Augustin Scalbert
email: scalbert@clermont.inra.fr

Sylvain Auclair

Gilles Chironi

Dragan Milenkovic

Peter C H Hollman

Catherine M G C Renard

Jean-Louis Mégnien

Jérôme Gariepy

Jean-Louis Paul

Alain Simon

Scalbert Augustin

The regular consumption of a polyphenol-rich apple does not influence endothelial function: a randomised double-blind trial in hypercholesterolemic adults

Keywords: apple, polyphenols, atherosclerosis, FMD

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

Introduction

A high consumption of fruit and vegetables have commonly been associated to a reduction of the risk of cardiovascular diseases [START_REF] Dauchet | Fruits, vegetables and coronary heart disease[END_REF][START_REF] Ellingsen | Consumption of fruit and berries is inversely associated with carotid atherosclerosis in elderly men[END_REF]. The consumption of plant foodstuff was shown to reduce cholesterolemia, oxidative stress, homocysteinemia, endothelial dysfunction and blood pressure [START_REF] Chopra | Influence of increased fruit and vegetable intake on plasma and lipoprotein carotenoids and LDL oxidation in smokers and nonsmokers[END_REF][START_REF] Dauchet | Fruits, vegetables and coronary heart disease[END_REF][START_REF] Silaste | Plasma homocysteine concentration is decreased by dietary intervention[END_REF]. In Western countries apples account for an important part of the fruit intake and their consumption were shown, in a human intervention study, to increase the plasma antioxidant capacity and uric acid levels [START_REF] Lotito | The increase in human plasma antioxidant capacity after apple consumption is due to the metabolic effect of fructose on urate, not apple-derived antioxidant flavonoids[END_REF]. Moreover, apple consumption has been associated with a reduced risk of cardiovascular diseases in the Women's Health Study since women ingesting apples had a 13-22% decrease in cardiovascular disease risk [START_REF] Sesso | Flavonoid intake and the risk of cardiovascular disease in women[END_REF]. Animal studies revealed that the consumption of apples exerts antioxidant effects [START_REF] Leontowicz | Comparative content of some bioactive compounds in apples, peaches and pears and their influence on lipids and antioxidant capacity in rats[END_REF], inhibits lipid oxidation [START_REF] Pearson | Apple juice inhibits human low density lipoprotein oxidation[END_REF] and lowers cholesterolemia [START_REF] Aprikian | Apple favourably affects parameters of cholesterol metabolism and of anti-oxidative protection in cholesterol-fed rats[END_REF][START_REF] Leontowicz | Comparative content of some bioactive compounds in apples, peaches and pears and their influence on lipids and antioxidant capacity in rats[END_REF].

Mechanisms involved in these protective effects can be attributed to their content in fibers as well as their content in polyphenols which are known to have preventive effects against cardiovascular diseases. Indeed, in a Finish study examining the link between flavonoid intake and coronary mortality, intake of flavonols with apple and teas as main contributors was inversely correlated with coronary mortality in women [START_REF] Knekt | Flavonoid intake and coronary mortality in Finland: A cohort study[END_REF]. In addition, in another study examining the impact of the consumption of flavonoid-rich foods, apples and pears were found to be associated with a decreased coronoary heart disease and cardiovascular disease mortality [START_REF] Mink | Flavonoid intake and cardiovascular disease mortality: a prospective study in postmenopausal women[END_REF].

Endothelial dysfunction is associated with early stages of atherosclerosis and can be detected before before the formation of atherosclerotic plaques notably by measuring the endothelium-dependent flowmediated vasodilation (FMD) [START_REF] Celermajer | Cigarette smoking is associated with dose-related and potentially reversible impairment of endothelium-dependent dilation in healthy young adults[END_REF][START_REF] Craiem | New monitoring software for larger clinical application of brachial artery flow-mediated vasodilatation measurements[END_REF]. Endothelial dysfunction is characterized by a decreased NO bioavailability and an enhancement of oxidative stress [START_REF] Celermajer | Endotheliumdependent dilation in the systemic arteries of asymptomatic subjects relates to coronary risk factors and their interaction[END_REF][START_REF] Heitzer | Endothelial dysfunction, oxidative stress, and risk of cardiovascular events in patients with coronary artery disease[END_REF]. Several studies have shown that brachial FMD is impaired by cardiovascular risk factors such as aging, male sex, smoking, hypercholesterolemia and diabetes mellitus, and is correlated with invasive coronary results [START_REF] Anderson | Close relation of endothelial function in the human coronary and peripheral circulations[END_REF][START_REF] Celermajer | Passive smoking and impaired endothelium-dependent arterial dilatation in healthy young adults[END_REF][START_REF] Chironi | Impact of shear stimulus, risk factor burden and early atherosclerosis on the time-course of brachial artery flow-mediated vasodilation[END_REF][START_REF] Levenson | Gender differences in wall shear-mediated brachial artery vasoconstriction and vasodilation[END_REF]. Moreover, brachial FMD is widely accepted to be a marker of cardiovascular disease risk [START_REF] Kuvin | Clinical utility of endothelial function testing: ready for prime time?[END_REF]Karas 2003, Verma et al., 2003). A recent meta-analysis of randomized-controlled trials with flavonoid-rich foods showed an improvement of endothelial function, assessed by flow-mediated vasodilation, and a reduced blood pressure following chronic intake of chocolate or cocoa [START_REF] Hooper | Flavonoids, flavonoid-rich foods, and cardiovascular risk: a meta-analysis of randomized controlled trials[END_REF]. Acute and chronic (4 weeks) black tea consumption were able to reverse endothelial dysfunction assessed by an improvement of FMD measurements [START_REF] Duffy | Shortand long-term black tea consumption reverses endothelial dysfunction in patients with coronary artery disease[END_REF]. The aim of the present study was to assess the effect of the consumption of two apple cultivars with different polyphenol contents, on the cardiovascular function notably by measuring the FMD and other linked cardiovascular parameters.

Methods

Materials

Cider apple fruits (Malus domestica Borkh.) from the Marie Ménard variety were harvested at maturity during the 2002 season in the experimental orchard of the Centre Technique des Productions Cidricoles (Sées, Orne, France). Golden delicious apples were obtained from a local wholesale grocery. They were drained and rapidly frozen prior to freeze-drying and ground. Polyphenols were measured by HPLC after thioacidolysis [START_REF] Guyot | Direct thiolysis on crude apple materials for high-performance liquid chromatography characterization and quantification of polyphenols in cider apple tissues and juices[END_REF]. The average degree of polymerisation was measured by calculating the molar ratio of all flavan-3-ol units (thioether adducts plus terminal units) to (-)-epicatechin and (+)-catechin terminal units. Nitrogen was analysed by the Kjeldahl method and protein was calculated as N x 6.25 . Cell walls were quantified as alcohol insoluble solids from the freeze-dried apple powders [START_REF] Renard | Variability in cell wall preparations: quantification and comparison of common methods[END_REF]. Starch was quantified by an enzymic-colorimetric method [START_REF] Missang | Changes in the pectic fraction of bush butter (Dacryodes edulis (G Don) HJ Lam) fruit pulp during ripening[END_REF]. Sugars (glucose, fructose and sucrose), and L-malic acid were determined by kit enzymatic assays (Boehringer, Mannheim and R-Biopharm AG, Darmstadt) according to producer's specifications.

The fiber content was determined as the sum of constituting sugars estimated as alditol acetate by GC [START_REF] Massiot | Composition, physico-chemical properties and enzymatic degradation of fibres prepared from different tissues of apple[END_REF]. The composition of the apple powders was balanced for simple sugars and dietary fibers, by addition of fructose ("La Vie Claire", Chaponost, France) to Marie Ménard powder and of icing sugar (St Louis Sucre, Paris, France, containing 97% saccharose and 3% starch), soluble apple fiber (POMELITE LV, Val-de-Vire Bioactives, Condé sur Vire, France) and insoluble cellulose (Avicel GP1030, FMC Biopolymers, Newark, USA) to Golden delicous powder. The Marie Ménard (20 g, HP) and Golden delicious (19.3 g, LP) apple powders were packed in sealed watertight bags.

