Teddy Grand

Sébastien L'hoste

David Mordasini

Nadia Defontaine

Mathilde Keck

Thomas Pennaforte

Mathieu Genete

Kamel Laghmani

Jacques Teulon

Stéphane Lourdel
email: stephane.lourdel@upmc.fr

John Wiley

Sons

Heterogeneity in the processing of ClC-5 mutants related to Dent disease

Keywords: Dent's disease, Chloride/proton exchanger, CLCN5, ClC-5, Processing Dent disease, Chloride/proton exchanger, CLCN5, ClC-5, processing

teaching and research institutions in France or abroad, or from public or private research centers.

INTRODUCTION

Dent disease is an X-linked recessive renal tubular disorder characterized by lowmolecular-weight proteinuria (LMWP), hypercalciuria, nephrocalcinosis, and progressive renal failure. Inactivating mutations of CLCN5 cause Dent disease (MIM# 300009). However, defects in OCRL1, the gene encoding a phosphatidylinositol-4,5-bisphosphate-5-phosphatase, have also been found in a subset of patients with Dent disease (MIM# 300555) [START_REF] Hoopes | Dent Disease with mutations in OCRL1[END_REF][START_REF] Ludwig | Recent advances in understanding the clinical and genetic heterogeneity of Dent's disease[END_REF].

CLCN5 encodes the electrogenic Cl -/H + exchanger ClC-5 [START_REF] Picollo | Chloride/proton antiporter activity of mammalian CLC proteins ClC-4 and ClC-5[END_REF][START_REF] Scheel | [END_REF]. In the kidney, ClC-5 expression has been observed in the proximal tubule, in the αand β-intercalated cells of the collecting duct, and at lower levels in the thick ascending limb of Henle's loop [START_REF] Devuyst | Intra-renal and subcellular distribution of the human chloride channel, CLC-5, reveals a pathophysiological basis for Dent's disease[END_REF]Gunther, et al., 1998]. Because ClC-5 colocalizes with v-type H + -ATPase in renal proximal tubular subapical endosomes, the central hypothesis advanced to explain the endocytosis defect in Dent disease is that ClC-5 may provide shunt conductance in early endosomes thus permitting intraluminal acidification by vtype H + -ATPase, and that loss-of-function of ClC-5 would therefore impair endosomal acidification, a crucial step in normal endosomal function [START_REF] Devuyst | Intra-renal and subcellular distribution of the human chloride channel, CLC-5, reveals a pathophysiological basis for Dent's disease[END_REF][START_REF] Dowland | Molecular cloning and characterization of an intracellular chloride channel in the proximal tubule cell line, LLC-PK1[END_REF]Gunther, et al., 1998;[START_REF] Piwon | ClC-5 Cl --channel disruption impairs endocytosis in a mouse model for Dent's disease[END_REF][START_REF] Sakamoto | Cellular and subcellular immunolocalization of ClC-5 channel in mouse kidney: colocalization with H + -ATPase[END_REF][START_REF] Suzuki | Intracellular localization of ClC chloride channels and their ability to form hetero-oligomers[END_REF]. However, recently, Jentsch's group has provided evidence that modulation of the chloride concentration during proton transport by the exchanger activity of ClC-5 plays a crucial role in endocytosis, rather than endosomal acidification [Novarino, et al., 2010]. A small fraction of ClC-5 is also present at the apical surface of proximal tubule cells where it may play a crucial role in mediating the protein-protein interactions required for receptormediated endocytosis [START_REF] Wang | ClC-5: role in endocytosis in the proximal tubule[END_REF]. ClC-5 has indeed been found to associate with cofilin, a protein involved in the depolymerization of actin in the vicinity of endosomes, with the PDZ-domain protein NHERF2, and with KIF3B, a member of the kinesin superfamily [START_REF] Hryciw | Cofilin interacts with ClC-5 and regulates albumin uptake in proximal tubule cell lines[END_REF][START_REF] Reed | CLC-5 and KIF3B interact to facilitate CLC-5 plasma membrane expression, endocytosis, and microtubular transport: relevance to pathophysiology of Dent's disease[END_REF]. It has also been shown to interact in heterologous expression systems with the ubiquitin protein ligases WWP2 and Nedd4-2, which by ubiquitinating ClC-5 at his PY-motif leads to its internalization by endocytosis [Hryciw, et al., 2006a;[START_REF] Hryciw | Nedd4-2 functionally interacts with ClC-5: involvement in constitutive albumin endocytosis in proximal tubule cells[END_REF][START_REF] Schwake | An internalization signal in ClC-5, an endosomal Cl-channel mutated in Dent's disease[END_REF]. However, the recent work of Rickheit et al. on different mouse models demonstrated that in vivo the PY-motif-dependent ubiquitylation of ClC-5 is not required for proximal tubular endocytosis [Rickheit, et al., 2010].

Recent studies of the functional consequences of naturally-occurring CLCN5 mutations revealed that several different mechanisms underlie the ClC-5 loss-of-function in patients with Dent disease [START_REF] Grand | Novel CLCN5 mutations in patients with Dent's disease result in altered ion currents or impaired exchanger processing[END_REF]Ludwig, et al., 2005;[START_REF] Smith | To further elucidate the mechanisms leading to altered currents, we next investigated the cell surface targeting of WT and mutant ClC-5 proteins in X. leavis oocytes. Levels of the S244L and Y272C mutants in the plasma membrane were comparable (Fig. 2). Thus, the REFERENCES[END_REF]. On the basis of functional data, mutations were classified into different groups in previous studies by us and others [START_REF] Grand | Novel CLCN5 mutations in patients with Dent's disease result in altered ion currents or impaired exchanger processing[END_REF][START_REF] Smith | To further elucidate the mechanisms leading to altered currents, we next investigated the cell surface targeting of WT and mutant ClC-5 proteins in X. leavis oocytes. Levels of the S244L and Y272C mutants in the plasma membrane were comparable (Fig. 2). Thus, the REFERENCES[END_REF]. A first group of mutations lead to endoplasmic reticulum retention, and degradation of the mutant proteins as they are misfolded. These amino acid substitutions were all located in helices forming the interface between the two subunits [START_REF] Smith | To further elucidate the mechanisms leading to altered currents, we next investigated the cell surface targeting of WT and mutant ClC-5 proteins in X. leavis oocytes. Levels of the S244L and Y272C mutants in the plasma membrane were comparable (Fig. 2). Thus, the REFERENCES[END_REF]. Very similarly, we demonstrated that mutations located along α-helices at quite some distance from the interface were also trapped in the endoplasmic reticulum [START_REF] Grand | Novel CLCN5 mutations in patients with Dent's disease result in altered ion currents or impaired exchanger processing[END_REF]. These mutations were associated with reduced protein expression and impaired N-glycosylation. A second group of mutations lead to ClC-5 proteins that are devoid of electrical activity and fail to enhance endosomal acidification [START_REF] Smith | To further elucidate the mechanisms leading to altered currents, we next investigated the cell surface targeting of WT and mutant ClC-5 proteins in X. leavis oocytes. Levels of the S244L and Y272C mutants in the plasma membrane were comparable (Fig. 2). Thus, the REFERENCES[END_REF]. A third group of mutations are associated with reduction of currents at the cell surface, reduction of plasma membrane expression and alterations in endosomal targeting [START_REF] Smith | To further elucidate the mechanisms leading to altered currents, we next investigated the cell surface targeting of WT and mutant ClC-5 proteins in X. leavis oocytes. Levels of the S244L and Y272C mutants in the plasma membrane were comparable (Fig. 2). Thus, the REFERENCES[END_REF]. These mutant proteins were nevertheless able to support endosomal acidification. Finally, we described another type of mutations that caused reduced currents. The mutant proteins trafficked normally to the cell surface and to early endosomes, and displayed complex glycosylation at the cell surface [START_REF] Grand | Novel CLCN5 mutations in patients with Dent's disease result in altered ion currents or impaired exchanger processing[END_REF]. These data do not rule out the possibility that other mechanisms may also contribute to the disease, because the functional aspects of numerous CLCN5 mutations have not yet been fully investigated. We therefore decided to investigate the functional consequences of nine previously-reported ClC-5 missense mutations, by focusing on amino acid substitutions, most of which are clustered at the interface between the two subunits or at the periphery of the subunit interface.

MATERIALS AND METHODS

Molecular Biology

ClC-5 mutants (Table 1) were synthesized from human wild-type ClC-5 (GenBank NG_007159.2) extracellularly HA tagged, and subcloned into the pTLN expression vector (a generous gift of Pr. T. J. Jentsch, MDC/FMP, Berlin, Germany) for expression in X. laevis oocytes, or into the peGFP expression vector for expression in HEK293 cells. The HA epitope (YPYDVPDYA) is introduced into the extracellular loop of ClC-5 between transmembrane domains B and C [START_REF] Dutzler | X-ray structure of a ClC chloride channel at 3.0 A reveals the molecular basis of anion selectivity[END_REF]. Experimental studies have shown that the HA epitope does not interfere with ClC-5 function [START_REF] Schwake | An internalization signal in ClC-5, an endosomal Cl-channel mutated in Dent's disease[END_REF]. Site-directed mutagenesis was performed with the Quickchange site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA). All constructs were fully sequenced.

