Vincent Chouraki

Guillaume Savoye

Luc Dauchet

Gwenola Vernier-Massouille

Jean-Louis Dupas

Veronique Merle

Eric Laberenne

Jean-Louis Salomez

Eric Lerebours

Dominique Turck

Gwénola Vernier-Massouille

Véronique Merle

Jean-Eric Laberenne

Antoine Cortot

Corinne Gower-Rousseau

Jean-Frédéric Colombel

The changing pattern of Crohn's disease incidence according to age in northern France: a continuing increase in the

Keywords: Crohn's disease < Disease-based, Inflammatory bowel disease < Disease-based, Ulcerative colitis < Disease-based, Epidemiology < Topics, Paediatric gastroenterology < Topics IBD, Inflammatory bowel disease, CD, Crohn's disease, UC, ulcerative colitis Inflammatory Bowel Disease, Incidence, Pediatric, Epidemiology,

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

Inflammatory bowel diseases (IBD) represent a major burden for developed countries. It has been estimated that as many as 1.4 million persons may be afflicted with IBD in the US and Canada, and 2.2 million in Europe [START_REF] Loftus | Clinical epidemiology of inflammatory bowel disease: Incidence, prevalence, and environmental influences[END_REF] Crohn's disease (CD) and ulcerative colitis (UC) are the major IBD that challenge gastroenterologists. Although significant advances have been made toward a better understanding of the pathophysiology of IBD [START_REF] Xavier | Unravelling the pathogenesis of inflammatory bowel disease[END_REF][START_REF] Budarf | GWA studies: rewriting the story of IBD[END_REF] , their etiology remains unclear [START_REF] Loftus | Clinical epidemiology of inflammatory bowel disease: Incidence, prevalence, and environmental influences[END_REF] .

Epidemiological studies of geographic and temporal variations in IBD provide important information on the natural history, health care burden and causal mechanisms of the disease. In particular, population-based epidemiological studies are more likely to reflect the true spectrum of illness than studies performed on patients seen at referral centers. Indeed, epidemiological studies have shown a continuing increase in [START_REF] Jacobsen | Increase in incidence and prevalence of inflammatory bowel disease in northern Denmark: a population-based study, 1978-2002[END_REF][START_REF] Vind | Increasing incidences of inflammatory bowel disease and decreasing surgery rates in Copenhagen City and County, 2003-2005: a population-based study from the Danish Crohn colitis database[END_REF][START_REF] Economou | Crohn's disease incidence evolution in North-western Greece is not associated with alteration of NOD2/CARD15 variants[END_REF] or stabilization [START_REF] Lapidus | Crohn's disease in Stockholm County during 1990-2001: an epidemiological update[END_REF][START_REF] Loftus | Update on the incidence and prevalence of Crohn's disease and ulcerative colitis in Olmsted County, Minnesota, 1940-2000[END_REF][START_REF] Gunesh | The incidence of Crohn's disease in Cardiff over the last 75 years: an update for 1996-2005[END_REF] of CD incidence rates in developed countries over the past 5 decades. In contrast, several studies have described a continuing increase in incidence rates of CD in children and adolescents [START_REF] Askling | Incidence of paediatric Crohn's disease in Stockholm, Sweden[END_REF][START_REF] Armitage | Increasing incidence of both juvenile-onset Crohn's disease and ulcerative colitis in Scotland[END_REF][START_REF] Hildebrand | Changing pattern of paediatric inflammatory bowel disease in northern Stockholm 1990-2001[END_REF][START_REF] Phavichitr | Increasing incidence of Crohn's disease in Victorian children[END_REF][START_REF] Perminow | Incidence and clinical presentation of IBD in children: comparison between prospective and retrospective data in a selected Norwegian population[END_REF][START_REF] Turunen | Incidence of inflammatory bowel disease in Finnish children, 1987-2003[END_REF][START_REF] Benchimol | Increasing incidence of paediatric inflammatory bowel disease in Ontario, Canada: evidence from health administrative data[END_REF] , suggesting that ongoing risk factors may exist in these populations. Nevertheless, these changes in the incidence of juvenile-onset CD remain subject to debate and need to be confirmed in larger population-based registries. The EPIMAD study records all incident cases of IBD since 1988 in northern France. Previously published studies covering the 1988-1999 period reported a significant increase in incidences rates of CD in the overall population [START_REF] Molinié | Opposite evolution in incidence of Crohn's disease and ulcerative colitis in Northern France (1988-1999)[END_REF] , as well as a non-significant increase in the pediatric population [START_REF] Auvin | Incidence, clinical presentation and localization at diagnosis of pediatric inflammatory bowel disease: a prospective population-based study in northern France (1988-1999)[END_REF] . The aim of the present study was therefore to conduct an updated analysis of the evolution of incidence rates and digestive sites of CD and UC for the 1988-2007 periods in a large population registry which included infants and young adults.

PATIENTS AND METHODS

Patient population

Cases included all patients from the EPIMAD registry who had a diagnosis of definite or probable IBD, including cases of CD and UC, between January 1988 and December 2007. The population density of 197/km2. Both rural and urban populations can be found in these areas (ratio urban/rural=8.9 in the Nord, 4.5 in the Pas-de-Calais, 1.4 in the Somme, and 3.0 in the Seine-Maritime). This region is a well-defined geographic entity bordering Belgium and the North Sea (Figure 1). The population is stable-that is, the percentages of the population moving per year for each area were 0.8 for Nord and Pas-de-Calais, 1.1 for Somme and 0.9 for Seine-Maritime (French National Statistical Institute).