Powder weights in bags were determined in order to provide identical quantities of fibers and simple sugars in both groups. Apples were freeze-dried so that they could be more easily transported and stored by the patients. Patients could thus leave the hospital at each visit with the 70 bags needed for a full experimental period (equivalent to about 70 apples). Very homogenous products were thus obtained. The ease of transport also contributed to the good compliance.

Study design

Thirty hypercholesterolemic men (total cholesterol > 6.2 mmol/L or LDL cholesterol > 4.1 mmol/L, with a mean age of 52.6 ± 5.5 y, a mean body mass index of 25.7 ± 2.6), without any cardiovascular diseases history and without hypocholesterolemic treatment for at least for 6 weeks, were included in the study. Exclusion criteria were as follows: familial homozygous hypercholesterolemia or LDL cholesterol > 6.2 mmol/L, diabetes (fasting blood glucose at or above 7mmol/L), hypertension (BP > 180mmHg), renal dysfunction (glomerular filtration < 60mL/min), alcohol addiction (> 3 glasses/day), current heavy smoking (Fragerström test > 6) and an excessive intake of other polyphenol-rich foods or beverages (coffee, wine, tea, soy). Volunteers were informed of the purpose and risks of the study and a written consent was obtained.

Investigators were blinded with regard to the nature of the apple samples. The study design was a double-blinded crossover. Patients received for two successive 4-week periods (4.3 ± 0.7 weeks) either the polyphenol-rich (HP) or the polyphenol-poor (LP) lyophilized apple with a 4-week washout (4.6 ± 3.2 weeks) period in-between. Patients made 4 visits, at the beginning and at the end of each experimental period (figure 1). At the first visit, they were given the exact number of apple bags required for 5 weeks. Unused bags were returned at the following visit and counted to check for compliance. Patients were asked to consume 2 bags of lyophilized apples per day, corresponding approximately to two fresh apples of 135 g each and maintained their usual diet during the whole study. The content of the two bags were consumed, one in the morning and one in the evening, after suspending the freeze-dried powder in half-a-glass of water. Blood samples were collected at the beginning and at the end of each experimental period, just before FMD measurements on either citrated or heparinized tubes. Plasma and serum fractions were prepared, aliquoted and stored at -80°C until further analysis. Spot urine samples were collected in the morning of each visit before FMD measurements and stored at -80°C.

Vascular function assessment

Endothelial-dependant flow-mediated dilation and endothelium-independent glyceryl trinitrate-mediated dilation were assessed at each of the four visits following the protocol previously described [START_REF] Chironi | Impact of shear stimulus, risk factor burden and early atherosclerosis on the time-course of brachial artery flow-mediated vasodilation[END_REF][START_REF] Craiem | New monitoring software for larger clinical application of brachial artery flow-mediated vasodilatation measurements[END_REF]. Patients were examined in the morning, after 12 h fasting, in a quiet and temperature controlled room (20±1°C). Dilatation response analysis was calculated as the ratio between the maximum change in diameter and baseline diameter and was expressed as a percentage change of the baseline diameter, as previously described [START_REF] Craiem | New monitoring software for larger clinical application of brachial artery flow-mediated vasodilatation measurements[END_REF].

Biochemical analyses

Glycemia, lipids, apolipoproteins, HDL-C and LDL-C were measured in the serum using standard laboratory procedures [START_REF] Fournier | Enhanced efflux of cholesterol from ABCA1-expressing macrophages to serum from type IV hypertriglyceridemic subjects[END_REF][START_REF] Mweva | Comparison of different cellular models measuring in vitro the whole human serum cholesterol efflux capacity[END_REF]. Urinary creatinine was determined using a commercial kit (ref: 61162 Créatinine Cinétique, Biomérieux, Marcy-l'Etoile, France) and a Kone automated apparatus (Progress Plus, Konelab, Thermo-Electron SA). Optical density was measured at 492 nm between 20-80 seconds and quality controls were also included in the analysis. (Unitrol,ref. 62 321,Biomérieux,France). Homocysteine was estimated in plasma as previously described [START_REF] Blacher | Influence of biochemical alterations on arterial stiffness in patients with end-stage renal disease[END_REF].

Ascorbic acid was determined in deproteinized plasma by HPLC [START_REF] Tessier | Validation of a micromethod for determining oxidized and reduced vitamin C in plasma by HPLC-fluorescence[END_REF]. α-Tocopherol was estimated in plasma by reverse-phase HPLC [START_REF] Simon | Study of vitamin E net mass transfer between alpha-tocopherol-enriched HDL and erythrocytes: application to asymptomatic hypercholesterolemic men[END_REF]. The ferric reducing ability of plasma (FRAP) was determined in plasma using the Benzie and Strain method [START_REF] Benzie | The ferric reducing ability of plasma (FRAP) as a measure of "antioxidant power": the FRAP assay[END_REF]. The oxygen radical absorbance capacity (ORAC) was determined on plasma samples [START_REF] Huang | High-throughput assay of oxygen radical absorbance capacity (ORAC) using a multichannel liquid handling system coupled with a microplate flourescence reader in 96-well format[END_REF]. Nitroso compounds [NOx; = NO, NO 2 -, and nitrosated and nitrosylated species (RNOs/ RSNOs)] were estimated by reductive release of NO with an iodide/triiodide containing reaction mixture and estimation of the NO formed by chemiluminescence [START_REF] Appeldoorn | Some phenolic compounds increase the nitric oxide level in endothelial cells in vitro[END_REF].

Phloretin content in urine was quantified by HPLC-ESI-MS-MS [START_REF] Ito | Polyphenol levels in human urine after intake of six different polyphenol-rich beverages[END_REF].

Statistical analysis

Data are presented as mean ± SD. Statistical tests were performed with SAS (SAS version 8.1, SAS Institute, Cary, NC). Values were log transformed before statistical analysis to compensate for unequal variance. The effects of treatment and treatment order were compared by two-way repeated measures ANOVA using SAS proc MIXED. SAS LSmeans function was used as post hoc means comparison test.

Results

Baseline characteristics

Thirty volunteers were included in this study with a mean age of 52.6 ± 5.5 years, a mean body mass index of 25.7 ± 2.6, systolic blood pressure was 123.2 ± 11 mmHg and diastolic blood pressure was 75.5 ± 7.6 mmHg.

Compliance

Compliance was assessed by measuring phloretin excretion in urine. Phloretin is a dihydrochalcone characteristic of apple and has been used as a marker of apple consumption [START_REF] Mennen | Urinary flavonoids and phenolic acids as biomarkers of intake for polyphenol-rich foods[END_REF].

Phloretin excretion in urine increased after apple consumption from 0.04-0.11 to 0.52-0.78 nmol/mg creatinine (P < 0.01). The difference in phloretin excretion levels between the two apple cultivars at the end of the supplementation (HP/LP ratio = 1.5) precisely reflected the difference in phloretin content in the two apples (Table 1; HP/LP ratio = 1.6). The exact duration of the periods of apple consumption was 4.3 ± 0.9 weeks for the HP apple and 4.2 ± 0.3 weeks for LP apple. The patients were asked to consume two apple bags per day. The number of bags actually consumed per day, calculated from the number of non-consumed returned bags, was 58.8 ± 9 bags for the HP period and 59.2 ± 7.7 bags for the LP period showing a good compliance of the subjects.

Brachial artery responses

As shown in table 1, no significance difference in FMD was observed between the LP and HP apples after one month of apple supplementation. No difference could neither be observed between baseline level at the beginning of each intervention period and the end of the apple supplementation period.

Figure 2 shows the flow-mediated dilation variability for the 30 patients before and after consuming the HP and LP apples. Apple consumption did not influence the endothelium-independent glyceryltrinitrate vasodilation.

Biochemical parameters

Consumption of the two apple cultivars during 4 weeks had no significant effect on all measured biochemical parameter such as lipids, glucose, antioxidant vitamin status or plasma antioxidant capacity (table 2). However, an increase in vitamin C concentration in plasma from 58 µM at baseline to 65 µM was observed after one month of apple consumption, whatever the nature of the apple consumed.

