Prevalence of Celiac Disease among Children in Finland
Markku Mäki, M.D., Ph.D., Kirsi Mustalahti, M.D., Jorma Kokkonen, M.D., Ph.D., Petri Kulmala, M.D., Ph.D., Mila Haapalahti, M.Sc., Tuomo Karttunen, M.D., Jorma Ilonen, M.D., Ph.D., Kaija Laurila, M.Sc., Ingrid Dahlbom, M.Sc., Tony Hansson, Ph.D., Peter Höpfl, Ph.D., and Mikael Knip, M.D., Ph.D.
Background Wheat, rye, and barley proteins induce celiac disease,an autoimmune type of gastrointestinal disorder, in geneticallysusceptible persons. Because the disease may be underdiagnosed,we estimated the prevalence of the disease and tested the hypothesisthat assays for serum autoantibodies can be used to detect untreatedceliac disease and that positive findings correlate with specificHLA haplotypes.
Methods Serum samples were collected from 3654 students (agerange, 7 to 16 years) in 1994 and screened in 2001 for endomysialand tissue transglutaminase antibodies. HLA typing was alsoperformed on stored blood samples. All antibody-positive subjectswere asked to undergo small-bowel biopsy in 2001.
Results Of the 3654 subjects, 56 (1.5 percent) had positiveantibody tests, as determined in 2001. Results of the two antibodytests were highly concordant. As of 1994, none of the subjectshad received a clinical diagnosis of celiac disease, but 10who had positive tests for both antibodies in serum obtainedin 1994 received the diagnosis between 1994 and 2001. Of the36 other subjects with positive antibody assays who agreed toundergo biopsy in 2001, 27 had evidence of celiac disease onbiopsy. Thus, the estimated biopsy-proved prevalence was 1 casein 99 children. All but two of the antibody-positive subjectshad either the HLA-DQ2 or the HLA-DQ8 haplotype. The prevalenceof the combination of antibody positivity and an HLA haplotypeassociated with celiac disease was 1 in 67.
Conclusions The presence of serum tissue transglutaminase andendomysial autoantibodies is predictive of small-bowel abnormalitiesindicative of celiac disease. There is a good correlation betweenautoantibody positivity and specific HLA haplotypes. We estimatethat the prevalence of celiac disease among Finnish schoolchildrenis at least 1 case in 99 children.
Celiac disease is a disorder induced by wheat, rye, and barleyproteins, and its classic form is characterized in childrenby malabsorption and failure to thrive. During the past twodecades, however, the clinical picture of the disease has changedto include milder forms, thus resulting in an upward shift ofthe age at diagnosis. Screening for active celiac disease withthe use of serum autoantibodies usually focuses on patientswith mild gastrointestinal symptoms, isolated iron deficiency,atypical or extraintestinal manifestations, or autoimmune diseasesor on the first-degree relatives of affected patients.1,2,3Screening programs within populations indicate that the diseaseis underdiagnosed,4,5,6,7 but because of the rather small numberof subjects studied, the confidence intervals for the true prevalenceare wide. In the United States, the disease is extremely rarewhen the criteria for diagnosis rely on classic symptoms suchas diarrhea and short stature.8 By broadening the clinical indication,however, antibody screening seems to indicate that the prevalencein the United States is similar to that in Europe.9
Approximately 90 percent of patients with celiac disease carrythe HLA-DQ2 heterodimer encoded by the HLA-DQA1*05 and DQB1*02genes. Such patients have at least one copy of the extendedHLA-DR3–DQ2 haplotype (encoding both the and chainsof the major histocompatibility complex [MHC]) common to manyautoimmune diseases, or they are heterozygous for the HLA-DR5–DQ7haplotype (encoding the chain of the MHC) and the HLA-DR7–DQ2haplotype (encoding the chain of the MHC), in which the heterodimermolecule is encoded in the trans position.10 Most of the remaining10 percent of patients have the HLA-DR4 –DQ8 haplotype.
