Journal of Pediatric Gastroenterology and Nutrition Publish Ahead of Print

DOI: 10.1097/MPG.0000000000001568

Reduced Bone Mineral Density in Children with Screening-detected Celiac Disease

Sara Björck, M.D., Ph.D.1, Charlotte Brundin M.Sc.1, Magnus Karlsson, M.D., Ph.D.2,

Daniel Agardh, M.D., Ph.D.1

Affiliations: 1Unit of Diabetes and Celiac Disease, Department of Clinical Sciences, Malmö,

Lund University, Sweden. 2 Clinical and Molecular Osteoporosis Research Unit, Department of

Clinical Sciences and Orthopedics, Lund University, Sweden.

Corresponding author: Daniel Agardh, Unit of Diabetes and Celiac Disease, CRC building 91

floor 10, Jan Waldenströms gata 35, S-205 02 Malmö, Sweden. Telephone number:

+4640332237 daniel.agardh@med.lu.se

Sources of support: The study was supported by the Faculty of Medicine, Lund University and

the Skåne County Council of Research and Development.

Disclosure of funding: All authors declare no financial relationships relevant to this work.

Conflict of Interest: All authors declare no conflict of interest.

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Word count: 3142

Number of tables: 4

Suppl Table 1. Supplemental table

Supplemental digital content is available for this article. Direct URL citations appear in the

printed text, and links to the digital files are provided in the HTML text of this article on

the journal’s Web site (www.jpgn.org).

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Abstract

Objectives: To assess if bone mass and metabolism are impaired in genetically at risk children

with screening-detected celiac disease.

Methods: Included were 71 children with screening-detected celiac disease diagnosed at

10.0+0.7 (mean+SD) years and 142 matched controls as well as 30 children with screening-

detected celiac disease diagnosed at 3.3±0.4 years of age presently on a gluten-free diet for

6.9+1.1 years and 60 matched controls. All participants were assessed for bone mineral density

(BMD) of total body and spine by Dual X-ray absorptiometry, serum 25(OH) vitamin D3,

parathyroid hormone (PTH), IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-13, IL-15,

IFNγ, and TNFα.

Results: At diagnosis, screening-detected celiac disease children as compared to controls had a

mean -0,03 g/cm2 reduced BMD of both total body and spine (p=0.009 and p=0.005

respectively), a mean -11.4 nmol/L lower level of 25(OH) vitamin D3 (p<0.001) and a mean

+1.0 pmol/L higher PTH level (p<0.001). Systemic levels of the cytokines IL-1β, IL-6, IL-8, IL-

10, IL12p70, IL13, and TNFα were all increased in screening-detected celiac disease as

compared to controls (p<0.001). No difference in BMD, 25(OH) vitamin D3, PTH and cytokine

levels were detected in children on a gluten-free diet compared to controls.

Conclusions: Children with screening-detected celiac disease have reduced BMD, lower levels

of vitamin D3, higher levels of PTH and signs of systemic inflammation compared with controls.

These differences were not found in celiac disease children on a gluten-free diet, indicating that

children with screening-detected celiac disease benefit from an early diagnosis and treatment.

Key words: Autoantibody, bone mass, HLA, inflammation, tissue transglutaminase.

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What is known

 Osteopenia and osteoporosis are long-term complications associated with untreated celiac

disease.

 A gluten-free diet heals reverses bone mineral changes in symptomatic celiac disease

patients.

 Majority of celiac disease patients are identified in screenings.

What is new

 Children with screening-detected celiac disease have lower bone mineral density at

diagnosis compared with healthy controls at 10-years of age.

 Children with screening-detected celiac disease on a gluten-free diet from 3 years of age

have normal bone mineral density at 10-years of age.

 Children with screening-detected celiac disease benefit from an early diagnosis and

treatment in order to restore bone health.

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Introduction

It has long been known that many adult celiac disease patients have reduced bone mineral

density (BMD) and risk for long-term complications such as osteoporosis with our without
1-5,6
associated fractures . The reasons are probably multifactorial, such as malabsorption with

hypocalcemia, hypovitaminosis D and secondary hyperparathyroidism, low physical activity

because of fatigue, autoimmune effects, and inflammation 7. It is also known that low BMD is

found in children with clinically-detected celiac disease at diagnosis 8, and that a gluten-free diet

reverses these changes 9. But, since the level of BMD does not correlate with the clinical
10,11
symptoms and the vast majority of individuals with celiac disease are only identified in
12
screenings by tissue transglutaminase autoantibodies (tTGA) , it is debated if children

identified through screenings, and therefore often having asymptomatic or subclinical disease,

also have low BMD. Neither is it recognized if a gluten-free diet in these children influences the

development of BMD.

