Judith M Boer

Eddy H J Van Roon

Noël F C C De Miranda

Merlijn P Van Nieuwenhuizen

Emile J De Meijer

Marjo Van Puijenbroek

Pearlly S Yan

H.-M Huang

Tom Van Wezel

J Morreau

M Huang

Hans Morreau
email: h.morreau@lumc.nl.

Tumour-specific methylation of PTPRG intron 1 locus in sporadic and Lynch syndrome colorectal cancer

Keywords: PTPRG, Colorectal cancer, CTCF, DNA methylation, Lynch syndrome

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INTRODUCTION

DNA methylation is a common mechanism in colorectal tumorigenesis. [START_REF] Grady | Genomic and epigenetic instability in colorectal cancer pathogenesis[END_REF][START_REF] Hiltunen | Hypermethylation of the APC (adenomatous polyposis coli) gene promoter region in human colorectal carcinoma[END_REF] Over the last decade, several genome-wide array-based methods have been developed, allowing the discovery of novel tumour-specific methylated loci. Enzymatic (HELP [START_REF] Khulan | Comparative isoschizomer profiling of cytosine methylation: the HELP assay[END_REF] , MMASS [START_REF] Ibrahim | MMASS: an optimized array-based method for assessing CpG island methylation[END_REF] , DMH [START_REF] Huang | Methylation profiling of CpG islands in human breast cancer cells[END_REF] , CHARM [START_REF] Irizarry | Comprehensive high-throughput arrays for relative methylation (CHARM)[END_REF] ) and ChIP methods [START_REF] Weber | Chromosome-wide and promoter-specific analyses identify sites of differential DNA methylation in normal and transformed human cells[END_REF] are most commonly used for genome-wide screening of DNA methylation, in combination with CpG island or promoter microarrays. An alternative genome-wide approach to identify genes silenced by DNA methylation detects expression differences in cell lines treated with DNA demethylating agents. [START_REF] Schuebel | Comparing the DNA hypermethylome with gene mutations in human colorectal cancer[END_REF][START_REF] Suzuki | A genomic screen for genes upregulated by demethylation and histone deacetylase inhibition in human colorectal cancer[END_REF] More recently, captured methylated DNA [START_REF] Down | A Bayesian deconvolution strategy for immunoprecipitation-based DNA methylome analysis[END_REF] and bisulphite-converted reduced representations [START_REF] Meissner | Genome-scale DNA methylation maps of pluripotent and differentiated cells[END_REF] are analyzed by high-throughput sequencing strategies. The unbiased approaches have indicated that transcription regulation associated with CpG methylation is not restricted to promoter CpG islands. [START_REF] Irizarry | The human colon cancer methylome shows similar hypo-and hypermethylation at conserved tissue-specific CpG island shores[END_REF][START_REF] Weber | Distribution, silencing potential and evolutionary impact of promoter DNA methylation in the human genome[END_REF] Conserved regions up to 2kb distant from the promoter, annotated as CpG island shores [START_REF] Irizarry | The human colon cancer methylome shows similar hypo-and hypermethylation at conserved tissue-specific CpG island shores[END_REF] , and promoter CpG islands of lesser density, annotated as intermediate-CpG islands [START_REF] Weber | Distribution, silencing potential and evolutionary impact of promoter DNA methylation in the human genome[END_REF] , undergo cancer-specific methylation more often than traditional promoter CpG islands. The methylation status of these regions is strongly related to gene expression and might have been underestimated in previous studies.

The aim of this study was to discover novel tumour-specific DNA methylation markers in right-sided colon cancer. These tumours have a higher frequency of the CpG island methylator phenotype (CIMP). Additionally, right-sided tumours are often clinically asymptomatic at early stages, thus, patients would greatly benefit from a reliable screening method. We employed differential methylation hybridization (DMH) combined with a 8.5k CpG clone library microarray for the initial identification of differential methylation in a cohort of colon cancers. [START_REF] Yan | Dissecting complex epigenetic alterations in breast cancer using CpG island microarrays[END_REF] This library is enriched for CG-rich areas throughout the genome, encompassing promoter CpG islands as well as CpGrich island shores and intermediate-CpG islands. [START_REF] Cross | Purification of CpG islands using a methylated DNA binding column[END_REF] We report tumour-specific methylation of the first intron of the receptor protein-tyrosine phosphatase gamma gene (PTPRG), in both sporadic and Lynch syndrome colorectal cancers. Additionally we demonstrate that methylation of this region affects its binding to the CCCTC-binding factor (zinc finger protein, CTCF).

MATERIALS AND METHODS

Tissue

Anonymized samples were obtained from patients who underwent surgery between 1988 and 2006 at the Leiden University Medical Center (Leiden, The Netherlands) or at the Rijnland Hospital (Leiderdorp, The Netherlands). Tumour sections were microdissected to minimize normal epithelium and stromal cells. DNA was isolated from fresh-frozen tissue using a previously described method [START_REF] Isola | Analysis of changes in DNA sequence copy number by comparative genomic hybridization in archival paraffin-embedded tumor samples[END_REF] , and from formalin-fixed paraffin-embedded (FFPE) tissue using the Wizard Genomic DNA Purification kit (Promega, Madison, WI, USA). We used available normal mucosa from the same individuals as control to correct for age-dependent methylation. Age, location and microsatellite instability (MSI) status for the sporadic tumours are listed in Supplementary Table S1, and for the Lynch syndrome-associated tumours in Supplementary Table S2. The colorectal cancer cell lines SW48, RKO, SW480, Caco2, SW837, and LS411 were obtained from the American Type Culture Collection (Manassas, VA, USA). DNA was isolated from these cell lines as described previously. [START_REF] Isola | Analysis of changes in DNA sequence copy number by comparative genomic hybridization in archival paraffin-embedded tumor samples[END_REF] RNA was isolated using TRIZOL (Invitrogen, Carlsbad, CA, USA) and subsequently purified with Qiagen RNeasy columns combined with the RNase-free DNase kit (Qiagen Sciences, Germantown, MD, USA). The present study was approved by the Medical Ethics committee of the LUMC (protocol P01-019). Cases were analyzed following the medical ethical guidelines described in the Code Proper Secondary Use of Human Tissue established by the Dutch Federation of Medical Sciences.

CpG island microarrays

CpG island clone inserts (8544) were amplified using vector-based primers as described previously. [START_REF] Yan | Dissecting complex epigenetic alterations in breast cancer using CpG island microarrays[END_REF][START_REF] Yan | Use of CpG island microarrays to identify colorectal tumors with a high degree of concurrent methylation[END_REF] The CpG island clone library was originally generated by the Sanger Centre from affinity-purified in vitro methylated DNA fragments. [START_REF] Cross | Purification of CpG islands using a methylated DNA binding column[END_REF] Clone sequence information was downloaded from the Toronto Microarray Facility. PCR products were spotted onto CodeLink (GE Healthcare, Munich, Germany) slides using an OmniGrid arrayer (Genomic Solutions, Ann Arbor, MI, USA) at the Leiden Genome Technology Center (www.lgtc.nl) as described. [START_REF] Knijnenburg | Insights from genomic microarrays into structural chromosome rearrangements[END_REF]

Differential methylation hybridization

DMH was performed according to Yan et al.. [START_REF] Yan | Dissecting complex epigenetic alterations in breast cancer using CpG island microarrays[END_REF] Cy5-labeled amplicons, representing methylated DNA fragments derived from tumours and paired normal mucosa samples, were co-hybridized to the CpG island microarrays with a Cy3-labeled common reference amplicon consisting of a pool of DNA from the six colorectal cancer cell lines described above. Detection was done on a G2565BA scanner (Agilent Technologies, Santa Clara, CA, USA) and image analysis using GenePix6.0 (Molecular Devices, Union City, CA, USA). Pre-processing, including normalization, was performed using the GenePix error model in Rosetta Resolver version 5.0 (Rosetta Biosoftware, Seattle, WA, USA). Microarray data for the tumour and normal samples were compared using an error-weighted ANOVA model and corrected for multiple testing [START_REF] Benjamini | Controlling the False Discovery Rate -A Practical and Powerful Approach to Multiple Testing[END_REF] in Rosetta Resolver. Microarray data are available on Gene Expression Omnibus with accession number GSE21181.

Bisulphite sequence analysis (BSA)

DNA samples (500 ng) were converted using the EZ DNA methylation Gold bisulphite kit (Zymo Research). Primers (Supplementary Table S3) for BSA of ten CpGs in the PTPRGint1 locus (Supplementary Figure S1) were designed using MethPrimer. [START_REF] Li | MethPrimer: designing primers for methylation PCRs[END_REF] Amplification was carried out in a DNA Engine Dyad Peltier Thermal Cycler (Bio-Rad, Hercules, CA, USA) using AmpliTaq Gold PCR buffer and enzyme (Applied Biosystems, Foster City, USA). For the direct BSA, PCR products were sequenced using the right primer, resulting in nine interpretable CpGs (CpG2-10). For additional clonal BSA, PTPRGint1 PCR fragments were cloned into TOP10 E. coli bacteria using a TOPO TA cloning kit (Invitrogen, Carlsbad, CA, USA). Sequence alignment analysis was performed using ClustalW [START_REF] Larkin | Clustal W and Clustal X version 2.0[END_REF] and BioEdit. 22 CpG dinucleotides in the direct BSA were scored as being methylated when the ratio of the cytosine/thymine peaks was above 0.4. The BiQ analyzer software was used for visualization. [START_REF] Bock | BiQ Analyzer: visualization and quality control for DNA methylation data from bisulfite sequencing[END_REF]