Discussion

The aim of this study was to assess the impact of the consumption of two apple varieties differing by their polyphenol content, on endothelial function and associated cardiovascular parameters. Volunteers were asked to consume 40 g of lyophilized apples per day corresponding to the consumption of about two apples per day. The patients ingested 214 mg polyphenols per day with the Golden Delicious (LP) apple and 1.43 g polyphenols per day with the Marie-Ménard (HP) apple, hence a difference of polyphenol intake of 1.21 g per day for the two apples. Compliance was assessed by measuring levels of apple phloretin in urine. A good compliance was observed as shown by the increase in phloretin excretion at the end of each intervention period.

The study was carried out in hypercholesterolemic patients known to have an impaired endothelial function [START_REF] Maas | Simultaneous assessment of endothelial function, nitric oxide synthase activity, nitric oxide-mediated signaling, and oxidative stress in individuals with and without hypercholesterolemia[END_REF]. A baseline value of 5-6% for FMD was measured in our subjects (Table 2). This value is similar to that measured by other groups, and significantly lower than the ~10% value commonly observed in normocholesterolemic subjects. Cut-off values for FMD or cholesterolemia at baseline, to observe effects on fasting FMD were determined in two meta-analyses. Cut-off for FMD was found to be less than 7% for intervention studies with arginine [START_REF] Bai | Increase in fasting vascular endothelial function after short-term oral L-arginine is effective when baseline flow-mediated dilation is low: a meta-analysis of randomized controlled trials[END_REF]. Cut-off for cholesterolemia was found to be more than 228 mg/dL [START_REF] Li | Effect of oral isoflavone supplementation on vascular endothelial function in postmenopausal women: a meta-analysis of randomized placebo-controlled trials[END_REF], a value compatible with the mild hypercholesterolemia of the present patients (6.4 mmol/L or 247 mg/dL; Table 2).

Flow-mediated vasodilation measured at the end of each intervention period did not differ between the HP and LP apples, neither any of the other cardiovascular disease risk factors estimated (Table 2). The power of the study would have been sufficient to detect a FMD difference of 2.3 percent units (for a power of 0.8 and a p value of 0.05), a value higher than the largest (non significant) difference between the groups (1.8 % for the difference between baseline and HP apple). In agreement with these data, NO did not change between treatments. Fifteen trials in which the effects of flavanoids or flavonoid-rich foods and beverages on FMD were examined, were recently analyzed in a metaanalysis [START_REF] Hooper | Flavonoids, flavonoid-rich foods, and cardiovascular risk: a meta-analysis of randomized controlled trials[END_REF]. Different results were observed depending in particular on the structure of the flavonoids. Only chocolate and tea, both rich in flavanols were shown to increase FMD in chronic studies lasting for more than two weeks. However, chronic studies on red wine or grape also rich in flavanols did not show any improvement of FMD. Improvement of FMD with flavanol-rich foods or beverages could be explained by either proanthocyanidins or the catechins which are systematically associated in these foods. Also, two FMD studies have been carried out with pure flavanols. An acute intake of either pure epicatechin (1 or 2 mg/kg body weight in one dose) or epigallocatechin gallate (EGCG; one dose of 300 mg) were shown to increase FMD in young healthy adults in the two hours following ingestion [START_REF] Schroeter | -)-Epicatechin mediates beneficial effects of flavanol-rich cocoa on vascular function in humans[END_REF][START_REF] Widlansky | Acute EGCG supplementation reverses endothelial dysfunction in patients with coronary artery disease[END_REF]. However, in a similar study with EGCG, the chronic administration of the same dose (300 mg/day) during two weeks failed to influence vascular reactivity [START_REF] Widlansky | Acute EGCG supplementation reverses endothelial dysfunction in patients with coronary artery disease[END_REF]. In the present work, the intake of flavanol monomers was 17 mg/day for the LP apple and 111 mg per day for HP apple consumption.

This level of intake might have induced a positive effect on FMD as observed in the Schroeter study [START_REF] Schroeter | -)-Epicatechin mediates beneficial effects of flavanol-rich cocoa on vascular function in humans[END_REF]. The lack of effect might therefore be explained by the too rapid elimination of flavanol monomers as suggested in the EGCG study. Similarly to this last study, FMD measurements were made in the morning about ten hours after consumption of the last apple bag. Catechin monomers if responsible for these effects would not be present anymore due to their rapid elimination (half-life of 2-3 hrs) (Manach et al., 2005b). However, this interpretation does not fit with the results of another chronic study on tea [START_REF] Duffy | Shortand long-term black tea consumption reverses endothelial dysfunction in patients with coronary artery disease[END_REF]. The regular consumption of black tea (900 ml/day) during 4 weeks still improved FMD measured one night after the last tea intake.

Another interpretation might also explain the lack of effects of apple polyphenols observed here.

Catechins in the apple powder might have been less well absorbed than catechins ingested as aqueous solutions due to interactions with the apple matrix. Tea catechins were shown to be better absorbed after an overnight fast on an empty stomach than when taken with foods resulting in a 3.5 fold increase of maximum plasma concentration of free epigallocatechin [START_REF] Chow | Effects of dosing condition on the oral bioavailability of green tra catechins after songle-dose administration of polyphenon E in healthy individuals[END_REF].

The main polyphenols consumed with the apples were procyanidins (Table 1). It is known that their bioavailability is low due to their high molecular weight [START_REF] Déprez | Transport of proanthocyanidin dimer, trimer and polymer across monolayers of human intestinal epithelial Caco-2 cells[END_REF][START_REF] Donovan | Procyanidins are not bioavailable in rats fed a single meal containing a grapeseed extract or the procyanidin dimer B3[END_REF], Gonthier et al., 2003). Only procyanidin dimers have been detected in human plasma but at very low concentrations [START_REF] Holt | Procyanidin dimer B2 [epicatechin-(4β-8)-epicatechin] in human plasma after the consumption of a flavanol-rich cocoa[END_REF]. Their poor bioavailability does not exclude systemic effects.

However, few authors have examined the in vivo biological effects of pure proanthocyanidins. Most often, their activity was tested using complex proanthocyanidin extracts which also contain catechin monomers. These catechin monomers might be responsible for the effects observed [START_REF] Schroeter | -)-Epicatechin mediates beneficial effects of flavanol-rich cocoa on vascular function in humans[END_REF]. Proanthocyanidins are also degraded in the colon into well absorbed low molecular weight phenolic acids which may exert systemic effects [START_REF] Rios | Chocolate intake increases urinary excretion of polyphenol-derived phenolic acids in healthy human subjects[END_REF]. The present study suggests that they do not influence vascular reactivity. Due to the relatively long exposure of procyanidins to the microbiota in the colon and slow degradation into phenolic acids, the plasma concentrations of their phenolic acid metabolites do not vary much over time [START_REF] Rios | Chocolate intake increases urinary excretion of polyphenol-derived phenolic acids in healthy human subjects[END_REF]. In contrast to catechins, they would still be present in the plasma in the morning following consumption of the last apple bag. The rapid elimination of catechins proposed to explain the lack of observed effects of the HP apple on FMD could not hold for phenolic acids which would therefore be truly inactive. Another FMD study with a cocoa drink regularly consumed over one week and providing about 900 mg flavanols per day showed an increase of FMD building up over the week, but FMD measurement was carried out 2 hrs after consumption of the cocoa drink [START_REF] Heiss | Sustained increase in flow-mediated dilation after daily intake of high-flavanol cocoa drink over 1 week[END_REF]. Two longer-term studies have been carried

out with flavanol-rich cocoa. The first one showed no effect of the consumption of flavanol-rich cocoa during 6 weeks on FMD [START_REF] Farouque | Acute and chronic effects of flavanol-rich cocoa on vascular function in subjects with coronary artery disease: a randomized double-blind placebo-controlled study[END_REF] and the second one only showed a modest effect of the chronic consumption of cocoa when measured in fasting conditions as in the present study [START_REF] Balzer | Sustained benefits in vascular function through flavanol-containing cocoa in medicated diabetic patients a double-masked, randomized, controlled trial[END_REF].