Data collection

The methodology of the EPIMAD registry has been previously described in detail [START_REF] Molinié | Opposite evolution in incidence of Crohn's disease and ulcerative colitis in Northern France (1988-1999)[END_REF][START_REF] Auvin | Incidence, clinical presentation and localization at diagnosis of pediatric inflammatory bowel disease: a prospective population-based study in northern France (1988-1999)[END_REF][START_REF] Gower-Rousseau | Incidence of inflammatory bowel disease in northern France (1988-1990)[END_REF] .

Interviewing practitioners collected data on all patients diagnosed between 1988 and 2007 from all adult and pediatric gastroenterologists (GE) (total: 248) practicing in the private or public sector.

Only patients who had been residents of the study areas at the time of diagnosis of IBD were included. Each GE reported any patient consulting for the first time with clinical symptoms compatible with IBD, and was contacted by phone at least three times a year by an interviewing practitioner. This interviewer went to the GE's consulting room and collected data from charts in a standardized questionnaire for each new case. Main data collected included age, gender, year of diagnosis, interval between onset of symptoms and diagnosis, and clinical, radiological, endoscopic and histological findings at the time of diagnosis. A final diagnosis of CD or UC was made by two expert gastroenterologists and was recorded as definite, probable or possible according to previously published criteria [START_REF] Gower-Rousseau | Incidence of inflammatory bowel disease in northern France (1988-1990)[END_REF] . For the purpose of this study, only patients with definite or probable For the study of phenotypes of CD, the site of disease was reported only for patients who underwent a complete bowel investigation (images of small and large bowel). The localizations of the lesions were coded according to the Montreal classification [START_REF] Satsangi | The Montreal classification of inflammatory bowel disease: controversies, consensus, and implications[END_REF] with modification for CD: L1 referred to ileal localization of lesions, L2 to colonic localization and L3 to ileocolonic localization, with the addition that an ileal localization with cecal involvement was considered as a L3 localization. For UC localization, ulcerative proctitis (E1) was defined as involvement limited to the rectum (i.e, the proximal extent of inflammation was distal to the rectosigmoid junction), left-sided UC (E2) was defined as involvement limited to the portion of the colorectum distal to the splenic flexure, and extensive UC (E3) was defined as involvement extending proximal to the splenic flexure.

Extraintestinal manifestations (EIMs) included joints, skin, ocular and hepato-biliary manifestations.

Statistical Analyses

Incidence rates were computed as the ratio of the number of incident cases with respect to the number of person-years for both sexes. Yearly estimations of population based on a procedure mixing exhaustive census before 2004 and random sampling after 2004 were obtained from the French Statistics Institute (INSEE). Three-year periods were used to ensure better statistical precision and robustness. Age standardization was performed using European standard population weights for 5-year age groups. Incidence rates were studied in the overall population and according to 20-year age brackets. Finally, 10-year category incidence rates were analyzed for patients diagnosed prior to the age of 20 years. Exact confidence intervals for standardized incidence rates were approximated using a gamma distribution [START_REF] Feuer | Confidence intervals for directly standardized rates: a method based on the gamma distribution[END_REF] . Contributions of age, sex, time period and region were tested for CD in a generalized linear model using Poisson distribution. Second-order interactions between these variables were selected using Akahike Information Criteria (AIC) [START_REF] Akaike | A new look at the statistical model identification[END_REF] . Linearity of the evolution of incidence rates of CD was assessed with a likelihood ratio test using nested models. Changes in CD localization and time were assessed with appropriate Chi-squared tests. For descriptions of age at diagnosis (years) and time intervals between symptoms and Comparisons of distribution of these variables across time were performed using the Wilcoxon, Kruskal-Wallis or Chi-squared tests for trend. Analyses were performed with R 2.12.1 [START_REF]R: A Language and Environment for Statistical Computing[END_REF] .

RESULTS

IBD cases

During the 1988-2007 period, 12,052 patients satisfying the criteria defined above were recorded in the EPIMAD registry, including 61% (n=7,409) of CD cases and 39% (n=4,643) of UC cases. Median age at diagnosis was 30 years (interquartile range IQR: 22-42) and was significantly higher in patients with UC than in patients with CD (35 vs. 26 years, respectively; p < 0.0001).

Median delay between first symptoms and diagnosis was 3 months (IQR: 1-6) and was significantly higher in CD cases than in UC cases (3 vs. 2 months, respectively; p < 0.0001). Five hundred and sixty cases of indeterminate colitis (IC) were recorded.

Incidence of CD

During the 1988-2007 period, 7,409 patients were diagnosed with either definite (35% n=2,570) or probable (65% n=4,839) CD, with a predominance of women (56% n=4,180). Median age at diagnosis was 26 years (IQR: 20-38) and was not statistically different between men and women (p=0.8). Median interval between first symptoms and diagnosis was 3 months (IQR: 1-8) for the entire period, with a significant decrease in the proportion of intervals longer than 9 months, from 28% in 1988-1990 to 22% in 2006-2007 (p<0.0001) (Table 1).

During the 1988-2007 period, incidence rate of CD was 6.4 (95% confidence interval (CI) 6.2-6.5).

CD incidence rate increased significantly, from 5.2 cases per 100,000 person-years in 1988-1990 to 6.7 in 2006-2007 (test for trend, p<0.0001) (Figure 2). Rates reached a maximum of 7.1 in 1997-1999 and then remained stable (test for non-linearity, p=0.0003). CD incidence rate was significantly higher in women (7.1; 95% CI 6.9-7.3) than in men (5.7; 95% CI 5.5-5.9) (p<0.0001), but analyses performed in each age group showed that this difference was present only in patients 8 aged 20 to 24 years (p for interaction between age and sex <0.0001) (Figure 3). This difference between women and men remained stable during the study period (p for interaction=0.21). Evolutions in incidence rates did not differ according to region (interaction, p=0.08).