Evidence suggests that celiac disease is underdiagnosed in children.Serologic testing has the potential to detect otherwise undiagnoseddisease. Evidence is also accumulating that daily ingestionof wheat, rye, and barley results in long-term extraintestinalsequelae in subjects with undiagnosed or untreated celiac disease.1,2Early detection of the disease and subsequent dietary eliminationof gluten might be the appropriate method for averting complicationslater in life.
We sought to determine the prevalence of celiac disease in Finlandand specifically to test the hypothesis that celiac diseasecan be identified by serologic testing in children who havenot previously received a clinical diagnosis. We used two serologictests simultaneously — endomysial and tissue transglutaminaseautoantibody tests — to screen a geographically defined,unselected population of schoolchildren. We also assessed whetherpositivity for disease-specific autoantibodies correlates withthe HLA haplotypes associated with celiac disease.
Methods
Subjects
We tested serum samples collected in 1994 as part of a studyof risk factors for type 1 diabetes among schoolchildren.11All 4280 schoolchildren who were 7 to 16 years old and who livedin five municipalities in northern Finland were invited to participatein the study. The cohort represents 8 percent of the area'sschool population. Whole blood for HLA typing and serum wereobtained from 3662 subjects (85.6 percent), and the sampleswere stored at –20°C until studied. Eight subjectswere excluded because the volume of their serum samples wasnot sufficient for analyses. The median age of the remainingstudy cohort of 3654 subjects (1826 of whom were boys) was 12years (range, 7 to 16) at the time of initial sampling.
The ethics committee of the Faculty of Medicine, Universityof Oulu, approved the original study protocol to screen forrisk factors associated with type 1 diabetes and for the collectionof blood samples in 1994. Written informed consent was alsoobtained from the subjects, their parents, or both. In 2001the new protocol, which included serologic screening, uppergastrointestinal endoscopy, and mucosal biopsies, was evaluatedand approved by the same committee. A new informed-consent documentregarding blood testing and small-bowel biopsies was signedby the subjects, their parents, or both.
Study Protocol
The cohort was screened for endomysial and tissue transglutaminaseantibodies in blood samples obtained in 1994, and all subjectswith a positive result who had not previously received a diagnosisof celiac disease were asked to undergo upper gastrointestinalendoscopy in 2001. At the visit for endoscopy, a second serumsample was obtained for antibody testing. Serum testing beganon August 31, 2000, and the last biopsy specimen was obtainedon December 14, 2001. Clinical symptoms were assessed with useof a semistructured questionnaire completed at the visit withthe study clinician in 2001. A clinical dietitian assessed thediet of the subjects.
Serum Antibody Tests
The serum samples from all 3654 schoolchildren were simultaneouslyassessed in a blinded fashion in two different laboratories:tests for endomysial antibodies were conducted in Tampere, Finland,and tests for tissue transglutaminase antibodies were performedin Freiburg, Germany. Serum IgA- and IgG-class endomysial antibodieswere determined by an indirect immunofluorescence method aspreviously described.3,12 Determinations of IgA-class tissuetransglutaminase antibodies were carried out with a Celikeyassay (Pharmacia Diagnostics) in accordance with the manufacturer'sinstructions. The limit of detection of the assay was 0.1 Uper milliliter, and we chose 5 U per milliliter as the cutoffpoint for positivity. Serum samples with IgA-class tissue transglutaminaseantibody levels below the limit of detection were further testedfor the determination of IgG-class tissue transglutaminase antibodieswith an enzyme-linked immunosorbent assay (Pharmacia Diagnostics),as previously described.13 The same microplates and test procedureused for IgA-class antibodies (Celikey) were used in this subgroupof serum samples. Values above 5 U per milliliter were consideredpositive.
In subjects who were positive for IgG-class tissue transglutaminaseantibodies, an IgG-class endomysial antibody test was performed.12In such subjects, the total serum IgA was determined nephelometrically,and serum levels below 0.05 g per liter were considered indicativeof selective IgA deficiency.
