Journal Pre-proof Lower Level of Plasma 25-Hydroxyvitamin D in Children at Diagnosis of Celiac Disease Compared with Healthy Subjects: A Case-Control Study Elena Lionetti, PhD, Tiziana Galeazzi, PhD, Vera Dominjanni, MD, Ilaria Acquaviva, MD, Giulia N. Catassi, MD, Mario Iasevoli, MD, Basilio Malamisura, MD, Carlo Catassi PII: S0022-3476(20)31125-2 DOI: https://doi.org/10.1016/j.jpeds.2020.08.089 Reference: YMPD 11756 To appear in: The Journal of Pediatrics Received Date: 16 May 2020 Revised Date: 17 July 2020 Accepted Date: 28 August 2020 Please cite this article as: Lionetti E, Galeazzi T, Dominjanni V, Acquaviva I, Catassi GN, Iasevoli M, Malamisura B, Catassi C, Lower Level of Plasma 25-Hydroxyvitamin D in Children at Diagnosis of Celiac Disease Compared with Healthy Subjects: A Case-Control Study, The Journal of Pediatrics (2020), doi: https://doi.org/10.1016/j.jpeds.2020.08.089. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Elsevier Inc. All rights reserved. Lower Level of Plasma 25-Hydroxyvitamin D in Children at Diagnosis of Celiac Disease Compared with Healthy Subjects: A Case-Control Study Elena Lionetti, PhD1, Tiziana Galeazzi, PhD1, Vera Dominjanni, MD1, Ilaria Acquaviva, MD1, Giulia N. Catassi, MD1, Mario Iasevoli, MD2, Basilio Malamisura, MD2, and Carlo Catassi,1,3 1 2 Department of Pediatrics, Marche Polytechnic University, 60123, Ancona, Italy; Pediatric Unit and Center for Celiac Disease - University Hospital of Salerno, Campus of Cava de' Tirreni, Italy; Center for Celiac Research, Mass General Hospital for Children, Boston, 02114, MA, USA ro of 3 Key-words: Vitamin D; deficiency; pediatric celiac disease; controls. -p Abbreviations: celiac disease (CD); 25-hydroxyvitamin D (25-OHD); gluten-free diet (GFD); lP mass Index (BMI); standard deviation (SD). re European Society for Pediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN); body na C.C. served as a consultant for Dr Schär SPA. The other authors declare no conflicts of interest. ur Corresponding author Elena Lionetti, PhD, Department of Pediatrics, Marche Polytechnic University, Via F Corridoni 11, Ancona, Italy. Telephone: Jo 60123, +39 0715962360, Fax: +39 07136281. E-mail: m.e.lionetti@univpm.it.com. 1 Objective: To evaluate the vitamin D status of children with a new diagnosis of celiac disease compared with healthy controls. Study design: This was a case-control study. Cases were consecutive children with newly diagnosed celiac disease. Controls were healthy children matched for age, sex, ethnicity, and month of blood testing. Plasma 25-hydroxyvitamin D (25-OHD) was measured as the index of vitamin D nutritional status. The Student t test were used for comparisons. Differences in frequencies were evaluated with the χ2 test. Associations between variables were estimated by calculating Pearson correlation coefficients. Results: 131 children with celiac disease were enrolled (62% females, mean age 8.1±1.1 years). ro of The control group included 131 healthy children (62% females, mean age 8.2±1.2). All were of European origin. Plasma 25-OHD levels were significantly lower in patients than in control subjects (25.3±8.0 and 31.6±13.7 ng/ml; P < .0001). The percentage of children with vitamin D deficiency -p (<20 ng/ml) was significantly higher in celiac disease children as compared with controls (31% vs re 12%; p<0.0001). The concentration of 25-OHD was significantly lower in patients than in controls during summer (p<0.01), and autumn (p<0.0001). lP Conclusion: In this case-control study, at diagnosis, children with celiac disease showed lower levels of plasma 25-OHD compared with healthy subjects. Vitamin D status should be checked at Jo ur na diagnosis of celiac disease, particularly during summer and fall months. 