No effect of a regular HP apple consumption on plasma antioxidant capacity or uric acid concentration was observed here. In a review of 12 intervention studies assessing the impact of the consumption of different polyphenol-rich foods or beverages during 1 to 12 weeks, no systematic improvement of plasma antioxidant capacity, uric acid concentration or in the status of antioxidant vitamins (vitamin C and E) could be observed (Manach et al., 2005a). No more consistent effect could be observed on lipid oxidation products or LDL oxidizability, also possibly explained by the too rapid elimination of polyphenols during the night prior to the collection of fasting plasma samples. The slight increase (x 1.1) in plasma vitamin C observed here for the two apple varieties when compared to baseline is most likely explained by the presence of vitamin C in apples. No effect of the HP apple on blood pressure could be observed. Quercetin, one of its phenolic constituents, was shown to reduce blood pressure in hypertensive subjects at a dose of 150 mg/day [START_REF] Egert | Quercetin reduces systolic blood pressure and plasma oxidised low-density lipoprotein concentrations in overweight subjects with a high-cardiovascular disease risk phenotype: a double-blinded, placebo-controlled cross-over study[END_REF]. The lack of effects observed here is most likely explained by the much lower intake of quercetin (about 12 mg/day).

This study may be limited by the lack of analysis of the kinetics of brachial artery dilation in response to hyperheaemia [START_REF] Chironi | Impact of shear stimulus, risk factor burden and early atherosclerosis on the time-course of brachial artery flow-mediated vasodilation[END_REF], which could not be studied due to the unavailability of the entire FMD time-course curve for some patients. However, the percentage change of FMD between baseline and maximal brachial artery diameter as measured here remains the gold standard for assessing endothelial function in clinical trials.

Another limitation of this study is the freeze-dried state of the apples. This contributed to the good compliance of the protocol but it may also have influenced the bioavailability of the polyphenols.

Although the bioavailability of polyphenols in fresh and freeze-dried fruits has not been compared, there is no indication that these would differ substantially. The bioavailability of organic acids of fresh and freeze-dried banana or sweet potato were compared in a recent publication, and no effect of freeze-drying could be seen [START_REF] Sabboh-Jourdan | Organic acid bioavailability from banana and sweet potato using an in vitro digestion and Caco-2 cell model[END_REF]. Freeze-dried fruits are also commonly used in animal studies with no notable effects on polyphenol bioavailability [START_REF] Felgines | Strawberry pelargonidin glycosides are excreted in urine as intact glycosides and glucuronidated pelargonidin derivatives in rats[END_REF].

Altogether, this first study comparing the effects of a polyphenol-rich and a polyphenol-poor fruit on FMD showed no improvement of the endothelial function after consuming the polyphenol-rich apple during one month. This result differs from several previous similar studies on endothelial function with cocoa or tea in which two foods or beverages differing in their flavanol content were compared.

However an acute effect on endothelial function in the few hours following ingestion cannot be excluded and should be examined. The present study was focused on apple polyphenols. However some effects of other apple constituents like fibers or vitamin C on endothelial function cannot be excluded [START_REF] Brock | A highcarbohydrate, high-fiber meal improves endothelial function in adults with the metabolic syndrome[END_REF][START_REF] Ting | Vitamin C improves endothelium-dependent vasodilation in forearm resistance vessels of humans with hypercholesterolemia[END_REF] and should also be further explored. Data are means ± SD. * P < 0.05; † P < 0.01; ‡ P < 0.001 as compared to baseline.

Figure legend Figure 1 :

 legend1 Figure legend Figure 1: Study protocol. Men were randomized and treated for 4 weeks with LP and HP lyophilized apples. Patients were studied four times (arrows): at baseline and at the end of each period. FMD: flow-mediated vasodilation.

Figure 2 :

 2 Figure 2: Effect of a one-month intervention with low polyphenol (LP) and high polyphenol (HP) apple varieties on flow-mediated vasodilation. Symbols represent individual subjects. Symbols on the left and right of each figure and brackets are means ± SD.

Table 2

 2 Brachial artery and biochemical parameters

	Parameters	Baseline	LP apple	Baseline	HP apple
	FMD (% compared to basal diameter)	6.1 ± 3.9	4.5 ± 3.0	5.7± 4.2	3.9 ± 3.2
	Glyceryl trinitrate
	(% compared to basal diameter)	15.6 ± 4.7	16.7 ± 4.3	16.1 ± 5.9	16.2 ± 6.1
	NOx (nM)	72.7 ± 23.4	88.7 ± 51.6	81.5 ± 27.1	77.8 ± 31.1
	Systolic blood pressure (mmHg)	120.0 ± 12.2	118.6 ± 10.0	119.0 ± 11.2	118.8 ± 8.1
	Diastolic blood pressure (mmHg)	76.1 ± 8.7	76.9 ± 7.5	76.7 ± 7.8	77.0 ± 7.5
	Pulse pressure (mmHg)	61.1 ± 9.5	60.1 ± 9.6	60.4 ± 7.6	60.2 ± 8.9
	Phloretin (nmol/mg creatinine)	0.04 ± 0.09	0.52 ± 0.67 †	0.11 ± 0.35	0.78 ± 1.17 ‡
	Glucose (mmol/L)	4.9 ± 0.4	5.1 ± 0.3	4.9 ± 0.5	5.1 ± 0.5
	Iron (µmol/L)	18.3 ±6.3	18.6 ± 5.7	18.2 ± 5.1	18.0 ± 4.3
	CRP (mg/L)	0.98 ± 1.06	0.78 ± 0.53	1.14 ± 1.08	1.16 ± 1.25
	FRAP (µM Fe 2+ /mL)	1047 ± 125	1021 ± 121	1026 ± 102	1057 ± 147
	ORAC (10 3 µmol Trolox equivalents/L)	14.1 ± 2.8	13.5 ± 2.5	14.1 ± 2.7	13.4 ± 2.4
	Vitamin C (µM)	57.3 ± 14.1	65.8 ± 11.4 ‡	59.3 ± 17.4	65.8 ± 14.9*
	Plasma Vit E (µM)	38.4 ± 5.8	37.3 ± 6.7	37.4 ± 6.0	36.6 ± 6.2
	Uric acid (µmol/L)	329 ± 85	316 ± 100	344 ± 47	321 ± 104
	Homocysteine (µmol/L)	11.3 ± 3.6	11.5 ± 4.6	12.1 ± 7.2	12.3 ± 7.8
	Total cholesterol (mmol/L)	6.3 ± 0.7	6.3 ± 0.9	6.5 ± 0.7	6.4 ± 0.9
	Triglycerides (mmol/L)	1.4 ± 0.7	1.4 ± 0.7	1.3 ± 0.5	1.4 ± 0.6
	LDL cholesterol (mmol/L)	4.3 ± 0.6	4.2 ± 0.7	4.5 ± 0,6	4.4 ± 0.7
	HDL cholesterol (mmol/L)	1.4 ± 0.4	1.4 ± 0.3	1.4 ± 0.3	1.3 ± 0.3
	APOA1 (g/L)	1.5 ± 0.3	1.5 ± 0.2	1.6 ± 0.2	1.5 ± 0.2
	APOB (g/L)	1.2 ± 0.2	1.2 ± 0.2	1.3 ± 0.2	1.2 ± 0.2
	ApoB/ApoA1	0.79 ± 0.25	0.82 ± 0.19	0.82 ±0.19	0.84 ± 0.21

Acknowledgement: This work was supported by the European Community (FLAVO-FOOD-CT-2004-513960).

Conflicts of interest:

The authors have no conflict of interest to declare. 2 Balanced by the addition of 2000mg apple fibers per bag.