Endoscopy
Upper gastrointestinal endoscopy was performed with an Olympusendoscope (model GIF-IT140) at the Department of Pediatrics,Oulu University Hospital. During the procedure, multiple duodenal-biopsysamples were obtained for routine histologic analysis. One samplewas prepared for immunohistochemical staining.
Diagnosis of Celiac Disease
Formalin-fixed biopsy specimens stained with hematoxylin andeosin were studied with the use of light microscopy and morphometrictechniques. Villous height and crypt depth were measured, andthe ratio of villous height to crypt depth was calculated. Aratio of less than 2 was considered to be indicative of celiacdisease (i.e., villous atrophy with crypt hyperplasia).
Immunohistochemical Staining
Frozen biopsy samples of the small intestine were stained forintraepithelial lymphocytes bearing / T-cell receptors, andcell densities were determined as previously described.14 Thebiopsy specimens were also stained for HLA class II molecules,and the expression of HLA-DR was considered to be enhanced whenepithelial staining was strong or was confined to crypt cells.14
HLA Typing
HLA typing was performed with the use of a screening test developedto detect alleles associated with an increased risk of type1 diabetes and those associated with protection against it.15Samples were analyzed for selected HLA-DQB1 alleles, includingDQB1*02 and DQB1*0302, and samples that were positive for theHLA-DQB1*02 allele were further analyzed for the presence ofassociated alleles: HLA-DQA1*0201 and DQA1*05.
Statistical Analysis
We calculated 95 percent confidence intervals for prevalencerates and odds ratios.16 The odds ratio was calculated withuse of the equation (a x d) ÷ (b x c), and the positivepredictive value was calculated with use of the equation a ÷(a + c) x 100. In these equations a and c represent the numbersof subjects with genotypic risk factors with and without celiacdisease–specific autoantibodies, respectively, and b andd represent the numbers of subjects without genotypic risk factorswho do and do not have autoantibodies, respectively.
Results
Serologic Tests
The correlation between the results of the two methodologicallydifferent autoantibody tests was almost perfect: 3651 of 3654results were concordant. Fifty subjects were positive for bothIgA-class endomysial antibodies (median titer, 1:500; range,1:5 to 1:4000) and IgA-class tissue transglutaminase antibodies(median titer, 70.3 U per milliliter; range, 8.8 to 680), and3601 were negative for both tests (Figure 1). One subject whowas negative for tissue transglutaminase antibodies (titer,4.8 U per milliliter) was positive for IgA-class endomysialantibodies at the lowest titer, 1:5. Two subjects who were negativefor IgA-class endomysial antibodies were positive for IgA-classtissue transglutaminase antibodies, with titers of 5.7 and 7.0U per milliliter.
Figure 1. Results of Screening for Autoantibodies Associated with Celiac Disease in a Population of Finnish Schoolchildren.
Blood samples obtained in 1994 were tested for serum endomysial antibodies and tissue transglutaminase antibodies in 2001. Subjects with positive results who had not previously been given a diagnosis of celiac disease were invited to undergo upper gastrointestinal endoscopy. The results of small-bowel biopsy and typing for HLA-DQ2 and DQ8 are shown. The overall prevalence of biopsy-proven celiac disease in the cohort was 1 case in 99 children.
Seventeen subjects had undetectable serum levels of IgA-classtissue transglutaminase antibodies (less than 0.1 U per milliliter).Fourteen were negative for IgG-class tissue transglutaminaseantibodies, with a median titer of 1.2 U per milliliter (range,0.8 to 3.1), and three were clearly positive, with serum titersof 160.6, 148.2, and 26.4 U per milliliter (Figure 1). All threeproved to have IgA deficiency.