To answer these questions, we used the Celiac Disease Prediction in Skåne (CiPiS) study, a

prospective population based cohort study with the aim to identify celiac disease by repeated

screening using tTGA in HLA genotyped children 13. Children participating in the CiPiS follow-

up study at 9 years of age were invited for measurement of BMD and bone related metabolism.

The aim of this case-control study was to evaluate if BMD is affected in children with screening-

detected celiac disease with and without a gluten-free diet. We hypothesized that children

diagnosed with screening-detected celiac disease have lower BMD as compared to their matched

controls and that children diagnosed at an earlier age already on a gluten-free diet do not present

with bone deterioration.

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Methods

Population based screening for celiac disease

Between June 2004 and August 2007, we invited 13,860 HLA-genotyped 3-year old children,
14
included in the Diabetes Prediction in Skåne (DiPiS) study , to participate in screening for

celiac disease in the CiPiS study using tTGA analysed by radioligand binding assays.

3435/13,860 (25%) children agreed to participate and 56/3435 (1.6%) were diagnosed with

celiac disease based on intestinal biopsy and recommended a gluten-free diet 13. Using the same

study protocol, we re-invited 13,024 children from the same birth cohort between June 2010 and

August 2013 to a repeated screening at 9 years of age. Children already diagnosed with celiac

disease at three years of age and clinically detected cases were excluded. Of the 4077/13,024

(31%) children who agreed to participate, 72/4077 (1.8%) were diagnosed with celiac disease 15.

The study was approved by the Ethics committee at Lund University and both parents gave their

informed consent.

Case-control study design

In this study, we invited children detected with tTGA in the follow-up screening at 9 years of age

to have their BMD investigated and anthropometrics measured at the time of intestinal biopsy. In

addition, a questionnaire concerning lifestyle factors potentially affecting bone mineral

metabolism was completed by their caregiver and a blood sample was collected from the child

for analysis of 25(OH) vitamin D3, parathyroid hormone (PTH) and the cytokines IL-1β, IL-2,

IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-13, IL-15, IFNγ, and TNFα. Children already

diagnosed with celiac disease in the first screening at 3 years of age who were on a gluten-free

diet were invited for equivalent measurements including analysis of tTGA. Two controls

matched for gender, HLA-DQ, and birth year per every child with celiac disease were selected

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from the tTGA negative children in the screening and included for the same measurements. If

identical HLA-DQ matching was not found, a control child having the same HLA-DQB1 allele

of *02 or *0302, the two known HLA-DQB1 risk alleles for celiac disease, was chosen.

Tissue transglutaminase autoantibodies

16
Both IgA-tTG and IgG-tTG were measured in separate radioligand binding assays and

expressed as units per milliliter (U/mL). Cut-off levels for a positive result were calculated using

QQ plots from 398 healthy blood donors and set at 16 U/mL for IgA-tTG and 4 U/mL for IgG-
16
tTG . Children with a positive result were considered for a follow-up sample after 3 months

and those with at least one value above borderline zones of 26 U/mL for IgA-tTG and 6.5 U/mL

for IgG-tTG in any of the two consecutive positive samples were defined as persistently tTGA

positive and qualified for intestinal biopsy.

Intestinal biopsy and celiac disease diagnosis

Intestinal biopsies were achieved from upper gastroscopy performed at the Endoscopy Unit at

Skåne University Hospital in Malmö, Sweden. Biopsies were evaluated at the Department of
17
Pathology at the same hospital and classified according to the modified criteria of Marsh .

Celiac disease diagnosis was, for the purpose of this study, defined as persistently positive tTGA

in combination with Marsh score 1 or higher.

Selection of cases and controls

Intestinal biopsies were performed in 77 children, thus being eligible for DXA measurement. Of

these 77 children, 5 had Marsh score 0, 7 had Marsh score 1, and 65 had Marsh 3. One child

(having Marsh 3) could not endure the DXA procedure and DXA measurement of children

having Marsh score 0 were excluded from further analysis. In all, 71 celiac disease children (age

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10.0+0.7 (mean+SD) years) and 142 matched controls (age 10.3+0.7 years) were included for the

final analysis (Table 1).