3 Balanced by the addition of 2200 mg fructose.

4 Balanced by the addition of 1100 mg fructose.

5 Balanced by the addition of 2200 mg sucrose.


Arianna Tucci

Mike A Nalls

Henry Houlden

Tamas Revesz

Andrew B Singleton

Nicholas W Wood

John Hardy

PhD Coro Paisán-Ruiz
email: c.paisan-ruiz@ion.ucl.ac.uk

Genetic variability at the PARK16 locus

Parkinson's disease is a complex neurodegenerative disease whose hallmark pathological features are loss of dopaminergic neurons in the substantia nigra and intracytoplasmic neuronal inclusions containing alpha-synuclein aggregations known as Lewy bodies. Although the majority of PD is idiopathic, pathogenic mutations in several mendelian genes have successfully been identified through linkage analyses. To identify susceptibility loci for idiopathic Parkinson's disease, several genome-wide association studies (GWAS) within different populations have recently been conducted in both idiopathic and familial forms of PD.

These analyses have confirmed SNCA and MAPT as loci harbouring PD susceptibility. In addition, the GWAS identified several other genetic loci suggestively associated with the risk of PD; among these, only one was replicated by two different studies of European and Asian ancestries. Hence, we investigated this novel locus known as PARK16 for coding mutations in a large series of idiopathic pathologically proven PD cases in addition to performing an association study in a case-control cohort from the UK. An association between a novel RAB7L1 mutation, c.379-12insT, and disease (Pvalue = 0.0325) was identified. Two novel coding variants present only in the PD cohort were also identified within the RAB7L1 (p.K157R) and SLC41A1 (p.A350V) genes. No copy number variation (CNV) analyses have yet been performed within this recently identified locus. We concluded that although both coding variants and risk alleles within the PARK16 locus seem to be rare, further molecular analyses in these genes within different populations are required in order to examine its biochemical role in the disease process.

Introduction

Parkinson's disease (PD; MIM #68600) is a common complex disease clinically characterized by resting tremor, bradykinesia, postural instability and rigidity, and pathologically by the presence of severe pars-compacta nigral-cell loss and an accumulation of aggregated α-synuclein in specific brain stem, spinal cord and cortical regions [START_REF] Lees | Parkinson's disease[END_REF] . Although the majority of PD is idiopathic, pathogenic mutations have successfully been identified in some mendelian forms [START_REF] Hardy | The genetics of Parkinson's syndromes: a critical review[END_REF] . Many of these mendelian genes have also been investigated in the idiopathic disease but only SNPs at the SNCA and LRRK2 loci have shown susceptibility for idiopathic PD (IPD): several SNPs at the SNCA locus have been characterized as risk factors for IPD in different populations [START_REF] Winkler | alpha-Synuclein and Parkinson disease susceptibility[END_REF] , a LRRK2associated haplotype showed an increased disease risk in the Chinese population [START_REF] Skipper | Comprehensive evaluation of common genetic variation within LRRK2 reveals evidence for association with sporadic Parkinson's disease[END_REF] , two LRRK2 mutations absent in European ancestry populations are overrepresented in PD in some Asian populations [START_REF] Fonzo | A common missense variant in the LRRK2 gene, Gly2385Arg, associated with Parkinson's disease risk in Taiwan[END_REF][START_REF] Ross | Analysis of Lrrk2 R1628P as a risk factor for Parkinson's disease[END_REF] and common LRRK2 variation may also contribute to the risk for IPD in the North American population [START_REF] Paisan-Ruiz | Parkinson's disease and low frequency alleles found together throughout LRRK2[END_REF] . Similarly, the frequency and distribution of GBA mutations in PD vary within populations, being more prevalent among Ashkenazi Jewish population and rare among Asians [START_REF] Sidransky | Multicenter analysis of glucocerebrosidase mutations in Parkinson's disease[END_REF] . Taken together, PD is a complex genetic disorder in which the prevalence of some pathogenic mutations may vary widely within ethnicities [START_REF] Sidransky | Multicenter analysis of glucocerebrosidase mutations in Parkinson's disease[END_REF][START_REF] Paisan-Ruiz | LRRK2 gene variation and its contribution to Parkinson disease[END_REF] .

Genome-wide (GW) SNP genotyping assays have been proven to be a powerful technique to identify genetic risk factors in many complex disorders [START_REF] Hardy | Genomewide association studies and human disease[END_REF] . Consequently, three large PDassociated genome-wide association studies (GWAS) from two European ancestry and one Asian populations have recently identified genetic risk underlying PD, of which SNCA (all three studies) and MAPT (only European ancestry studies) loci showed the strongest evidences of association with PD [START_REF] Pankratz | Genomewide association study for susceptibility genes contributing to familial Parkinson disease[END_REF][START_REF] Satake | Genome-wide association study identifies common variants at four loci as genetic risk factors for Parkinson's disease[END_REF][START_REF] Simon-Sanchez | Genome-wide association study reveals genetic risk underlying Parkinson's disease[END_REF] ; these associations have recently been corroborated by a meta-analysis carried out in European ancestry PD-associated GWAS [START_REF] Edwards | Genome-Wide Association Study Confirms SNPs in SNCA and the MAPT Region as Common Risk Factors for Parkinson Disease[END_REF] . In addition, one out of three additional genetic risk loci for PD was independently identified by two studies [START_REF] Pankratz | Genomewide association study for susceptibility genes contributing to familial Parkinson disease[END_REF][START_REF] Satake | Genome-wide association study identifies common variants at four loci as genetic risk factors for Parkinson's disease[END_REF][START_REF] Simon-Sanchez | Genome-wide association study reveals genetic risk underlying Parkinson's disease[END_REF] . Therefore, we here investigate whether novel genetic variants within this locus designated as PARK16 may predispose to the risk for PD in a British cohort of pathologically proven PD cases and neurologically normal individuals. PARK16 located on chromosome 1q32 comprises 169.6kb and contains 5 different genes (Table 1).

Materials and Methods

Subjects:

The PD cohort was collected from brain tissues at The Queen Square Brain Bank for Neurological Disorders in the UK. Cases (n=453) were clinically and pathologically diagnosed according to the PD Brain Bank criteria [START_REF] Gibb | The relevance of the Lewy body to the pathogenesis of idiopathic Parkinson's disease[END_REF][START_REF] Hughes | Accuracy of clinical diagnosis of idiopathic Parkinson's disease: a clinico-pathological study of 100 cases[END_REF] . The mean age at onset was 59 years (ranged from 35 to 86 years) and the average of death was 78 years (ranged from 51 to 94 years). The maleto-female ratio was 3.5: 1. Family history was reported in < 1% individuals. DNA samples from 82 PD cases reporting positive family history were also employed. Positive family history was compatible with the diagnosis of PD in at least one first or second degree relative. The mean age of disease onset in these familial cases was 57 years (ranged from 29 to 71 years). Patients and all relatives of patients gave informed consent for scientific research. The control cohort (n=483) analyzed here was the "1958 British birth cohort" whose individuals were all born in March 1958 in England, Scotland or Wales and which is used in all disease-related studies carried out by the Wellcome Trust Case Control Consortium (WTCCC; http://www.b58cgene.sgul.ac.uk).

PCR and sequencing analyses: In first instance, PCR and sequencing analyses of all open-

reading frames (ORFs) of NUCKS1 (RefSeq NM_022731, 7 exons), RAB7L1 (RefSeq NM_003929, 5 exons) and SLC41A1 (RefSeq NM_173854.4, 11 exons) genes were performed in 182 PD cases.

Later, each variant identified in the PD cohort (n=9) was also analyzed in 351 neurologically normal individuals. Hereafter, every SNP showing association with the disease (n=1; c.379-12insT) and each coding variant absent in controls (n=2; p.K157R and p.A350V) were further analyzed in larger sample size compiling a total of 454 PD cases and 483 controls analyzed. In addition, the two coding variants absent in control population were also tested in 82 familial PD cases. All PCR analyses were performed using both forward and reverse genomic primers (all primer-sequences are available upon request) previously designed by ExonPrimer (http://ihg.gsf.de/ihg/ExonPrimer.html) and FastStart Taq DNA polymerase (www.rocheapplied-science.com). Each purified product was sequenced using both forward and reverse primers with Applied Biosystems BigDye terminator v3.1 sequencing chemistry as per the manufacturer's instructions; the resulting reactions were then resolved on an ABI3730XL genetic analyzer (Applied Biosystems) and analyzed with Sequencher software 4.9 (Gene Codes Corporation).