Celiac Disease Detected on the Basis of Symptoms
As of 1994, no cases of celiac disease had been identified inthis cohort. Between 1994 and 2001, 10 cases were detected onthe basis of abdominal symptoms and confirmed by biopsy (medianage, 16 years; range, 14 to 20) (Figure 1). Nine of the subjectswere positive for both serum IgA-class endomysial antibodies(median titer, 1:1000; range, 1:500 to 1:4000) and IgA-classtissue transglutaminase antibodies (median titer, 119 U permilliliter; range, 37.9 to 634.4), and 1 had IgA deficiencybut was positive for IgG-class endomysial antibodies (titer,1:2000) and IgG-class transglutaminase antibodies (26.4 U permilliliter). The antibody results were obtained retrospectivelyfrom tests of serum samples obtained in 1994, before the clinicaldiagnosis had been made.
Celiac Disease Detected by Screening
In 2001 the remaining 46 antibody-positive subjects who hadnot previously received a diagnosis of celiac disease were invitedto undergo small-bowel biopsy and antibody testing. Thirty-six(78.3 percent) agreed to undergo biopsy, and 27 had mucosalvillous atrophy with crypt hyperplasia typical of celiac disease.Morphometric studies revealed a mean ratio of villous heightto crypt depth of 0.87 (range, 0.11 to 1.68; a ratio of lessthan 2 is indicative of celiac disease). In addition, the intraepithelialdensities of / T-cell receptor–bearing lymphocytes exceededthe threshold for positivity (3.5 cells per millimeter), witha mean of 20 cells per millimeter (range, 3.5 to 71.6). Aberrantup-regulation of the expression of HLA-DR was seen in 25 of27 mucosal specimens.
A second blood sample was obtained at the time of biopsy in2001. Altogether 24 of 27 patients with celiac disease detectedby screening had both IgA-class endomysial antibodies (mediantiter, 1:1000; range, 1:50 to 1:4000) and IgA-class tissue transglutaminaseantibodies (median titer, 37.8; range, 6.8 to 624). Both antibodytests were negative in one subject. Two subjects had IgA deficiencybut were positive for IgG-class antibodies alone. A gluten-freediet was prescribed for all 27 subjects with newly diagnosedceliac disease, and 25 agreed to follow the diet.
Normal Mucosal Morphology
Table 1 summarizes the findings in the nine subjects with normalmucosal architecture on small-bowel biopsy. Five subjects whowere initially antibody-positive were negative for antibodiesduring follow-up in 2001. All but one of them had an increaseddensity of / T-cell receptor–bearing lymphocytes, andall had enhanced expression of HLA-DR, indicating ongoing mucosalinflammation in the morphologically normal mucosa. Two subjectswho were initially negative for endomysial antibodies but whohad low levels of tissue transglutaminase antibodies had negativetests for both types of antibodies in 2001. Eight of the ninesubjects were positive for HLA-DQ2.
Table 1. Results of Serum Antibody Tests and Small-Bowel Biopsy in Nine Subjects with Normal Mucosal Architecture on Small-Bowel Biopsy.
Antibody-Positive Subjects Who Declined to Undergo Biopsy
Ten of the antibody-positive subjects declined to undergo small-bowelbiopsy. In the 1994 serum samples, the median endomysial antibodytiter was 1:200 (range, 1:5 to 1:4000), and the median levelof tissue transglutaminase antibodies was 70.6 U per milliliter(range, 4.8 to 169.7). Four subjects agreed to provide a secondblood sample in 2001, and all four had increased levels of IgA-classendomysial antibodies (1:1000, 1:2000, 1:2000, and 1:4000) andIgA-class tissue transglutaminase antibodies (66.7, 89.0, 91.1,and 101 U per milliliter, respectively).