2 Celiac disease is a systemic immune-mediated disorder caused by the ingestion of gluten-containing grains in genetically susceptible individuals.1 It is one of the most common lifelong disorders, affecting approximately 1% of the population worldwide.2 The prevalence of the disease has increased in developed countries over recent decades; this finding points to the role of one or more possible environmental triggers other than gluten.3 Vitamin D and CD may be connected by a two-way relationship. It has been suggested that earlylife vitamin D deficiency may contribute to the development of several autoimmune diseases, including CD.4-6 Vitamin D has been examined as a potentially protective factor because it has an active role in the regulation of the immune system.4 The variable incidence of CD according to the ro of season of birth could be explained, at least in part, by seasonal fluctuations in vitamin D levels.7-12 A relationship between sun exposure and CD pathogenesis has also been suggested by the finding of a higher frequency of CD in individuals living at northern latitudes.13 On the other hand, vitamin -p D deficiency could be secondary to the intestinal malabsorption caused by CD. Low-levels of re vitamin D may be responsible for the alteration of bone metabolism, resulting in low bone mineral density and high fracture risk, that are well known complications of CD.14 However, indications for lP vitamin D measurement in CD patients and the need of supplementation are still controversial and vary among different scientific societies.5 na Recent studies reported conflicting results about serum vitamin D levels in CD patients at diagnosis ur and on a gluten-free diet.14-25 Previous studies have mostly been performed in adults, with small sample sizes, lack of a control group, retrospective methods of recording, and rarely took into Jo account the possible cofactors influencing vitamin D level, such as month of blood testing, ethnicity or type of diet., we aimed to evaluate the vitamin D status of children with newly diagnosed CD by a large case-control study. Methods Study design and population This study was conducted at the Center for Celiac Disease of the Polytechnic University of Marche in Ancona, Italy, from October 2018 to January 2020. Ancona is located in central Italy, and, on average has around 2,220 sunshine hours per year. July is the most sunny month, and November has the lowest amount of sunshine. Cases were consecutive children aged 5 to 11 years with newly diagnosed CD (according to the European Society for Pediatric Gastroenterology, Hepatology and Nutrition – ESPGHAN criteria)26, investigated before starting treatment with the gluten-free diet (GFD). Controls were 3 healthy children not affected with CD (on the basis of a negative result of the IgA class antitransglutaminase test), participating in a previously described mass screening program for CD,27 and matched for age, sex, ethnicity, and month of blood testing. Exclusion criteria were any of the following: (1) associated chronic conditions (including type 1 diabetes or inflammatory bowel disease) or genetic disorders (Down or Turner syndrome); (2) special diet (vegetarian, vegan, milkfree diet, GFD); (3) use of vitamin D supplements during the last 6 months. Clinical presentation of CD The pattern of clinical presentation of CD was defined as "classical," if the patient presented the classical picture of malabsorption (diarrhea, weight loss and abdominal distension), “atypical”, in ro of presence of other clinical manifestations including iron deficiency, short stature, aphthous stomatitis, recurrent abdominal pain, etc, or "silent," in individuals apparently asymptomatic, diagnosed as part of the screening program.1 -p Anthropometric measurements re For all children, anthropometric measurements were collected by the same-trained operator. Body weight was measured using the same mechanical balance (SECA 200); height was measured to the lP nearest 5 mm using a stadiometer (SECA 220). Body mass Index (BMI) was calculated from weight and height (Kg/m2). The BMI values