Expression in Xenopus laevis oocytes

Capped cRNA were synthesized in vitro from wild-type and mutant ClC-5 expression vectors linearized with MluI using the SP6 mMessage mMachine Kit (Ambion, Austin, TX, USA). Defolliculated Xenopus leavis oocytes were injected with 20 ng of the different cRNAs. The oocytes were then kept at 17°C in modified Barth's solution containing (in mM): 88 NaCl, 1 KCl, 0.41 CaCl 2 , 0.32 Ca(NO 3 ) 2 , 0.82 MgSO 4 , 10 HEPES, pH 7.4 and gentamycin (20 µg/ml).

Electrophysiology

Five days after injection, two-electrode voltage-clamp experiments were performed at room temperature using a TEV-200A amplifier (Dagan, Minneapolis, MN, USA) and PClamp 8 software (Axon Instruments, Union City, CA, USA). Pipettes were pulled from borosilicate glass (Harvard Apparatus, Edenbridge, Kent, UK) using a puller (Sutter Instrument Co., Novato, CA, USA), and filled with 3 M KCl. Pipette resistances were less than 1 MΩ.

Currents were recorded in ND96 solution containing (in mM): 96 NaCl, 2 KCl, 1.5 CaCl 2 , 1 MgCl 2 , 5 HEPES, pH 7.4. Currents were recorded in response to a voltage protocol consisting of 20 mV steps from -100 mV to +100 mV during 800 ms from a holding potential of -30 mV.

Surface labelling of oocytes

Experiments were essentially performed according to the method of Zerangue et al.

with slight modifications [START_REF] Zerangue | A new ER trafficking signal regulates the subunit stoichiometry of plasma membrane K(ATP) channels[END_REF]. Oocytes were incubated for 30 minutes in ND96 with 1% Bovine Serum Albumin (BSA) at 4°C to block unspecific binding, and were then incubated for 60 minutes with a rat monoclonal anti-HA antibody (1 µg/ml, 3F10, Roche Diagnostics, Meyland, France) in 1% BSA/ND96 at 4°C. The oocytes were then washed eight times with 1% BSA/ND96 at 4°C, before being incubated for 45 minutes with a peroxidaseconjugated affinity-purified F(ab')2 fragment goat anti-rat antibody (2 µg/ml, Jackson ImmunoResearch, West Grove, PA, USA) in 1% BSA/ND96 at 4°C. The oocytes were washed six times with 1% BSA/ND96 at 4°C, and then six times in ND96 without BSA at 4°C. Individual oocytes were placed in 50 µl of SuperSignal Elisa Femto Maximum Sensitivity Substrate Solution (Pierce, Rockford, IL, USA) and, after an equilibration period of 1 minute, chemiluminescence was quantified in a Turner TD-20/20 luminometer (Turner Designs, Sunnyvale, CA, USA) by integrating the signal over a period of 10 seconds. (GIBCO, Invitrogen, CA, USA) supplemented with 10% fetal bovine serum, penicillin (100 IU/ml), and streptomycin (100 µg/ml) at 37°C in 5% CO 2 . The cells were transiently transfected using Fugene 6 according to the Manufacturer's instructions (Roche Diagnostics, Meyland, France).

Immunocytochemistry

Transfected HEK293 cells were plated on 12-mm diameter Petri dishes. Cells were then fixed in 4% paraformaldehyde, and permeabilized with 0.3% Triton. Nonspecific binding sites were blocked with 16% goat serum solution. The primary antibodies used were mouse anti-HA (Sigma, St Louis, MO, USA), rabbit anti-EEA1 (Sigma, St Quentin Fallavier, France), rabbit anti-calnexin (Stressgen, Ann Arbor, MI, USA). FITC-conjugated goat antimouse (Jackson ImmunoResearch, West Grove, PA, USA), TRITC-conjugated goat antirabbit (Jackson ImmunoResearch, West Grove, PA, USA), or Cy5-conjugated streptavidin (Sigma, St Quentin Fallavier, France) were added to the cells as secondary antibodies.

Labeled cells were analyzed with a Zeiss LSM 510 confocal laser scanning microscope.

Image analysis was performed by using ImageJ and Photoshop CS2 (Adobe, San Jose, CA, USA).

Surface biotinylation of HEK293 cells

Forty-eight hours after transfection, cells were placed on ice and rinsed twice with a cold rinsing solution containing PBS, 100 µM CaCl 2 and 1 mM MgCl 2 . The cells were then incubated at 4°C for 1 h with PBS and 1.5 mg/ml NHS-biotin (Pierce, Rockford, IL, USA).

They were incubated in quenching solution containing 0.1% BSA diluted in PBS, and rinsed 3 EDTA, 2 mM EGTA, 30 mM NaF, 30 mM NaPPi, 1% Triton, 0.1% SDS and a protease inhibitor mix (Complete, Roche Diagnostics, France), equal amounts of proteins were precipitated at 4°C overnight using streptavidin-agarose beads (Pierce, Rockford, IL, USA).

Samples were then centrifuged at 2,500 x g for 2 min at 4°C with TLB solution containing 50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 5 mM EDTA and the protease inhibitor mix.

Protein isolation

For the isolation of total cell lysates from HEK293, the cells were incubated for 10 min on ice with the lysis solution. Samples were centrifuged at 13,000 rpm for 5 minutes.

The protein concentration in the supernatant was quantified using a protein assay quantification kit (BCA Protein Kit Assay, Pierce, Rockfort, IL, USA).

Western blot analysis

The proteins were separated on an 8% SDS-PAGE gel and transferred to PVDF membranes. Primary rat anti-HA monoclonal antibody (3F10, Roche Diagnostics, Meyland, France), rabbit anti-GAPDH monoclonal antibody (Abcam, Cambridge, UK), and secondary peroxidase-conjugated goat anti-rat antibody (Jackson ImmunoResearch, West Grove, PA, USA) were diluted in TBS-blocking solution. Detection was performed using the ECL Western Blotting Substrate (Pierce, Rockford, IL, USA).

Pulse-chase assays

Prior to the experiment, HEK 293 cells were transiently transfected with plasmid DNA using Fugene 6 reagent as described above. 24 h later they were incubated in cysteine-and methionine-free DMEM starvation media for 1 h. The starvation medium was removed and Cells were then rinsed three times with PBS, and another three times with normal growth medium before being returned to normal growth medium for the duration of the chase to the specified time points. Cells were washed twice with ice-cold PBS, and incubated on ice for 1 h in lysis buffer with a mixture of protease inhibitors, after which solubilized extracts were collected for immunoprecipitation. Proteins were immunoprecipitated with mouse anti-HA antibody (Sigma, St Louis, MO, USA), resolved with SDS-PAGE, blotted onto nitrocellulose, and revealed by autoradiography.

Statistics

Results are shown as mean ± SEM. n indicates the number of experiments. Statistical significance was analyzed by applying a paired Student's t-test using SigmaStat software (SPSS, Erkrath, Germany). P < 0.05 was considered significant. significant decrease in current amplitudes in the S244L mutant, and the loss of electrical activity in the Y272C mutant (Fig. 1A andB, Fig. 2) cannot be ascribed to reduced protein trafficking to the cell surface. In contrast, the G260V, L278F, K546E, and W547G mutants all showed reductions of 25-50% in their surface expression, and no surface expression at all was detected with the L225P, N340K, and G513R mutants. The change in electrical activity observed with these latter ClC-5 mutants could be explained by the abolition or impairment of cell-surface expression due to mis-targeting (Fig. 1A andB, Fig. 2) or altered protein expression.

To check their protein expression, total cell lysates isolated from HEK293 cells transiently transfected with either WT or mutant ClC-5 were subjected to a western blot analysis (Fig. 3). In our previous study, we reported positive western blot staining for the 90-kDa core-glycosylated and the 100-kDa complex glycosylated forms of WT ClC-5 [START_REF] Grand | Novel CLCN5 mutations in patients with Dent's disease result in altered ion currents or impaired exchanger processing[END_REF]. We also showed that the 90-kDa ClC-5 protein displays high mannose glycosylation, and is retained in the endoplasmic reticulum, and that the plasma membrane ClC-5 component displays complex glycosylation [START_REF] Grand | Novel CLCN5 mutations in patients with Dent's disease result in altered ion currents or impaired exchanger processing[END_REF]. Here, when an equivalent amount of proteins was loaded in each lane, no significant difference in density or size could be detected between WT ClC-5 and the S244L and Y272C mutants. In contrast, the 100-kDa complex glycosylated form/90-kDa core-glycosylated form ratio was significantly reduced by ~50% for the G260V, L278F, K546E and W547G mutants versus WT ClC-5.

Thus, the decreased currents in the S244L and Y272C mutants were not attributable to differing levels of protein expression. However, the altered currents in the L278F, G260V, K546E and W547E mutants could be attributable to reduced amounts of the complex glycosylated component, which could result from changes in the post-translational processing or from an increase in the turnover of the mutant proteins (Fig. 3). In contrast, only the 90-kDa core-glycosylated form of ClC-5 was detected in the L225P, N340K, and G513R mutants (Fig. 3). The abolition of conduction and surface expression in these latter mutants can be the consequence of a rapid degradation of the proteins within the cell. These results are similar to what we had previously reported for other ClC-5 mutants [START_REF] Grand | Novel CLCN5 mutations in patients with Dent's disease result in altered ion currents or impaired exchanger processing[END_REF].

