MAGI2 Gene Region and Celiac Disease

Celiac disease (CD) patients present a loss of intestinal barrier function due to structural alterations in the tight junction (TJ) network, the most apical unions between epithelial cells. The association of TJ-related gene variants points to an implication of this network in disease susceptibility. This work aims to characterize the functional implication of TJ-related, disease-associated loci in CD pathogenesis. We performed an association study of 8 TJ-related gene variants in a cohort of 270 CD and 91 non-CD controls. The expression level of transcripts located in the associated SNP region was analyzed by RT-PCR in several human tissues and in duodenal biopsies of celiac patients and non-CD controls. (si)RNA-driven silencing combined with gliadin in the Caco2 intestinal cell line was used to analyze the implication of transcripts from the associated region in the regulation of TJ genes. We replicated the association of rs6962966*A variant [p = 0.0029; OR = 1.88 (95%1.24–2.87)], located in an intron of TJ-related MAGI2 coding gene and upstream of RP4-587D13.2 transcript, bioinformatically classified as a long non-coding RNA (lncRNA). The expression of both genes is correlated and constitutively downregulated in CD intestine. Silencing of lncRNA decreases the levels of MAGI2 protein. At the same time, silencing of MAGI2 affects the expression of several TJ-related genes. The associated region is functionally altered in disease, probably affecting CD-related TJ genes.


INTRODUCTION
Celiac disease (CD) is a chronic enteropathy, characterized by villous atrophy and inflammation of the intestinal mucosa, that develops in genetically predisposed individuals. The disease is caused by an inappropriate immune response to ingested gluten, a protein consisting of soluble gliadin and insoluble glutenin components that is found mostly in wheat, but also in barley, rye and oat. There is evidence supporting that various environmental factors, such as repeated gastrointestinal infections (1,2) or early infant feeding (3,4) may also contribute to triggering the disease. A lifelong gluten-free diet (GFD) is the only effective treatment for CD that achieves complete remission of the symptoms.
CD is a complex genetic disorder, and multiple genes contribute to disease risk. The most important genetic determinants, Human Leukocyte Antigen (HLA) molecules HLA-DQ2 and HLA-DQ8, are necessary but not sufficient to explain the predisposition to CD. Genome wide association (GWA) and follow-up genetic studies have identified several additional genomic regions associated to the disease, which probably contain CD susceptibility genes, but our understanding of the disease pathogenesis is still limited (5).
In fact, the majority of single-nucleotide polymorphisms (SNPs) associated with increased disease risk are located in non-coding regions of the genome (6)(7)(8). Some of them map to long non-coding RNAs (lncRNAs) transcripts longer than 200 bases in length, with no protein-coding potential, that have been described to participate in genomic regulation (9). The analysis of the expression profiles of lncRNAs on autoimmune disease-associated regions has shown that they are enriched in autoimmune-disease loci, suggesting that they may be crucial to explain GWAS findings (10,11). Although their mechanisms of action are diverse and still not very well-characterized, lncRNAs regulate gene expression at transcriptional and post-transcriptional levels and are involved in a range of developmental processes, adding complexity to our understanding of how gene expression is regulated (12). On the other hand, expression quantitative trait loci (eQTL) analyses have demonstrated that certain SNPs associated with complex diseases can affect the expression of lncRNAs, and thus lncRNA genes can be considered candidate genes for diseasesusceptibility (13). In the case of CD, it has recently been shown that a lncRNA in the IL18RAP locus that harbors a diseaseassociated SNP is a key regulator of genes involved in the NFκB pathway, which is known to be constitutively activated in the small intestine of patients with CD (14,15).
Apart from GWAS studies, pathway analyses contribute to the reconstruction of altered biological networks that are potentially pathological and could help identify functional candidates that participate to the genetic susceptibility. In the case of CD, although an aberrant immune response is the main force driving the disease, whole genome expression analyses have identified important dysfunctions of other complex biological processes that could be involved in CD development, including alterations in the expression of genes related to intestinal permeability (16)(17)(18)(19)(20). Impairment of the epithelial barrier and increased permeability have been shown to be implicated in the development of CD (20) and other gastrointestinal inflammatory diseases like Crohn's Disease (21) and Ulcerative Colitis (22).
The permeability of the intestinal epithelium is dependent on the regulation of intercellular tight junctions (TJ), a continuous, circumferential, belt-like structure at the luminal end of the intercellular space that functions as a barrier. Changes in the expression, distribution, and phosphorylation of TJ proteins have been observed in CD and malfunction of this pathway could have an important role in the augmented intestinal permeability observed in the disease (23,24). These alterations persist in asymptomatic CD patients who are on GFD (25) and ultrastructural and functional abnormalities in TJs appear also in antibody-negative, asymptomatic first-degree relatives (26), supporting a genetic origin of the pathway alterations and a possible role in the initial stages of the disease. Different studies in European populations have found polymorphisms in several TJ genes, including adapter proteins MAGI2 and PARD3, together with MYO9B, a cytoskeleton protein involved in TJ assembly, that are associated with CD (27)(28)(29).
The MAGI proteins (Membrane Associated Guanylate-kinase proteins with Inverted domain arrangement) are evolutionarily conserved scaffolding proteins with several protein-protein interaction domains to coordinate signaling complexes (30). MAGI-2 protein (also known as AIP1/S-SCAM/ARIP1) is mainly expressed in the brain at synaptic junctions but has also been identified in the TJ plaque, where it controls epithelial integrity. Interestingly, apart from the association with CD, linkage analyses have shown that MAGI2 is located in a genomic region harboring susceptibility genes for IBD (31).
With this on mind, we hypothesized that TJ genes could be relevant candidates for gut disorders and specifically for CD. Thus, the aim of the study was to replicate previous association results and to perform gene expression and functional analyses of the potential candidate genes in the associated regions.