Evolution of incidence differed according to age group (Figure 4a). Incidence rates in the 0-19 age group increased steadily over the entire study period, from 3.4 cases per 100,000 person-years in 1988-1990 to 5.9 in 2006-2007 (p for trend <0.0001). Evolution of incidence rates in the 0-19 age group did not differ according to sex or region (interaction, p=0.10 and p=0.22 respectively).

Further analyses showed that the 10-19-year-old age group was mainly responsible for this increase, since the incidence rates increased continuously, from 6.5 cases per 100,000 person-years in 1988-1990 to 11.1 in 2006-2007 (p for trend <0.0001), whereas the incidence rates in the 0-9-year-old age group only slightly increased during the same period (p for trend=0.02, Figure 4b). Furthermore, the incidence rate in the age group 20-29 was similar to incidence rates in the overall population, with stabilization beginning from the 1997-1999 period, whereas incidence rates in patients aged 30 and over remained stable or only slightly increased. whereas other sites remained stable (Table 1).

CD phenotypes

During

The proportion of patients with complete bowel investigation was stable in the 0-19 age group (test for trend, p=0.12) and increased from 77.9% to 88.8% (test for trend, p <0.0001), from 68.6% to 83.9% (test for trend, p=0.003) and from 52.5% to 74.4% (test for trend, p=0.013) in the 20-39, 40-59 and 60 and over age groups, respectively. Similarly to the overall population, ileo-colonic (L3 and L3+L4) forms of CD were predominant in children and adolescents, but a higher proportion of extended lesions (L3+L4) than in other age groups was observed. The decreasing proportion of pure L2 localizations observed in the overall population was found in the 20-39-year-old and the 40-59year-old age groups, as it decreased from 22.5% to 5.8% (test for trend, p <0.0001) and from 40.3% to 4.8% (test for trend, p=0.004) respectively, but not in the other age groups.

Incidence of UC

From 1988 to 2007, 4,643 incident cases of UC were recorded in the EPIMAD study, with a predominance of men (55%; n=2,566). Median age at diagnosis was 35 (IQR: 26-47) years and was significantly higher in men than in women: 38 versus 32, respectively (p < 0.0001). The median interval between first symptoms and diagnosis was 2 months (IQR: 1-6) for the entire period, with stable proportions over the time (Table 2). During the total period, UC incidence rate was 4.1 (95%CI 4.0-4.2) and was significantly higher in men (4.7; 95%CI 4.5-4.9) than in women (3.6; 95%CI 3.4-3.7) (p<0.0001) (Figure 2). This difference in UC incidence rates according to gender was observed for patients over 35 (Figure 5). A significant decrease in UC incidence rate was observed from 4.3 (95%CI 4.0-4.7) in 1988-1990 to 3.4 (95%CI 3.1-3.7) in 2006-2007 (test for trend, p=0.001). Time trends of UC incidence rates differed significantly according to age group (interaction, p<0.0001) with a more important decrease in the patients aged 60 and over than the other age groups.

UC phenotype

During the 1988-2007 period, 3,411 patients underwent complete colonic investigation. The proportion of complete colonic investigations carried out significantly increased, from 66.1% in 1988-1990 to 82.6% in 2006-2007 (p for trend <0.0001). E2 localizations constituted most UC cases, followed by E1 and E3 localizations (45%, 29% and 26% respectively). These proportions remained stable during the study period (Table 2).

DISCUSSION

In Northern France, from 1988 to 2007, incidence rates of CD stabilized following an initial increase, while those of UC decreased during that period. Conversely, CD incidence rates increased in the 0-19-year-old age category, mainly due to the 10-19-year-old category whereas they remained stable or stabilized after initial increase in the other age groups. For CD localization at diagnosis, ileo-colonic forms (L3) of CD were predominant in the overall population, with an increase of extended forms (at least 3 affected segments). In the 0-19-year-old category, a higher proportion of L3+L4 localizations than in the whole population was observed, with similar evolution. Incidence rates of CD have stabilized in northern France since 1999. These results are coherent with previous studies in Minnesota8, the United Kingdom9 and Sweden 7 . Conversely, other studies in Denmark [START_REF] Jacobsen | Increase in incidence and prevalence of inflammatory bowel disease in northern Denmark: a population-based study, 1978-2002[END_REF][START_REF] Vind | Increasing incidences of inflammatory bowel disease and decreasing surgery rates in Copenhagen City and County, 2003-2005: a population-based study from the Danish Crohn colitis database[END_REF] and Greece 6 have shown a continuing increase. Since only definite and probable cases of CD were included in the EPIMAD study, underestimation of incidence rates due to incomplete CD case ascertainment cannot be ruled out. Potential IBD cases likely to be reclassified as definite or probable CD, such as possible CD, UC or IC cases, represented 2,436 patients in the entire database. Indeed, only continuous surveillance of these populations will enable us to more precisely determine the stabilization of incidence rates of CD. It might be speculated that accuracy of case recording has improved with time in the EPIMAD registry following a learning curve from the time of registry creation. However, in our opinion, this is unlikely since, in this case, the same variation (i.e. modification in the slope beginning in 1997-1999) in incidence rates of UC would have been observed. Our earlier data [START_REF] Molinié | Opposite evolution in incidence of Crohn's disease and ulcerative colitis in Northern France (1988-1999)[END_REF] as well as the present report did not support this hypothesis; indeed, a significant decrease in UC incidence over time was observed.