were categorized as underweight, normal weight, overweight na and obesity, using the standards of the International Obesity Task Force.28 ur Vitamin D assessment The plasma concentration of 25-hydroxy vitamin D (25-OHD) was quantitatively determined by Jo chemiluminescent-immunoassay (CLIA) (LIAISON, DiaSorin, Italy). Vitamin D status was defined according to the ESPGHAN criteria as follows: severe deficiency (<10 ng/ml), deficiency (<20 ng/ml), and sufficiency/adequacy (≥20 ng/ml).29 Outcome measures Primary outcome measure was the plasma 25-OHD levels in patients and controls and the percentage of children with vitamin D deficiency (<20 ng/ml) in both groups. Secondary outcome measures were the correlation between plasma 25-OHD levels and age in both groups; the percentage of children with vitamin D deficiency according to BMI and to CD clinical presentation; the comparison of 25-OHD levels during the four seasons between patients and controls. Statistical analyses Results are reported as means ± standard deviation (SD). The Student t test (for normally distributed data) or the Mann–Whitney U test (for non-normally distributed data) were used for comparisons between patients with CD and control subjects. Differences in frequencies were evaluated with the 4 χ2 test. Associations between variables were estimated by calculating Pearson correlation coefficients. Significance was accepted for p values <0.05. Data were analyzed using SPSS (Chicago, IL, USA). The Institutional Review Board of the Polytechnic University of Marche approved this study protocol. Results Study population Overall, 137 children with celiac disease were contacted for participation but 3 patients (2%) ro of refused to participate, and 3 were excluded because of an underlying disease (n=1 affected with diabetes) or a special diet (n=2 on a milk-free diet). Among the 131 participants, there were 81 females (62%), with a mean age of 8.1 (± 1.1). The control group included 131 healthy children, 81 -p females (62%), with a mean age of 8.2 (±1.2). All cases and controls were of European origin. re Clinical and demographical characteristics of the two groups are reported in Table I (available at www.jpeds.com). lP Clinical presentation of CD Twenty-one (16%) children were diagnosed because of typical intestinal manifestations (classical na form) of CD, 46 (35.1%) children presented with atypical manifestations of CD, and 64 (48.9%) Anthropometric data ur children were asymptomatic and diagnosed after a screening program. Jo No differences were found between children with celiac disease and controls in anthropometric measurements. The mean BMI was 17.4 in children with celiac disease and 17.1 in the control group and the prevalence of underweight, overweight and obesity was similar in both groups (Table 1). Vitamin D status Table 2 shows 25-OHD results in CD children and controls. Overall, plasma 25-OHD levels were significantly lower in patients than in control subjects (25.3±8.0 vs 31.6±13.7 ng/ml; p<0.0001) (Figure 1). The percentage of children with vitamin D deficiency was significantly higher in the CD group as compared with controls (31.2% vs 12.2%; p<0.001, Table 2). There was no significant correlation between plasma 25-OHD levels and age, both in celiac (r=0.09; p=0.3) and control group (r=0.14; p=0.09). The 25-OHD status did not change according to CD clinical presentation (classical, atypical or silent form). In both groups, there was no difference in plasma 25-OHD levels according to sex, mean BMI, and classes of BMI (Table 2). 