We then compared the stability and the maturation of one out of the four similar mutants showing a reduction of ~50% of the 100-kDa complex glycosylated form/90-kDa core-glycosylated form of ClC-5 (G260V, Fig. 3) and one out of the three similar mutants showing only the 90-kDa core-glycosylated form of ClC-5 (L225P, Fig. 3) with those of WT ClC-5, by tracing the sorting and delivery of newly synthesized proteins from the endoplasmic reticulum to Golgi compartments by means of a pulse-chase analysis. We observed that the WT ClC-5 protein migrated as the 90-kDa core-glycosylated form immediately after pulse labeling (Fig. 4A). The 100-kDa complex glycosylated form, which was barely visible at the end of the pulse period, increased in amount during the first hour of chase, and became the predominant form by 2 hours (Fig. 4A). Kinetic analysis (Fig. 4B-C) revealed a progressive decrease in the 90-kDa immature form of the WT ClC-5 protein, with half-life of 1.5 hours (n = 3), and a progressive increase in the 100-kDa mature form of the WT ClC-5 protein (n = 3). The decrease represents the conversion of the immature form to the mature form as well as the degradation of the immature form of ClC-5 protein. Likewise, the G260V mutant protein was initially converted from the core-glycosylated form to the complex glycosylated form, like WT ClC-5 (Fig. 4A). The degradation of the immature form of WT ClC-5 and the G260V mutant followed the same kinetic (Fig. 4B, n = 3), but the conversion of the G260V mutant into its mature, complex glycosylated form was significantly slower than that of WT ClC-5 (Fig. 4B-C, n = 3). Furthermore, there was significant reduction between WT ClC-5 and the G260V mutant in the maximum amount of the mature form at 4 hours of chase (Fig. 4B, n = 3). In contrast to the G260V mutant, the L225P mutant was initially synthesized as the 90-kDa core-glycosylated form, but had not acquired complex sugars at any of the time intervals, and so was not converted into the mature form during the chase (Fig. 4A). The half-life of the immature form of L225P was 50% shorter than that of WT ClC-5 (Fig. 4B, n = 3).

Overall, our results demonstrate that the G260V, L278F, K546E and W547G mutations lead to a delay in the maturation and to a decrease of the stability of the mature, complex glycosylated form of ClC-5, whereas the L225P, N340K and G513R mutations abolish the maturation of ClC-5.

In the light of the above findings, we next investigated the behavior of the different ClC-5 mutants at the cellular level by assessing their subcellular distribution by means of immunostaining and confocal microscopy in transiently-transfected HEK293 cells. As expected from previous reports, WT ClC-5 colocalized with biotinylated cell-surface proteins, and with the early endosomes marker EEA1 (Fig. 5) [START_REF] Dowland | Molecular cloning and characterization of an intracellular chloride channel in the proximal tubule cell line, LLC-PK1[END_REF][START_REF] Grand | Novel CLCN5 mutations in patients with Dent's disease result in altered ion currents or impaired exchanger processing[END_REF][START_REF] Smith | To further elucidate the mechanisms leading to altered currents, we next investigated the cell surface targeting of WT and mutant ClC-5 proteins in X. leavis oocytes. Levels of the S244L and Y272C mutants in the plasma membrane were comparable (Fig. 2). Thus, the REFERENCES[END_REF][START_REF] Suzuki | Intracellular localization of ClC chloride channels and their ability to form hetero-oligomers[END_REF]. A similar distribution was found for the Y272C and the W547G mutants (Fig. 5), and for the S244L, G260V, L278F, and K546E mutants (data not shown). In contrast to WT ClC-5, the L225P, N340K, and G513R mutants were retained in the endoplasmic reticulum compartment, as indicated by their colocalization with the endoplasmic reticulum marker calnexin, and were excluded from the plasma membrane (Fig. 5). To further explore the plasma membrane expression of the mutant ClC-5, we performed cell surface biotinylation experiments (Fig. 6). No significant difference could be detected between the surface fraction containing WT ClC-5, and that containing the S244L mutant (n = 3). In contrast, the abundance of the G260V mutant at the cell surface was significantly lower than that of WT ClC-5 (n = 3). Surface biotinylation experiments also showed that the G513R was excluded from the surface biotinylated protein fraction (n = 3) (Fig. 6). Overall, the endoplasmic reticulum retention of the L225P, N340K, and G513R mutants was compatible with abolition of their conduction and surface expression, which is The functional effects of the CLCN5 mutations we studied are summarized in Table 1.

DISCUSSION

We performed functional, biochemical, and cell-biology analyses in X. laevis oocytes and in HEK293 cells of nine CLCN5 pathogenic missense mutations that had been described before [START_REF] Anglani | Family history may be misleading in the diagnosis of Dent's disease[END_REF][START_REF] Hoopes | Evidence for genetic heterogeneity in Dent's disease[END_REF][START_REF] Igarashi | [END_REF][START_REF] Lloyd | [END_REF][START_REF] Ramos-Trujillo | A missense mutation in the chloride/proton ClC-5 antiporter gene results in increased expression of an alternative mRNA form that lacks exons 10 and 11. Identification of seven new CLCN5 mutations in patients with Dent's disease[END_REF]Tosetto, et al., 2006].

The G260V, Y272C, L278F, G513R, K546E, and W547G mutations are located in helix H, in the loop between helices H and I, in helix I, and in helices O and Q, respectively (Fig. 7). Helices H, I, O and Q are involved in the formation of the dimer interface, as are helices B and P [START_REF] Dutzler | X-ray structure of a ClC chloride channel at 3.0 A reveals the molecular basis of anion selectivity[END_REF][START_REF] Dutzler | Gating the selectivity filter in ClC chloride channels[END_REF][START_REF] Wu | Modeling study of human renal chloride channel (hCLC-5) mutations suggests a structuralfunctional relationship[END_REF]. Previous published modeling studies of ClC-5 based on the crystal structures of two prokaryotic ClCs suggested that ClC-5 missense mutations clustering at the dimer interface would cause a loss of electrical activity by disrupting the assembly of the homodimers [START_REF] Smith | To further elucidate the mechanisms leading to altered currents, we next investigated the cell surface targeting of WT and mutant ClC-5 proteins in X. leavis oocytes. Levels of the S244L and Y272C mutants in the plasma membrane were comparable (Fig. 2). Thus, the REFERENCES[END_REF][START_REF] Wu | Modeling study of human renal chloride channel (hCLC-5) mutations suggests a structuralfunctional relationship[END_REF]. This would result in the formation of misfolded proteins in the endoplasmic reticulum, and their rapid degradation within the cell. Alternatively, mutations of residues positioned in a subgroup at the periphery of the dimer interface would be associated with residual currents at the cell surface, because they would induce a relatively minor disruption of the protein structure. Our results using the L278F and the G513R mutations are consistent with these modeling studies. On the one hand, despite reduced cell surface expression, the L278F mutant exhibited residual activity and its early endosomal distribution was similar to that of WT ClC-5. It should be noted that the amino acid substitution is located in helix I at the periphery of the subunit interface. On the other hand, the G513R mutant showed no functional expression in electrophysiological recordings, and was improperly N-glycosylated due to being retained on the endoplasmic reticulum. However, in contrast to this mutant, the Y272C mutants were normally N-glycosylated, properly targeted to the plasma membrane Furthermore, the G260V, K546E and W547G mutants also escaped endoplasmic reticulum retention and subsequent degradation, as shown by their complex glycosylation and plasma membrane expression. Like the L278F mutant, the cell surface expression of the mature, complex glycosylated form of these mutants was, however, reduced. Our results indicate that this may be explained by a delay in the maturation and by a decreased stability of the mature form of ClC-5. These mutant proteins were also distributed in the early endosomes.

Interestingly, the reduction of currents correlated with the decreased surface expression of the mature forms for the L278F and the W547G mutants. In contrast, despite a comparable decreased level expression of their mature form at the plasma membrane like the L278F and W547G mutants, we failed to record any currents with the G260V and K546E mutants, indicating that these mutants ClC-5 displayed abolished electrical activity. Taken together, these results demonstrate that mutation of a residue located at the interface may cause less protein-folding defects than expected from previous published modeling data even though they are positioned at the interface between the two subunits [START_REF] Smith | To further elucidate the mechanisms leading to altered currents, we next investigated the cell surface targeting of WT and mutant ClC-5 proteins in X. leavis oocytes. Levels of the S244L and Y272C mutants in the plasma membrane were comparable (Fig. 2). Thus, the REFERENCES[END_REF][START_REF] Wu | Modeling study of human renal chloride channel (hCLC-5) mutations suggests a structuralfunctional relationship[END_REF].

The S244L mutation was shown to severely reduce currents, and was associated with unaffected trafficking to the plasma membrane and to the early endosomes, and unaltered Nglycosylation. This mutation is located in helix G (Fig. 7), a helix that is not involved in the formation of the transporter interface, or the transport pathways of Cl -or H + [START_REF] Chen | CLC-0 and CFTR: chloride channels evolved from transporters[END_REF][START_REF] Dutzler | X-ray structure of a ClC chloride channel at 3.0 A reveals the molecular basis of anion selectivity[END_REF][START_REF] Dutzler | Gating the selectivity filter in ClC chloride channels[END_REF]. Further studies are required to assess the exact mechanism responsible for the reduced electrical activity of the S244L mutant.