HLA Typing
The distribution of the HLA genotypes associated with celiacdisease in the 3627 subjects who underwent genotyping is shownin Table 2. Altogether 655 schoolchildren (18.1 percent) carriedthe HLA-DQA1*05-DQB1*02 (HLA-DR3–DQ2) haplotype, and 1411(39 percent) carried either the HLA-DR3–DQ2 or the HLA-DQ-A1*03-DQB1*0302(HLA-DR4–DQ8) haplotype. All but two of the antibody-positivesubjects were positive for one or both of these haplotypes,irrespective of the findings on small-bowel biopsy (Figure 1).The majority (85.7 percent) carried the HLA-DR3–DQ2 haplotype.There was no correlation between the various haplotypes andthe severity of mucosal abnormalities.
Table 2. Prevalence of HLA-DR and DQ Genotypes Associated with Celiac Disease among the 3627 Schoolchildren Who Underwent Genotyping, Including the 56 Subjects Who Were Positive for Serum Endomysial or Tissue Transglutaminase Antibodies.
Clinical Aspects of Celiac Disease Detected by Screening
All 27 subjects with newly diagnosed, biopsy-proved celiac diseaseand the 9 subjects with normal mucosal architecture on small-bowelbiopsy completed a questionnaire concerning symptoms relatedto celiac disease. None had another autoimmune disease. Fourof the 27 patients with newly diagnosed celiac disease had anaffected first-degree relative. When specifically asked, 11reported recurrent abdominal pain and intermittent diarrheaor constipation, 1 reported tiredness, and 1 had skin symptoms.Ten subjects had clinically silent celiac disease. Two of thesubjects with normal mucosal architecture on small-bowel biopsyreported abdominal pain.
Prevalence of Celiac Disease
Among the 3654 schoolchildren, 37 had biopsy-proven celiac disease,for a minimum prevalence of 1 case in 99 children (95 percentconfidence interval, 1 in 146 to 1 in 75) (Figure 1). The prevalenceof subjects who were positive for both antibodies and HLA-DQ2or DQ8 was 1 in 67 (95 percent confidence interval, 1 in 89to 1 in 52).
Discussion
This population-based screening study showed that celiac diseaseis underdiagnosed. Simple, noninvasive serologic tests detectedceliac disease in schoolchildren who had not previously beengiven a diagnosis of the disease.
Clinical celiac disease represents the tip of the iceberg.17,18According to our findings, the prevalence of biopsy-proven celiacdisease is at least 1 case in 99 children. One explanation forthis high prevalence might be that the population studied mayhave had an unusually high genetic risk of celiac disease. However,the fact that the distribution of HLA genotypes in this populationcorresponds to that in the Finnish population in general suggeststhat the study population was representative of the Finnishpopulation as a whole. The true prevalence of celiac diseaseis likely to be even higher than 1 in 99. Not all our antibody-positivesubjects consented to undergo biopsy, and some may have hada mucosal lesion or gluten-induced disease despite the presenceof morphologically normal mucosa.19,20,21 The presence of gluten-inducedautoantibodies in subjects with initially normal villous architectureon small-bowel biopsy who are eating normal amounts of glutenpredicts subsequent mucosal deterioration and celiac disease.22,23Five subjects who were initially positive for such autoantibodieswere negative on follow-up testing, despite the fact that theywere eating a normal, gluten-containing, diet. This findingmight point to a minor variant of the natural history of celiacdisease, in which gluten sensitivity fluctuates over time.24We have previously observed that intolerance to cereals is nota specific sign of celiac disease, and only 10 percent of patientswho spontaneously report abdominal symptoms after consumingcereals are found to have celiac disease.25
An important finding of this study is that most antibody-positivesubjects carried the HLA-DQ2 or DQ8 molecules that are characteristicof celiac disease.10 The HLA-dependency of the production ofautoantibodies associated with celiac disease has been reportedin a study of the first-degree relatives of patients with celiacdisease.3,26 We found that the risk associated with the HLA-DR3–DQ2haplotype was much greater than that associated with the HLA-DR4–DQ8haplotype, and it is striking that a compound heterozygote (onewho carried both haplotypes) had a lower risk than a personwho carried only the HLA-DR3–DQ2 haplotype.