Duodenal Biopsies and Cell Culture
CD was diagnosed at the Pediatric Gastroenterology Clinic (Cruces University Hospital), according to the European Society for Pediatric Gastroenterology, Hepatology, and Nutrition criteria in force at the time of recruitment. The study was approved by the ethics committee of Cruces University Hospital (ref. CEIC-E08/59, CEIC-E13/20, and CEIC-E16/46) and informed consent was obtained from patients or their parents. A total of 48 duodenal biopsy samples were taken from 32 patients divided in 3 categories: 16 biopsies from CD patients at the time of diagnosis (symptomatic and on a gluten-containing diet; 10 girls/6 boys; mean age at diagnosis 2.9 years, range 1.3-9.3 years), 16 biopsies from the same patients after at least 2 years on strict GFD (asymptomatic, antibody negative, and with a recovered intestinal epithelium) and 16 samples from non-celiac individuals (5 girls/11 boys; mean age 7.7 years, range 1.1-13.0 years) who had been subjected to endoscopy but had no signs of gut inflammation. After collection, samples were immediately frozen and stored in liquid nitrogen until use.
The C2BBe1 human colon carcinoma cell line (clone of Caco-2) was selected as a well-established in vitro model to study the tight junctions in CD (32,33). Cells were maintained in DMEM supplemented with 10% heat inactivated FBS, 1% nonessential amino acids and 1% penicillin-streptomycin) in tissue culture flask.

Association Study
DNA samples from 270 celiac patients and 91 unselected blood donors from the CEGEC (Spanish Consortium for Genetics of Celiac Disease) collection were used for the association study. DNA samples were extracted from whole blood using conventional methods and stored at −80 • C until use. The CEGEC study was approved by the Ethics Committees or Institutional Review Board of all participating institutions. Samples were genotyped in the BioMark HD TM platform at the Sequencing and Genotyping Unit of the University of the Basque Country (UPV/EHU). Fluidigm SNP assays were designed according to the protocols recommended by the manufacturer (Fluidigm Corporation, San Francisco, CA, USA) (Table S1). We selected 3 SNPs in the MAGI2 locus, 2 in PARD3, and 3 in MYO9B, based on previously published association results (29,34,35). The SNP Genotyping Analysis software by Fluidigm was used for genotype calling. Clusters were evaluated manually, and doubtful calls were recalled or rejected. To ensure population homogeneity, an identity-by-state analysis and multidimensional scaling were performed before standard χ2 association analysis using Plink 1.07 (36).

Analysis of Coding Potential of RP4-587D13.2
The protein-coding capacity of RP4-587D13.2 was evaluated in silico. All possible open reading frames (ORFs) were identified using ORF Finder (NCBI) (37) and the coding potential of the transcript was assayed with Phylogenetic Codon Substitution Frequencies (PhyloCSF) based on phylogenetic conservation (38), as well as with the coding potential assessment tool (CPAT) that uses a model built with open reading frame size and coverage together with codon and hexamer usage bias (39).