Finally, the L225P and N340K mutants showed no functional expression in electrophysiological recordings, and were improperly N-glycosylated as a result of being for these mutations, which are located in helices F and J (Fig. 7), are in accordance with our previous data and show that amino acid substitutions located within α-helices at quite some distance from the transporter interface enhance protein degradation, probably by distorting the structure of the α-helices, and preventing the proper folding of the monomer [START_REF] Grand | Novel CLCN5 mutations in patients with Dent's disease result in altered ion currents or impaired exchanger processing[END_REF]. Defects in protein folding and processing have been shown to be involved in the pathogenesis of several inherited disorders [START_REF] Gregersen | Protein misfolding and human disease[END_REF]. One well-known example is cystic fibrosis, which is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene encoding a chloride channel. Many mutant forms of the CFTR are misfolded, achieve only partial N-glycosylation, and are retained and degraded within the endoplasmic reticulum. Thus, the mechanism of transporter dysfunction identified for the type-II mutations appears to be similar to the mechanisms responsible for most cases of cystic fibrosis.

Recently, we found that two types of ClC-5 mutants can be distinguished [START_REF] Grand | Novel CLCN5 mutations in patients with Dent's disease result in altered ion currents or impaired exchanger processing[END_REF]. Here, in the line with our functional analysis, we demonstrated that five mutations (L225P, S244L, Y272C, N340K and G513R) corresponded to these two types of CLCN5 mutations (Table 1). However, in addition, our data also revealed that four mutations (G260V, L278F, K546E and W547G) could be grouped to form a novel type of CLCN5 mutations.

These type-III mutations (Table 1) enhance a delay in the processing of ClC-5, a decrease of the stability of its mature, complex glycosylated form, a reduction of its cell surface expression, and a reduction of currents at the plasma membrane. The distribution of these mutants in the early endosomes is normal.

Another interesting aspect of our study is that 6 out of the 9 mutants were associated with expression at the cell surface in HEK293 transfected cells. Although ion conduction through ClC-5 is thought not to be physiologically relevant at the cell surface, several recent studies in transfected proximal tubule cells have demonstrated that the ClC-5 population at the brush border is a rate-limiting step in receptor-mediated endocytosis by mediating proteinprotein interactions with a variety of proteins essential for renal reabsorption [Hryciw, et al., 2006b]. In the case of the type-III mutants, one could speculate that their lower abundance at the plasma membrane could severely reduce the endocytosis by altering protein-protein interactions that are required in the formation of the endocytic macromolecular complex. The ClC-5 type-I mutants should not prevent these interactions, because they displayed normal protein expression and subcellular localization. However, these mutants displayed reduced (S244L) and abolished currents (Y272C). As a consequence, one may explain the presence of Dent disease in patients carrying these mutations by altered endosomal acidification leading to impaired receptor-mediated endocytosis, due to alteration of the function of v-type H + -ATPase caused by reduced or abolished chloride shunt conductance. Alternatively, one may also hypothesize that chloride accumulation in early endosomes could be compromised due to decreased electrical activity of ClC-5. In the view of the recent work of Novarino et al., this could also lead to altered endocytosis. Further experiments are needed to confirm these hypothesis.

Numerous in vitro studies have shown that dysfunctional channels or transporters can be rescued by pharmacological therapy. For example, this is the case for CFTR in patients with cystic fibrosis [START_REF] Kerem | Pharmacological induction of CFTR function in patients with cystic fibrosis: mutation-specific therapy[END_REF]. Chemical and molecular chaperones have been shown to allow class-II CFTR mutants to escape from degradation in the endoplasmic reticulum and to be transported to the cell surface. Several compounds have also been found to directly activate class-III and class-IV CFTR mutants that display defective regulation and conduction at the plasma membrane, respectively. The findings of this study, together with previous data from the functional analysis of ClC-5 mutations in Dent disease, provide new clues for future [START_REF] Grand | Novel CLCN5 mutations in patients with Dent's disease result in altered ion currents or impaired exchanger processing[END_REF]Ludwig, et al., 2005;[START_REF] Smith | To further elucidate the mechanisms leading to altered currents, we next investigated the cell surface targeting of WT and mutant ClC-5 proteins in X. leavis oocytes. Levels of the S244L and Y272C mutants in the plasma membrane were comparable (Fig. 2). Thus, the REFERENCES[END_REF]. A major challenge for such studies will be to find specific therapeutic drugs that are able to restore sufficient ClC-5 function in patients with Dent disease.

  grown in Dulbecco's modified Eagle's medium (DMEM)

  rinsing solution. After lysis in a solution containing 20 mM Tris HCl, 2 mM

  labeling medium containing [ 35 S]methionine/cysteine labeling mix.

  in rapid degradation of these proteins within the cell, as shown by our pulsechase analysis conducted on the L225P mutant in HEK293 cells.

  early endosomes like WT ClC-5, and displayed reduced electrical activity.

  endoplasmic reticulum by quality control systems. Moreover, pulse-chase analysis of the L225P mutant demonstrated early degradation of the mutant protein. Results

Figure 1 .

 1 FIGURE LEGENDS

Figure 2 .

 2 Figure 2. Currents/cell surface expression relationship for WT and mutants ClC-5 in X. laevis

Figure 3 .

 3 Figure 3. Western blot analysis of total expression levels of WT and mutants ClC-5 in

Figure 4 .

 4 Figure 4. Pulse-chase analysis of WT and mutant ClC-5 in HEK293 transfected cells. A: WT

Figure 5 .

 5 Figure 5. Immunocytochemical localization of WT and mutant ClC-5 in HEK293-transfected

Figure 6 .

 6 Figure 6. Cell surface expression of WT and mutant ClC-5 in HEK293 transfected cells.

Figure 7 .

 7 Figure 7. Amino acid sequence alignment of several ClCs showing the positions of the

John Wiley & Sons, Inc.Human Mutation

ACKNOWLEDGMENTS

This work was supported in part by grants from the French ANR program (ANR-05-MRAR-033-01), and the Bonus Qualité Recherche from the UPMC-Université Paris 06. We thank Prof. T.J. Jentsch for kindly providing the HA-tagged ClC-5, and C. Klein for excellent technical assistance in confocal microscopy. T. Grand holds a PhD fellowship from the Ministère de l'Enseignement supérieur et de la Recherche. The English text was edited by M. Ghosh.

F o r P e e r R e v i e w


Zhong-Zheng Zhu

Lifang Hou

Valentina Bollati

Letizia Tarantini

Barbara Marinelli

Laura Cantone

Andrea Baccarelli
email: andrea.baccarelli@unimi.it

Allen S Yang

Pantel Vokonas

Jolanta Lissowska

Silvia Fustinoni

Angela C Pesatori

Matteo Bonzini

Pietro Apostoli

Giovanni Costa

Pier Alberto Bertazzi

Wong-Ho Chow

Joel Schwartz

Predictors of global methylation levels in blood DNA of healthy subjects: a combined analysis

Keywords: Blood, DNA methylation, epigenetics, meta-analysis, repetitive elements Blood, DNA methylation, epigenetics, meta-analysis, repetitive elements

ou non, émanant des établissements d'enseignement et de recherche

Introduction

DNA methylation is a well-defined epigenetic mechanism involved in the regulation of a wide variety of biological processes, including gene expression, genomic stability, and parental imprinting. [START_REF] Bird | DNA methylation patterns and epigenetic memory[END_REF] In mammals, methylation involves addition of methyl groups to cytosine to form 5-methyl-cytosine (5mC). It is estimated that more than one-third of DNA methylation occurs in repetitive elements, [START_REF] Kochanek | DNA methylation in the Alu sequences of diploid and haploid primary human cells[END_REF][START_REF] Schmid | Does SINE evolution preclude Alu function?[END_REF] which represent a large portion of the human genome. [START_REF] Lander | Initial sequencing and analysis of the human genome[END_REF] Among these repetitive sequences, Alu and LINE-1 are the most plentiful families representing approximately 30% of the human genome. [START_REF] Grover | Alu repeat analysis in the complete human genome: trends and variations with respect to genomic composition[END_REF][START_REF] Babushok | Progress in understanding the biology of the human mutagen LINE-1[END_REF][START_REF] Kazazian | LINE drive: retrotransposition and genome instability[END_REF] Because of their high representation throughout the genome, Alu and LINE-1 have been used as surrogate markers for estimating global DNA methylation level. [START_REF] Weisenberger | Analysis of repetitive element DNA methylation by MethyLight[END_REF][START_REF] Yang | A simple method for estimating global DNA methylation using bisulfite PCR of repetitive DNA elements[END_REF] Methodologically, a high-throughput quantitative method based on bisulfite treatment of DNA and subsequent PCR pyrosequencing [START_REF] Yang | A simple method for estimating global DNA methylation using bisulfite PCR of repetitive DNA elements[END_REF] or real-time PCR [START_REF] Weisenberger | Analysis of repetitive element DNA methylation by MethyLight[END_REF] has been developed for estimating global DNA methylation level of Alu and LINE-1 elements. This method is amenable to use in screening large sets of samples because it is cost-effective and requires relatively small amounts of genomic DNA. As a result, DNA methylation analysis of Alu and/or LINE-1 methylation has been used in a number of epidemiological investigations. [START_REF] Baccarelli | Rapid DNA methylation changes after exposure to traffic particles[END_REF][START_REF] Baccarelli | Low blood DNA methylation determines higher risk and mortality from ischemic heart disease and stroke among elderly individuals[END_REF][START_REF] Bollati | Changes in DNA methylation patterns in subjects exposed to low-dose benzene[END_REF][START_REF] Rusiecki | Global DNA hypomethylation is associated with high serum-persistent organic pollutants in Greenlandic Inuit[END_REF][START_REF] Bollati | Decline in genomic DNA methylation through aging in a cohort of elderly subjects[END_REF][START_REF] El-Maarri | Gender specific differences in levels of DNA methylation at selected loci from human total blood: a tendency toward higher methylation levels in males[END_REF][START_REF] Ogino | A cohort study of tumoral LINE-1 hypomethylation and prognosis in colon cancer[END_REF][START_REF] Tarantini | Effects of particulate matter on genomic DNA methylation content and iNOS promoter methylation[END_REF][START_REF] Figueiredo | Global DNA hypomethylation (LINE-1) in the normal colon and lifestyle characteristics and dietary and genetic factors[END_REF][START_REF] Smith | DNA global hypomethylation in squamous cell head and neck cancer associated with smoking, alcohol consumption and stage[END_REF][START_REF] Richards | Genome-wide hypomethylation in head and neck cancer is more pronounced in HPV-negative tumors and is associated with genomic instability[END_REF][START_REF] Yamamoto | LINE-1 hypomethylation is associated with increased CpG island methylation in Helicobacter pylori-related enlarged-fold gastritis[END_REF][START_REF] Woloszynska-Read | Intertumor and intratumor NY-ESO-1 expression heterogeneity is associated with promoter-specific and global DNA methylation status in ovarian cancer[END_REF][START_REF] Geli | Global and regional CpG methylation in pheochromocytomas and abdominal paragangliomas: association to malignant behavior[END_REF][START_REF] Ogino | LINE-1 hypomethylation is inversely associated with microsatellite instability and CpG island methylator phenotype in colorectal cancer[END_REF][START_REF] Iacopetta | Methylation levels of LINE-1 repeats and CpG island loci are inversely related in normal colonic mucosa[END_REF][START_REF] Choi | Hypomethylation of LINE-1 and Alu in welldifferentiated neuroendocrine tumors (pancreatic endocrine tumors and carcinoid tumors)[END_REF][START_REF] Estecio | LINE-1 hypomethylation in cancer is highly variable and inversely correlated with microsatellite instability[END_REF] Because of the growing interest in DNA methylation, as well as the wide availability of the technology necessary to conduct DNA methylation analyses, the number of epidemiological investigations including DNA methylation measures will increase rapidly in the next few years. Information about how DNA methylation measures vary in relation with subjects' characteristics and other information commonly collected in epidemiological investigations will be relevant for effective design and statistical analysis of such studies.