5 Figure 2 shows the seasonal fluctuation in plasma 25-OHD level, with significantly higher values during summer and autumn (from June to November) as compared with winter and spring (from December to May) in both patients (p<0.0001) and control subjects (p<0.0001). However, the concentration of 25-OHD was significantly lower in patients than in controls during summer (p<0.01), and autumn (p<0.0001) (Figure 3). Discussion Nutritional status of D vitamin is best represented by the circulating concentration of plasma 25OHD.30 The present case-control study shows that the plasma 25-OHD level is lower in children ro of with celiac disease at the time of diagnosis as compared with a control group of healthy children. The percentage of CD children with vitamin D deficiency was 31%, significantly higher than healthy controls (12%). -p Data from literature on the serum level of 25-OHD in CD patients at either diagnosis or on a GFD re are controversial.14-25 adult patients with celiac disease at diagnosis show 25-OHD deficiency in 20%-59% of cases,14-17 with case-control studies showing a lower level of vitamin D in CD patients lP at diagnosis compared with controls.14,18-19,25 Zanchi et al showed lower levels of 25-OHD in 54 children with celiac disease as compared with 60 controls.20 However, Lerner et al showed vitamin na D deficiency (<20 ng/ml) in 17/51 (33%) Israeli children with celiac disease at diagnosis as compared with 30% of Israeli children with recurrent abdominal pain, and concluded that ur hypovitaminosis D is common, irrespective of CD.21 In 59 Spanish children and 22 adults with CD Jo at diagnosis, vitamin D deficiency was found in 16% and 54% of cases, respectively, suggesting that age is the major factor affecting vitamin D levels.21 Margoni et al assessed vitamin D levels in 45 CD patients at diagnosis and 36 patients after one year of a GFD, showing a deficiency (<8 ng/ml) in 35% of both groups.22 Similarly, Wessels et al, by a retrospective study, showed a 25OHD deficiency (<20 ng/ml) in 8/30 (27%) children with celiac disease and in 7/28 (25%) children with celiac disease after 5 years of a GFD, concluding that hypovitaminosis D is not directly linked to CD, but merely represents its frequency in the general population.24 Worth noting, most previous studies had several limitations, such as retrospective study design; small sample size lack of control on possible cofactors influencing vitamin D level, such as type of diet, type of laboratory test used in the study, ethnicity, and most importantly the month of blood testing; and different definition of vitamin D deficiency (<8 ng/ml or <20 ng/ml). Our study evaluated 25-OHD levels in a large group of children with celiac disease at diagnosis as compared with a control group of healthy children matched for age, ethnicity, sex, and month of 6 blood testing, all variables that may affect vitamin D status. We defined vitamin D deficiency as 25OHD levels <20 ng/ml, according to the recent ESPGHAN recommendations.26 Our findings of lower 25-OHD levels and significantly higher frequency of 25-OHD deficiency in children with celiac disease as compared with controls, clearly demonstrates that low levels of vitamin D is an important clue no correlation was found between vitamin D levels and age in both groups, showing that the effect of age may be negligible in children. Based on current literature, indications for 25-OHD measurement in CD patients and the need for supplementation vary among different societies. The North American Society for Pediatric Gastroenterology, Hepatology and Nutrition in 2016 recommended serum vitamin D evaluation in ro of children with celiac disease at diagnosis and annually after symptom resolution and normalization of CD serology,31 while the ESPGHAN does not give any recommendation. Our findings are in favor of testing vitamin D status in children with celiac disease at diagnosis, and to supplement it -p until normalization. Indeed, vitamin D deficiency is considered one of the main causes of low bone re mineral density and consequent risk of fractures.5 Vitamin D has other significant extra-skeletal effects, with a possible role in several conditions, including infectious diseases, asthma or levels in children and adolescents.5 lP cardiovascular