Our main result, indicating that incidence rates of CD continue to increase in young persons in Northern France, is of interest. Indeed, there existed stable incidence rates in the 0-9 year age group but increasing incidence rates in the 10-19-year-old age group. Similar results have been observed in Norway [START_REF] Perminow | Incidence and clinical presentation of IBD in children: comparison between prospective and retrospective data in a selected Norwegian population[END_REF] and Scotland [START_REF] Armitage | Increasing incidence of both juvenile-onset Crohn's disease and ulcerative colitis in Scotland[END_REF] , and in other studies in Finland 15 , Sweden [START_REF] Askling | Incidence of paediatric Crohn's disease in Stockholm, Sweden[END_REF][START_REF] Hildebrand | Changing pattern of paediatric inflammatory bowel disease in northern Stockholm 1990-2001[END_REF] , Australia 13 and Canada [START_REF] Benchimol | Increasing incidence of paediatric inflammatory bowel disease in Ontario, Canada: evidence from health administrative data[END_REF] .

Those studies showed an overall increase in the incidence of childhood-onset CD. Better diagnostic procedures and greater awareness by parents of the risk of CD may have led to reducing the time lapse between symptoms and diagnosis, thereby increasing the incidence rates of CD in younger patients. In the EPIMAD study, the median time between symptoms and diagnosis was 3 months, which is low. Even if a decreasing proportion of intervals longer than 9 months had been observed over time, it would not easily confirm a decrease of several years of age at diagnosis. We therefore feel that a true increase in the incidence rates of CD is occurring in adolescents in Northern France.

Moreover, if our observations had relied only on advances in diagnosis, then this would have impacted the incidence rate of the older age groups, which was not the case here. The increase in CD incidence in the 10-19 years group could also reflect an earlier presentation of CD in predisposed patients as proportion of familial history of CD increased and at the same time proportions of extra intestinal manifestations and anoperineal lesions decreased between 1988 and 2007. It might be hypothesized that, in Northern France over time, a change in smoking habits, a well known risk factor for CD [START_REF] Lakatos | Smoking in inflammatory bowel diseases: good, bad or ugly?[END_REF] , would explain the differential evolution in incidence rates of CD according to age. However, accurate regional data supporting this hypothesis are scarce. At a national level, two population-based studies concerning teenagers showed a 30% decrease in the proportion of 17-year-old subjects reporting daily tobacco consumption, from 41.1% in 2000 to 28.9% in 2008 [START_REF] Legleye | Drug use among 17 years old. Results of the 2008 ESCAPAD survey[END_REF] , and a 45% decrease among 15-and 16-year-old subjects, from 31% in 1999 to 17% in 2007 [START_REF] Legleye | Alcohol, tobacco and cannabis at 16 years old -Initial results of the French part of the ESPAD 2007 survey[END_REF] . Moreover, other national data on smoking habits indicate a decrease in the prevalence of smoking in every age group [START_REF] Grizeau | Epidemiology of tobacco smoking in France[END_REF][START_REF] Brucker | Smoking behavior in France: recent developments[END_REF] during the study period. Change of diet has also been incriminated as a risk factor for IBD. A recent French cohort study of middle aged women showed that protein intake was associated with a higher risk of IBD, CD in particular [START_REF] Jantchou | Animal protein intake and risk of inflammatory bowel disease: The E3N prospective study[END_REF] .

Analysis of the localizations of CD lesions at diagnosis showed a predominance of ileo-colonic (L3) forms, with increasing proportions of extended forms, including ileo-colonic and upper gastrointestinal lesions (L3+L4), from 1988 to 2007. These results are unusual compared to the literature. Some [START_REF] Jacobsen | Increase in incidence and prevalence of inflammatory bowel disease in northern Denmark: a population-based study, 1978-2002[END_REF][START_REF] Vind | Increasing incidences of inflammatory bowel disease and decreasing surgery rates in Copenhagen City and County, 2003-2005: a population-based study from the Danish Crohn colitis database[END_REF][START_REF] Economou | Crohn's disease incidence evolution in North-western Greece is not associated with alteration of NOD2/CARD15 variants[END_REF][START_REF] Lapidus | Crohn's disease in Stockholm County during 1990-2001: an epidemiological update[END_REF][START_REF] Gunesh | The incidence of Crohn's disease in Cardiff over the last 75 years: an update for 1996-2005[END_REF] , but not all [START_REF] Loftus | Crohn's disease in Olmsted County, Minnesota, 1940-1993: incidence, prevalence, and survival[END_REF] , population-based studies reported a higher proportion of pure colonic localizations (L2) in CD patients. A possible explanation for these results might lie in the 12 fact that we reported CD sites only in patients who had undergone a complete bowel investigation (with imaging of both large and small bowel). In other studies, the proportion of pure colonic localizations (L2) may have been overestimated; as they were calculated from the entire population regardless of whether or not small bowel investigation had been performed. Moreover, the recent use of wireless capsule endoscopy (WCE) may have impacted the proportion of patients diagnosed with extensive small bowel involvement. In France, however, use of WCE remained scarce before 2007 so it is unlikely to have influenced diagnostic of more extensive forms of CD.

Among patients aged 0 to 19, an ileo-colonic (L3) localization constituted most CD lesions at diagnosis and this was stable over time. This is coherent with recent studies reporting extensive lesions of CD in childhood at diagnosis, either an ileo-colonic localization [START_REF] Perminow | Incidence and clinical presentation of IBD in children: comparison between prospective and retrospective data in a selected Norwegian population[END_REF][START_REF] Cucchiara | Polymorphisms of tumor necrosis factor-alpha but not MDR1 influence response to medical therapy in pediatric-onset inflammatory bowel disease[END_REF][START_REF] Gupta | Gender differences in presentation and course of disease in pediatric patients with Crohn disease[END_REF] or an association of ileo-colonic and upper gastrointestinal lesions [START_REF] Vanlimbergen | Definition of phenotypic characteristics of childhood-onset inflammatory bowel disease[END_REF] . These differences in phenotypes between adults, children and adolescents may reflect the influence of distinct causal factors or a more systematic investigation of the gastrointestinal tract in younger patients. One strength of this study lies in the number of registered cases, which enabled precise estimates of incidence rates of CD and adjustment for covariates in statistical models. Systematic follow-up of newly diagnosed patients at 18 months allowed for potential reclassification and well-documented final diagnoses of CD. In contrast, questions concerning the completeness of case registration may be raised. The EPIMAD registry relied only on gastroenterologists' declarations of incident cases, thereafter confirmed by experts. In France, gastroenterologists have a monopoly in their profession, especially in terms of administrative authorization to perform endoscopy. As a consequence, there is a strong probability that each new IBD patient will consult a gastroenterologist and therefore be included in the study.