Abnormal DNA methylation patterns have been associated with various human diseases, including cancer, cardiovascular disease and autoimmune disease. [START_REF] Lucchinetti | Inhibition of LINE-1 expression in the heart decreases ischemic damage by activation of Akt/PKB signaling[END_REF][START_REF] Castro | Increased homocysteine and S-adenosylhomocysteine concentrations and DNA hypomethylation in vascular disease[END_REF][START_REF] Wilson | DNA hypomethylation and human diseases[END_REF] In healthy populations, inter-individual methylation variations at Alu and LINE-1 elements from blood DNA have F o r R e v i e w O n l y also been associated with risk factors for cancer, neurological and cardiovascular diseases. [START_REF] Baccarelli | Rapid DNA methylation changes after exposure to traffic particles[END_REF][START_REF] Baccarelli | Low blood DNA methylation determines higher risk and mortality from ischemic heart disease and stroke among elderly individuals[END_REF][START_REF] Bollati | Changes in DNA methylation patterns in subjects exposed to low-dose benzene[END_REF][START_REF] Rusiecki | Global DNA hypomethylation is associated with high serum-persistent organic pollutants in Greenlandic Inuit[END_REF] As recently discussed by Foley et al., [START_REF] Foley | Prospects for epigenetic epidemiology[END_REF] the identification and validation of epigenetic markers measured in easily obtainable tissues will be required for epidemiologic studies to benefit from incorporation of epigenetics. Among the biospecimens suitable for DNA methylation analysis, blood DNA has been the most widely collected in epidemiology investigations.

Although global DNA methylation levels have been proposed to be influenced by a number of endogenous and exogenous parameters, [START_REF] Jaenisch | Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals[END_REF] determinants of blood DNA methylation in repetitive elements in healthy individuals are still largely unexplored.

Although a number of demographic and lifestyle factors have been associated with global hypomethylation, the results have been inconsistent including those from our own studies. [START_REF] Bollati | Changes in DNA methylation patterns in subjects exposed to low-dose benzene[END_REF][START_REF] Rusiecki | Global DNA hypomethylation is associated with high serum-persistent organic pollutants in Greenlandic Inuit[END_REF][START_REF] Bollati | Decline in genomic DNA methylation through aging in a cohort of elderly subjects[END_REF] Because of the moderate inter-individual differences in global methylation measures in blood leukocyte DNA, the limited sample size of each investigation may have contributed to the inconsistencies. In a study of elderly individuals in the VA Normative Aging Study (NAS), we have shown that repetitive element methylation was cross-sectionally associated with age, particularly in Alu element. [START_REF] Bollati | Decline in genomic DNA methylation through aging in a cohort of elderly subjects[END_REF] In the present work on a combined analysis of 1465 subjects from five investigations, we sought to determine the association of age, gender, smoking habits, alcohol drinking, and body mass index (BMI) with the methylation status of Alu and LINE-1 elements among healthy individuals. Because DNA methylation was analyzed on DNA from white blood cells in all studies, whether differences in white blood cell count were associated with Alu and LINE-1 methylation was also explored.

Methods

Study populations

Individual data from five studies were combined including a total 1465 healthy subjects: [1] 722 subjects from the VA Normative Aging Study (NAS) in Eastern Massachusetts, USA; [2] [START_REF] Bell | The normative aging study: an interdisciplinary and longitudinal study of health and aging[END_REF] The men, aged 55 to 100 during 1999-2007, are given a health examination every 3-5 years and asked to donate a 7 ml blood sample at each visit. DNA methylation data from only the first blood sample of each study participant was included in our analysis.

Study #2 was a population-based case-control study of gastric cancer conducted in Warsaw, Poland during 1994-1996. [START_REF] Chow | Risk of stomach cancer in relation to consumption of cigarettes, alcohol, tea and coffee in Warsaw, Poland[END_REF] Four hundred and twenty-one subjects included in the present analysis were non-cancer controls randomly selected from a computerized registry of the Warsaw population, frequency-matched by age and gender to the cases.

Study #3 was carried out among residents of Milan, Italy, from October 1999 to June 2000. [START_REF] Bollati | Changes in DNA methylation patterns in subjects exposed to low-dose benzene[END_REF] This study included 77 gasoline station attendants, 77 traffic-control officers (both

F o r R e v i e w O n l y
groups exposed to low-benzene levels), and 57 office workers. All subjects were free of major illnesses.

Study #4 included 63 healthy male workers from a steel production plant in Brescia, Italy. [START_REF] Tarantini | Effects of particulate matter on genomic DNA methylation content and iNOS promoter methylation[END_REF] Between April and May 2006, every subject was asked to donate a blood sample at two different times. In the current study, only blood samples of every participant collected in the morning of the first work week day (after 2 days off work) before the beginning of any work activity were analyzed.

Study #5 included 48 male workers who were employed in PFOA production during 1978-2007. [START_REF] Costa | Thirty years of medical surveillance in perfluooctanoic acid production workers[END_REF] Blood samples were collected in 2007, when 35 were still active workers in the PFOA department, whereas 13 were no longer exposed after retirement or being transferred to other departments.

Completeness of the data

Some of the variables evaluated in our analysis were not available in all studies. BMI of the 211 subjects from Study #3 and information on alcohol drinking about the 63 subjects from Study #4 were not recorded. Detailed information on smoking except for smoking status (nonsmoker, former and current smoker) was not collected from the 48 subjects from Study #5. In addition, white blood cell count data were not available on the 421 subjects from Study #2, and only data on lymphocyte and monocyte counts, but not on neutrophils, eosinophils, and basophils, were available on the 63 subjects from Study #4.

Definition of the variables of interest

All variables used in the present analysis were defined for all studies using the same criteria.

An ever-smoker was defined as a smoker of at least 1 cigarette per day for 6 months or longer.

Pack-years of smoking were calculated as the product of packs of cigarettes smoked per day and total years of smoking. An ever-drinker was defined as a person who reported drinking

Alu and LINE-1 methylation analyses

Alu and LINE-1 methylation analyses were conducted using the method developed by Yang et al. [START_REF] Yang | A simple method for estimating global DNA methylation using bisulfite PCR of repetitive DNA elements[END_REF] for all the studies, as previously described. [START_REF] Bollati | Changes in DNA methylation patterns in subjects exposed to low-dose benzene[END_REF][START_REF] Bollati | Decline in genomic DNA methylation through aging in a cohort of elderly subjects[END_REF][START_REF] Tarantini | Effects of particulate matter on genomic DNA methylation content and iNOS promoter methylation[END_REF] One of the study collaborators (V.B.) participated in all DNA methylation analyses, either by directly performing them or supervising the laboratory protocol application. Blood leukocyte DNA was extracted, bisulfite treated, and PCR amplified. Pyrosequencing was done using the PSQHS96 Pyrosequencing System (Pyrosequencing). The degree of methylation was expressed for both Alu and LINE-1 as percentage of methylated cytosines (%5mC). For all assays built-in controls were used to verify bisulfite conversion. Each marker was tested in at least two replicates and their average was used in the statistical analysis. The within-sample coefficient variations in duplicate runs were 7.0% and 5.3% for Alu and LINE-1, respectively. Samples were dropped from analysis when replication failed or replicates were not within a specified range (5%). Pyrosequencing data for each marker were successful obtained for ≥98.6% of the samples.