The Alamut mutation interpretation software was used to look for amino acid properties and for predictions of the functional and structural effects of novel coding mutations (http://www.interactive-biosoftware.com/alamut.html). Multiple alignments for RAB7L1 and SLC41A1 encoding proteins were performed through NCBI-associated homoloGene database by using the MUSCLE program [START_REF] Edgar | MUSCLE: multiple sequence alignment with high accuracy and high throughput[END_REF] . The Human Protein Reference Database (HPRD; http://www.hprd.org/) was employed to search for predicted protein motifs and domains. And NCBI-BLAST database was also used to search for sequence similarities between Rab proteins; the RAB7L1 protein sequence (RefSeq: NP_001129134.1) was aligned with, RAB1 (RefSeq: NP_004152.1), RAB3A (RefSeq: NP_002857.1), RAB7 (RefSeq: NP_004628.4) and RAB8A (RefSeq: CAG38820.1) proteins (http://blast.ncbi.nlm.nih.gov/Blast.cgi).

Statistical analyses: All statistical analyses (chi square tests of association and permutation analyses) were performed using the Haploview 4.1 software (http://www.broad.mit.edu/haploview/). In order to compare PARK16-associated allelic frequencies between diverse populations, HapMap data corresponding to the PARK16 locus from Yoruba (YRI), Japan (JPT), Han Chinese (CHB) and Northern and Western European (CEU-Utah residents) populations was also analyzed through haploview software (www.hapmap.org).

Results

To try and identify novel genetic variants underlying risk for PD in a British case-control cohort, the genomic area harboring PARK16 locus was deeply investigated through sequencing analyses. In first instance, it was decided to perform sequencing analyses of all coding regions and exon-intron boundaries of genes located within the genomic area shared by both PARK16 loci identified in European ancestry and Asian populations, respectively [START_REF] Satake | Genome-wide association study identifies common variants at four loci as genetic risk factors for Parkinson's disease[END_REF][START_REF] Simon-Sanchez | Genome-wide association study reveals genetic risk underlying Parkinson's disease[END_REF] ; this area flanked by rs823128 (203,980,001 bp) and rs11240572 (204,074,636 bp) SNPs contained four genes (NUCKS1, RAB7L1, SLC41A1 and PM20D1) (Table 1). However, NUCKS1, RAB7L1 and SLC41A1 genes were located in the same LD block and were suggestively reported as the best candidates for the etiology of PD according to their functional roles [START_REF] Satake | Genome-wide association study identifies common variants at four loci as genetic risk factors for Parkinson's disease[END_REF] . In addition, the minor allele frequency of rs11240572 located in intron 10 of PM20D1 is < 0.03 in European ancestry population (Table 3). Hence, only the coding regions of NUCKS1, RAB7L1 and SLC41A1 were analyzed in our 182 PD cases. PCR analyses of all ORFs revealed the presence of 9 different genetic variants within RAB7L1 (n=5) and SLC41A1 (n=4) genes, while no genetic variation was identified across the NUCKS1 gene. There were two coding variants (p.Gln104Glu (s41302139) and p.Lys157Arg (novel)), two novel intronic variants (c.197-49insG and c.379-12insT) and one UTR-5' variant (rs708755) among the variants identified within the RAB7L1 gene; whereas three coding mutations (p.Thr113Thr (rs11240569), p.Asn252Asn (rs708727) and p.Ala350Val (novel)) and a known intronic variant (rs41264905) were identified within the SLC41A1 gene. All genetic variants, with the exception of both novel coding mutations which were identified in one PD patient each, were found present in both cases and controls (Table 2). The coding mutations were a heterozygous c.470A>G transition causing p.Lys157Arg and a heterozygous c.1049C>T transition causing p.Ala350Val which were respectively located within RAB7L1 (exon 4) and SLC41A1 (exon 8) genes (supplemental figure 1). In order to inspect whether these novel coding mutations may or may not be the disease-causing mutations, both were tested in larger sample size of additional pathologically proven idiopathic PD cases (n =272, n (total) =454), 82 familial cases clinically diagnosed of PD, and 483 neurologically normal individuals; failing to detect any other mutation carrier in neither PD nor control populations. Both variants are also conserved among species (supplemental figure 1). Contradictory results were obtained with respect to the functional consequences for both novel mutations; however, the K157 amino acid of RAB7L1 was predicted to be highly conserved whereas the A350 amino-acid of SLC41A1 was shown to be moderately conserved (Alamut software). Both clinical and pathological features of K157R and A350V mutation carriers are described in the supplemental material 1.

To test the hypothesis whether the remaining seven genetic variants identified may predispose to the risk for PD, they were additionally tested in 351 neurologically normal individuals. A single-marker chi square test of association was then performed. This analysis revealed a slightly significant association between the c.379-12insT mutation within the RAB7L1 gene (intron 3) and PD (frequentist P value = 0.0325), which remained significant after one million of iterations of permutation testing to adjust for multiple comparisons (permuted P value = 0.0399; Table 2).

Discussion

Sequencing analyses of the coding region of NUCKS1, RAB7L1 and SLC41A1 genes in a British cohort of 182 pathologically proven PD cases revealed the presence of two novel mutations, in one patient each, within RAB7L1 (K157R) and SLC41A1 (A350V) genes. PARK16 locus was not examined for the presence of large CNVs. Both mutations carriers showed typical IPD and Lewy body pathology. However, even the non-occurrence of both mutations in a large sample of ethnicity-matched control individuals (n =483) does not fully disclose their pathogenecity.

Seven additional intronic and exonic variants were also identified during the sequencing process. Therefore, an association study which revealed a weak association between the c.379-12insT mutation and IPD was carried out. Curiously, no intronic variation was previously reported within the RAB7L1 locus, suggesting that genetic variability within this locus is rare.

Given the presence of a rare novel mutation and slightly associated risk allele within RAB7L1, further investigations within this gene are warranted in order to determine its precise biochemical role in the pathogenesis of PD. The RAB7L1 encoding protein is a member of the Rab GTPases subfamily which includes a large number of small GTPases involved in intracellular cell signaling processes and vesicle trafficking. The K157 amino acid of RAB7L1 lies in the Rab domain (8 -176 amino acids) of the protein which is predicted to be highly conserved among species and is also conserved among other Rab proteins, such as RAB1A, RAB3A, RAB7A and RAB8A proteins (data not shown). Molecular links between PD and Rab proteins were already suggested: mutations in the Ras-like GTPase domain of dardarin cause PD [START_REF] Paisan-Ruiz | LRRK2 gene variation and its contribution to Parkinson disease[END_REF][START_REF] Greggio | Leucine-rich repeat kinase 2 mutations and Parkinson's disease: three questions[END_REF] and elevated expression of RAB1, RAB3A and RAB8A proteins protect against alpha-syn-induced dopaminergic neuron loss in animal models of PD [START_REF] Cooper | Alpha-synuclein blocks ER-Golgi traffic and Rab1 rescues neuron loss in Parkinson's models[END_REF][START_REF] Gitler | The Parkinson's disease protein alpha-synuclein disrupts cellular Rab homeostasis[END_REF] . SLC41A1 is a Mg (2+) transporter that may play role in magnesium homeostasis. Brain metal dyshomeostasis has often been speculated as cause for neurodegeneration; nevertheless, the precise nature of its biochemical mechanisms underlying neurodegeneration is still vague [START_REF] Bolognin | Metal ion physiopathology in neurodegenerative disorders[END_REF][START_REF] Kolisek | SLC41A1 is a novel mammalian Mg2+ carrier[END_REF] .