Cellular Fractionation
To analyze the distribution of RP4-587D13.2 RNA in the nuclear and cytoplasmic cell compartments of C2BBe1 cells, nuclei were isolated using hypotonic lysis buffer (10 mM Hepes, 10 mM KCl, 0.1 mM EDTA and %0.8 Triton-X) as previously described. For relative quantification, the amounts of nuclear RP4-587D13.2, Lnc13 (nuclear control) and RPLP0 (cytoplasmic control) RNA were measured by RT-QPCR and compared to the amount of each RNA in whole cells.

Protein Extraction and Immunoblot Analysis
Whole cell extracts were directly lysed into 2x Laemmli Sample buffer containing 5% β-mercaptoethanol and stored at −20 • C until use. Total protein extracts were heat-treated at 95 • C for 5 min, fractionated by SDS-PAGE and transferred to nitrocellulose membranes using the Trans-Blot Turbo Transfer apparatus (BioRad, Hercules, California, USA). After incubation with 5% non-fat milk in Tris-buffered saline with 0.05% Tween (TBS-T) for 1 h, the membrane was incubated at 4 • C overnight with polyclonal antibodies against MAGI2 (1:500) (ABCAM, Cat. No. ab97343) and β-Tubulin (1:1,000) (ABCAM, Cat. No. ab18207). Membranes were washed three times for 10 min and incubated with horseradish peroxidase-conjugated anti-mouse (1:2,000) or anti-rabbit (1:10,000) antibodies for 1 h at room temperature in 5% BSA in TBS-T (Jackson ImmunoResearch Laboratories, Inc.; Cat No. 111-035-003 and 115-035-003, respectively). Blots were washed with TBS-T three times and visualized using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher, Cat No. 34094) on the ChemiDoc MP system (BioRad, Hercules, California, USA). The quantification of band intensities was done using the Image Lab 4.1 software and results were normalized to β-tubulin.

RNA and DNA Isolation From Tissue Samples and Cultured Cells
Total RNA and DNA were prepared with the NucleoSpin RNA kit and NucleoSpin Blood kit, respectively (Macherey-Nagel, Düren, Germany) according to the protocols provided. Frozen biopsy samples were disrupted with disposable plastic pellet pestles prior to nucleic acid extraction. Cells were lysed and homogenized using the buffers supplied with the kits. The concentration and purity of the RNA and DNA were determined by UV absorbance at 260 nm on a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and samples were stored at −80 • C until use. To determine the expression of MAGI2 and RP4-587D13.2 across different human tissues, a commercial Human Total RNA Master Panel II (Clontech Laboratories, Inc., Cat. No. 636643) was used.  Table S2). The reverse transcribed cDNA from each sample was preamplified using the Fluidigm Gene Expression Specific Target Amplification kit (Fluidigm Corporation, San Francisco, CA, USA) and amplified using TaqMan Universal PCR master mix (Thermo Fisher Scientific, Waltham, MA, USA). Complete results are available at the Gene Expression Omnibus (https://www.ncbi.nlm.nih. gov/geo/) with accession number GSE84729. The expression of the housekeeping gene RPLP0 (large ribosomal protein) was quantified in each experiment and used as an endogenous control of input RNA. The relative expression of each gene was calculated using the accurate Ct method as previously described (44).
The Commercial TaqMan assays probes (Thermo Fisher Scientific, Waltham, MA, USA) used for expression analyses are listed in Table S2. Probes that spanned an exon-exon junction were chosen to avoid the detection of genomic DNA.

SNP Genotyping
In order to determine whether the risk attributed to the newly identified variant rs6962966 was related to gene expression in CD patients, the tag SNP rs2691527 (r 2 = 1 with associated SNP rs6962966) was genotyped in the duodenal biopsy samples on a Eco Real-Time PCR System (Illumina, Inc., San Diego, CA, USA) using a predesigned TaqMan Genotyping assay (Thermo Fisher Scientific, Assay ID 4351379) and TaqMan genotyping Mastermix (Thermo Fisher Scientific, Waltham, MA, USA).