In Northern France, incidence rates of CD continue to rise among younger patients, with a more extensive phenotype than in adults. These findings suggest that different causal factors may exist in patients aged 10 to 20. It is therefore of great importance to focus etiological research on comparisons of trends in incidence rates and phenotypes between adult-and childhood-onset CD.

Number of words: 3149

  The changing pattern of Crohn's disease incidence according to age in northern France: a continuing increase in the 10-19-year-old age bracket (1988-2007). Docteur Corinne Gower-Rousseau, Epidemiology Unit and Registre EPIMAD, Parc Eurasanté, 154 rue du Dr Yersin, CH et U de Lille, 59037 Lille Cedex, France; Tel: (33) 3 20 44 55 18; Fax: (33) 3 20 44 69 45; corinne.gower@chru-lille.fr

  the northern part of France, which has 5,790,526 inhabitants (1999 national population census) residing in four regions: Nord, Pas-de-Calais, Somme and Seine-Maritime. This part of France represents 9.3% of the total French population and is composed of four sectors: (a) Nord, with 2,554,449 inhabitants and a population density of 445/km2; (b) Pas-de-Calais, with 1,441,422 inhabitants and a population density of 216/km2; (c) Somme, with 555,479 inhabitants and a population density of 90/km2; and (d) Seine-Maritime, with 1,239,176 inhabitants and a

  ), median and interquartile ranges (25th and 75th percentiles) are given.

  the 1988-2007 periods, 5,790 patients underwent complete bowel investigation. The proportion of complete bowel investigations significantly increased, from 76.9% in 1988-1990 to 86.5% in 2006-2007 (p for trend <0.0001). An ileo-colonic localization (L3 and L3+L4) was predominant in the overall population and increased during the study period, from 52.0% in 1988-1990 to 68.3% in 2006-2007 (p for trend=0.0004). Extended phenotypes (L3+L4) significantly increased, from 8.1% in 1988-1990 to 23.2% in 2006-2007 (p for trend <0.0001), whereas an ileocolonic localization without upper GI lesions (L3) remained stable. Colonic site forms (L2) significantly decreased, from 22.6% in 1988-1990 to 5.4% in 2006-2007 (p for trend < 0.0001),

Figure 1 :

 1 Figure 1: Geographic situation in Europe of the EPIMAD Registry area.

Figure 2 :

 2 Figure 2: Trends in standardised incidence (95% confidence interval) of Crohn's disease (CD) and

Figure 3 :

 3 Figure 3: Incidence rate of Crohn's disease (CD) by sex and age in Northern France from 1988 to

Figure 4a :

 4a Figure 4a: Evolution of the incidence of Crohn's Disease in Northern France from 1988-1990 to

Figure 4b :

 4b Figure 4b: Evolution of the incidence of Crohn's Disease in Northern France from 1988-1990 to

Figure 5 :

 5 Figure 5: Incidence rate of Ulcerative Colitis (UC) by sex and age in Northern France from 1988 to

  Geographic situation in Europe in the area of the EPIMAD registry.80x49mm (600 x 600 DPI)

Table 1 : Trends in Crohn's disease phenotype modifications at diagnosis from 1988 to 2007

 1

		1988-1990	1991-1993	1994-1996	1997-1999	2000-2002	2003-2005	2006-2007
	Age at diagnosis (years)*	27 (20-38)	27 (20-38)	27 (21-38)	27 (20-38)	27 (20-38)	26 (20-39)	25 (19-37)
	Interval before diagnosis (months)*	3 (1-12)	3 (1-11)	3 (1-8)	3 (1-7)	3 (1-6)	3 (1-6)	3 (1-8)
	Family history of IBD % (n)**	9.4 (70)	11 (95)	11.6 (99)	14.4 (150)	13.9 (136)	15.6 (163)	14.8 (107)
	EIMs % (n)**	15.6 (121)	14.5 (146)	11 (113)	13.7 (163)	11.4 (133)	9.9 (118)	11.8 (90)
	Complete gut investigation % (n)**	76.9 (618)	72 (752)	73.3 (809)	79.4 (997)	79 (953)	81.2 (987)	86.5 (674)
	Disease localization % (n)**
	L1	18.4 (100)	14.8 (102)	15.9 (117)	16 (144)	18 (144)	15 (118)	15.5 (84)
	L2	22.6 (123)	16.6 (115)	12.6 (93)	13 (117)	9 (72)	9 (71)	5.4 (29)
	L3	43.9 (239)	48.3 (334)	49.3 (363)	46.1 (416)	44.4 (356)	48.3 (380)	45 (244)
	L1+L4	3.7 (20)	3.8 (26)	5.3 (39)	4.7 (42)	3.6 (29)	5 (39)	7.6 (41)
	L2+L4	3.3 (18)	3 (21)	2.7 (20)	3.2 (29)	2.9 (23)	1.7 (13)	3.3 (18)
	L3+L4	8.1 (44)	13.5 (93)	14.1 (104)	17.1 (154)	22.2 (178)	21 (165)	23.2 (126)
	Anoperineal lesions % (n)**	17.8 (135)	15.4 (148)	12.1 (122)	11.2 (132)	11.4 (128)	12.2 (141)	12 (91)

* Continuous variables: median (interquartile range); **Categorical variables: % (n) EIMs : Extra intestinal manifestations

Alimentary Pharmacology & Therapeutic

Acknowledgements

The authors wish to thank the interviewing practitioners who collected data: B. Lemaire, N. Guillon, I. Rousseau, A Pétillon, B. Turck, P. Fosse, M. Leconte, C. Le Gallo, C. Dias and S.