MS-MLPA assay

Custom MS-MLPA probes (Supplementary Table S3) for the PTPRGint1 locus were designed in Primer3 [START_REF] Rozen | Primer3 on the WWW for general users and for biologist programmers[END_REF] and included the HhaI site in CpG9 (see Figure 1B). As a control we used a BRCA2 probe set from the SALSA MS-MLPA KIT ME001B Tumour suppressor-1 kit (MRC-Holland, Amsterdam, The Netherlands). Fragment analysis was performed on an ABI 3130 (Applied Biosystems, Foster City, US). The MS-MPLA was performed as described using 50 ng of genomic DNA. [START_REF] Nygren | Methylation-specific MLPA (MS-MLPA): simultaneous detection of CpG methylation and copy number changes of up to 40 sequences[END_REF] A negative, unmethylated control (human semen DNA) and a 100% methylated DNA control (CpGenome Universal methylated DNA, Chemicon, Millipore, Billerica, MA, USA), were included in every experiment to asses HhaI cleavage and PCR. Fragment analysis was performed in GeneMapper (Applied Biosystems). PTPRGint1 peak heights were normalized by comparison with the BRCA2 peak height from the same run. Subsequently, the PTPRGint1/BRCA2 ratio from the digested reaction was divided by the PTPRGint1/BRCA2 ratio from the undigested reaction resulting in one ratio per sample that represented the fraction of methylated CpG9. Ten independent measurements provided a ratio distribution for unmethylated (mean 0.108, sd 0.037) and fully methylated (mean 0.833, sd 0.148) control DNA. Samples were typed as being unmethylated or methylated when they were within three standard deviations of the mean of the unmethylated and methylated reference samples, respectively. Samples with ratios in between these standard deviation boundaries were scored as partially methylated. For specificity and sensitivity calculations, partially methylated samples were considered methylated. Determination of specificity between normal and tumour tissue was performed by a Chi-square test.

Real-time RT-PCR

cDNA was generated using the random primer protocol from the Transcriptor First Strand cDNA Synthesis Kit (Roche Applied Science, Indianapolis, IN, USA) using 1 µg of RNA. Intron-spanning primers (Supplementary Table S3) were designed to target exon 1 and 2 of the main PTPRG transcript in Primer3. [START_REF] Rozen | Primer3 on the WWW for general users and for biologist programmers[END_REF] Reactions were performed in duplicate on a Light Cycler 480 (Roche Applied Science) using IQ SYBR Green SuperMix (Biorad). High resolution melting curve analysis was performed to check primer specificity. Reactions with more than one peak in the melting curve were discarded, as were samples where the standard deviation between technical replicates was above one Ct value. A standard curve was generated using five 1:10 dilutions of pooled cDNA from colon cancer cell lines (SW48, RKO, SW480, Caco2, SW837, and LS411), showing an efficiency of 100%. The Ct values that were used for analysis were between 23 and 33. Relative mRNA concentrations were calculated from this standard curve. Stably expressed control genes for normalization were selected with the GeNorm applet; the two most stably expressed genes were used for normalization of each tumour cDNA (CPSF6 and EEF1). [START_REF] Vandesompele | Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes[END_REF]

CTCF ChIP and quantitative PCR

The primary colon cancer cell line KP7038t, established at the Department of Pathology at the LUMC, was grown in GIBCO RPMI 1640 with glutamax (Invitrogen, Carlsbad, CA, USA), 10% foetal calf serum, and penicillin/streptomycin (50µg/ml). Tumourassociated fibroblasts (KP7038f), collected from the primary tumour, as well as cell lines RKO and SW480 were grown under identical conditions. Cells at approximately 80% confluency were fixed with 1% formaldehyde for 10 minutes at room temperature.

The formaldehyde was quenched with glycine (0.125 M) and the cells were harvested by scraping. Chromatin immunoprecipitations were performed using the SimpleChIP Enzymatic Chromatin IP kit (Cell Signaling Technology, Danvers, MA, US) with 10 µl anti-CTCF antibody (D31H2 XP; Cell Signaling Technology). Normal colon mucosa was collected from a patient who underwent a colon colonoscopy unrelated to colon cancer. Twenty 50µm sections, cut from macro-dissected frozen tissue, were fixed in PBS with 1% formaldehyde. After quenching, the tissue was micro-dissected and processed for ChIP followed by duplicate PCR reactions as above. Primers (Supplementary Table S3) targeting control regions were selected from Kim et al. [START_REF] Kim | Analysis of the vertebrate insulator protein CTCF-binding sites in the human genome[END_REF] .

The Ct values that were used for analysis were between 25 and 40. Standard curves were generated using four consecutive 1:5 dilutions of input DNA for both cultures (non-immunoprecipitated, cross-linked DNA) to determine relative DNA concentrations. For comparison between pull-downs, relative DNA concentrations of the CTCF and IgG antibody pull-downs were normalized with the corresponding relative concentration from the histone H3 antibody pull-down.

RESULTS

Locus PTPRGint1 is methylated in colorectal adenomas and carcinomas

Methylation profile comparison by ANOVA of 15 carcinomas, three adenomas and eight paired normal mucosa samples identified 20 differentially methylated loci for the three tissue groups (false discovery rate < 0.01%). For all but one of these loci methylation in the adenomas was comparable to the normal samples while carcinomas showed increased methylation (Figure 1A). The most significant CpG island clone was 47B02 that showed increased methylation in both adenomas and carcinomas compared to normal mucosa. Therefore, we performed validation experiments for the corresponding locus, which mapped to the first intron of the PTPRG gene (chr3: 61524993-61525363, UCSC assembly: March 2006, see Supplementary Figure S1), referred to as PTPRGint1.

Analysis at single CpG resolution using direct BSA showed that 17 out of 18 tumour samples were fully methylated in the PTPRGint1 region, while one carcinoma was unmethylated for CpG dinucleotides 8-10 (Figure 1B, bottom panel). In contrast, normal colon samples were mostly unmethylated (Figure 1B, upper panel). CpGs 7-10 were most informative to distinguish between tumour and normal in this set of samples.

These results were confirmed at the single chromosome level using clonal BSA (Supplementary Figure S2). In summary, the microarray-based finding of differential PTPRGint1 methylation in right-sided tumours was confirmed, and extended to leftsided adenomas and carcinomas.

Methylation of PTPRGint1 CpG9 has high sensitivity and specificity

To assess the sensitivity and specificity of PTPRGint1 methylation to discriminate between cancer and normal tissue, we developed a high-throughput MS-MLPA assay (Figure 2A,2B). We tested an FFPE cohort of 103 tumours and 58 corresponding normal tissues, which allowed us to assess the possibility of age-related methylation often seen in aging mucosa. Of the 67 carcinoma samples 94% showed methylation of the targeted CpG dinucleotide (61 fully, two partially methylated), while 46 (95.8%) of the 48 corresponding normal samples were unmethylated, and the remaining two partially methylated (Figure 2C, Table 1). Comparing the methylation numbers between normal and tumour tissue by Chi-square test provided a highly significant p-value of 9.8x10 -110 . PTPRGint1 methylation was independent of MSI status in the sporadic carcinomas, as both MSI-High and stable cases were methylated. A relatively small group of 18 sporadic adenomas (13 low grade dysplastic and five high grade dysplastic) and neighbouring normal mucosa of 10 of these showed PTPRGint1 methylation in all adenomas, but not in the available normal mucosa (Figure 2C, Table 1). To assess whether PTPRGint1 methylation is an early event in colorectal carcinogenesis, several colon lesions preceding the adenoma/carcinoma stages were studied. All six hyperplastic polyps and 11 out of 12 serrated adenomas tested showed PTPRGint1 methylation (Figure 2C).

PTPRGint1 CpG9 methylation in Lynch syndrome associated colorectal cancer

The initial cohort for MS-MLPA validation contained two Lynch syndrome associated colorectal carcinomas that were both methylated. Therefore, we further investigated PTPRGint1 methylation in 63 carcinomas from patients with a DNA mismatch repair (MMR) gene mutation (14 MLH1 mutations, 19 MSH2 mutations and 30 MSH6 mutations). Of these 92.1% showed methylation (58 fully, 3 partially methylated), while 62 of the 65 (95.7%) corresponding normal samples were unmethylated (Figure 2C, Table 1; Chi-square p-value 3.3x10 -153 ). In conclusion, methylation of PTPRGint1 CpG9 has similarly high sensitivity and specificity in Lynch syndrome associated colorectal carcinomas as in sporadic colorectal cancer.

No silencing of PTPRG gene expression in methylated samples

To assess if hypermethylation of PTPRGint1 affected expression of the gene, relative mRNA levels of PTPRG were studied in 15 right-sided colon carcinomas, three adenomas and 18 corresponding normal mucosa samples. The PTPRG gene encodes six protein coding isoforms (Source: HGNC Symbol; Acc:9671, aligned to Ensembl GRCh37). Using intron 1 spanning primers transcripts ENST00000474889, ENST00000295874, ENST00000394461 and ENST00000420974 were analyzed (Figure 3). Two transcripts ENST00000383711 and ENST00000394462, starting at exon 3, were analyzed using intron 26 spanning primers and gave comparable results (data not shown). Expression of the PTPRG gene was detected in all samples. To asses the effects of PTPRGint1 methylation on PTPRG expression, we compared the MS-MLPA methylation ratio with the PTPRG relative expression (Figure 3). We found that PTPRG was expressed at similar levels in the tumour and normal samples independent of methylation status. One patient (No. 28) showed increased PTPRG expression in both normal and tumour samples, thought to reflect individual expression differences and a possible copy number effect in the tumour.