Analysis of Results
Statistical analyses were carried out using GraphPad Prism version 5.0 software (GraphPad Software Inc, La Jolla, CA, USA) to assess differences among groups. Non-parametric, paired (Wilcoxon matched-pairs test) tests were performed for the comparison of the expression levels between CD patients at diagnosis and after 2 years on GFD, and the Mann-Whitney U test was used to compare each disease group to the controls and unpaired t test to in the silencing experiments. Coexpression was assessed with Spearman's correlation test. Heat-maps were constructed using the FiRe Macro v2.2 for Microsoft Excel from the University of Fribourg available at http://www.unifr.ch/ plantbio/FiRe/main.html. Only 2-tailed p-values below 0.05 were considered significant.

An Intronic Polymorphism in MAGI2 Is Associated to CD
We analyzed 8 SNPs located in MAGI2, PARD3, and MYO9B, previously associated with CD. Their frequencies in CD patients and controls are listed on Table 1. We detected an association between SNP rs6962966 (GRCh38/hg38; chr7:77,804,123) in an intronic region of MAGI2 and CD risk. The SNP was in Hardy-Weinberg equilibrium in the control group and the frequency of the minor allele was higher in patients, augmenting the risk to develop CD 1.88-fold (95% CI = 1.24-2.87; p = 0.0029). No other SNPs in MAGI2, PARD3, or MYO9B were associated with CD.
In order to characterize the effects of the associated intronic SNP on disease risk, we analyzed its genomic context in search for putative regulatory elements that could be affected  causing alterations in gene regulation. An analysis of current UCSC Genome browser annotations showed that proteincoding gene MAGI2 hosts different types of non-coding genes, including processed transcripts, pseudogenes, and RNA genes. The great majority of them are located in introns and no specific function or effect on their host gene have been described so far. As an exception, the antisense gene MAGI2-AS3 overlaps the first exon of MAGI2 and has been recently linked to breast cancer (45). Regarding the intron where the CD-associated rs6962966 SNP falls, we found that it also harbors a non-characterized transcript, named RP4-587D13.2 (Figure 1).

RP4-587D13.2 Is a Long Non-coding Transcript That Can Regulate MAGI2 Expression
In order to characterize the region around the associated SNP, we performed expression analysis of both RP4-587D13.2 and MAGI2 across different human tissues. The results showed that these transcripts are ubiquotiusly expressed in all human tissues analyzed, including the intestine, the CD target tissue, and are predominantly expressed in brain (Figure 2A). It can be observed that both present a similar expression pattern and are positively correlated (p = 0.0007) across tissues. We next evaluated the protein-coding capacity of RP4-587D13.2 in silico using three different approaches. Overall, the lack of functional ORFs as well as the low phylogenetic conservation and coding probability of the region suggests that RP4-587D13.2 is a long non-coding transcript ( Table S3).
The cellular localization of the lncRNA was evaluated in C2BBe1 intestinal human cells, and results revealed that RP4-587D13.2 accumulates preferentially in the nucleus ( Figure 2B). The nuclear localization of RP4-587D13.2 together with its coexpression with the neighboring MAGI2 suggests that this lncRNA could contribute to the regulation of MAGI2 expression. To test this hypothesis, we performed siRNA silencing of RP4-587D13.2 in C2Bbbe1 intestinal cells using two different siRNAs. We managed to reduce the expression of RP4-587D13.2 by 46% in intestinal cells (Figure S1) which in turn resulted in a 56% decrease in MAGI2 protein (Figures 2C,D), confirming that this lncRNA is somehow affecting the regulation of the coding gene.

PTG Stimulation Alters the Expression of TJ-Related Genes in MAGI2-Silenced Cells
In order to determine the functional significance of the MAGI2 region in the CD characteristic dysregulation of the TJ pathway, the expression of a TJ-related gene panel was analyzed in the  C2BBe1 intestinal cell line. Due to the difficulty of efficiently silencing the lncRNA, probably because of its nuclear location, the effects in downstream gene expression were assessed by MAGI2 silencing. As expected, the efficiency of MAGI2 silencing was above 59% (Figures 3A,B) and silenced cells showed altered expression of CLDN1, F11R, PARD6A, PPP2R3A, TICAM2, and ZAK ( Figure 3C). Interestingly, upon PTG stimulation, there was an additional decrease of MAGI2 and a stronger effect on the TJ-related genes in MAGI2-silenced cells: the changes observed in PARD6A and ZAK were more pronounced, and alterations extended to other genes of the pathway, including GNAI1 and CLDN2.