In addition, 56 children already diagnosed with celiac disease in the first screening at 3.3±0.4

years of age who were on a gluten-free diet were invited to the study, of whom 39 (70%)

accepted participation. Nine of these children stated regular intake of gluten-containing diet

and/or had tTGA values above cut-off for a positive result, and were thus excluded from further

analysis. Consequently, DXA measurements of 30 children (age 10.5+0.8 years) on a gluten-free

diet for 6.9+1.1 years and 60 matched controls (age 11.1+ 0.9 years) were included in the final

analysis (Table 1).

Dual X-ray absorptiometry (DXA)

Bone mineral content (BMC) (g), bone mineral density (BMD) (g/cm2), lean body mass (kg),

and body fat mass (kg) were measured using Dual X-ray absorptiometry (DXA). The first 24

children investigated were analyzed using Lunar Prodigy DXA (GE Lunar Corp., Madison, WI,

USA) and the following 279 children were measured utilizing Lunar iDXA (software version

13.60, GE Healthcare, Madison, WI, USA). Cross-calibration measurements were made and

differences were considered to be negligible. BMC and BMD were measured in total body by a

total body scan, in spine (L1-L4) by a lumbar spine scan, and in femoral neck by a hip scan using

standard software. Lean body mass and body fat mass were calculated from the total body scan.

Assessment of 25(OH) vitamin D3 and parathyroid hormone (PTH) levels

Serum was analyzed for levels of 25(OH) vitamin D3 using liquid-chromatography-mass-

spectrometry (Model Sciex API 4000 LC/MS/MS, MA, USA) and expressed as nmol/L. Since

vitamin D levels vary according to season of sampling in the northern hemisphere, the values

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were adjusted for being collected during the winter (October-March) or during the summer
18
(April-September) . The Swedish Pediatric Society Guidelines recommend treatment of

vitamin D deficiency in children with levels < 50 nmol/L 19. Plasma was analyzed for levels of

PTH using an Electro Chemi Luminiscence Immunoassay (Cobas 8000 (E601), Roche, Basel,

Switzerland) and expressed as pmol/L. Values were adjusted for season of sampling in the same
20
way as in vitamin D analysis . Analysis-specific normal reference was set at 1.6-6.9 nmol/L

according to manufacturer’s protocol. All analyses were performed at the Department of

Laboratory Medicine at Skåne University Hospital, Malmö.

Cytokine measurements

All blood samples in the CiPiS study were immediately stored in -20ºC after arrival. After

samples were assessed for tTGA, all samples were kept frozen in -20ºC until being analyzed for

cytokines (pg/mL) using an Multiplex Electro Chemiluminescent Sandwich Immunoassay for the

analysis of IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, IFNγ, and TNFα supplemented

with two Singleplex assays measuring IL-5 and IL-15 respectively (Meso Scale Diagnostics,

Rockville, MD, USA). Cytokine selection was based on former analysis21 and measurements

were adjusted for season of sampling due to the seasonality of virus infections in Sweden22.

Anthropometrics and life style questionnaires

Height (cm) and weight (kg) were measured by the same standard equipment in all children at

the day of the DXA examination, and body mass index (kg/m2) was calculated. A questionnaire

concerning diet, chronic diseases, medication, fractures, and physical activity in school and

outside school modified from Löfgren et al 23 was sent to all participants and their caregivers for

registration at the time of DXA measurement. Children on a gluten-free diet were asked about

duration of gluten-free diet and grading of compliance. Adherence to a strict gluten-free diet was

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defined as children stating gluten-free diet for more than 1 year in the questionnaire, in

combination with tTGA levels below the cut-off level for a positive result at time of DXA

measurement.

Statistical methods

Descriptive data are presented as numbers (n), proportions (%), and mean with standard

deviation (SD) as distribution measurement. Results are presented as mean with 95% confidence

interval (95% CI). Group differences in categorical data were tested using Pearson Chi Square

test or Fishers exact test if small sample sizes. Group differences in numerical continuous data

were measured using ANCOVA to adjust for covariates. Correlations between BMD and tTGA,

Marsh scores, levels of 25(OH) vitamin D3, PTH and cytokines were assessed by the Spearman

coefficient. All p-values are two-sided and <0.05 was considered to indicate statistical

significance. All statistical analysis was performed using SPSS version 22.0

(www.ibm.com/software/se/analytics/spss/).