Although no association between the PARK16 locus and PD was identified in a GWAS metaanalysis [START_REF] Edwards | Genome-Wide Association Study Confirms SNPs in SNCA and the MAPT Region as Common Risk Factors for Parkinson Disease[END_REF] , analyses of the PARK16-associated SNPs within the HapMap data revealed marked differences in the minor allelic frequencies between populations; thus, affecting analytic power (Table 3). Likewise, population differences at the BST1 and MAPT loci were recently reported [START_REF] Satake | Genome-wide association study identifies common variants at four loci as genetic risk factors for Parkinson's disease[END_REF][START_REF] Simon-Sanchez | Genome-wide association study reveals genetic risk underlying Parkinson's disease[END_REF] and the haplotype H2 of MAPT reported to be almost exclusively of Caucasian origin is low in all populations [START_REF] Evans | The tau H2 haplotype is almost exclusively Caucasian in origin[END_REF] . By and large, different genetic markers should be used when investigating different populations as some may be not relevant to all populations. Thereafter, we conclude that although pathogenic mutations and risk alleles within the PARK16 locus seem to be rare in European ancestry populations, further molecular analyses within different populations are required in order to examine its biochemical role in the PD and prior to undertake any functional work on the encoded proteins associated with this locus.

Acknowledgements

We thank the patients for taking part of this study and to all families who support the donation of tissue for research. We would like also to thank The Medical Research Council (MRC; HH: MRC fellowships G108/638 and G0802760 and JH: Start up funds), and The Michael J Fox Foundation (HH and CPR) for support. This study was also supported by the NIHR UCLH/UCL Comprehensive Biomedical Research Centre. Participation by MAN and ABS in this research was supported in part by the Intramural Research Program of the NIH, National Institute on Aging (AG000957-07 (2009) Assessment of Candidate Loci in Neurological diseases).

Conflict of Interest Statement

The authors declare they have no conflict of interest.

Legend to supplemental figure 1

Supplemental figure 1 . The SNPs showing the highest P value for each independent study are highlighted in bold.

Legends to tables

RAB7L1 chromosomal localization: 204,003,738 -204,011,233bp. RAB7L1 is located in the 27.6kb interval between rs823122 (at ~12kb away) and rs947211 (at ~8kb away) SNPs.

Table 2:

Chi square associations tests for common variants identified within PARK16 locus performed by haploview software. The only variant (c.379-12insT) which showed association with the disease is highlighted in bold; the 1 x 10 6 permutation value associated to this variant is also shown in brackets. The case/control frequencies for each variant are also shown.


Laurent Villard
email: laurent.villard@univmed.fr

Pierre Cacciagli

Marie-Reine Haddad

Cecile Mignon-Ravix

Bilal El-Waly

Anne Moncla

Chantal Missirian

Brigitte Chabrol

Cécile Mignon-Ravix

Disruption of the ATP8A2 gene in a

Keywords: chromosome aberrations, mental retardation, Atp8a2, 10p12, 13q12

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

INTRODUCTION

Mental retardation is a frequent condition that is clinically and genetically highly heterogeneous [START_REF] Leonard | The epidemiology of mental retardation: challenges and opportunities in the new millennium[END_REF]. Mutations in hundreds of different genes have been identified in patients suffering from mental retardation and these genes encode proteins performing a wide range of molecular functions inside and outside the central nervous system [START_REF] Chelly | Genetics and pathophysiology of mental retardation[END_REF].

Despite its important prevalence, ranging between 0,5 and 3% of the population, the majority of mental retardation cases remain unexplained.

The presence of de novo chromosomal rearrangements in patients with an abnormal phenotype can be used to identify disease-causing genes [START_REF] Higgins | Characterization of apparently balanced chromosomal rearrangements from the developmental genome anatomy project[END_REF]. Cloning of the breakpoints of balanced chromosomal translocations in affected patients has led to the identification of a large number of genes, including genes implicated in different types of mental retardation phenotypes [START_REF] Vandeweyer | Balanced translocations in mental retardation[END_REF].

Here, we describe a female patient with moderate mental retardation, severe hypotonia, and a de novo balanced translocation 46,XX,t(10;13)(p12.1;q12.13)dn. We cloned and sequenced the two translocation breakpoints and showed that the translocation is perfectly balanced at the molecular level. Analysis of the breakpoint regions reveals that the coding sequence of a P-type ATPase called ATP8A2 on chromosome 13q12.13 is interrupted by the translocation whereas there is no known transcription unit located in the immediate vicinity of the chromosome 10 breakpoint. We analyzed the expression pattern of the ATP8A2 transcript in mice using in situ hybridization and in human using RT-PCR. We show that ATP8A2 is highly expressed in the brain and testis both in human and mouse. We hypothesize that haploinsufficiency of the ATP8A2 gene could be involved in the severe neurological phenotype presented by the patient with the balanced translocation.

MATERIALS AND METHODS

Cell lines, karyotyping, FISH and array CGH

Karyotypes were determined by analysis of R-banded chromosomes at a minimum resolution of 550 bands using standard protocols. FISH was performed on metaphase chromosomes of the patient using BAC clones DNA (provided by the Wellcome Trust Sanger Institute, Hinxton, Cambridgeshire CB10 1SA, UK) and commercial probe CEP10 (Abbott Molecular Laboratories, USA). Hybridization was performed using the standard procedures. Array-CGH was performed using the Agilent Human Genome 244K CGH Microarray (Agilent Technologies, Inc., Santa Clara, CA). The microarray chip was scanned on an Agilent Microarray Scanner. Data were processed by Agilent Feature Extraction software 9.5 and analyzed with Agilent CGH Analytics 3.4

Software.

RNA extraction, reverse transcription and PCR

Total RNA was extracted using TRIzol reagent (Invitrogen Corp., Carlsbad, CA).

Reverse transcription of 5 μg of total RNA was performed in 20 μl of Superscript reaction buffer (Invitrogen Corp., Carlsbad, CA) containing 0.01M dithiothreitol (DTT), 7.5 ng/μl of dN6, 40 U of Rnase inhibitor (Invitrogen Corp., Carlsbad, CA), 0,5 mM dNTP and 200 U of Superscript reverse transcriptase (Invitrogen Corp., Carlsbad, CA).

Real-time PCR reactions were performed in the LightCycler 480 system (Roche) using the SYBR Green I Master Kit (Eurogentec, Seraing, Belgium) with 2 μl of cDNA and 200 nM of each primer. Each reaction was performed in triplicate.

In situ hybridization on mouse embryos

The antisense and sense riboprobes used in this study correspond to nucleotides 2943-previously described [START_REF] Andrieu | Expression of the Prader-Willi gene Necdin during mouse nervous system development correlates with neuronal differentiation and p75NTR expression[END_REF]. Control experiments were performed using corresponding sense riboprobes on adjacent sections, giving either no signal or a uniformly low background as expected.

RESULTS

Case report

The patient is the first child of non consanguineous, healthy parents. During pregnancy, the mother was given Zovirax for 5 days because she had been in contact with a patient suffering from chicken pox. The patient was born at term and with normal growth parameters (height 50cm, weight 3180g, OFC 36cm). The first clinical signs were noted after one month when she was noticed not to be able to hold her head. CT-scan, muscle enzymes, amino acids, organic acids and thyroid hormones were normal in the blood of the patient. At one year of age she had major axial hypotonia. She was not able to hold her head and could not sit. She was demonstrating incessant abnormal movements. She had a normal EEG. Nerve conduction velocity and electromyogram were normal, as were her visual and hearing parameters. The brain MRI performed at 13 months of age was reported to be normal. At three years of age, she had normal growth parameters but was still severely hypotonic and was not able to hold her head. She could speak a few words.

Cytogenetic and molecular characterization of the translocation

The routine karyotype revealed a translocation between chromosomes 10p12 and 13q12 (data not shown). Serial FISH experiments lead to the identification of two BAC clones overlapping the translocation breakpoints (supplementary figure S1). BAC clone RP11-164A7 overlaps the chromosome 10 breakpoint at 10p12.1. BAC clones RP11-111G7 and RP11-467D10 overlap the chromosome 13 breakpoint at 13q12.13. A map of the translocation breakpoint regions on chromosomes 10 and 13 is presented in Figure 1.

Array comparative genomic hybridization (a-CGH) was performed using an Agilent 244K CGH array and did not reveal any genomic imbalance at the resolution level provided by the array (data not shown).