The Expression of MAGI2 and RP4-587D13.2 Is Reduced in CD and Could Explain Alterations of Other Tight Junction Genes
Since the region harboring RP4-587D13.2 and MAGI2 is associated to CD and we have observed that both genes can influence the expression of other TJ-related genes, we analyzed their expression in biopsies from CD patients and controls. Figures 4A,B, both MAGI2 and RP4-587D13.2 show significantly lower expression levels in the duodenum of active CD patients, and this downregulation persisted after GFD treatment. The correlation between MAGI2 and RP4-587D13.2 gene expression previously observed in different human tissues (Figure 2A) was also present in duodenal biopsies (r = 0.4033; p = 0.027) (Figure S2).

As shown in
In order to assess whether the TJ genes that are altered by the downregulation of the MAGI2 region are also altered in CD, we analyzed their expression in CD and control biopsies. Two of the genes, CLDN2 and ZAK, are overexpressed in the intestine of active CD patients (on a gluten containing diet) compared to treated and control samples (Figures 4C,D), confirming what had been previously observed in vitro (Figure 3C).
Despite the limited number of biological samples representing all three genotypes in our study, we also searched for relationships between the risk genotypes and the expression level of the MAGI2 region genes. As the presence of all three genotypes in every group was not possible, heterozygous, and risk-allele homozygous samples were combined. The results of eQTL analysis showed that the presence of the risk allele was not significantly associated with decreased expression of either transcript in the MAGI2 region (Figures 5A,B). However, when we analyzed the correlation of both transcripts based on the genotype we observed that the samples harboring the risk allele showed a stronger correlation (r = 0.6364; p = 0.0402) than those with no risk allele (r = 0.4286; p = 0.4194) (Figures 5C,D) suggesting that the risk allele strengthens the interaction of both genes thus affecting the regulation of downstream genes.