Results

There were no differences in lifestyle factors in children with screening-detected celiac disease

when compared to matched controls. In children on a gluten-free diet (GFD), no differences were

found in lifestyle factors when compared with their matched controls, except for the gluten-free

diet (Supplemental Digital Content 1, Table, http://links.lww.com/MPG/A944).

Bone mineral density (BMD)

At diagnosis, children with screening-detected celiac disease had lower BMD in total body and

spine, compared to controls (Table 2). In children with screening-detected celiac disease, there

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was no correlation between BMD of total body and spine and levels of IgA-tTGA (r=-0.09, p=

0.46 and r=-0.11, p= 0.38), IgG-tTGA (r=-0.14, p= 0.23 and r=-0.19, p=0.12) and Marsh score

(r=0.03, p= 0.83 and r=-0.06, p= 0.65). Children with celiac disease on a gluten-free diet did not

differ in BMD when compared to controls (Table 2).

Levels of 25(OH) vitamin D3 and parathyroid hormone (PTH)

After adjustment for season of sampling, 25(OH) vitamin D3 levels were lower in untreated

celiac disease children, compared to controls and PTH levels were higher in untreated children

when compared to controls (Table 3). In 11 of 66 (17%) children with screening-detected celiac

disease, we found vitamin D values of <50 nmol/L, compared to 11 of 140 (8%) in control

children (p=0.056). In children with screening-detected celiac disease, there was a weak

correlation between BMD of total body and levels of vitamin D (r=0.28; p=0.03) but no

correlation between BMD in spine and vitamin D levels (r=0.23; p=0.06). There was no

correlation between BMD of total body and spine and levels of PTH (r=0.16 and r=0.22, p=0.20

and p=0.08). Children with celiac disease on a gluten-free diet did not differ from control in

levels of 25(OH) vitamin D3 and PTH (Table 3).

Levels of systemic cytokines

In children with screening-detected celiac disease, after adjustment for season of sampling,

systemic levels of the cytokines IL-1β, IL-6, IL-8, IL-10, IL12p70, IL13, and TNFα were

increased compared to matched controls (Table 4). There was a weak negative correlation

between BMD and the cytokines IL-10 (r=-0.29, p= 0.02) and IL-13 (r=-0.25, p= 0.05). On the

contrary, levels of IL-15 were decreased in children with screening-detected celiac disease

compared to controls (Table 4) and correlated positively to BMD (r=0.28, p= 0.04). No

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differences between groups were found for the cytokines IL-2, IL-4, IL-5 and IFN-γ. Children on

a gluten-free diet did not differ from controls in any measured systemic cytokine (Table 4).

Discussion

Reduced BMD has previously been well described in clinically-diagnosed celiac disease children
24-26
of different ages . Reports from other clinical cohort studies suggest that a proportion of

children investigated for associated conditions and diagnosed with subclinical or asymptomatic

celiac disease also display decreased BMD and/or Vitamin D levels and increased PTH levels
10,11, 27
. Albeit screening-detected children have similar degree of mucosal damage than those
27
found in clinical practice , it has not previously been shown whether screening-detected

children have reduced BMD as a consequence of an unrecognized disease already at diagnosis.

This present study extend previous investigations to include celiac disease children and matched

controls prospectively identified in a population-based screening. We found that children with

screening-detected celiac disease had lower total body and spine BMD already at 10 years of age

whereas children with already screening-detected celiac disease put on a gluten-free diet at an

early age had a BMD corresponding healthy controls. This is line with a previous screening
28
study and give further strength to the hypothesis that children detected in population-based

screening, may benefit from an early diagnosis in order to institute treatment to restore bone

health.

The laboratory finding of lower 25 (OH) vitamin D3 levels in cases as compared to controls also

gave indication of a reduced BMD 29. In addition, a weak correlation between vitamin D levels

and BMD in the same group of children were found, although the prevalence of vitamin D

deficiency between the two groups did only reach borderline statistical significance, probably

because of lack of power since very few actually had values below <50 nmol/ L. The findings of

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low vitamin D levels are in contrast to recent studies indicating vitamin D levels in celiac disease
30,31
children being comparable to the normal population . Nevertheless, the lower level of

vitamin D could indicate inadequate intake, but this was not reflected in presence of dairy

elimination or low milk product intake which was the same in cases and controls. It could also

reflect some other form of malnutrition, albeit this was not shown is the measured

anthropometrics, including total body lean mass and total body fat mass that were no different

compared to control children. This is in line with a previous study on growth parameters in

screening-detected celiac disease which have shown that growth-related factors cannot be

considered reliable tools for identifying celiac disease during early childhood in the general

population 32. Subclinical hypovitaminosis D and malabsorption leading to a reduced uptake of

calcium is probably reflected in the higher levels of PTH found in children with screening-

detected celiac disease and secondary hyperparathyroidism has been found in children with

clinically-detected celiac disease 24,33,34.