Statistical methods

Multivariable linear regression models were used to evaluate Alu and LINE-1 methylation levels in relation to i) subjects' characteristics (age, gender, BMI, smoking status, current cigarettes/day, past cigarettes/day, total smoking years, pack-years, age smoking started, age smoking stopped and alcohol drinking) with adjustment for age (as a continuous variable), gender (male vs. female), and study (using indicator variables), and to ii) total number of white blood cells and the proportions of white-blood-cell types (neutrophils, lymphocytes, monocytes, eosinophils and basophils) with adjustment for age (as a continuous variable), gender (male vs. female), alcohol drinking (ever-drinker vs. nondrinker) and study (using

F o r R e v i e w O n l y
indicator variables). To evaluate whether differences in differential blood counts confounded the potential associations between subjects' characteristics and DNA methylation, multivariable models were fitted adjusting for percent lymphocytes, in addition to age, gender and study. Since blood count data were missing in Study #2 (Warsaw, Poland), percent lymphocyte data were imputed using linear prediction from a multiple-equation (ME) regression model [START_REF] Mccleary | Full-information models for multiple psychometric tests: annualized rates of change in normal aging and dementia[END_REF] that included age, gender, smoking status, current cigarettes/day, packyears and alcohol drinking. All statistical analyses were conducted using Stata 10.1 (Stata Corporation, College Station, TX).

Results

The characteristics of the study populations, as well as of the combined data, are shown in Table 1. In the combined data, mean blood DNA methylation levels, expressed as percentage of 5-methylcytosine (%5mC), were 26.0 (SD = 1.8) for Alu, and 76.2 (SD = 6.2) for LINE-1.

The associations between the subjects' characteristics and Alu and LINE-1 methylation are shown in combination, as well as within each of the studies (Table 2). In the combined data, age (β = -0.011 %5mC/year, 95% CI -0.020 to -0.001 %5mC/year) was negatively associated with DNA methylation in Alu elements. Compared with females, males had a lower Alu methylation levels (β = -0.385, 95% CI -0.665 to -0.104), but a higher LINE-1 methylation levels (β = 0.796, 95% CI 0.261 to 1.330). As Studies #1, 4 and 5 contained males only, further analyses of gender in relation to Alu and LINE-1 methylation were repeated using only subjects from Studies #2 and 3. In this analysis (Supplementary Table S1), no major differences in effect estimates were observed from the results reported above. In addition, alcohol drinkers tended to have a lower Alu methylation levels than nondrinkers (β = -0.214, 95% CI -0.415 to -0.013). Age and alcohol drinking showed no association with LINE-1 methylation level. DNA methylation in both Alu and LINE-1 elements was not 2), current cigarettes/day, past cigarettes/day, total smoking years, pack-years, age smoking started or age smoking stopped (data not shown). Further analyses on the associations of age, gender, and BMI with Alu and LINE-1 methylation were conducted on subsets of the study population by including alcohol drinkers only (n = 246), smokers only (n = 371), or individuals who were both alcohol drinkers and smokers (n = 481). These sets of analyses produced comparable results (data not shown). As smoking habits, alcohol drinking, and BMI differed between males and females (P < 0.001 for all, Table 1), we also performed gender-stratified analyses of smoking habits, alcohol drinking, and BMI, which showed no major differences in their associations with Alu or LINE-1 methylation (data not shown). Since 265 subjects (18.1% of the total number of subjects) were exposed to certain occupational pollutants, including benzene, [START_REF] Bollati | Changes in DNA methylation patterns in subjects exposed to low-dose benzene[END_REF] particulate matter [START_REF] Tarantini | Effects of particulate matter on genomic DNA methylation content and iNOS promoter methylation[END_REF] and PFOA, [START_REF] Costa | Thirty years of medical surveillance in perfluooctanoic acid production workers[END_REF] which may exert some effects on global DNA methylation and thus modify the effects of characteristics on global DNA methylation, further association analyses of subjects' characteristics with DNA methylation were repeated by excluding these exposed subjects. In this analysis (Supplementary Table S2), only minor departures of the effect estimates were observed from the results based on the total number of subjects.

As DNA methylation was measured on white blood cells, whether the differential white blood cell count was associated with Alu and LINE-1 element methylation was examined (Table 3). The total number of white blood cells was not associated with either Alu or LINE-1 methylation. Percent neutrophils and lymphocytes, the two major types of white blood cells, were inversely correlated with each other (r = -0.936, P < 0.001) and exhibited a positive (β = 0.036, 95% CI 0.010 to 0.061) and negative (β = -0.038, 95% CI -0.065 to -0.012) association with LINE-1 methylation, respectively. No association between the differential blood count and Alu element methylation was found. Adding percent lymphocytes into multivariable linear regression models produced no major difference in the effects of 4). When these analyses were repeated without imputation of percent lymphocytes for Study #2 (i.e., by excluding Study #2), the results were generally similar to the ones reported (data not shown). The only noticeable difference was that alcohol drinking was not associated with Alu methylation in this set of models (β = -0.178, 95% CI -0.433 to 0.076). However, this appeared to result from the exclusion of Study #2, which was the study with the highest number of ever drinkers (n=283), rather than from adjustment for percent lymphocytes, as also demonstrated in a model adjusting for age, gender and study, but not for percent lymphocytes, fitted without including Study #2 (β for alcohol drinking = -0.177, 95% CI -0.428 to 0.073).

Discussion

In a combined analysis of blood DNA methylation from five investigations, we showed that age, gender and alcohol drinking are predictors for global methylation levels in blood DNA from healthy subjects. In particular, age and alcohol were inversely associated with Alu element methylation. Gender had opposite associations with Alu and LINE-1 methylation levels, with males having lower Alu and higher LINE-1 methylation levels than females. We also showed that the neutrophil and lymphocyte proportions of white-blood-cell types modified LINE-1 methylation level measured in blood DNA, but did not confound the association between global methylation levels and subjects' age, gender, and alcohol drinking.

Although Alu and LINE-1 methylation levels were shown to be correlated with each other in tumor tissue DNA, [START_REF] Choi | Hypomethylation of LINE-1 and Alu in welldifferentiated neuroendocrine tumors (pancreatic endocrine tumors and carcinoid tumors)[END_REF] no such correlation has been found in surrounding normal tissues, [START_REF] Choi | Hypomethylation of LINE-1 and Alu in welldifferentiated neuroendocrine tumors (pancreatic endocrine tumors and carcinoid tumors)[END_REF] or in non-target tissues such as blood and buccal cells. [START_REF] Bollati | Changes in DNA methylation patterns in subjects exposed to low-dose benzene[END_REF][START_REF] Rusiecki | Global DNA hypomethylation is associated with high serum-persistent organic pollutants in Greenlandic Inuit[END_REF][START_REF] Li | Differential stress induction of individual Alu loci: implications for transcription and retrotransposition[END_REF] In this respect, the differences between Alu and LINE-1 in methylation regulation mechanisms, [START_REF] Jones | The fundamental role of epigenetic events in cancer[END_REF] responses to cellular stressors and environmental exposures, [START_REF] Bollati | Changes in DNA methylation patterns in subjects exposed to low-dose benzene[END_REF][START_REF] Rusiecki | Global DNA hypomethylation is associated with high serum-persistent organic pollutants in Greenlandic Inuit[END_REF][START_REF] Li | Differential stress induction of individual Alu loci: implications for transcription and retrotransposition[END_REF] and average levels of DNA methylation [START_REF] Yang | A simple method for estimating global DNA methylation using bisulfite PCR of repetitive DNA elements[END_REF][START_REF] Rusiecki | Global DNA hypomethylation is associated with high serum-persistent organic pollutants in Greenlandic Inuit[END_REF] The effect of age on global DNA methylation has been widely discussed. [START_REF] Fraga | Cross-talk between aging and cancer: the epigenetic language[END_REF][START_REF] Liu | Aging, cancer and nutrition: the DNA methylation connection[END_REF] In analysis of blood DNA derived from healthy subjects, several studies based on relatively small study samples (between 76 and 237 subjects) reported an inverse association between age and genomic 5mC content, [START_REF] Fuke | Age related changes in 5-methylcytosine content in human peripheral leukocytes and placentas: an HPLC-based study[END_REF][START_REF] Shimabukuro | Global hypomethylation of peripheral leukocyte DNA in male patients with schizophrenia: a potential link between epigenetics and schizophrenia[END_REF] concluding that age is an independent predictor for genomic 5mC content. However, several other similarly sized or larger studies (between 32 and 526 samples) reported no association of age with genomic 5mC content [START_REF] Moore | Genomic DNA hypomethylation as a biomarker for bladder cancer susceptibility in the Spanish Bladder Cancer Study: a case-control study[END_REF] or other estimates of global DNA methylation obtained from various genomic components, including LINE-1, Alu, and CCGG sequences. [START_REF] Rusiecki | Global DNA hypomethylation is associated with high serum-persistent organic pollutants in Greenlandic Inuit[END_REF][START_REF] Chalitchagorn | Distinctive pattern of LINE-1 methylation level in normal tissues and the association with carcinogenesis[END_REF][START_REF] Hsiung | Global DNA methylation level in whole blood as a biomarker in head and neck squamous cell carcinoma[END_REF][START_REF] Bjornsson | Intra-individual change over time in DNA methylation with familial clustering[END_REF] Our finding that Alu, but not LINE-1, methylation levels decreased in association with age suggests that age-related decreases in DNA methylation are not occurring homogenously across the human genome. Also, our recent longitudinal analysis in the VA Normative Aging Study, [START_REF] Bollati | Decline in genomic DNA methylation through aging in a cohort of elderly subjects[END_REF] along with analyses in the present study and in a previous investigation, [START_REF] Fuke | Age related changes in 5-methylcytosine content in human peripheral leukocytes and placentas: an HPLC-based study[END_REF] showed that age-related methylation alteration are small, particularly compared with the inter-individual variability in DNA methylation. Thus, the lack of association with age in several studies [START_REF] Rusiecki | Global DNA hypomethylation is associated with high serum-persistent organic pollutants in Greenlandic Inuit[END_REF][START_REF] Chalitchagorn | Distinctive pattern of LINE-1 methylation level in normal tissues and the association with carcinogenesis[END_REF][START_REF] Hsiung | Global DNA methylation level in whole blood as a biomarker in head and neck squamous cell carcinoma[END_REF][START_REF] Bjornsson | Intra-individual change over time in DNA methylation with familial clustering[END_REF] could be partially due to smaller sample size, insufficient age-range evaluated, and/or limitations of the analytical methods used.