PTPRGint1 is a methylation-dependent CTCF binding site

The PTPRGint1 locus overlapped with an experimentally defined CTCF binding site from a CTCF ChIP-chip study in fibroblasts [START_REF] Kim | Analysis of the vertebrate insulator protein CTCF-binding sites in the human genome[END_REF] displayed in the genome web browsers USCS [START_REF] Kuhn | The UCSC Genome Browser Database: update 2009[END_REF] and Ensembl. [START_REF] Hubbard | Ensembl[END_REF] This CTCF binding region of 750bp (OREG0015647; chr3: 61525101-61525851, UCSC assembly: March 2006) has a 262 bp overlap with the 3' part of the PTPRGint1 locus ( Supplementary Figure S1). We studied CTCF binding by ChIP in the primary tumour culture KP7038t that carried PTPRGint1 methylation (MS-MLPA ratio 0.97, data not shown), unmethylated tumour-derived fibroblasts KP7038f from the same patient (MS-MLPA ratio 0.07, data not shown), as well as normal colon mucosa. We detected CTCF binding to PTPRGint1 in the normal mucosa and KP7038f fibroblasts, but little binding to the primary tumour cells (Figure 4A).

To control for possible differences in the amount of CTCF protein between the samples, we compared the qPCR results for the PTPRGint1 region with a positive control locus 7.9 Mb distant from PTPRGint1 that was shown to bind CTCF and does not contain a CpG in its putative 20 bp consensus sequence. [START_REF] Kim | Analysis of the vertebrate insulator protein CTCF-binding sites in the human genome[END_REF] This locus was enriched in pull-downs of all samples, including the primary tumour cells, indicating that lack of CTCF binding to PTPRGint1 was not due to lack of CTCF protein. CTCF binding to PTPRGint1 was comparable to the positive control in both KP7038f fibroblasts (ratio 1.2) and normal colon mucosa (ratio 1.1). However, the PTPRGint1/positive control ratio was 0.06 in the primary tumour culture KP7038t (Figure 4B). CTCF binding to PTPRGint1 was similarly decreased in colorectal cancer cell lines RKO and SW480 (Figure 4B). These results indicate a significant decrease of bound CTCF to the methylated PTPRGint1 region in tumour cells.

DISCUSSION

We describe colorectal tumour-specific methylation of a locus in the first intron of the putative tumour suppressor gene PTPRG in both proximal and distal carcinomas and adenomas, including Lynch syndrome tumours. For these high-risk individuals, who are advised to undergo regular colonoscopies, no molecular markers have been described so far. [START_REF] Alemayehu | Redundant DNA methylation in colorectal cancers of Lynch-syndrome patients[END_REF] Assuming that a successful faecal or blood DNA test for PTPRGint1 methylation could be developed, this is a promising discovery that would aid the early detection of colorectal tumours, independent of their aetiology.

PTPRGint1 is not located in a promoter CpG island but in the first intron, about 3kb from the transcriptional start site. We did not find a relation between the methylation status of PTPRGint1 and PTPRG expression, indicating that PTPRGint1 methylation does not lead to loss of function of PTPRG as has been observed for mutations in colon cancer, and deletions in lung carcinomas and renal carcinoma cell lines. [START_REF] Laforgia | Receptor protein-tyrosine phosphatase gamma is a candidate tumor suppressor gene at human chromosome region 3p21[END_REF][START_REF] Wang | Mutational analysis of the tyrosine phosphatome in colorectal cancers[END_REF] However, the identification of a methylation-sensitive CTCF binding site overlapping with PTPRGint1 suggests that tumour-specific methylation may have a more distant effect. Differential binding of the insulator protein CTCF could have a major influence on expression of distant genes through alternative loop formation, as has been observed in β-globin expression in mouse models. [START_REF] Hou | CTCF-dependent enhancer-blocking by alternative chromatin loop formation[END_REF] A recent study has shown that loss of CTCF binding to a boundary region upstream of CDKN2A resulted in spreading of repressed chromatin and DNA methylation into the p16 promoter with sequential downregulation of p16 expression. [START_REF] Witcher | Epigenetic silencing of the p16(INK4a) tumor suppressor is associated with loss of CTCF binding and a chromatin boundary[END_REF] The same study described that loss of upstream CTCF binding resulted in promoter DNA methylation of RASSF1 and CDH1. [START_REF] Witcher | Epigenetic silencing of the p16(INK4a) tumor suppressor is associated with loss of CTCF binding and a chromatin boundary[END_REF] Contradictory to the finding that CTCF binding abrogation was shown to be causative of heterochromatin spreading and DNA methylation [START_REF] Witcher | Epigenetic silencing of the p16(INK4a) tumor suppressor is associated with loss of CTCF binding and a chromatin boundary[END_REF][START_REF] Tiwari | Breaching the boundaries that safeguard against repression[END_REF] , is the observation that DNA methylation of CTCF binding sites is suggested to regulate CTCF binding. [START_REF] De | Epigenetic regulation of the human retinoblastoma tumor suppressor gene promoter by CTCF[END_REF][START_REF] Xu | CpG island methylation affects accessibility of the proximal BRCA1 promoter to transcription factors[END_REF] Interestingly, aberrant DNA methylation that excludes CTCF binding to intronic regulatory DNA was shown to promote expression of an oncogene, BCL6, in B cell lymphomas. [START_REF] Lai | DNA methylation prevents CTCFmediated silencing of the oncogene BCL6 in B cell lymphomas[END_REF] Although the sequence of events is unknown, the age-related aberrant hypermethylation often seen in colon cancer hints towards the latter.

We excluded differential peptidic abundance of CTCF between tumour and normal samples by successfully amplifying a positive control CTCF binding site on all samples. It remains to be demonstrated if CTCF protein modifications and its cellular and nuclear distribution are maintained in all tumour cells, both of which can influence CTCF activity and the binding to specific regions. More insight into the role of aberrant DNA methylation of PTPRGint1 in the aetiology of cancer requires a better understanding of whether aberrant CTCF binding is caused by inhibition of protein activity or by initial aberrant methylation of the CTCF binding site.

Hypermethylation of the CpG island in the PTPRG gene promoter has been previously described in cutaneous T-cell lymphomas, melanoma cell lines, and gastric cancer. [START_REF] Van Doorn | Epigenetic profiling of cutaneous Tcell lymphoma: promoter hypermethylation of multiple tumor suppressor genes including BCL7a, PTPRG, and p73[END_REF][START_REF] Furuta | Silencing of Peroxiredoxin 2 and aberrant methylation of 33 CpG islands in putative promoter regions in human malignant melanomas[END_REF][START_REF] Wang | Metastatic suppressor genes inactivated by aberrant methylation in gastric cancer[END_REF] Transcriptional downregulation was shown to be associated with PTPRG promoter methylation in the cutaneous T-cell lymphomas study. [START_REF] Van Doorn | Epigenetic profiling of cutaneous Tcell lymphoma: promoter hypermethylation of multiple tumor suppressor genes including BCL7a, PTPRG, and p73[END_REF] This study used a similar microarray for identification of differential methylation as the present study. We did not find differential methylation of the PTPRG promoter region between normal and colon tumour samples on the microarray. Moreover, BSA of colorectal cancer cell lines showed that the PTPRG promoter region was unmethylated (R. van Doorn, personal communication). Therefore, we have no indication for upstream spreading of DNA methylation from the CTCF binding region at PTPRGint1 towards the promoter.

In conclusion, this study provided evidence for tumour-specific hypermethylation of a CTCF binding site located in the first intron of PTPRG. The high specificity and sensitivity imply a possible utility for PTPRGint1 methylation in new or existing colonspecific methylation marker panels. Especially the high level of PTPRGint1 methylation in Lynch syndrome associated colorectal tumours is unique and could prove to be a valuable addition. Methylation-dependent absence of CTCF binding to the PTPRGint1 locus suggests a possible effect on chromatin density or conformation that could play a role in colon tumorigenesis.

TITLES AND LEGENDS TO FIGURES

Figure 1

 1 Figure 1 Identification and validation of the differentially methylated locus PTPRGint1.

Figure 2

 2 Figure 2 PTPRGint1 methylation detected by MS-MLPA. (A) GeneMapper output of

Figure 3

 3 Figure 3 Scatter plot of the relative PTPRG expression against the PTPRGint1

Figure 4 (

 4 Figure 4 (A) CTCF binding to PTPRGint1, positive-and negative control regions in

  [START_REF] Lewis | Genomic imprinting: CTCF protects the boundaries[END_REF]

Table 1

 1 TABLES Sensitivity and specificity of the PTPRGint1 locus CpG9 methylation in sporadic tumours and tumours associated with a specific MMR mutation. The mutational analysis of the MMR genes was incomplete for four unpaired normal samples in the Lynch syndrome cohort. Therefore, these were only included in the total specificity calculations.

		Sensitivity	Specificity I
	CpG9	Methylated tumours /	Unmethylated normals / Total
		Total tumours	normals
	Sporadic adenomas	100% (18/18)	100% (10/10)
	Sporadic carcinomas	94% (63/67)	95.8% (46/48)
	MLH1 mutated	100% (14/14)	87% (20/23)
	MSH2 mutated	96% (18/19)	100% (24/24)
	MSH6 mutated	86.7% (26/30)	100% (14/14)
	Total Lynch	92.1% (58/63)	95.4% (62/65)

I

ACKNOWLEDGMENTS

The authors thank Remco van Doorn and Wim Zoutman for sharing protocols and unpublished data and for technical assistance with the library amplification. The authors would also like to thank Jaap van Eendenburg for establishing primary culture KP7038.

This study was supported by grants from the Stichting prof. A.A.H. Kassenaar fund and

the Centre for Medical Systems Biology within the framework of the Netherlands Genomics Initiative (NGI)/ Netherlands Organization for Scientific Research (NWO).