health. Therefore, it is highly recommended to maintain normal plasma 25-OHD na The pathogenesis of vitamin D deficiency in CD patients remains unclear. Traditionally, it is ur thought to result from fat-soluble vitamin malabsorption.32 However, an alternative, intriguing hypothesis is that early-life vitamin D deficiency could be primarily involved in the pathogenesis of Jo CD rather than being a consequence of the disease.32 In our study, by comparing 25-OHD levels in celiac patients and controls during the four seasons, we found a significant difference between the two groups only during summer and autumn, when the endogenous production of vitamin D after sun exposure increased in controls much more than in patients. This finding suggests that vitamin D deficiency may be the result of an altered pathway of endogenous production. Meta-analyses of genome-wide association studies have identified single-nucleotide polymorphisms in genes involved in cholesterol synthesis, hydroxylation, and vitamin D transport that affect vitamin D status.33 Polymorphism in genes encoding the vitamin D binding protein or enzymes involved in vitamin D synthesis (DHCR7, CYP2R1 and CYP27B1) may result in the reduced production of vitamin D after sun exposure observed in CD, as well as in other autoimmune diseases.33-35 The composition of gut microbiota can influence the expression of vitamin D receptor, vitamin D binding protein and activator enzymes.35 Patients with CD show intestinal dysbiosis36 that may in 7 turn influence the endogenous production of vitamin D and, almost in part, explain the lower 25OHD levels observed in celiac disease. Studies have reported low serum vitamin D levels in several autoimmune diseases, while higher vitamin levels seem to be inversely correlated with the incidence of them.6,37 Animal models have shown that vitamin D suppresses several autoimmune pathways including the Th1, B cells, Th-17, dendritic cell, and co-stimulatory molecule systems.37 The main limitations of our study are the cross-sectional design, with no information available on plasma 25-OHD levels before diagnosis, and the lack of data on other biomarkers of bone metabolism, such as parathyroid hormone, total calcium, phosphate, 1,25-OHD level. However, ro of current guidelines recommend the measurement of 25-OHD in blood as the preferred test for the assessment of vitamin D status,38 based on numerous studies demonstrating significant associations of 25-OHD with biochemical, functional and clinical indices, such as parathyroid hormone, -p neuromuscular function, bone mineral density and fracture risk.30,39 Furthermore, 25-OHD re represents the sum of vitamin D intake and dermal production, and because of a long half-life of 2– 3 weeks, serum levels vary very little within short periods. Therefore, 25-OHD is considered the lP best biomarker of vitamin D status.30,39 Further limitations of the present study are the lack of information on the effect of the GFD on vitamin D levels, and on the use of UV-blockers on the na skin of enrolled children, although there is no reason why CD patients (recruited at diagnosis) ur should have used more UV-blockers than controls. In conclusion, children with celiac disease at diagnosis showed lower 25-OHD level compared with Jo controls. Future research is required to clarify the correct vitamin D dosing regimen to treat vitamin D deficiency in children with celiac disease, and to prospectively evaluate the effect of a GFD on vitamin D status. 8 References 1. Fasano A, Catassi C. Celiac disease. N Engl J Med 2012;367:2419-26. 2. Green PH, Cellier C. Celiac disease. N Engl J Med 2007;357:1731-43. 3. Lionetti E, Catassi C. New clues in celiac disease epidemiology, pathogenesis, clinical manifestations, and treatment. Int Rev Immunol 2011;30:219-31. 4. Rewers M, Ludvigsson J. Environmental risk factors for type 1 diabetes. Lancet 2016;387:2340-8. 