To further refine the localization of the translocation breakpoints, Southern blots were prepared using the patient's DNA. Series of probes were prepared using PCR on the chromosome 13 breakpoint overlapping BAC DNA and the Southern blots were hybridized with these probes until a junction fragment could be identified. A 4.8 kb junction fragment was identified on the patient's DNA using the StuI restriction endonuclease and a probe located at position 70508-70999 on the RP11-164A7 BAC DNA sequence (supplementary figure S2). This junction fragment was cloned and sequenced (supplementary document S3). Analysis of the sequence of this fragment allowed us to map the chromosome 10 breakpoint at position 28,846,023 bp and the chromosome 13 breakpoint at position 25,231,633 bp (according to reference sequence Ensembl54) and to show that the translocation is perfectly balanced with no gain or loss of genomic material.

The chromosome 10 breakpoint does not interrupt a known transcription unit. A gene called WAC (WW domain containing adaptor with coiled-coil) is located 15,7 kb towards the centromere of chromosome 10. A gene called MPP7 is located 235 kb away on the telomeric side of the chromosome 10 breakpoint. The chromosome 13 breakpoint is located between exons 27 and 28 of a gene called ATP8A2, recently shown to encode a putative phosphatidylserine flippase [START_REF] Coleman | Localization, purification, and functional reconstitution of the P4-ATPase Atp8a2, a phosphatidylserine flippase in photoreceptor disc membranes[END_REF]. In order to test if a chimaeric transcript could be produced between ATP8A2 and MPP7 on the der(13), we used primers located in ATP8A2 (exon 24) and MPP7 (exon 10) and tried to amplify a chimaeric transcript using cDNA prepared from RNA extracted from the lymphocytes of the patient. We were not able to detect any chimaeric transcript.

Expression of ATP8A2 in human and mouse tissues

In order to study the expression profile of ATP8A2, we performed RT-PCR on a panel of 13 human tissues. ATP8A2 was found to be highly expressed in testis and brain (Figure 2A). To refine the brain expression profile, we next used a panel of RNAs prepared from 12 human brain areas to perform RT-PCR. This experiment revealed that ATP8A2 is expressed similarly in the different brain regions tested.

The human ATP8A2 and mouse Atp8a2 proteins share 97% homology. We thus performed in situ hybridization in the mouse with a murine Atp8a2 antisense probe.

These experiments were performed in E14.5, E15.5, E18.5 embryos and at postnatal day 3 (P3). We observed a strong and homogeneous brain expression at all studied stages with the highest levels of Atp8a2 transcripts detected before birth (Figure 2B). Because ATP8A2 is not expressed in lymphocytes, we were not able to quantify its expression in the cells of the patient. The expression of MPP7 could not be tested either because it is not expressed in the lymphocytes (data not shown). We could quantify the expression of WAC in the lymphocytes of the patients, her two parents and a control cell line using real-time quantitative PCR. We found no difference in the amount of WAC transcript in the four samples tested (data not shown).

Screening of the ATP8A2 gene for mutations

We sequenced the 37 exons of the ATP8A2 allele located on the normal chromosome 13 in the patient with the balanced translocation but found no mutation. We also sequenced all ATP8A2 exons on 38 additional patients selected because they were suffering both from mental retardation and severe hypotonia. Again, we found no deleterious mutation (data not shown). This group of patient had been investigated using 105K array CGH and no gross rearrangement was found (31 oligonucleotides cover the ATP8A2 gene on these arrays).

DISCUSSION

We have studied a patient with mental retardation, severe hypotonia and a de novo balanced translocation. We have cloned and sequenced the two translocation breakpoints and we have shown that the translocation is molecularly balanced. Based on the results obtained, we find it reasonable to hypothesize that the breakpoint on chromosome 10 is not involved in the phenotype of our patient. This hypothesis is based on the fact that this region of the genome does not contain any gene. The closest gene to the breakpoint on chromosome 10 is the WAC gene, located 15,7 kb away towards the centromere. The function of the protein encoded by the WAC gene is unknown. We have shown that the expression of WAC is not altered in the patient's lymphoblastoid cell line, ruling out a major position effect, although the expression in the brain of the patient is unknown.

If the phenotype of the patient is caused by the presence of the translocation, then we propose that it is due to the haploinsufficiency of the ATP8A2 gene on chromosome 13.

A possibility exists that a chimaeric transcript could be produced using ATP8A2 exons 1 3 to 27 and the MPP7 exons, in the brain. In this later case however, ATP8A2 dosage would also be altered. The interruption of the coding region of the gene by the translocation breakpoint is a strong argument in favour of the pathogenicity of the chromosomal rearrangement. Expression analysis in mouse and human shows that ATP8A2 is strongly expressed in the brain and during development. Several studies have addressed the function of the ATP8A2 gene. Injection of an antisense ATP8A2 cDNA construct was shown to cause a loss of growth control and to convert non tumorigenic AIG cells into tumorigenic cells [START_REF] Sun | Changes in levels of normal ML-1 gene transcripts associated with the conversion of human nontumorigenic to tumorigenic phenotypes[END_REF]. A study of the mouse Atp8a2 gene revealed that it was highly expressed in the central nervous system and that it could transport aminophospholipids [START_REF] Halleck | Differential expression of putative transbilayer amphipath transporters[END_REF]. More recently, ATP8A2 was confirmed to be involved in the energy-dependent translocation of phosphatidylserine across the cell membrane of photoreceptors [START_REF] Coleman | Localization, purification, and functional reconstitution of the P4-ATPase Atp8a2, a phosphatidylserine flippase in photoreceptor disc membranes[END_REF]. Phosphatidylserine plays important roles in several biological processes including apoptosis or cell signalling. Alteration of phosphatidylserine receptor (PSR) in the mouse reveals that it is necessary for proper brain development [START_REF] Li | Phosphatidylserine receptor is required for clearance of apoptotic cells[END_REF].

Terminal deletions of chromosome 13 are rare but they represent a well known clinical entity [START_REF] Walczak-Sztulpa | Chromosome deletions in 13q33-34: report of four patients and review of the literature[END_REF]. Deletions of 13q14 have extensively been studied in the context of retinoblastoma [START_REF] Nichols | Recent advances in retinoblastoma genetic research[END_REF]. On the contrary, proximal deletions affecting the 13q12 region have not been frequently reported. An interstitial 13q12.1q14.1 deletion was reported in a patient with developmental delay, dysmorphic features and hypotonia [START_REF] Drummond-Borg | Maternal complex chromosome rearrangement ascertained through a del (13)(q12.1q14.1) detected in her mildly affected daughter[END_REF]. The rearrangement was inherited from a normal mother with a complex chromosomal rearrangement. This later case was not characterized molecularly and it is not known if the ATP8A2 gene was involved. Deletions in the 13q13-q31 region cause growth retardation but are usually not associated with major malformations [START_REF] Brown | Preliminary definition of a "critical region" of chromosome 13 in q32: report of 14 cases with 13q deletions and review of the literature[END_REF].

The screening of additional patients revealed no mutations in the ATP8A2 gene. However, it is very difficult to select patients for mutation screening because the phenotype of the patient carrying the balanced translocation is not very specific, although severe. Mental retardation and severe hypotonia are found in several other syndromes such as Prader-Willi patients when they are young. However, in many instances, the clinical course of these disorders when the patients grow older usually reveals additional features. Given that our patient is still rather young, we were not able to refine the clinical diagnosis enough to select a clinically matched patient cohort for molecular screening.

ACKNOWLEDGEMENTS

We thank the Centre de Ressources Biologiques of La Timone Children's Hospital for access to the biological samples used in this study, and the microarray core facility of the Department of Medical Genetics of La Timone Children's Hospital. We thank Dr.

Jean-Christophe Roux for helpful discussions. The authors declare no competing interest.

CONFLICT OF INTEREST STATEMENT

The authors have no conflict of interest to disclose.

TITLES AND LEGENDS TO FIGURES

Top panel : ideograms of human chromosomes 10 (Hs.10) and 13 (Hs.13)

Supplementary document S3.

Sequence of the 4864 bp StuI junction fragment.