CONFLICT OF INTEREST

The authors (EHJvR, HM, and JB) have filed a patent application pertaining to intellectual property related in part to this report (P78692EP00 Submitted December 31, 2007). All other authors have nothing to disclose. methylated CpG. White dot: unmethylated CpG. Grey dot: sequence not readable. In a partially methylated normal sample (ID127) not more than three out of ten alleles were found methylated for several CpGs. In addition, complete methylation of 9/9, 8/10 and 4/7 sequenced tumour alleles was found respectively, roughly corresponding to the estimated percentage of tumour cells observed in HE-stained tissue sections. S1 Overview of the sporadic colorectal tumour samples and paired controls used in this study. Location, histology, age, sex and microsatellite instability status are listed, as well as the different techniques used on each sample. A gray box behind a sample in one of the last five columns indicates that the sample was tested with the technique listed in the header. S2 Overview of Lynch cohort used. Location, histology, mismatch repair gene mutation, age and sex are listed for each sample as well as the different techniques used in this study. A gray box in the last column indicates that the sample was tested with the technique listed in the header. S3 Overview of the primers used in this study.

Supplementary Information accompanies the paper on European

Supplementary Table

Supplementary Table

Supplementary Table


T Jung

T J Simat

W Altkofer

Study on mass transfer ways of UV printing ink ingredients into foodstuffs

Keywords: Survey, Chromatography -HPLC Additives/Contaminants: Packaging -inks UV printing inks, migration, photoinitiator, mass transfer ways, yoghurt, 50% ethanol, isopropylthioxanthone, 2-methyl-4´-(methylthio)-2-morpholinopropiophenone, ethyl-4-(dimethylamino)benzoate

The ITX case (isopropylthioxanthone) showed conclusively that ingredients of UV inks may migrate into packaged foodstuffs. For multi-layered materials like beverage cartons the only way that mass transfer can occur is by the so called set-off effect. In contrast, in the case of rigid plastics like yoghurt cups, two other methods of mass transfer, permeation and gas phase, have to be considered. In cooperation with producers of ink, plastic cups and yoghurt, a project was conducted in order to elucidate the mass transfer of ink ingredients. In addition, the influence of storage time and age of UV lamps on the migration level was examined. The suitability of 50% ethanol as a simulant for yoghurt was tested, as well. ITX was chosen as a model migrant, as it is easily detectable. Furthermore, the migration of two other substances, the photoinitiator 2-methyl-4´-(methylthio)-2-morpholinopropiophenone (MTMP) and the amine synergist ethyl-4-(dimethylamino)benzoate (EDAB), which may be used in combination with ITX, was studied. Prior to being filled with yoghurt or 50% ethanol the printed cups were stored under different contact conditions, with and without contact between the inner layer and printed surfaces, in order to distinguish between the possible mass transfer ways. All analyses were performed by means of HPLC-DAD/FLD. It was shown that contamination with ITX and EDAB occurs via set-off and that the degree of migration increases with lamp age and storage time of the unfilled cups. Migration of MTMP was not detectable. The results show, that besides the careful selection of the appropriate raw materials for printing ink, a close monitoring of the process also plays a major role in migration control. In addition, the results proved that 50% ethanol is a suitable simulant for yoghurt.

Introduction

Recently, migration of printing ink and varnish ingredients into foodstuffs has become a focus of interest. This was especially caused by the detection of isopropylthioxanthone (ITX) in baby milk, milk products and cloudy juices packaged in beverage cartons in 2005. But also the latest findings of 4methylbenzophenone and benzophenone migrating from printed carton packages into cereals highlight that contamination of foodstuffs through printing inks is still a problem. All these packages were printed with ultraviolet (UV) inks which contain monomers and oligomers, pigments, additives and photoinitiators such as ITX. Under UV radiation photoinitiators start the polymerization of monomers and oligomers, thereby causing curing of the ink. UV printing inks and varnishes have a broad range of application in food packaging and are used in all kinds of materials like paper and cartonboard, plastics and multi-layered materials. [START_REF] Rothenbacher | 2-Isopropylthioxanthone (2-ITX) in food and food packaging materials on the German market[END_REF] showed that besides multi-layered cartons, also rigid plastic materials like yoghurt cups or plastic foils for sausages are printed with UV inks containing ITX, where contamination of the foodstuffs could take place. Several other studies also deal with the migration of ITX and/or photoinitiators and amine synergists like benzophenone, 4hydroxybenzophenone, 4,4'-bis(dimethylamino)benzophenone, 4,4'-bis(diethylamino)benzophenone, 1-hydroxycyclo-hexyl-1-phenyl ketone, dimethylbenzyl ketal, 2-ethylhexyl-4-dimethylaminobenzoate, 2-methyl-1(4-(methylthio)phenyl)-2-morpholino propan-1-one, ethyl-4-dimethylaminobenzoate from various packaging materials into foodstuffs [START_REF] Anderson | Benzophenone in cartonboard packaging materials and the factors that influence its migration into food[END_REF][START_REF] Fsa | Survey of benzophenone and 4-hydroxybenzophenone migration from food packaging into foodstuffs[END_REF], MAFF 1995[START_REF] Sagratini | Determination of ink photoinitiators in packaged beverages by gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry[END_REF], Sanches-Silva et al. 2008 a+b). For example, migration into foodstuffs of up to 7.3 mg kg -1 benzophenone and up to 214 µg kg -1 2-ethylhexyl-4-dimethylaminobenzoate was shown [START_REF] Anderson | Benzophenone in cartonboard packaging materials and the factors that influence its migration into food[END_REF], Sanches-Silva et al. 2008 b).

The reported results in literature conclusively show that ingredients of printing inks and varnishes especially with low molecular weights which are not fully incorporated into the polymer matrix, e.g. photoinitiators and monomers/oligomers like acrylates, may migrate from the packaging into the packaged foodstuffs under certain conditions [START_REF] Anderson | Benzophenone in cartonboard packaging materials and the factors that influence its migration into food[END_REF][START_REF] Papilloud | Analysis of food packaging UV inks for chemicals with potential to migrate into food simulants[END_REF]. Contamination of the packed food can take place by three different mass transfer ways: set-off, permeation and gas phase. Set-off may occur during the storage of printed packaging material on reels or in stacks. The internal layer comes into contact with the external layer and ink ingredients can then be transferred to the food contact surface and finally migrate into the foodstuff after packaging. An example is the storing of printed multi-layered material in reels before the production of single beverage cartons. In the case of permeation the ink ingredients migrate from the outside through the packaging material to the inside, if there is no functional barrier like aluminum, e.g. yoghurt cups. Indirect transfer via the gas phase is possible if the substances are (semi-)volatile, e.g. migration of benzophenone and 4-methylbenzophenone into foodstuffs packaged in carton packages with inner plastic bags [START_REF] Efsa | EFSA statement on the presence of 4-methylbenzophenone found in breakfast cereals[END_REF][START_REF] Hartman | Profiling of volatile and semivolatile out-gas products from radiation (UV/EB)-cured packaging using automated short path thermal desorption gas chromatography-mass spectrometry (GC-MS) analysis[END_REF], Pastorelli et al. 2008[START_REF] Rothenbacher | 2-Isopropylthioxanthone (2-ITX) in food and food packaging materials on the German market[END_REF], Sanches-Silva et al. 2008 b). For composite materials with an internal aluminum layer, e.g. for beverage cartons, contamination with packaging ink ingredients can only occur by set-off. In contrast, in the case of rigid plastics like yoghurt cups, the two other methods of mass transfer, permeation and gas phase, have to be considered. In cooperation with producers of ink, plastic cups and yoghurt, a project was conducted to elucidate the mass transfer of ink components. In addition, the influence of other factors of the packaging production and filling process on the migration level such as the age of the UV lamps during printing and the storage time of the cups before being filled with yoghurt were examined. ITX was chosen as a model migrant, as it can easily be detected and some analytical methods exist. Another photoinitiator, 2-methyl-4´-(methylthio)-2-morpholinopropiophenone (MTMP), and the amine synergist ethyl-4-(dimethylamino)benzoate (EDAB), which are often used together with ITX were examined, as well. Furthermore, 50% ethanol was used as a migration simulant, according to Council Directive (EEC) [START_REF] Eec | Council Directive 85/572/EEC of 19 December 1985 laying down the list of simulants to be used for testing migration of constituents of plastic materials and articles intended to come into contact with foodstuffs[END_REF]/572 (EEC 1985) and its amendments.

Materials and methods

Chemicals

HPLC-grade acetonitrile and tert-butyl methyl ether, isohexane for gas chromatography, and ethanol absolute and toluene for analysis were purchased from Merck (Darmstadt, Germany). Nanograde acetone was purchased from Mallinckrodt Baker (Griesheim, Germany). Demineralized water was produced by a Milli-Q water purification system (Millipore, Schwalbach, Germany). Standards of 2isopropylthioxanthone (ITX, CAS 5495-84-1, purity 99%) were purchased from IGM Resins (Krefeld, Germany), 2-methyl-4´-(methylthio)-2-morpholinopropiophenone (MTMP, CAS 71868-10-5, purity 98%) from Sigma-Aldrich (Steinheim, Germany), ethyl-4-(dimethylamino)benzoate (EDAB, CAS 10287-53-3, purity 98%) and thioxanthone (TX, CAS 492-22-8, purity 98.5%) as the internal standard for ITX from Fluka (Sigma-Aldrich, Steinheim, Germany) and 2-ethylhexyl-4-dimethylaminobenzoate (EHDAB, CAS 21245-02-3, purity 98.5%) as the internal standard for EDAB and MTMP from Merck (Darmstadt, Germany). All other chemicals were, at least, of analytical quality. To prepare the 0.1 M citrate phosphate buffer (pH 6.0), 21 g citric acid monohydrate and 14.2 g disodium hydrogen phosphate were dissolved in approximately 900 ml water. After adjusting to pH 6.0 with concentrated sodium hydroxide, the solution was diluted to 1000 ml with water. Solutions of analytical standards were prepared in acetonitrile and stored at 4 °C in the dark.

