OF INTEREST

The authors declare no conflict of interest.