Increased levels of systemic cytokines in children with screening-detected celiac disease have

been investigated in this study before in earlier screening at three years of age21. Both screenings

confirm increased levels of systemic cytokines IL-10, IL-12p70 and IL-13 at diagnosis of

screening-detected celiac disease but at ten years of age we found no difference in levels of IL-5

and INF-γ. In this study IL-10 and IL-13 was inversely correlated to BMD indicating that these

cytokines should be investigated further for a possible role in bone metabolism in celiac disease.

In addition, IL-1β, IL-6 and TNFα, well known to play a role in inflammation interaction in bone

resorption in inflammatory bowel disease35, was increased at diagnosis of screening-detected

celiac disease at ten years of age which also have been found in adults with symptomatic celiac

disease36. The altered pattern of systemic cytokines in screening-detected celiac disease

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highlights the fact that the disease is by far not only located in the intestinal mucosa and that

cytokines could be a link between the immunologically derived intestinal inflammation and

alterations in other parts of the body such as the skeleton.

In line with our hypothesis, we found no difference in BMD when comparing children who were

detected at a young age (i.e. diagnosed in the screening at 3 years of age) and consequently put

on a gluten-free diet early, and controls. The children with a gluten-free diet were restricted to

children stating strict gluten-free diet and having tTGA values below cut-off levels for a positive

result in order to evaluate only strict compliant children since lack of compliance is known to

affect BMD 37. The effect of a gluten-free diet on BMD in childhood celiac disease has revealed

diverging results with both total recovery of BMD after 1 year of treatment 9, as well as
38
incomplete recovery after a follow up time of 2 years . In addition, we found that 25(OH)

vitamin D3 levels in children with a gluten-free diet were the same as control children. It is

important to notice that 25(OH) vitamin D3 levels and BMD were not measured before the

institution of the diet in these children and therefore the effect of gluten-free diet between 3 and

10 years of age could not be evaluated. However, their consistency with control children could

indicate a benefit from the treatment.

The accumulation of bone mass during childhood and puberty is thought to be important

determinants of the common end points in studies of skeletal health; peak bone mass,
39
osteopenia/osteoporosis, and fracture risk in adult life . A newly published meta-analysis
40
revealed the association between clinically-diagnosed celiac disease in adults and fractures .

Furthermore, the same group demonstrated an association between tTGA and increased fracture
41
risk, especially hip fractures . Children with screening-detected celiac disease with a reduced

BMD could therefore be of potential risk of lower peak bone mass and future fractures later in

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life. Our findings stress the importance of considering screening for celiac disease already in

young children when bone mass is being acquired.

The strength of this study, in contrast to previous similar studies, is the design of including

screening-detected cases at time of diagnosis and the matching of controls participating in the

same screening. In order to exclude potential confounders we also studied variables associated

with affected BMD and bone metabolism. A limitation of our study was the lack of estimation of

pubertal maturation. Timing of puberty affects bone mineral acquisition and adolescents with a

later onset of maturation may have lower peak bone mass 42. Since delayed puberty is considered

a common extra-intestinal symptom of celiac disease it cannot be ruled out that this condition

affected the outcome.

This study shows that celiac disease children identified by population-based screening have

lower BMD in total body and spine and lower levels of 25 (OH) vitamin D3 compared to control

children. It also shows that children with screening-detected celiac disease diagnosed at an

earlier age already on a gluten-free diet have the same BMD and vitamin D3 levels as controls.

Although the finding of affected BMD is considered a subclinical symptom and the long-term

clinical consequences of this finding were not investigated in our study, it suggests that children

with screening-detected celiac disease benefit from early identification and treatment. Additional

studies are required to determine the rate and quality of bone health recovery in rapidly growing

adolescents with celiac disease.

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Table 1. Matching variables and age at measurement in children with screening-detected celiac disease compared to matched controls and celiac
disease children on a gluten free diet (GFD) compared to matched controls.