Samples and migration tests

For all tests, deep drawn polypropylene cups with a capacity of 250 g were used. Printing was performed by a dry offset process. To examine the influence of the printing ink on migration, the cups were printed with two different model UV inks, a yellow and a black one, with different quantities of photoinitiators and curing behavior. A banderole was chosen as the print layout to have a nearly fully printed surface as worst case. In addition, the cups were printed using two different UV lamps and reflectors, new and used (ca. 1500 h), and the printed, unfilled cups were stored for two different time periods, 1 and 21 days, at room temperature. These storage times were chosen according to the information of the plastic cup producer who delivers the printed cups normally just in time or in between three weeks after production. For migration tests, the cups were filled with 250 g yoghurt (fat content ca. 3.8%) and 50% ethanol (v/v), respectively, covered with aluminum foil and stored for 10 days at 5 °C. At the end of the storage time the migrates were bottled into glass flasks and stored at -18 °C and 5 °C for yoghurt and ethanol, respectively, prior to analysis. All tests were carried out fivefold. To examine the mass transfer four different methods of cup contact were used. First, printed cups were stacked into each other as in the normal production process, i.e. the inner layer was in partial contact with the printed outer layer (Figure 1a). Second, single printed cups were covered with a piece of aluminum foil, so that the inner layer had no contact with a printed surface and no contamination via the gas phase could occur (Figure 1b). Third, a printed cup was stacked into an unprinted one and the unprinted one was subjected to analysis at end of the storage time (Figure 1c). Fourth, to simulate the possible transfer of ingredients via the gas phase, a printed cup was stacked into an unprinted one in such a way that the surfaces had no contact. This construct was stored in a preserving glass for 10 days at 40 °C prior to being filled with 50% ethanol (Figure 1d).

[Insert Figure 1 about here] Preparation of the model ink Ca. 50 mg of the ink was diluted with 50 ml acetonitrile with the addition of the internal standards. For HPLC analysis this solution was diluted again at 1:100 with acetonitrile.

Preparation of ink solutions from not cured cups

To determine the quantity of photoinitiators and the amine synergist brought onto the cups with the printing ink, uncured printed cups were taken from the offset printing press. These were put into screw-top jars containing ca. 150 ml acetone, where the ink was washed off of the cups by swiveling the jars around. The solution was subsequently poured into 500 ml volumetric flasks, and the jar and cup were washed several times with acetonitrile. An aliquot with the addition of internal standards was subjected to HPLC analysis.

Preparation of cup extracts

The cup was cut into small pieces (ca. 1 x 1 cm) and subsequently extracted with 100 ml toluene containing internal standards in an ultrasonic bath for 2 h. After filtration of the extract the solvent was evaporated in a rotary evaporator at 77 mbar and 40 °C water bath temperature. The residue was extracted threefold with acetonitrile and filtered through glass wool into a 50 ml volumetric flask. For HPLC analysis, 100 µl and 200 µl of the black and yellow cup extracts, respectively, were filled up to 1 ml with acetonitrile.

Preparation of yoghurt migrates

Extraction of the yoghurt was performed with acetonitrile, following a liquid-liquid clean-up according to a method published by [START_REF] Rothenbacher | 2-Isopropylthioxanthone (2-ITX) in food and food packaging materials on the German market[END_REF], with the following modifications. Instead of 2,4diethylthioxanthone TX was used as internal standard and EHDAB was added as the second internal standard. Furthermore instead of filter paper glass wool was used due to problems with the internal standard TX in case of filter paper and at the end the solvent was evaporated to dryness under a gentle stream of nitrogen at 60 °C.

To approximately 5 g of homogenized yoghurt, the internal standards and 5 ml 0.1 M citrate phosphate buffer solution (pH 6.0) were added and the sample was extracted by shaking intensively for 1 min. After addition of 30 ml acetonitrile, the mixture was shaken for 10 min and quantitatively filtered through glass wool. Flask and glass wool were rinsed with 10 ml acetonitrile/water (3:1, v/v). After addition of 1.5 g sodium chloride and intensively shaking for 30 s, the filtrate was mixed with 20 ml tert-butyl methyl ether/isohexane (80:20, v/v) before gently shaking again. The lower aqueous phase was discarded. The organic layer was washed two times with 20 ml water. After addition of 10 ml tertbutyl methyl ether/isohexane (50:50, v/v) and, if necessary, separation of the aqueous phase, the organic layer was dried over anhydrous sodium sulfate. The solvent was evaporated to dryness under a gentle stream of nitrogen at 60°C. Finally, the residue was dissolved in 1 ml acetonitrile and directly subjected to HPLC analysis.

Preparation of 50% ethanol migrates

Internal standards were added to approximately 10 g ethanol migrate. Then the solvent was reduced to 1 ml under a gentle steam of nitrogen at 60 °C. After addition of 1 ml acetonitrile, the mixture was shaken extensively for 1 min and then subjected to HPLC analysis.

High performance liquid chromatography with diode array and fluorescence detection (HPLC-DAD/FLD)

Analysis was performed on an Agilent 1100 high performance liquid chromatograph (Agilent, Waldbronn, Germany) equipped with a Luna C18(2) (100 mm; 2 mm I.D.; 3 µm) column (Phenomenex, Aschaffenburg, Germany) coupled with a diode array (260 nm and 310 nm; spectra recorded from 190 to 400 nm) and a fluorescence detector (excitation 264 nm/emission 440 nm) connected in a series. The system was run at 40 °C (stop time 18 min) in gradient mode with demin. water/acetonitrile: 0 min 40% acetonitrile, 12 min 85% acetonitrile, 16 min 100% acetonitrile, 18 min 100% acetonitrile with a post time of 6 min, a flow rate of 0.3 ml/min and an injection volume set to 10 µl.

Limits of detection and quantification, as well as the range of linearity, were determined for MTMP and EDAB by HPLC-DAD analysis, and for ITX by HPLC-FLD analysis using standard solutions in acetonitrile in the absence of matrix interferences, according to DIN standard 32645 (DIN 2008). Recovery tests were conducted by adding standard solutions directly into yoghurt and 50% ethanol, respectively, both of which were proven to be free of the photoinitiators and the amine synergist being investigated. The spiked matrices were shaken briefly and left to stand for 5 min before extraction and concentration, respectively, to enable dispersion. Yoghurt was spiked five times at levels of 10, 30, 50, 70 and 90 µg kg -1 ; 50% ethanol was spiked five times at levels of 10, 20, 40, 60, 80 and 100 µg kg -1 .

Results and discussion

Limits of detection and quantification for yoghurt

All calculations were carried out by internal standard correction. TX was used for ITX and EHDAB for MTMP and EDAB. ITX was linear in the range of 25 to 500 µg l -1 for the FLD-Signal, with a correlation coefficient of 0.9999. These values corresponded to 5 and 100 µg kg -1 yoghurt. Limits of detection and quantification were determined at 2 and 13 µg l -1 (0.4 and 2.5 µg kg -1 yoghurt), respectively, in acetonitrile, according to DIN standard 32 645 (DIN 2008). Linearity could also be shown for MTMP and EDAB for the DAD Signal in the same range as for ITX, with correlation coefficients of 0.9998 and 0.9993. Limits of detection and quantification were determined at 19 and 60 µg l -1 (3.8 and 11.9 µg kg -1 yoghurt) for MTMP, and 18 and 54 µg l -1 (3.5 and 10.7 µg kg -1 yoghurt) for EDAB. Confirmation was achieved by comparing the UV spectra of the samples with the standard solutions spectra.

Limits of detection and quantification for 50% ethanol

Calculations for ITX and EDAB were made by internal standard correction. For MTMP the calculations were carried out by external calibration since it was shown that MTMP is not as much lost as EHDAB.

All substances showed linearity in the range of 50 to 500 µg l -1 , corresponding to 10 and 100 µg kg -1 50% ethanol. Correlation coefficients were 0.9998 for ITX, 0.9998 for MTMP and 0.9994 for EDAB.

Limits of detection and quantification were determined at 7 and 29 µg l -1 (1.4 and 5.8 µg kg -1 50% ethanol) for ITX, 8 and 27 µg l -1 (1.6 and 5.4 µg kg -1 50% ethanol) for MTMP and 12 and 41 µg l -1 (2.5 and 8.2 µg kg -1 50% ethanol) for EDAB.

Recoveries

Recovery rates for ITX in yoghurt were achieved at between 91 and 93% with a relative standard derivation (RSD) of ≤4% (Table I). For MTMP and EDAB the values were 87-93% with a RSD of ≤11% and 76-102% with a RSD of ≤10%, respectively (Table I). Mean recovery rates for the internal standards TX and EHDAB in yoghurt were 86% with a RSD of ≤4% and 88% with a RSD of ≤6%, respectively. Recovery rates for ITX in 50% ethanol were 83-101% with a RSD of ≤9%, for MTMP 89-93% with a RSD of ≤4% and for EDAB 77-97% with a RSD of ≤10% (Table II). For the internal standards TX and EHDAB mean recovery rates in 50% ethanol were 66% with a RSD of ≤8% and 60% with a RSD of ≤18%, respectively. It is assumed that the recovery of TX, ITX, EHDAB and EDAB in 50% ethanol is low due to a increased volatility or a decreased solubility under the concentration conditions. Nevertheless, the recovery rates of ITX and EDAB, as well as the relative standard derivations, corrected by the internal standards are satisfactory.