5. Saggese G, Vierucci F, Prodam F, Cardinale F, Cetin I, Chiappini E, et al. Vitamin D in pediatric age: consensus of the Italian Pediatric Society and the Italian Society of Preventive ro of and Social Pediatrics, jointly with the Italian Federation of Pediatricians. Ital J Pediatr 2018;44:51. 6. Aranow C. Vitamin D and the immune system. J Investig Med 2011;59: 881-6. -p 7. Ivarsson A, Hernell O, Stenlund H, Persson LA. Children born in the summer have re increased risk for coeliac disease. J Epidemiol Community Health 2003;57:36-9. 8. Lewy H, Meirson H, Laron Z. Seasonality of birth month of children with celiac disease lP differs from that in the general population and between sexes and is linked to family history and environmental factors. J Pediatr Gastroenterol Nutr 2009;48:181-5. na 9. Lebwohl B, Green PH, Murray JA, Ludvigsson JF. Season of birth in a nationwide cohort of ur coeliac disease patients. Arch Dis Child 2013;98:48-51. 10. Tanpowpong P, Obuch JC, Jiang H, McCarty CE, Katz AJ, Leffler DA, et al. Multicenter Jo study on season of birth and celiac disease: evidence for a new theoretical model of pathogenesis. J Pediatr 2013;162:501-4. 11. Capriati T, Francavilla R, Castellaneta S, Ferretti F, Diamanti A. 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Aliment Pharmacol Ther 2000;14:35-43. 16. Chakravarthi SD, Jain K, Kochhar R, Bhadada SK, Khandelwal N, Bhansali A, et al. Prevalence and predictors of abnormal bone mineral metabolism in recently diagnosed adult celiac patients. Indian J Gastroenterol 2012;31:165-70. 17. Posthumus L, Al-Toma A. Duodenal histopathology and laboratory deficiencies related to bone metabolism in coeliac disease. Eur J Gastroenterol Hepatol 2017;29:897-903. 18. Corazza GR, Di Sario A, Cecchetti L, Tarozzi C, Corrao G, Bernardi M, et al. Bone mass ro of and metabolism in patients with celiac disease. Gastroenterology 1995;109:122-8. 19. Corazza GR, Di Sario A, Cecchetti L, Jorizzo RA, Di Stefano M, Minguzzi L. Influence of pattern of clinical presentation and of gluten-free diet on bone mass and metabolism in adult -p coeliac disease. Bone 1996;18:525-30. disease. J Pediatr 2008;153:262-5. re 20. Zanchi C, Di Leo G, Ronfani L, Martelossi S, Not T, Ventura A. Bone metabolism in celiac lP 21. Lerner A, Shapira Y, Agmon-Levin N, Pacht A, Ben-Ami Shor D, López HM, et al. The clinical significance of 25OH-vitamin D status in celiac disease. Clin Rev Allergy Immunol na 2012;42:322-30. ur 22. Margoni D, Chouliaras G, Duscas G, Voskaki I, Voutsas N, Papadopoulou A, et al. Bone health in children with celiac disease assessed by dual x-ray absorptiometry: effect of Jo gluten-free diet and predictive value of serum biochemical indices. J Pediatr Gastroenterol Nutr 2012;54:680-4. 23. Erdem T, Ferat Ç, Nurdan YA, Halime E, Muhammed Selçuk S, Hamza K, et al. Vitamin and mineral deficiency in children newly diagnosed with celiac disease. Turk J Med Sci 2015;45:833-6. 24. Wessels MM, van Veen II, Vriezinga SL, Putter H, Rings EH, Mearin ML. Complementary serologic investigations in children with celiac disease is unnecessary during follow-up. J Pediatr 2016;169:55-6. 25. 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J Pediatr Gastroenterol Nutr 2013;56:692-701. 30. Herrmann M, Farrell CL, Pusceddu I, Fabregat-Cabello N, Cavalier E. Assessment of vitamin D status - a changing landscape. Clin Chem Lab Med 2017;55:3-26. -p 31. Hill ID, Fasano A, Guandalini S, Hoffenberg E, Levy J, Reilly N, et al. NASPGHAN Gastroenterol Nutr 2016;63:156-65. re clinical report on the diagnosis and treatment of gluten-related disorders. J Pediatr lP 32. Tanpowpong P, Camargo CA. Early-life vitamin D deficiency and childhoodonset coeliac disease. Public Health Nutr 2014;17:823-6. na 33. Wang TJ, Zhang F, Richards JB, Kestenbaum B, van Meurs JB, Berry D, et al. Common 2010;376:180-8. ur genetic determinants of vitamin D insufficiency: a genome-wide association study. Lancet Jo 34. San-Pedro JI1, Bilbao JR, Perez de Nanclares G, Vitoria JC, Martul P, Castaño L. Heterogeneity of vitamin D receptor gene association with celiac disease and type 1 diabetes mellitus. Autoimmunity 2005;38:439-44. 