We found that gender had opposite associations with Alu and LINE-1 methylation levels, with males having lower Alu but higher LINE-1 methylation levels. Previous studies have also observed that males, compared with females, had elevated LINE-1 methylation in blood DNA, [START_REF] El-Maarri | Gender specific differences in levels of DNA methylation at selected loci from human total blood: a tendency toward higher methylation levels in males[END_REF][START_REF] Hsiung | Global DNA methylation level in whole blood as a biomarker in head and neck squamous cell carcinoma[END_REF] consistent with our data for LINE-1. Yet, our observation that males had lower Alu methylation levels may seem paradoxical. One of the two X chromosomes in females is transcriptionally inactive and heavily methylated. This, together with differences in Alu element density and composition between chromosome X and Y, [START_REF] Grover | Alu repeat analysis in the complete human genome: trends and variations with respect to genomic composition[END_REF] reflecting a net effect of increase and decrease of methylation of various components in genomic DNA, [START_REF] Dunn | Hypomethylation: one side of a larger picture[END_REF] revealed a higher global 5mC content of blood DNA in males than in females. [START_REF] Fuke | Age related changes in 5-methylcytosine content in human peripheral leukocytes and placentas: an HPLC-based study[END_REF][START_REF] Shimabukuro | Global hypomethylation of peripheral leukocyte DNA in male patients with schizophrenia: a potential link between epigenetics and schizophrenia[END_REF] Our data seemed to be consistent with these findings, since male gender was associated with higher β estimates for LINE-1 (β = 0.796) than for Alu (β = -0.385), which may contribute to an elevated net global methylation levels in males.

Smoking has been associated with a state of global hypomethylation in genomic DNA derived from tumor tissues in head and neck squamous cell carcinoma (HNSCC). [START_REF] Smith | DNA global hypomethylation in squamous cell head and neck cancer associated with smoking, alcohol consumption and stage[END_REF][START_REF] Hsiung | Global DNA methylation level in whole blood as a biomarker in head and neck squamous cell carcinoma[END_REF][START_REF] Furniss | Line region hypomethylation is associated with lifestyle and differs by human papillomavirus status in head and neck squamous cell carcinomas[END_REF][START_REF] Guerrero-Preston | Global DNA methylation: a common early event in oral cancer cases with exposure to environmental carcinogens or viral agents[END_REF] Vaissiere et al. [START_REF] Vaissiere | Quantitative analysis of DNA methylation profiles in lung cancer identifies aberrant DNA methylation of specific genes and its association with gender and cancer risk factors[END_REF] found that smoking was associated with increased methylation in lung cancer tissues of MTHFR, a gene participating in the methyl donor pathway, and that in turn MTHFR methylation showed a marked inverse correlation with LINE-1 methylation. We hypothesized that smoking would exert effects on global methylation levels in genomic DNA derived from blood of healthy subjects, but failed to find any association between smoking habits (smoking status, current cigarettes/day, past cigarettes/day, total smoking years, packyears, age smoking started and age smoking stopped) and Alu or LINE-1 methylation levels.

Consistent with our data, other studies of smoking effects on global methylation in blood DNA of healthy subjects also reported negative results. [START_REF] Rusiecki | Global DNA hypomethylation is associated with high serum-persistent organic pollutants in Greenlandic Inuit[END_REF][START_REF] Moore | Genomic DNA hypomethylation as a biomarker for bladder cancer susceptibility in the Spanish Bladder Cancer Study: a case-control study[END_REF][START_REF] Hsiung | Global DNA methylation level in whole blood as a biomarker in head and neck squamous cell carcinoma[END_REF] The lack of association between smoking habits and global methylation in genomic DNA from blood of healthy subjects, which are at variance with inverse associations found in tumor tissues, [START_REF] Smith | DNA global hypomethylation in squamous cell head and neck cancer associated with smoking, alcohol consumption and stage[END_REF][START_REF] Hsiung | Global DNA methylation level in whole blood as a biomarker in head and neck squamous cell carcinoma[END_REF][START_REF] Furniss | Line region hypomethylation is associated with lifestyle and differs by human papillomavirus status in head and neck squamous cell carcinomas[END_REF][START_REF] Guerrero-Preston | Global DNA methylation: a common early event in oral cancer cases with exposure to environmental carcinogens or viral agents[END_REF] may suggest that smoking acts differently in different tissue types, or acts differently in healthy versus tumor tissues. Although we did not observe a direct effect of smoking on global DNA methylation among healthy subjects, a possible involvement of smoking in modifying global DNA methylation cannot be excluded. Hillemacher et al. [START_REF] Hillemacher | Global DNA methylation is influenced by smoking behaviour[END_REF] when both were non-smokers, and absent when the offsprings smoked. [START_REF] Hillemacher | Global DNA methylation is influenced by smoking behaviour[END_REF] In addition, Breton et al. [START_REF] Breton | Prenatal Tobacco Smoke Exposure Affects Global and Gene-Specific DNA Methylation[END_REF] reported that prenatal exposure to tobacco smoke was associated with decreased global methylation level in buccal cell DNA in children. Because DNA methylation is largely established in utero or in early life, [START_REF] Gluckman | Effect of in utero and early-life conditions on adult health and disease[END_REF] these findings may reflect higher susceptibility to smoking in specific periods of life.

Several studies have examined the association between alcohol drinking and global 5mC content or LINE-1 element methylation in both normal and tumor tissue DNA, with most studies reporting no association. [START_REF] Moore | Genomic DNA hypomethylation as a biomarker for bladder cancer susceptibility in the Spanish Bladder Cancer Study: a case-control study[END_REF][START_REF] Hsiung | Global DNA methylation level in whole blood as a biomarker in head and neck squamous cell carcinoma[END_REF][START_REF] Furniss | Line region hypomethylation is associated with lifestyle and differs by human papillomavirus status in head and neck squamous cell carcinomas[END_REF][START_REF] Guerrero-Preston | Global DNA methylation: a common early event in oral cancer cases with exposure to environmental carcinogens or viral agents[END_REF] Only one study of HNSCC tumor tissues reported that alcohol use was inversely associated with LINE-1 methylation levels in univariate analysis, but this significance disappeared in multivariate analysis. [START_REF] Smith | DNA global hypomethylation in squamous cell head and neck cancer associated with smoking, alcohol consumption and stage[END_REF] In our data, no association was found between alcohol drinking and LINE-1 methylation, while we showed for the first time that drinking was inversely associated with Alu methylation levels. Alcohol drinkers might have lower intakes as well as lower serum levels of vitamin B 12 and folate, [START_REF] Gibson | Alcohol increases homocysteine and reduces B vitamin concentration in healthy male volunteers--a randomized, crossover intervention study[END_REF] and thus have lower availability of methyl sources, as well as decreased efficiency of the enzymatic pathways responsible for DNA methylation. [START_REF] Gibson | Alcohol increases homocysteine and reduces B vitamin concentration in healthy male volunteers--a randomized, crossover intervention study[END_REF][START_REF] Purohit | Role of S-adenosylmethionine, folate, and betaine in the treatment of alcoholic liver disease: summary of a symposium[END_REF] In addition, animal models have shown that chronic alcohol exposure can decrease cytosine methyltransferase mRNA levels in sperm [START_REF] Bielawski | Paternal alcohol exposure affects sperm cytosine methyltransferase messenger RNA levels[END_REF] and DNA methylation in tissues of liver and colon mucosa. [START_REF] Fiala | Effects of chronic dietary ethanol on in vivo and in vitro metabolism of methylazoxymethanol and on methylazoxymethanol-induced DNA methylation in rat colon and liver[END_REF] It should be noted that most of the studies included in the present analysis did not have detailed data on alcohol drinking, which hindered our ability to further evaluate alcohol dose-related effects on global DNA methylation. Moreover, our present analysis, together with all other studies on the association between alcohol drinking and global DNA methylation, [START_REF] Smith | DNA global hypomethylation in squamous cell head and neck cancer associated with smoking, alcohol consumption and stage[END_REF][START_REF] Moore | Genomic DNA hypomethylation as a biomarker for bladder cancer susceptibility in the Spanish Bladder Cancer Study: a case-control study[END_REF][START_REF] Hsiung | Global DNA methylation level in whole blood as a biomarker in head and neck squamous cell carcinoma[END_REF][START_REF] Furniss | Line region hypomethylation is associated with lifestyle and differs by human papillomavirus status in head and neck squamous cell carcinomas[END_REF][START_REF] Guerrero-Preston | Global DNA methylation: a common early event in oral cancer cases with exposure to environmental carcinogens or viral agents[END_REF] mostly included social drinkers. Additional studies on alcohol abusers and dependence are required.