[Insert Tables I andII about here] Analytical results of the model inks Table III summarizes the concentrations of the two photoinitiators and the amine synergist in the printing inks and their quantity on the yoghurt cups before curing. The comparison of the concentration of the photoinitiators in the yellow and black ink shows that the black contains more of the two photoinitiators: 1% more ITX and 2.9% more MTMP. This is in order to correct for the UV absorption of the carbon black pigments. Especially MTMP is recommended for a good through curing of printing ink films [START_REF] Rahn | Datasheet GENOCURE * PMP[END_REF]. Regrettably, the analyzed concentrations on the yoghurt cups are not consistent with the determined concentrations in the ink. For instance, in the case of the yellow ink the concentration in the inks are nearly the same but the concentrations on the yoghurt cups show considerable fluctuation. A possible cause could be a variation in the production of the model printing inks since the inks for the direct analysis and for the manufacture of the uncured cups were not derived from the same lot. The high concentration of the photoinitiators and of the amine synergist on the black cups can only be explained with a higher color application in order to achieve a sufficient covering of the cups.

[Insert Table III about here]

Migration tests with yoghurt

Figure 2 shows the results of the ITX migration into yoghurt depending on the different production and storage conditions for the black cups. These can be considered as a worse case, in comparison with the yellow cups, since the concentration of the photoinitiators is higher and the curing is more difficult.

Under the chosen contact conditions, no migration was observed in the printed cups whose insides had no contact with a printed surface (all values below the detection limit of 0.4 µg kg -1 ). For the cups whose inside had contact with a printed surface the migration of ITX into the yoghurt was nearly the same under equal conditions, i.e. lamp age and storage time. Furthermore, there was no obvious difference between migration levels in cups with a printed surface and those without. These results clearly indicate that the contamination of the yoghurt with ITX occurred by set-off and not by permeation in this experiment. In case of the yellow cups the ITX migration showed the same tendency (data not shown). For MTMP all migration values in yoghurt were below the detection limit (< 3.8 µg kg -1 ), independent of printing ink, stacking method, lamp status and storage time (data not shown). This reveals that MTMP is a photoinitiator which does not migrate as easily as ITX. EDAB, in contrast, showed migration behavior similar to ITX (Figure 3, only data for black cups shown). This supports The dependency of the migration level of ITX on (a) the printing inks used, (b) lamp age and (c) storage time before filling, is depicted in Figure 4. In this experiment the cups were stacked as in the normal production process. In order to evaluate the influence of the three variables (a-c) on the migration of ITX into yoghurt, analysis of variance (ANOVA) for a three-factor experiment was performed. A comparison of the migration levels after 1 and 21 days' storage time under the same conditions, i.e. ink and lamp, shows that in all cases the migration level after 21 days was higher than after 1 day of storage time (α < 0.001). The influence of lamp age can be deduced when controlling for ink and storage time. It can be seen that migration is higher for the old lamp (α < 0.001). With respect to color of ink, the larger quantity of ITX in the black ink starts to play a role as the lamp gets older. For the yellow and black cups cured with the new UV lamp, the degree of ITX migration is nearly the same when controlling for storage time, while for the old lamp ITX migration is very different (interaction 'lamp age' x 'color of ink', α < 0.001). Due to the lower UV intensity of the old lamp, the extent of polymerization and crosslinking decreases and the diffusion of ITX through the polymer network increases.

Furthermore, the effect of storage time on ITX migration is stronger on cups cured with the old lamp than on those cured with the new lamp (interaction 'lamp age' x 'storage time', α < 0.001). Thus, all three factors have an influence on the migration level. To simplify the result: the older the lamp and the longer the storage time, the greater the degree of ITX migration into the yoghurt.

For EDAB the migration levels in yoghurt were mainly under or only slightly above the limit of quantification (see Figure 3). Nevertheless, according to the results for ITX, also for EDAB could be shown that the lamp age clearly influences the migration level: it is higher for the old lamp. However, data for the storage time are not consistent with the findings for ITX.

For the producers of cups and food the results above imply that through an accurate process control, in particular lamp age and storage time of the unfilled printed cups, the migration of substances into foodstuffs can be minimized. Another option for minimization could be using a different cup shape, so that no set-off can take place.

[Insert Figures 2-4 about here]

Comparison of the migration levels into yoghurt and 50% ethanol

As a result of the widespread detection of ITX in various foodstuffs, it was proven that, contrary to of the project was to see whether 50% ethanol would be an adequate simulant for the migration of the two photoinitiators and the amine synergist into yoghurt. Figure 5 illustrates the comparison of the migration of ITX and EDAB into yoghurt with that into 50% ethanol for all cups whose insides had contact with printed outsides. In most cases the values for 50% ethanol were above those for yoghurt, i.e. 50% ethanol overestimates the migration. This clearly shows that 50% ethanol can be used here as a simulant. Migration of MTMP into 50% ethanol was mainly below either the detection or the quantification limit, which is in accordance with the findings for yoghurt (data not shown).

[Insert Figure 5 about here]

Gas phase transfer

Since it was proven that 50% ethanol can be used as a simulant for the migration into yoghurt, this test was only conducted with ethanol. Outside printed cups which had been cured with the old lamp were used, as they can be considered as a worst case in terms of migration. The analytical results in Table IV show that contamination via the gas phase is negligible. Only for MTMP could a value slightly above the detection limit be observed. However, since 50% ethanol overestimates in most cases, it can be concluded that no migration into yoghurt takes place.

[Insert Table IV about here]

Degree of migration

To determine the degree of migration, the quantities of ITX, MTMP and EDAB on the surface of the cups prior to and after UV irradiation were compared to the quantities migrated into yoghurt (Figures 678). Here once again the different characteristics of the two photoinitiators and the amine synergist is demonstrated. While, on average, about 22% of the initial quantity of ITX disappeared during the UV curing process, 60% and 54% of MTMP and EDAB, respectively, reacted completely. This means that MTMP and EDAB are more reactive under UV irradiation than ITX. The degree of migration into yoghurt was 2% for ITX and about 3% for EDAB while for MTMP no migration could be detected (< 4%) under the chosen conditions.

[Insert Figures 678about here]

Conclusions

Contamination of yoghurt with the photoinitiator ITX and the amine synergist EDAB occurred under the chosen contact conditions via set-off and not by permeation or by gas phase transfer. Migration of MTMP could not be detected in this study. Exemplary for ITX it was shown that lamp age and storage time of the printed cups have an influence on the migration level of the printing ink component: the older the lamp and the longer the storage time, the greater the degree of ITX migration into the yoghurt. However, only 2% and about 3% of the available quantity of ITX and EDAB, respectively, on the cups migrated into the yoghurt under the experimental conditions. These results show that by a careful selection of the type of photoinitiators (ITX vs. MTMP) and accurate process control (lamp age and storage time) migration of substances into foodstuffs can be minimized. Another option for minimization could be using a different cup shape, so that no set-off can take place. In addition, the suitability of 50% ethanol as a simulant for yoghurt was demonstrated for the three examined substances. ; values corrected by mean recovery rate (92%)).

  under the chosen contact conditions the migration in the yoghurt cups occurred via set-off and not by permeation.

  Council Directive (EEC)[START_REF] Eec | Council Directive 85/572/EEC of 19 December 1985 laying down the list of simulants to be used for testing migration of constituents of plastic materials and articles intended to come into contact with foodstuffs[END_REF]/572 (EEC 1985) distilled water was not an adequate simulant for milk and milk products. It was replaced by 50% ethanol, which was found to better simulate the fatty character of these products (Commission Directive (EC)No 2007/19 (EC 2007)). The aim of this part

  List of figures with captions for the paper "Study on mass transfer ways of UV printing ink ingredients into foodstuffs".

Figure 1 .Figure 2 .

 12 Figure1. Methods of cup contact a) printed cups whose insides were in contact with printed outsides (as in normal production process) b) printed cups whose insides were not in contact with printed outsides c) unprinted cups whose insides were in contact with printed outsides d) unprinted cups whose insides were not in direct contact with printed outsides, only via the gas phase

Figure 6 .

 6 Figure6. Comparison of the quantity of ITX on the outside of the cups with quantity of ITX migrated into the yoghurt (cups printed outside and insides in contact with printed outsides (see Fig.1a)).

  Figure 4. Influence of the lamp age and storage time on the ITX migration level in yoghurt (cups outside printed and inside in contact with printed outsides (see Fig. 1a).Figure 5. Comparison of the quantities of ITX (a) and EDAB (b) migrated into yoghurt and 50% ethanol (values corrected by mean recovery rates (see TableI+II)). Limits of quantification (LOQ) for EDAB are 10.7 µg kg -1 for yoghurt and 8.2 µg kg -1 for 50% ethanol. Analyzed values below the LOQ are only rough estimations of the expected value, but nevertheless support the applicability of 50% ethanol as a simulant.

		24 24
	ITX in µg kg -1 yoghurt -1 yoghurt EDAB in µg kg	0 4 8 12 16 20 20 0 4 8 12 16	9 F 8 <LOD o r 16 20 14 <LOQ <LOD F o r F o r	<LOD 16 <LOD	13	9 13	<LOD <LOQ <LOD	19	19 12	<LOD 14 <LOD	outside not printed, outside not printed, inside with contact (see Fig. 1c) outside printed, inside with contact (see Fig. 1a) outside printed, inside without contact (see Fig. 1b) inside with contact (see Fig. 1c) outside printed, inside with contact (see Fig. 1a) outside printed, inside without contact (see Fig. 1b)
			1 d, new lamp 1 d, new lamp	1 d, old lamp P 1 d, old lamp P P	21 d, new lamp 21 d, new lamp	21 d, old lamp 21 d, old lamp
	e e r Figure 3. EDAB migration into yoghurt filled in black cups (LOD = limit of detection < 3.5 µg kg -1 e e ; LOQ = limit of quantification < 10.7 µg kg -1 ; values corrected by mean recovery rate (89%)). e r e r
						R R R
							e e e
								v i e v i e v i e
										w w w
												O n l O n l O n l
												y y y

  Figure 7. Comparison of the quantity of MTMP on the outside of the cups with quantity of MTMP migrated into the yoghurt (cups printed outside and insides in contact with printed outsides (see Fig. 1a); LOD = limit of detec-Figure 8. Comparison of the quantity of EDAB on the outside of the cups with quantity of EDAB migrated into the yoghurt (cups printed outside and insides in contact with printed outsides (see Fig. 1a); LOQ = limit of quantification < 10.7 µg kg List of tables with captions for the paper "Study on mass transfer ways of UV printing ink ingredients into foodstuffs".