Richon. The authors thank all gastroenterologists and other specialists who participated in this study.

Conflict of interest: None

Guarantor of the article: Jean-Frédéric Colombel.


Cédric Ramassamy
email: cedric.ramassamy@univ-nantes.fr

Vincent Ricordel
email: vincent.ricordel@univ-nantes.fr

Oana Cramariuc
email: oana.cramariuc@tut.fi

Bogdan Cramariuc
email: bogdan.cramariuc@citst.ro

LATTICE VECTOR QUANTIZATION FOR THE ANALYSIS OF MOLECULAR DATA

Keywords: computational chemistry, electron density, simplification, tree-structure, lattice, vector quantization, multiresolution, molecular similarity, MQPA, NAPAP, 4-TAPAP

We introduce a novel simplification scheme of molecular data using Tree-Structured Lattice Vector Quantization (TSLVQ). The method, based on the embedding of truncated lattices, permits also hierarchical description of the 3-D volume through a tree-structure. We apply TSLVQ to simplify the electron densities maps of three thrombin inhibitors (MQPA, NAPAP,.

INTRODUCTION

During the last decades signal processing methods have been employed beyond their traditional application domains and have started to contribute significantly to advancements in biology, biochemistry and biomedicine. What was earlier viewed as digital signal processing represents nowadays only a small part of the new concept of signal processing which can be described as the collection of methods for analyzing, manipulating and presenting natural information. One underlying cause of this expansion is the exponentially growing volume of numerical data obtained through modeling and simulation techniques or by employing modern highthroughput experimental investigation tools such as DNA-, protein-, cellular-and antibody-microarrays. One other cause is the substantial effort employed in developing systemic models of processes taking place in living organisms, models which combine individual processes into a larger coherent picture.

The computing power has now increased to a level high enough to process large amounts of molecular data. Nowadays large systems of 10.000 -50.000 atoms are approached computationally. Beyond the mere resources limits of processing such a huge amount of data, the major task is to detect and extract semantic knowledge in the molecular system. Thus, more than data compression, a simplification of the molecular topology can help in this task. Additionally, Critical points (CP) and singularities, computed from the simplified molecular data, become representatives of groups of atoms rather than atoms themselves [START_REF] Leherte | Application of multiresolution analyses to electron density maps of small molecules: Critical point representations for molecular superposition[END_REF].

With specific molecular properties conferred by functional groups (e.g. hydroxyl -OH and carboxyl -COOH) efficient ways to identify these groups are sought [START_REF] Leherte | Application of multiresolution analyses to electron density maps of small molecules: Critical point representations for molecular superposition[END_REF]. This process can lead to simplified representations of the molecules, for e.g. amino acid residues in proteins can be depicted using a lower level representation, i.e. two or three pseudo-atoms representing the whole residue. Such representations are of importance for drug design applications and to overcome structural inaccuracies issued from experimental data such as Xray analysis. In protein studies, for instance, researches focus on the detailed characterization of macromolecular surfaces and topologies [START_REF] Burton | Can Descriptors of the Electron Density Distribution Help To Distinguish Functional Groups?[END_REF]. Synthesis of drug-like molecules is driven by the 3D structural characteristics of molecules [START_REF] De-Alarcón | Modeling shape and topology of low-resolution density maps of biological macromolecules[END_REF].

Previous works on simplification focus on a crystallography-based formalism, whereas a recent trend aims at adapting methods taken from signal processing domain. Exactly, the electron densities are decomposed into successive resolution of wavelets. CP analysis on low wavelet resolution showed relevant results [START_REF] Leherte | Application of multiresolution analyses to electron density maps of small molecules: Critical point representations for molecular superposition[END_REF]. In addition, from the image processing domain, Vector Quantization (VQ) [START_REF] Gersho | Vector quantization and signal compression[END_REF] was adapted to electron density simplification, in particular a neural network method based on a cost function [START_REF] Burton | Can Descriptors of the Electron Density Distribution Help To Distinguish Functional Groups?[END_REF]. The technique of VQ represents the data into a set of reproduction vectors or codewords while keeping the sensible shape of the source density map.

In the present work we employ Vector Quantization, a method typically used in transmission (source coding) and data classification, as a first step in processing of molecular electron densities for further molecular similarity analysis, topological exploration and visualization. The considered electron densities are obtained from quantum mechanical calculations which, as opposed to previous attempts, allow for a full control of the input data quality and a good understanding of the structure-density relationship. The paper is organized as follows: In the first part the Tree-Structured Lattice VQ (TSLVQ) approach is detailed, the second part deals with the simplification of the Electron Density Maps (EDM) using TSLVQ, results are described in the third part and a conclusion is given at the end.

QUANTIZATION

Vector Quantization

A concise description defines quantization as the computation of an approximation for a given signal [START_REF] Gersho | Vector quantization and signal compression[END_REF]. VQ consists in representing, an ordered set of numbers (or vectors) by a more reduced set called the codebook [START_REF] Gersho | Vector quantization and signal compression[END_REF][START_REF] Ricordel | Etude d un schéma de quantification vectorielle algébrique et arborescente. Application à la compression de séquences d'images numériques[END_REF]. VQ is mainly applied to source encoding in order to shrink the volume of data needed to represent the information [START_REF] Ricordel | Etude d un schéma de quantification vectorielle algébrique et arborescente. Application à la compression de séquences d'images numériques[END_REF]. Our method deals with decreasing the granularity of the source representation in order to characterize hierarchically the information.