Celiac disease GFD

Cases Controls Cases Controls
Matching Variable n=71 n=142 p-value n=30 n=60 p-value
Gender:
-Boys 26 (37%) 52 (37%) 10 (33%) 20 (33%)
-Girls 45 (63%) 90 (63%) Na 20 (67%) 40 (67%) Na

HLA DQB1 genotype (allele 1//allele 2):

*02a//*X 23 (32%) 47 (33%) 18 (60%) 43 (72%)
*02b//*X/*02a 6 (9%) 11 (8%) 2 (7%) 0
*02c//*03:01/*06:02/*06:03/*06:04 13 (18%) 26 (18%) 5 (17%) 7 (12%)
*02c//*03:02 14 (20%) 28 (20%) 2 (7%) 4 (7%)
*03:02//*X 11 (16%) 22 (16%) 3 (10%) 6 (10%)
*03:02//*03:01/*06:02/*06:03/*06:04 4 (6%) 8 (6%) Na 0 0 Na

Mean age, years (SD):

- at tTGA measurement 9.4 (0.6) 9.2 (0.3) 0.06 10.5 (0.8) 9.3 (0.5) <0.001*
- at DXA measurement 10.0 (0.7) 10.3 (0.7) 0.001** 10.5 (0.8) 11.1 (0.9) 0.007**
DXA, Dual X-ray absorptiometry; Na, not applicable; tTGA, tissue transglutaminase autoantibodies.
a
DQA1*05:01-DQB1*02; bDQA1*02:01-DQB1*02; cDQA1*02:01-DQB1*02 or DQA1*05:01-DQB1*02.
*Since measurement of tTGA was performed in GFD-controls at time of screening and in GFD-cases at time of DXA measurement the GFD-cases were older compared to
GFD-controls at time of tTGA measurement.
**DXA measurements in controls were performed after matching procedure, which regarding celiac disease controls were at time of qualification for intestinal biopsy for
cases and regarding GFD-controls were at time of cases accepting participation, which lead to controls being older in both groups.

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Table 2. Anthropometrics and bone mineral parameters, measured by Dual X-ray absorptiometry, in screening-detected celiac disease children
compared to matched controls and children with celiac disease on a gluten-free diet (GFD) compared to matched controls.

Celiac disease GFD

Cases Controls Cases Controls
Variable n=71 n=142 p-value n=30 n=60 p-value

Anthropometrics:
- Weight (kg) 37.2 (35.3 to 39.1) 38.6 (37.3 to 40.0) 0.23 41.6 (37.9 to 45.2) 41.7 (39.1 to 44.2) 0.96
- Height (cm) 143.4 (141.9 to 144.9) 144.2 (143.1 to 145.2) 0.43 147.8 (144.9 to 150.7) 149.6 (147.6 to 151.6) 0.33
- BMI (kg/m2) 17.9 (17.2 to 18.6) 18.5 (18.0 to 19.0) 0.22 18.8 (17.6 to 20.0) 18.4 (17.5 to 19.2) 0.57
- Lean mass (kg) 24.8 (24.0 to 25.6) 25.5 (24.9 to 26.1) 0.15 27.0 (25.3 to 28.7) 27.5 (26.3 to 28.7) 0.64
- Fat mass (kg) 11.0 (9.7 to 12.3) 11.7 (10.8 to 12.6) 0.39 13.1 (10.8 to 15.4) 12.7 (11.1 to 14.3) 0.78

BMC (g):
- Total body 1335 (1291 to 1379) 1388 (1357 to 1419) 0.06 1457 (1363 to 1551) 1525 (1460 to 1590) 0.25
- Spine L1-L4 28.0 (26.9 to 29.1) 28.8 (28.0 to 29.6)* 0.29 31.3 (28.3 to 34.2) 33.2 (31.2 to 35.3) 0.29
- Femoral neck 3.1 (3.0 to 3.2) 3.2 (3.1 to 3.3) 0.17 3.3 (3.1 to 3.6) 3.5 (3.3 to 3.7) 0.24

BMD (g/cm2):
- Total body 0.84 (0.82 to 0.86) 0.87 (0.86 to 0.88) 0.009 0.85 (0.82 to 0.88) 0.87 (0.85 to 0.89) 0.25
- Spine L1-L4 0.76 (0.74 to 0.77) 0.79 (0.78 to 0.80)* 0.005 0.81 (0.77 to 0.85) 0.83 (0.80 to 0.86) 0.48
- Femoral neck 0.80 (0.78 to 0.82) 0.83 (0.81 to 0.84) 0.05 0.83 (0.79 to 0.87) 0.86 (0.83 to 0.89) 0.23

Data expressed as age-adjusted mean (95%CI) and comparisons adjusted for age using ANCOVA.
BMI; Body mass index, BMC; Bone mineral content, BMD; Bone mineral density
*N=140.