35. Singh P, Kumar M, Al Khodor S. Vitamin D Deficiency in the Gulf Cooperation Council: Exploring the Triad of Genetic Predisposition, the Gut Microbiome and the Immune System. Front Immunol 2019;10:1042. 36. Cenit MC, Olivares M, Codoñer-Franch P, Sanz Y. Intestinal microbiota and celiac disease: cause, consequence or co-evolution? Nutrients 2015;7:6900-23. 37. Dankers W, Colin EM, van Hamburg JP, Lubberts E. Vitamin D in Autoimmunity: Molecular Mechanisms and Therapeutic Potential. Front Immunol 2017;7:697. 38. Holick MF, Binkley NC, Bischoff-Ferrari HA, Gordon CM, Hanley DA, Heaney RP, et al. Evaluation, treatment, and prevention of vitamin D deficiency: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab 2011;96:1911-30. 11 39. Tsuprykov O, Chen X, Hocher CF, Skoblo R, Lianghong Yin, Hocher B. Why should we Jo ur na lP re -p ro of measure free 25(OH) vitamin D? J Steroid Biochem Mol Biol 2018;180:87-104. 12 Figure legends Figure 1. Box plot of plasma 25-hydroxyvitamin D (25-OHD) levels in patients with celiac disease and in control group. Data are presented as box plots showing the 10th, 25th, 50th (median), 75th and 90th percentiles. Figure 2. Box plot of plasma 25-hydroxyvitamin D (25OHD ) levels in patients with celiac disease (grey) and in control subjects (white) according to the month of blood testing. Data are presented as box plots showing the 10th, 25th, 50th (median), 75th and 90th percentiles. Months are expressed as numbers. In the month of August (n. 8) only two celiac disease patients were diagnosed and two ro of matched controls were enrolled. Figure 3. Box plot of plasma 25-hydroxyvitamin D (25OHD) levels in patients with celiac disease (grey) and in control subjects (white) according to the season of blood testing. Data are presented as -p box plots showing the 10th, 25th, 50th (median), 75th and 90th percentiles. Season are expressed as Jo ur na lP re number: 1= Winter; 2= Spring; 3= Summer; 4= Autumn. 13 Table 1; online. Demographical and clinical characteristics of children with celiac disease and healthy controls. Celiac disease (n=131) Healthy p controls (n=131) Mean age (range) - years 8.1 (5-11) 8.2 (5-11) NS Female gender – no. (%) 81 (62) 81 (62) NS 131 (100%) NS 36 (27.5) 36 (27.5) NS 35 (26.7) 35 (26.7) NS 22 (16.8) 22 (16.8) NS 38 (29) 38 (29) NS 17.4 (2.9) 17.1 (2.4) NS Underweight 90 (68.7) 100 (76.3) NS Normal weight 41 (31.3) 29 (22.1) NS Overweight 0 2 (1.6) NS Obese 0 0 NS ro of Origin – no (%) 131 (100%) -p European Season of blood testing – no. (%) re Winter Spring lP Summer ur Jo Mean Body mass index (SD) na Autumn Body mass index class – no. (%) Clinical presentation – no. (%) Typical 21 (16) Atypical 46 (35) Silent 64 (48.9) Table 2. Plasma 25-hydroxyvitamin D (25-OHD) levels in children with celiac disease and healthy controls. Celiac disease (n=131) Insufficiency Deficiency 2 (1.5) 0.5a 41 (31.2) 16 (12.2) <0.001a 90 (68.7) 113 (86.2) 0.001a 13 (32) 5 (17.2) NSb,c 28 (31.1) 13 (13) NS b,c 0 0 NS b,c 0 0 NS b,c re lP 25-OHD deficiency according to BMI class – no. (%) Underweight na Normal weight Overweight Jo ur Obese (n=131) 0 -p Adequacy controls <0.0001a ro of 25-OHD status – no. (%) p 31.6 (13.7) 25.3 (8) Mean 25-OHD – ng/ml (SD) Healthy 25OHD deficiency according to CD clinical presentation– no. (%) Typical 7 (33.3) NSd Atypical 15 (32.6) NSd Silent 19 (29.7) NSd a: comparison between celiac disease and healthy controls; b: comparison of 25-OHD levels between BMI classes in celiac disease; c: comparison of 25-OHD levels between BMI classes in healthy controls; d: comparison of 25-OHD levels according to clinical presentation in celiac disease. ro -p re lP na ur Jo of ro -p re lP na ur Jo of ro -p re lP na ur Jo of