Lattice Vector Quantization 2.2.1. General

The goal of Lattice VQ (LVQ) is to strongly structure the codebook in order to reduce the computing complexity involved in the codebook design [START_REF] Ricordel | Etude d un schéma de quantification vectorielle algébrique et arborescente. Application à la compression de séquences d'images numériques[END_REF]. LVQ does not require a training step nor an exhaustive search in order to build the codebook. The codewords are points of a truncated lattice regularly scattered into space [START_REF] Conway | Spherepackings, lattices, and groups[END_REF]. The codewords are also the centres of the Voronoi cells, the duals of lattices. The space embedding of the lattices will determine their properties. Unlike in a LBG-based (Linde, Buzo, Gray algorithm) method [START_REF] Gersho | Vector quantization and signal compression[END_REF][START_REF] Linde | An Algorithm for Vector Quantizer Design[END_REF], in LVQ the encoding is related to the coordinates of the vector. Thus, it is only based on rounding and scaling operations. Therefore, the coding is very simple [START_REF] Ricordel | Etude d un schéma de quantification vectorielle algébrique et arborescente. Application à la compression de séquences d'images numériques[END_REF][START_REF] Conway | Spherepackings, lattices, and groups[END_REF].

Lattices

In [START_REF] Conway | Spherepackings, lattices, and groups[END_REF] it is shown that all lattices have not necessarily an optimal space embedding. Moreover, the efficiency of the quantization is directly linked to the geometry of the chosen polytope which describes the Voronoï cell. The choice of the best lattice in the molecular situation leads to an orientation independence feature. The EDMs are three-dimensional matrices, thus we need 3-D lattices.

There are only three lattices for dimension 3 for which fast quantization algorithms are known: Z3 Cubic lattice, D3 the Rhombic dodecahedron and D3* the Truncated Octahedron. Figure 1 shows a 2-D lattice embedding example with the hexagon.

Figure 1: A sub optimal embedding -the hexagonal

Lattice [START_REF] Ricordel | Vector Quantization by packing of embedded truncated lattices[END_REF].

Tree-Structured Vector Quantization

Tree Structured VQ is a gathering of many quantization approaches where the quantization is processed through a decision tree [START_REF] Gersho | Vector quantization and signal compression[END_REF]. Its advantages are reduced computation with the use of simpler sub-codebooks, and a structure adapted to progressive representation.

Tree-Structured Lattice Vector Quantization

Tree Structured LVQ (TSLVQ) [START_REF] Ricordel | Tree-Structured Lattice Vector Quantization[END_REF] aims at using a hierarchical set of embedded lattices which is achieved such as it is possible to embed a lower scale truncated lattice into a cell of the next higher scale truncated lattice. So a scaling factor between consecutive lattices of the hierarchy has to be set (Figure 2). The principles of the TSLVQ are [START_REF] Ricordel | Vector Quantization by packing of embedded truncated lattices[END_REF]: 1. A source vector is projected into a first truncated lattice; 2. To get a finer quantization, another lower scale truncated lattice is embedded into the Voronoï cell where lies the input vector;

3. The previous operation can be repeated.

It is more convenient to deal with the input vector scale than to use several lattices with different scales. The principles of the encoding are shown at Figure 3. Here, the scaling factor b=3 [START_REF] Ricordel | Vector Quantization by packing of embedded truncated lattices[END_REF]. Where we have: the scaling factor F used to project the input vector x into the first truncated lattice:

where ρ is the corresponding packing radius, and L 2max the maximal L 2 norm of x. So, all the inputs are projected into a hyper-sphere whose radius equals b × ρ.

In the normalized space, the scaling factor used to project each translated vector into the next truncated lattice of the hierarchy is b. The reproduction vector of the truncated lattice for the j th level is y j. The final value of the reproduction vector associated with x will be then: with j the level of the quantization. At each step, the same fast quantization algorithm is used. We introduce a novel simplification scheme of molecular data based on TSLVQ. This kind of VQ is very fast thanks to the LVQ and its tree structured codebook. It is, therefore, well adapted to the analysis of the distribution of the source.

METHODS

The data source is a Gaussian cube file [START_REF] Bourke | Gaussian Cube Files specifications[END_REF], the output data are a set of cube files, from the TSLVQ step. Output cube files are snapshots of the different levels of quantization (Figure 4).

Electron densities

The electron density of a chemical entity plays a critical role in explaining and understanding its properties. In fact, it was proven in 1964 by Hohenberg and Kohn that the electron density determines all properties of the ground state of a chemical system, including the ground state energy. Accordingly, the work of Popelier, based on Bader's Atoms in Molecules theory, proves that it is relevant to exploit 3D electron density data by taking advantage of its topology [START_REF] Popelier | Molecular similarity and complementarity based on the theory of atoms in molecules[END_REF][START_REF] Popelier | Integration of atoms in molecules: A critical examination[END_REF]. It has also been shown that the topological analysis of electron density allows simplification of the 3D distribution in reduced representations without losing significant information. In this work, we employ quantum mechanical calculations at the density functional theory level to calculate the EDMs of the compounds described in section 4. The Per-dew-Burke-Ernzerhof exchange-correlation functional to-gether with a TZVP basis set were used as implemented in the Gaussian 09 computational package www.gaussian.com.

Vector Quantization of the electron densities.