		1800 1000
		1600 900			Quantity of ITX in non-cured ink on cups in µg, which would come into Quantity of MTMP in non-cured ink Quantity of EDAB in non-cured ink
		1400 800			contact with 1 kg yoghurt. on cups in µg, which would come into contact with 1 kg yoghurt. on cups in µg, which would come into contact with 1 kg yoghurt.
	quantity of ITX in µg quantity of MTMP in µg quantity of EDAB in µg	200 400 600 800 1000 1200 100 200 300 400 500 600 700	F o r F o r F o r	Quantity of ITX in cured ink on cups in µg, which would come into contact with 1 kg yoghurt. Quantity of ITX in yoghurt after 21 days' storage of the empty cups in µg per kg yoghurt; % refers to the ratio of ITX in the cured ink to the amount migrated into yoghurt. Quantity of MTMP in cured ink on cups in µg, which would come into contact with 1 kg yoghurt. Quantity of MTMP in yoghurt after 21 days' storage of the empty cups in µg per kg yoghurt. Quantity of EDAB in cured ink on cups in µg, which would come into contact with 1 kg yoghurt. Quantity of EDAB in yoghurt after 21 days' storage of the empty cups in µg per kg yoghurt; % refers to the ratio of EDAB in the cured ink to the amount migrated into yoghurt.
		0 0	yellow, new lamp 1.6% yellow, new lamp <LOD yellow, new lamp <LOQ	yellow, old lamp 2.2% P e e black, new lamp 0.6% r yellow, old lamp black, new lamp <LOD <LOD P e yellow, old lamp black, new lamp <LOQ <LOQ P e e r e r	black, old lamp 1.3% black, <LOD 2.8% black, old lamp old lamp
	tion < 3.8 µg kg -1 ).	-1 ).	R R R
					e e e
					v i e v i e v i e
					w w w
					O n l O n l O n l
					y y y

Table I .

 I Mean recovery rates and relative standard derivations (RSD) for the two photoinitiators and the amine synergist in yoghurt (n = 5). The bracketed data are the absolute mean recovery rates and RSD without internal standard correction.

		ITX		MTMP		EDAB
	Fortification lev-els (µg kg -1 )	Mean (%)	RSD (%)	Mean (%)	RSD (%)	Mean (%)	RSD (%)
	10	91	2	88	11	102	4
	30	93	4	93	3	91	10
	50	92	3	87	5	76	4
	70 90 Mean	93 91 92 (80) F o r	2 3 3 (3)	87 90 89 (79)	7 6 7 (9)	91 85 89 (79)	3 4 11 (11)
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Table II .

 II Mean recovery rates and relative standard derivations (RSD) for the two photoinitiators and the amine synergist in 50% ethanol (n = 5). The bracketed data are the absolute mean recovery rates and RSD without internal standard correction.

	Page 21 of 23	Food Additives and Contaminants
		ITX		MTMP		EDAB
	Fortification lev-els (µg kg -1 )	Mean (%)	RSD (%)	Mean (%)	RSD (%)	Mean (%)	RSD (%)
	10	97	3	89	3	78	6
	20	100	1	91	2	77	4
	40	101	2	93	4	79	8
	60	101	3	92	3	81	9
	80	96	8	91	2	84	6
	100	83	9	89	3	97	10
	Mean	96 (66) F o r	8 (9)	91	3	82 (48)	6 (8)
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Table III .

 III Concentrations of the two photoinitiators and the amine synergist in the model printing inks and on yoghurt cups before curing

		Food Additives and Contaminants	Page 22 of 23
		Concentration in the ink	Concentration on a yoghurt cup before curing
		(%)		(µg per cup)
		Yellow	Black	Yellow	Black
	ITX	3.3	4.3	217	444
	MTMP	3.5	6.4	145	406
	EDAB	3.4	2.6	136	215
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Table IV .

 IV Gas phase transfer of the two photoinitiators and the amine synergist from cups printed with the old lamp (see Fig.1d): migration into 50% ethanol (* = limit of detection, # = limit of quantification)

	Page 23 of 23
		Migration into 50% ethanol (µg kg -1 )
		Yellow cups	Black cups
	ITX	< 1.4*	< 1.4*
	MTMP	< 1.6*	< 5.4	#
	EDAB	< 2.5*	< 2.5*

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Food Additives and Contaminants

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Acknowledgements

We would like to thank Dr. Schweiger from Zeller+Gmelin GmbH & Co. KG, Mr. Hölz from Uniplast Knauer GmbH and Dr. Brüger from Campina GmbH for providing the inks, the printed yoghurt cups and valuable technical information, as well as the team from the commodities laboratory at the CVUA Stuttgart for skillful technical assistance and valuable discussions.


Dr Anders Wallensten
email: anders.wallensten@smi.setel.

Isabel Oliver

Katherine Ricketts

George Kafatos

James M Stuart

Carol Joseph

Affiliations

Windscreen wiper fluid without added screenwash in motor vehicles: a newly identified risk factor for Legionnaires' disease

Keywords: Legionnaires' disease, Legionella, Legionellosis, Driving, Motor vehicles, Transmission

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

Introduction

Legionnaires' disease is caused by inhalation of subspecies of the bacteria Legionella, which grow in aquatic environments. The incubation period is usually 2-10 days [START_REF]Control of communicable diseases manual[END_REF] and the case fatality can be as high as 15% [START_REF] Diederen | Legionella spp. and Legionnaires' disease[END_REF]. Males, the elderly, smokers, the immunosuppressed, and people with chronic disease are at higher risk [START_REF] Diederen | Legionella spp. and Legionnaires' disease[END_REF][START_REF] Marston | Surveillance for Legionnaires' disease. Risk factors for morbidity and mortality[END_REF].

Outbreaks of Legionnaires' disease are well documented, the source commonly being traced to water aerosol systems such as air conditioning units, cooling towers, showers, fountains, and whirlpools [START_REF]Control of communicable diseases manual[END_REF]. Sporadic cases are not infrequent, and can represent more than half the annual number of cases in England and Wales [START_REF]Epidemiological data on Legionnaires' disease, legionnaires' disease in residents of England and Wales -nosocomial, travel or community acquired cases, 1980 -2008[END_REF].

Identified risk factors include overnight stays in a hotel, or other accommodation site [START_REF] Boer | Risk factors for sporadic communityacquired Legionnaires' disease. A 3-year national case-control study[END_REF][START_REF] Che | Sporadic community-acquired Legionnaires' disease in France: a 2-year national matched case-control study[END_REF], and exposure to industrial aerosols [START_REF] Che | Sporadic cases of community acquired legionnaires' disease: an ecological study to identify new sources of contamination[END_REF], however a source is rarely identified.

Following the report of an unusually high number of cases of Legionnaires' disease in England and Wales during the summer of 2006 [START_REF]National increase in cases of legionnaires' disease[END_REF], a study was undertaken to identify high risk groups. The occupation of sporadic cases with onset between 2001 and 2006 was compared to data from the Office for National Statistics on the UK workforce [9]. A total of 145 cases were reported among 851,000 professional drivers (including heavy goods vehicle, van, bus and taxi drivers), while 782 cases were reported among 27,061,000 persons with other occupations. This suggests that professional drivers were at increased risk of Legionnaires' disease (RR=5.90, 95%CI: 5.04-6.90 p<0.0001). Previous research also suggests that drivers may be at higher risk. A recent Dutch study found that professional drivers had a three times increased risk [START_REF] Boer | Risk factors for sporadic communityacquired Legionnaires' disease. A 3-year national case-control study[END_REF] and a Turkish serological study identified a higher risk for long distance bus drivers [START_REF] Polat | Investigation of Legionella pneumophila seropositivity in the professional long distance drivers as a risky occupation[END_REF]. In addition, a case report from the USA reported the isolation of Legionella bacteria in the case's car which had a malfunctioning air conditioning unit [START_REF] Pinar | The use of heteroduplex analysis of polymerase chain reaction products to support the possible transmission of Legionella pneumophila from a malfunctioning automobile air conditioner[END_REF].

We conducted a case control study to corroborate this finding and to examine whether specific vehicle-related exposures were associated with an increased risk of Legionnaires' disease.

Methods

Case definition: Sporadic community acquired cases of Legionnaires' disease in residents of England and Wales aged 19-69 years, reported through the national enhanced surveillance scheme and with onset of disease between 15 July 2008 and 9 March 2009. Cases that had travelled abroad in the 2-10 days before onset of illness, those with a suspected nosocomial transmission link and those that were part of an outbreak were excluded. Cases that had died or were unable to give informed consent were also excluded.

We attempted to recruit three controls per case, matched on sex, age group (19-49y or 50-69y), and area of residence. In the analysis these were also classified by season (cold weather Oct-Mar and warm weather Apr-Sep). Controls were excluded if they had travelled abroad in the 2-10 days before completion of the questionnaire and if they had been hospitalised in the two weeks before completion of the questionnaire.

Cases were contacted by telephone and asked if they wished to participate. Controls were consecutively recruited through sequential digit dialling using the case's telephone area code to link to the same geographic area. Calls were made in the evenings or at weekends. Cases and controls who consented to participate by telephone were sent a questionnaire, information about the study and a pre-paid return envelope. A reminder was sent after three weeks if they had not responded.