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Table 3. Serum levels of 25 (OH) vitamin D3 and parathyroid hormone (PTH) in children with screening-detected celiac disease compared to
matched controls and children with celiac disease on gluten-free diet (GFD) compared to matched controls at time of Dual X-ray absorptiometry.

Celiac disease GFD

Variable Cases Controls p-value Cases Controls p-value

25 (OH) vitamin D3 (nmol/L) 65.7 (60.9 to 70.4)a 77.1 (73.8 to 80.3)c <0.001 75.5 (61.8 to 89.1)e 66.5 (58.6 to 74.3)f 0.38
PTH (pmol/L)  4.2 (3.9 to 4.5)b 3.2 (3.0 to 3.4)d <0.001 3.6 (2.7 to 4.5)e 3.1 (2.6 to 3.6)f 0.46

Data expressed as mean (95%CI) adjusted for season of sampling (Oct-March vs. April-Sept) and comparisons adjusted for season using ANCOVA.
a
n=66, bn=65, cn=140, dn=139, en=30, fn=57

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Table 4. Cytokine levels among children with screening-detected celiac disease (CD) compared to matched controls and among children with
screening-detected celiac disease having a gluten-free diet (GFD) compared to matched controls.

Cytokine CD Controls GFD Controls

n=71 n=140 n=30 n=60

(pg/mL) Median (Q1, Q3)* Median (Q1, Q3)* p-value** Median (Q1, Q3)* Median (Q1, Q3)* p-value

IL-1β 38.49 (0.22, 297.16) 0.00 (0.00, 0.09) <0.001 0.00 (0.00, 0.07) 0.00 (0.00, 0.06) 0.85
IL-2 0.36 (0.16, 0.96) 0.14 (0.08, 0.22) 0.05 0.16 (0.10, 0.29) 0.12 (0.08, 0.19) 0.98
IL-4 0.11 (0.04, 0.26) 0.02 (0.01, 0.07) 0.06 0.02 (0.01, 0.05) 0.02 (0.00, 0.05) 0.63
IL-5 0.42 (0.25, 0.74) 0.43 (0.26, 0.82) 0.78 0.43 (0.18, 0.98) 0.51 (0.22, 0.87) 0.62
IL-6 1.73 (0.38, 16.78) 0.38 (0.24, 0.58) <0.001 0.37 (0.22, 0.73) 0.36 (0.25, 0.55) 0.95
IL-8 155.33 (13.88, 519.00) 10.14 (8.41, 13.04) <0.001 10.20 (7.89, 15.04) 11.15 (8.65, 14.01) 0.25
IL-10 0.79 (0.42, 1.69) 0.43 (0.34, 0.60) <0.001 0.41 (0.36, 0.48) 0.43 (0.33, 0.60) 0.93
IL-12p70 0.23 (0.14, 0.47) 0.13 (0.07, 0.21) <0.001 0.13 (0.10, 0.23) 0.14 (0.08, 0.23) 0.42
IL-13 1.26 (0.47, 3.21) 0.32 (0.00, 0.61) <0.001 0.27 (0.00, 0.53) 0.28 (0.00, 0.54) 0.99
IL-15 1.68 (1.33, 2.08) 1.92 (1.52, 2.31) 0.02 2.22 (1.92, 2.53) 2.14 (1.76, 2.54) 0.62
TNF-α 7.70 (3.71, 21.04) 3.61 (3.05, 4.08) <0.001 3.95 (3.40, 4.58) 3.72 (3.08, 4.26) 0.80
IFN-γ 7.84 (4.90, 12.10) 5.65 (4.06, 8.18) 0.30 6.30 (4.76, 10.05) 5.65 (3.81, 9.21) 0.70
*
Q1= first quartile, Q3= third quartile. ** Comparisons adjusted for season of sampling (Oct-March vs. April-Sept) using ANCOVA.
IL= Interleukin; TNF= Tumour necrosis factor; IFN= Interferon

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