Our data showed that the proportions of white-blood-cell subsets affected LINE-1 methylation levels measured in blood DNA, with percent neutrophils and lymphocytes, the major types of white blood cells, exhibiting opposite effects on LINE-1 methylation levels. Previous studies have shown that there exist between-tissue methylation variations, which contribute to regulating functions that are specific to each tissue or cell types. [START_REF] Eckhardt | DNA methylation profiling of human chromosomes 6, 20 and 22[END_REF] In the present work, further analysis showed that the addition in multivariable regression models of percent lymphocytes did not modify the associations of the subjects' characteristics we evaluated with repetitive-element methylation levels. However, the association we observed between differential white blood count and LINE-1 methylation indicates that findings from epidemiological studies based on DNA methylation analyses on blood DNA might be confounded by differences in the proportion of white blood cell types. This would be a concern for exposures or disease states that are associated with inflammatory responses that may produce shifts in white blood cell type proportions. [START_REF] Essen | The respiratory inflammatory response to the swine confinement building environment: the adaptation to respiratory exposures in the chronically exposed worker[END_REF][START_REF] Requena | Erythema nodosum[END_REF] Our study had the advantages of being based on a large sample size and accurate quantitative analysis using pyrosequencing methodology, which is suitable for measuring subtle changes in DNA methylation. In the present work, all DNA methylation analyses were performed using the same laboratory protocol, and each variable used in the present analysis was defined using the same criteria. To account for potential confounding due to differences between the five studies, all our analyses were adjusted by study using indicator variables in the regression models, thus also minimizing the potential effect on methylation caused by between-study differences in ethnic background, which has been shown to be associated with global DNA methylation levels, [START_REF] Figueiredo | Global DNA hypomethylation (LINE-1) in the normal colon and lifestyle characteristics and dietary and genetic factors[END_REF][START_REF] Piyathilake | Race-and age-dependent alterations in global methylation of DNA in squamous cell carcinoma of the lung (United States)[END_REF] as well as potential analytical batch effects. One limitation of our analysis is that results on the association of gender with LINE-1 or Alu methylation could be confounded by the origin of the study and by the difference in lifestyle factors between males and females. Only two (Studies #2 and 3) of the five studies used in the combined analysis included females (only 14.1% of the study subjects were females). In addition, smoking habits, alcohol drinking, and BMI differed between males and females. However, similar effect estimates were observed after excluding studies containing males S1), or by further adjusting for variables in multivariable analyses that might have confounded the associations, including smoking habits, alcohol drinking, BMI and percent lymphocytes (data not shown).

Nonetheless, it cannot be excluded that other unmeasured lifestyle or personal factors might underline the gender-related differences in DNA methylation levels we observed. Secondly, dietary differences were not investigated here, which may also influence methylation levels in genomic DNA. [START_REF] Suzuki | Roles and causes of abnormal DNA methylation in gastrointestinal cancers[END_REF] Lastly, we assumed that DNA methylation in Alu and LINE-1 repetitive elements may be used as a surrogate for global DNA methylation content. However, methylation levels of both repetitive elements are not equivalent to, albeit vastly represents, global DNA methylation content. [START_REF] Yang | A simple method for estimating global DNA methylation using bisulfite PCR of repetitive DNA elements[END_REF] Also, measures of repetitive element-based methylation analyses as used in the present study, representing a pool of multiple unique Alu or LINE-1 repeats in the entire genome, are different from measures of gene-specific or genome-wide array-based (genomic site-specific) methylation analyses, and thus the extent to which our conclusions apply to site-specific methylation measures remains to be determined. In particular, whereas the present of associations of the variables we evaluated with Alu and/or LINE-1 methylation might suggest that there may also exist associations with gene-specific methylation, lack of association with Alu and LINE-1 methylation does not exclude associations with specific loci. Both Alu and LINE-1 methylation levels represent an average measure of methylation at multiple sites across the genome. Opposite concurrent changes of hypo-and hyper-methylation might offset each other without being reflected in these average measures of methylation.

In conclusion, this combined analysis of blood DNA methylation showed that study participant data that are routinely collected in most epidemiological investigations, including age, gender and alcohol drinking, are predictors of global DNA methylation levels. We also showed that the neutrophil and lymphocyte proportions of white-blood-cell types modified We showed that study participant data that are generally collected in most epidemiological investigations, including age, gender, alcohol drinking, and blood neutrophil and lymphocyte proportions, are predictors of global DNA methylation levels.

These findings need to be considered in designing epidemiological investigations aiming at identifying associations between DNA methylation and health outcomes. -0.049 (-0.425 to 0.327) Abbreviations: %5mC, percentage of 5-methyl cytosine. a Adjusted for age, gender, percent lymphocytes in blood count, and study. b Beta for age were obtained using continuous variables.

F o r R e v i e w O n l y

  controls of a population-based case-control study of gastric cancer conducted in Warsaw, Poland;[3] 211 subjects from a cross-sectional study of low-benzene exposure conducted in Milan, Italy;[4] 63 healthy workers from a steel plant in Brescia, Italy; and[5] 48 healthy workers from a perfluooctanoic acid (PFOA) production facility in Trissino, Italy. All participants in studies #2, 3, 4 and 5, and 98.2% in study #1 were Caucasians. Detailed information about gender, age, height, weight, drinking and smoking was obtained by structured questionnaires, either self-administered (studies #1, 3 and 4) or through in-person interviews (studies #2 and 5). White blood cell counts were performed on fresh blood using automated methods (Coulter Cell Counter, Abbott or Bayer) within 3 hours from the time of blood drawing for all studies. Informed consent was obtained from all participants and the ethical committees of all participating centers approved the studies. Details on each of the studies are provided below. Study #1 included 722 men who were evaluated between March 1999 and October 2007 as part of the Normative Aging Study (NAS), a longitudinal study of normal aging established in 1963 by the U.S. Veterans Administration.

  least 1 time per month for 6 months or longer. The age and BMI at blood draw were used for all analyses.

  with BMI, smoking status (Table

  and LINE-1 methylation level (Table

  may account for the different findings for Alu and LINE-1 methylation in association with the in the present study, such as age, gender, alcohol drinking and the proportions of white-blood-cell types.

  may partly account for the lower Alu methylation levels observed in males. Two investigations of global 5mC content,

  recently reported that smoking modifies the correlation between paternal and offspring DNA methylation. Although neither paternal smoking nor offspring smoking had effect on global DNA methylation estimated in CCGG sequences, correlation between offspring and paternal DNA methylation was strongest

  #1, 4 and 5) from the analyses (Supplementary Table

16 LINE- 1

 161 methylation level measured in blood DNA. These findings provide valuable information that can be used in planning future epidemiological studies, as well as in analyzing existing datasets. on how global methylation measured in blood leukocyte DNA varies in relation with subjects' characteristics and other factors commonly collected in epidemiological investigations will be relevant for effective study design and statistical analysis.

F

Table 1

 1 Subject characteristics of the five studies included in a combined analysis of determinants of blood DNA methylation in repetitive elements in healthy individuals [mean or n (%)]

	Variable	Study #1 Boston, USA	Study #2	Study #3	Study #4	Study #5	All studies

Table 2

 2 Association of Alu and LINE-1 methylation with the characteristics of the study subjects, in combination as well as within each of the five studies

	Alu

Abbreviations: %5mC, percentage of 5-methyl cytosine; NA, not available. a Adjusted for age, gender and study. b Beta for age and body mass index were obtained using continuous variables.

Table 3

 3 Effect of blood count on Alu and LINE-1 methylation (combined analysis of the five studies) Adjusted for age, gender, alcohol drinking and study.

	Alu	LINE-1

a

Table 4

 4 Effects of age, gender and alcohol drinking on Alu and LINE-1 methylation in multivariable models including percent lymphocytes in blood count, age, gender and study as independent variables (combined analysis of the five studies)

	Alu

Funding

This work was supported by grants as follows: Study #1: National Institute of Environmental Health Sciences [ES015172-01]; and Environmental Protection Agency [R83241601, R827353]. The VA Normative Aging Study is supported by the Cooperative Studies Program/Epidemiology Research and Information Center of the U.S. Department of Veterans Affairs and is a component of the Massachusetts Veterans Epidemiology Research and Information Center (MAVERIC). Study #2: Intramural Research Program (Division of Cancer Epidemiology and Genetics, NCI) of the National Institutes of Health; Diane Belfer Program for Human Microbial Diversity; and the National Institutes of Health [R01GM63270]. Study #3: Italian Association for Cancer Research (AIRC) (2008-2010) [6016]. Study #4: Cariplo Foundation [2007-5469]; and Italian Ministry of Scientific Research (MIUR) [PRIN-20072S2HT8]. Study #5: Milan University intramural funding.

Conflict of interest: None declared.