Data loading

A Gaussian cube file describes the 3-D maps of EDMs or of electrostatic potentials but the present work focuses on EDMs only. The latter stands for a grid that slices the space of the studied molecule. Three data sets of Gaussian cube [START_REF] Bourke | Gaussian Cube Files specifications[END_REF] files were used with the new TSLVQ method. We focused our study on three anticoagulants, thrombin inhibitors (MQPA, NAPAP, 4-TAPAP), which were previously used by Leherte as test systems for the wavelet based simplification [START_REF] Leherte | Application of multiresolution analyses to electron density maps of small molecules: Critical point representations for molecular superposition[END_REF]. Each of the molecules is described in three resolutions of electron densities through their dedicated cube file. Given the amount of data, the EDMs need to be batch processed. Flaws of the data slicing should introduce sets of non-trivial errors. Indeed, the size of the batch directly impacts the precision of the quantization.

Quantization with TSLVQ

TSLVQ with cube files source needs two parameters: scaling (or boxing) factor and quantization level. There are two ways of constructing the tree: a) "Merging" way consists in building the whole tree in a first step. Then the tree has to be fully browsed in order to merge leaves successively. b) "Splitting" way constructs the tree progressively at each iteration of the quantization. At the first level all points are combined in one single node, this is the most degraded representation of the source, and at the next levels, if the processing rule requires it; a leaf is splitted in a range of children leaves and so on. Using a higher number of quantization levels, we get a more detailed (and voluminous) description of the molecule.

For obvious reasons of resources saving, we have implemented the splitting way. The coordinates of the density are rounded to be aligned on the current lattice reproduction vectors. Once all data are processed a mean is calculated between all the densities of the current codeword. The scaling factor controls the number of possible children for the current node (e.g.: for the cubic lattice, if b=3 in 3-D → 3 3 = 27 children possible for each node but only those whose dual Voronoï cell contains densities are created). The existing lattice is rescaled for the current child node, and all the densities that belong to the parent node are quantized. The process stops when it reaches the quantization level given in parameter. The quantization tree resulting is the input of the next step.

Visualization and analysis of the results

The method produces as result a set of cube files (one file per level). This allows the visualization and analysis with common computational chemistry tools (e.g.: Gaussian [START_REF] Bourke | Gaussian Cube Files specifications[END_REF]).

RESULTS

The compounds have a star shape with three branches completed by a cyclic substructure bound to sulfonyl function, piperidine ring, or amidino group (Figure 5).

Figure 5: 2-D structures of compounds MQPA (A), NAPAP (B), and 4-TAPAP (C)

In Figures 678the EDMs are visualized using a blue (low)to-red (high) colour map and a transparency coefficient proportional to the voxel value. When analyzing Figures 678, it is important to keep in mind that the EDMs obtained by TSLVQ have a level of detail proportional to the number of quantization steps. Subsequently, with 5 levels of quantization, the obtained EDMs are closest to the source data. In each case the oxygen atoms of the sulfony, carbonyl and/or carboxy groups lead to peaks in the EDMs obtained after 5 levels of quantization. Such groups should thus easily match together in a superposition algorithm using a density based similarity measure. Other atoms in the functional groups like for e.g. nitrogen and sulfur (see Figure 5) are not detected at this resolution. The terminal amidino groups become visible in the third quantization level in which they bear clear EDM peaks. At the same time also the other nitrogen atoms like the ones in the hetero-cycles become visible. Independent on the quantization level no peaks are identifiable with the alkyl part of MQPA implying that chemical groups composed of only C atoms can remain undetected. We do expect however that aromatic rings connected to functional groups to produce clear peaks in the EMS due to the disruption in πconjugation. Further studies are needed to justify this assumption. The order of the peak density values can be approximately summarized as: sulfonyl ≥ (CO(O) > amidino/N > alkyl chain.

The above analysis, although performed at a qualitative level, agrees well with the results obtained from employing wavelet quantization [START_REF] Leherte | Application of multiresolution analyses to electron density maps of small molecules: Critical point representations for molecular superposition[END_REF]. Some differences exist though like for e.g. the identification of the sulfonyl groups which is done in our case based on the peaks created by the oxygen atoms rather than by sulfur. This is why in our case sulfonyl groups become less distinguishable from other oxygen bear-ing functional groups like carboxyl. Nevertheless, we can conclude that the same atoms or functional groups can be identified at certain quantization levels of all molecules considered in this study which could in principle make the theoretical assignment of a chemical group to a peak possible. However, this assertion needs to be validated through further work implementing for e.g. critical points analysis and graph representation of the electron density topology.

Figure 2 :

 2 Figure 2: Hierarchical set corresponding to the cubic lattice.Here, the scaling factor b=3[START_REF] Ricordel | Vector Quantization by packing of embedded truncated lattices[END_REF].

Figure 3 :

 3 Figure 3: The principle of TSLVQ [8]: x is the source vector, y n the successive reproduction vector, F the first scaling factor and b the next scaling factors.

Figure 4 :

 4 Figure 4: General scheme of the method.

Figure 6 :

 6 Figure 6: 3-D MQPA EDM (resolution 113×96×93) with 5, 3 and 2 levels of TSLVQ (Z3, b2), respectively.

Figure 7 :

 7 Figure 7: 3-D NAPAP EDM (resolution 120×98×85) with 5, 3 and 2 levels of TSLVQ (Z3, b2), respectively.

Figure 8 :

 8 Figure 8: 3-D 4-TAPAP EDM (resolution 119×94×89) with 5, 3 and 2 levels of TSLVQ (Z3, b2), respectively.

CONCLUSION

In this paper we showed that TSLVQ is well adapted to the simplification of EDMs. Using a cubic lattice we obtain a set of descriptions hierarchically organized in levels (with one cube file per level): the deeper the level the finer the resolution. Result are presented for three molecules (MQPA, NAPAP, 4-TAPAP) and compared qualitatively with the ones obtained by Leherte [1]. The next step will be to include the computation of critical points directly into the 3-D hierarchical description (the tree-structure).