DISCUSSION
Polymorphisms in TJ-related genes MAGI2, PARD3, and MYO9B that potentially influence the homeostasis of the intestinal barrier have been associated with risk of gastrointestinal disorders like CD and IBD (27,29,46). However, attempts to replicate those associations in different cohorts have sometimes failed, highlighting the heterogeneity and complexity of common human disorders (34,35,47). In this study, we have been able to replicate the association with rs6962966 (an intronic variant in MAGI2), previously identified in Dutch and British cohorts (29), in a Spanish CD cohort.
Human MAGI2 is a relatively large gene (∼1.5 Mbps) that maps to chromosome 7q21 and encodes the membraneassociated, guanylate kinase inverted 2 tight junction protein, involved in epithelial integrity. The CD-associated allele has been proposed to be involved in MAGI2 gene function, but no studies have been performed to determine the possible effects of the CD-associated polymorphism. The candidate gene selection that follows SNP association studies is often aprioristic, and functional studies are the only unbiased approach to characterize the associated region. Therefore, in order to define the role of this intronic variant in CD pathogenesis, we scrutinized the genomic region around rs6962966 and found that, apart from the coding gene MAGI2, the region also harbors RP4-587D13.2, a long RNA transcript represented by a cluster of expressed sequence tags (ESTs) covering 721 bp that maps 3,9 kb upstream (centromeric) of the CD-associated SNP. In silico analysis showed that this transcript is a long non-coding intronic RNA of unknown biological function. Recent studies have evidenced that intronic regions are key sources of regulatory non-coding RNAs that are able to regulate the expression of other genes through diverse mechanisms (48). Interestingly, the antisense lncRNA MAGI2-AS3 overlapping the first exon of MAGI2 has been recently related to tumor suppression in several cancers including breast, lung and hepatocellular carcinoma (45,49,50). In the case of lncRNA RP4-587D13.2, located within the intron harboring the CD-associated rs6962966, both its differential tissue distribution and preferential nuclear localization suggest that it is a functional, regulatory transcript. Interestingly, RP4-587D13.2 and MAGI2 show a concordant expression profile across human tissues, including the small intestine, indicating that RP4-587D13.2 could be processed from the same pre-mRNA and could be functionally related to MAGI2. Moreover, MAGI2 shows decreased protein levels upon silencing of this intronic lncRNA, suggesting that RP4-587D13.2 could be involved in the transcriptional (or posttranscriptional) regulation of MAGI2 expression and thus in the coordination of the expression of genes that are relevant to the TJ pathway. However, these regulatory relationships between the intronic lncRNA and the protein transcribed by its host locus MAGI2 must be confirmed and the mechanisms that underlie such regulatory behaviors remain to be fully understood.
We observed that the expression of several TJ-related genes is altered in an in vitro model that mimics the context of CD, where the expression of MAGI2 is repressed and intestinal cells are exposed to digested gliadin (PTG), suggesting that both MAGI2, and the upstream RP4-587D13.2 are necessary for a correct function of the pathway. Moreover, the decrease in expression of both genes, together with the alteration observed in several other TJ genes in the disease tissue suggests that the MAGI2 region could be involved in the regulation of the network. Interestingly, in the in vitro model, gliadin stimulation enhanced the effects of gene-silencing, resembling what is observed for CLDN2 and ZAK in biopsy samples from active and treated CD patients. On the other hand, the expression of MAGI2 and RP4-587D13.2 is downregulated in the duodenum of active CD patients and persists even after more than 2 years on GFD and an apparent recovery from intestinal atrophy, supporting a constitutive alteration that could have a genetic origin. However, several other TJ genes that are altered in the PTG-challenged in vitro model (and in active CD) are normalized in GFD-treated patients, stressing the role of gluten-mediated induction of some of the alterations. The constitutive alteration of gene expression in the MAGI2 region could promote an increased response to ingested gluten, probably leading to a higher disruption of TJ structures and to an enhanced intestinal permeability. Overall, the constitutive alteration of gene expression, together with the presence of an associated genetic variant point to a dysfunction that is present before the clinical onset of the disease, as has been previously suggested for the enhanced intestinal permeability that occurs in CD (25).
Several studies have described that more than half of the disease-associated SNPs correlate with the expression of at least one adjacent gene (51) acting as cis-eQTL. The eQTL analysis in our samples did not find a link between the risk allele and the reduction of MAGI2 or RP4-587D13.2 expression in intestinal biopsy samples. However, there was a stronger correlation between the expression of both genes in the presence of the risk allele, suggesting that the associated SNP could be influencing the regulation of MAGI2 exerted by RP4-587D13.2. The effects of disease-associated SNPs go far beyond the simplistic idea of transcriptional control of an adjacent locus and the complexity of the mechanisms involved in the functional translation of associated genetic variants make it difficult to explain their role in the disease. Further functional studies and large metaanalyses might be able to pinpoint the causal variants within a disease-associated locus and determine their downstream effects in greater detail (52).
In summary, we have demonstrated that the MAGI2 genomic region is associated with CD and presents transcriptional alterations that seem to be relevant for disease pathogenesis. We postulate that RP4-587D13.2 is regulating MAGI2 expression, that this relationship seems to be affected by the associated SNP and that the constitutive reduced expression of both transcripts in patients could affect the TJ pathway, which is crucial for intestinal permeability, and enhance the response to the ingested gluten. Further studies are needed to verify the functionality of this intronic lncRNA and the complex regulatory relationships between RP4-587D13.2, its host loci MAGI2 and disease. All this will help to understand the complexity of the interactions between the environmental trigger, genetic polymorphisms and gene expression.

DATA AVAILABILITY STATEMENT
The datasets generated for this study can be found in the Gene Expression Omnibus GSE84729.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by Ethics Committee of Cruces University Hospital (ref. CEIC-E08/59, CEIC-E13/20 and CEICE16/46). Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.

AUTHOR CONTRIBUTIONS
AJ-M designed and performed experiments, analyzed and interpreted data, and wrote the manuscript. IS contributed with the experimental design and procedures and data analysis. KG-E analyzed and interpreted data. AO-G performed experiments and analyzed data. IR-G and MS performed experiments and collected samples. II performed sample collection. AC-R conceived the ideas of experimental design of the study, performed experiments, analyzed and interpreted data, and provided revisions to scientific and grammatical content of the manuscript. JB conceived the ideas of experimental design of the study, interpreted data, provided revisions to scientific, and grammatical content of the manuscript and provided funding.  (Universidad de  Valladolid). Consortium participants, other than JB, did not participate in the study design, analysis and interpretation of data, writing of the report and in the decision to submit the report for publication. Technical and human support provided by SGIker (UPV/EHU, MICINN, GV/EJ, ERDF, and ESF) is gratefully acknowledged.