Questionnaire content

The questionnaire included questions on exposures related to driving or being a passenger in a car or other vehicle, distance and time travelled in a vehicle, time driven with windows open, time driven in rural, urban and industrial areas, and plausible risk exposures in vehicles, such as air conditioning and air re-circulation use. The questions related to average use or average exposure in the two weeks before onset of illness in cases and in the two weeks before completing the questionnaire for controls. Information was also collected on the vehicle type, make, age, service history, content of wind screen wiper fluid reservoir, and whether driving was work or socially related, or both. In addition, questions on the known risk factors for Legionella infections such as smoking, underlying illness, and the use of showers were included.

Data manipulation and statistical analysis

Data verification, data manipulation and data analysis were carried out using Stata 10.1 (StatCorp, Texas, USA). Initially, risk factors were grouped to create summary variables and single variable analysis was carried out. Any variables with p<0.1 were included in a multivariable model. The least significant factors were dropped from the model in a stepwise fashion until all the remaining exposures exhibited a significant association of p<0.05. Personal information variables such as sex, age, season, and smoking were not part of the stepwise process and were included in the multivariable model at all times. Interactions were tested at a stricter criterion of p<0.01.

Ethics approval

Ethics approval was granted for the study through the Oxford Research Ethics Committee B. Reference number 08/H0605/75. Informed content was obtained from all study participants.

Results

Case recruitment: During the study period 123 sporadic cases were reported. Ten of these did not have contact details and a further six were not contacted due to staff limitations. Fourteen were excluded according to the specified criteria. The remaining 93 cases agreed to be sent questionnaires, 75 of whom responded (80.6%); 62 men and 13 women. Nineteen cases were aged under 50 years, 56 were aged 50 -69 years.

Control recruitment: Telephone contact was established with 672 people who were asked to participate as controls; 388 did not fulfil the inclusion criteria. Of the 284 people eligible for inclusion as controls, 117 agreed to be sent a questionnaire (41.2%). Two questionnaires were not sent due to administrative reasons. Of the remaining 115, 70 controls responded (60.9%), giving a final response rate of 70/284 (24.6%). Three of those who returned questionnaires did not meet the inclusion criteria, leaving 67 controls in the study, 63 men and 4 women. Twelve controls were aged less than 50 years, 55 were aged 50-69 years. Fifteen cases were matched with one control, 17 with two controls and six with three controls. 38 cases were not matched to any control.

Analysis

The case and control recruitment thus identified a total of 142 individuals that were entered in the analysis. Several exposures linked to driving or being in a vehicle were associated with illness at the p<0.1 level in the single variable analysis (Table 1).

Those with higher odds ratios (>2) were; driving / being a passenger in a van, driving through industrial areas, number of hours as well as ever driving with the window open, hours in vehicle as part of job, vehicle year of manufacture older than 2000, lack of screenwash (pre-diluted or concentrated screenwash) in windscreen wiper fluid, and not taking showers at home. Those which suggested a protective effect (OR <0.5) were use of car, bus, train and underground as well as air conditioning in the vehicle especially when at a warm temperature.

In multivariable analysis, after adjusting for sex, age, season and smoking, "driving through industrial area" (OR=7.17; 95% CI: 1.52-33.73) and "using no screenwash in the windscreen fluid" (OR=47.24; 95% CI: 3.70-603.63) were associated with illness (Table 2). "Shower at home", "driving a van", and "year of make" were of borderline statistical significance but dropped out of the model.

The population attributable risk for driving or being a passenger in a car that did not use screenwash in the windscreen wiper fluid among community acquired sporadic cases aged under 70 years was calculated as 21.8%; 95% CI (21.0-22.5%). The calculation was carried out using the odds ratio as an approximation to the relative risk assuming 30% (15/49) of the cases were exposed [START_REF] Armitage | Attributable risk. Statistical methods in medical research[END_REF].

Discussion

We found evidence in the surveillance data for England and Wales that professional drivers may be at increased risk of acquiring Legionnaires' disease. Our case control study identified two previously unrecognised potential risk factors that may explain this increased risk among professional drivers and also a proportion of the sporadic cases among non professional drivers who spend time in vehicles. The strength of these associations was high; the relative risk from not using screenwash in the windscreen wiper fluid was nearly 50. The increased risk of driving through industrial areas is more likely to explain cases in professional drivers and may be linked to a greater exposure to outside sources of Legionella, for example from cooling towers. The finding that not adding screenwash to the water used as windscreen wiper fluid poses a risk is biologically plausible; it is likely that Legionella bacteria would grow in the stagnant water of the windscreen wiper fluid reservoir, which is intermittently sprayed onto the windscreen, creating an aerosol. Some of the aerosol may be sucked into the passenger compartment through the ventilation intake. It is also plausible that screenwashes, as they usually contain biocidal agents like propranolol, inhibit the growth of Legionella. Although we restricted this study to those under 70 years, it is also likely that risk linked to not using screenwash would also apply to older age groups who are at higher risk of Legionnaires' disease.

In addition to the above findings, the single variable analysis also identified driving with the windows down, driving long hours for work and driving a van as associated with illness. These may all have been linked to the risk of driving through industrial areas. Also associated with an increased risk were driving an older vehicle and not using showers at home. The single variable analysis found some associations that appeared to be protective: travelling by car, bus and train and using a vehicle with air conditioning. None of these factors retained their significance in the multivariable model.

Ultimately, recruiting three controls per case as planned was not possible with the resources and time available as the selected method of control recruitment turned out to be very resource intensive. Due to the number of cases (n=38) without any matched controls, applying matched analysis techniques using conditional logistic regression was no longer ideal. We therefore did an unmatched analysis using logistic regression that included adjustment for the matching variables of age and sex. Adjustment for area of residence was not possible due to the low numbers of cases and controls in the same local area. Unmatched analysis of a matched study may well introduce some selection bias but this is likely to underestimate the true strength of association [START_REF] Rothman | Design strategies to improve study accuracy[END_REF].

These findings offer a possible explanation for the higher risk among professional drivers identified in the Netherlands [START_REF] Boer | Risk factors for sporadic communityacquired Legionnaires' disease. A 3-year national case-control study[END_REF]. During the course of our study, a Japanese study was published [START_REF] Sakamoto | Is driving a car a risk for Legionnaires' disease?[END_REF] whose results were in line with our findings and those from the Netherlands. The Japanese study showed a disproportionately high number of cases among transportation industry workers. They also identified evidence of Legionella bacteria in air conditioning evaporators of discarded cars suggesting that air conditioning units may be a source of infection. We did not find evidence of this in our multivariable analysis and our single variable analysis actually found a lower risk for those using cars with air conditioning systems. In a second paper the same research group hypothesised that contaminated aerosols may enter the car when a car drives through puddles as they managed to isolate Legionella bacteria from rain puddles on roads [START_REF] Sakamoto | Legionella pneumophila in rainwater on roads[END_REF].

Of the identified risk exposures, the risk found to be associated with not using screenwash in the windscreen wiper fluid was the most intriguing. This exposure pathway needs further investigation. In a pilot study carried out by the UK Health Protection Agency, the windscreen wiper fluid was tested in a sample of cars with and without added screenwash. Legionella bacteria were isolated from the windscreen wiper fluid of one car out of five that did not use screenwash while none were identified from 16 cars that did use screenwash (unpublished data). We estimate that around 20% of community acquired sporadic cases under the age of 70 years in England and Wales could be prevented by using screenwash. This simple public health advice may be of worldwide relevance in reducing morbidity and mortality from Legionnaires' disease.

Table 1 Risk factors for sporadic cases of Legionnaires' disease; single variable analysis (p < 0.1) 1a

 1 . Exposures relating to time spent in vehicle as a driver/passenger Two controls who added washing up liquid as screenwash were not included in the analysis of this exposure.

	Exposure
	(n. available for analysis)		Cases	% Controls %	OR	95% CI	P-value
	Use of car	No	14		3		1.00	baseline
	(130)	Yes	54	79.4	59	95.2	0.20	(0.05, 0.72)	0.006
	Use of van	No	49		57		1.00	baseline
	(130)	Yes	19	27.9	5	8.1	4.42	(1.54, 12.71)	0.003
	Use of bus	No	61		48		1.00	baseline
	(129)	Yes	6	9.0	14	22.6	0•34	(0.12, 0.94)	0.031
	Driving through industrial
	areas	No	44		51		1.00	baseline
	(142)	Yes	31	41.3	16	23.9	2.25	(1.09, 4.64)	0.026
	Daily hours with window
	open	Never	4	5.9	17	27.4	1.00	baseline
	(130)	<2	36	53.0	39	62.9	3.92	(1.21, 12.76)
		>=2	28	41.2	6	9.7	19.83	(4.88, 80.54)	<0.001
	Ever having window open	No	4		17		1.00	baseline
	(130)	Yes	64	94.1	45	72.6	6.04	(1.91, 19.17)	0.001
	Driving as part of job	No	38		43		1.00	baseline
	(131)	Yes	31	44.9	19	30.6	1.85	(0.90, 3.79)	0.092

*

Table 2 Vehicle related risk factors for Legionnaires' disease: multivariable analysis

 2

	Exposure		OR*	95% CI	p-value
	Driving through industrial area	No	1.00	baseline
		Yes	7.17	(1.52, 33.73)	0.007
	Use of screenwash in
	windscreen wiper fluid	Yes	1.00	baseline
		No	47.24	(3.70, 603.63)	<0.001
	* adjusted for age, sex, season and smoking

Acknowledgements

The study was funded entirely by the Health Protection Agency. We would like to thank all staff at the Health Protection Agency who assisted in the investigation, in particular: Rebecca Close, Magdalene Mbanefo, Esam Gharish, Falguni Naik, and Anitra Jones. We would also like to thank Nick Phin for his support of the study, and John V Lee and Sandra Lai for carrying out the pilot study into the sampling of windscreen wiper fluid.