We used the IgA-class serum endomysial antibody test, whichhas been validated in Europe,27 to identify untreated celiacdisease, but the drawback of this test remains its subjectivity.After the identification of tissue transglutaminase as the targetof celiac disease–specific autoantibodies in both rodentand primate tissues,28 a non–observer-dependent enzyme-linkedimmunosorbent assay was developed to detect the antibodies.29,30,31We found that this assay was as reliable and sensitive as theendomysial antibody test, which is based on indirect immunofluorescence.
Our findings suggest that these assays are a reliable and simplemeans of screening children for clinically silent celiac diseaseand genetically inherited gluten intolerance before symptomsor signs of chronic malabsorption develop. The crucial questionnow is whether population-based screening should be consideredoutside research programs. In our study, one third of the subjectsin whom celiac disease was detected by screening had no symptomsand did not have any risk factors for celiac disease. In otherclinical settings, even those in which serologic testing forceliac disease is routine, prevalence figures similar to oursare not obtained.32 Nonetheless, whether treatment benefitsclinically silent celiac disease should be thoroughly assessed.Undetected celiac disease increases the risk of several complications,including osteoporosis.1,2,18 On the other hand, the lifelongneed to follow a gluten-free diet may be burdensome, especiallyif the patient is asymptomatic.33 Further studies of the effectof asymptomatic celiac disease, including cost-effectivenessevaluations, are needed before population-based screening studiescan be recommended. However, given our finding that celiac diseaseis underdiagnosed, clinicians should keep in mind the complexclinical picture of the disease and have a high index of suspicionand a low threshold for ordering serologic tests.
In summary, we found that celiac disease was highly prevalentin an unselected population of schoolchildren and adolescents.The results of serum endomysial and tissue transglutaminaseantibody tests were highly correlated with the HLA genotype.The tissue transglutaminase antibody test offers an objectivemeans of detecting celiac disease early, when it is clinicallysilent.
The Celiac Disease Study Group is supported by grants from thePäivikki and Sakari Sohlberg Foundation, the Foundationof the Friends of the University Children's Hospitals in Finland,the Medical Research Fund of Tampere University Hospital, andthe Academy of Finland Research Council for Health (73489).The study was also funded by the Commission of the EuropeanCommunities in the form of the Research and Technology Developmentprograms "Quality of Life and Management of Living Resources"(QLRT-1999-00037) and "Evaluation of the Prevalence of CeliacDisease and its Genetic Components in the European Population."The study does not necessarily reflect the current views orfuture policies of the Commission of European Communities.
Dr. Höpfl, Dr. Hansson, and Ms. Dahlbom are employees ofPharmacia Diagnostics. Pharmacia did not sponsor the study butdid provide the immunoassays for the detection of tissue transglutaminaseantibodies.
Source Information
From the Pediatric Research Center, Medical School, University of Tampere, Tampere, Finland (M.M., K.M., K.L.); the Department of Pediatrics, Tampere University Hospital, Tampere, Finland (M.M., K.M., M.K.); the Departments of Pediatrics (J.K., P.K., M.H.) and Pathology (T.K.), Oulu University Hospital, Oulu, Finland; Turku Immunology Center and Department of Virology, University of Turku, Turku, Finland (J.I.); Pharmacia Diagnostics, Uppsala, Sweden (I.D., T.H.); Pharmacia Diagnostics, Freiburg, Germany (P.H.); and the Hospital for Children and Adolescents, University of Helsinki, Helsinki, Finland (M.K.).
Address reprint requests to Professor Mäki at the Celiac Disease Study Group, Pediatric Research Center, Medical School, Bldg. FM3, FIN-33014 University of Tampere, Tampere, Finland, or at markku.maki{at}uta.fi.
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and 
chains of the major histocompatibility complex [MHC]) common to many autoimmune diseases, or they are heterozygous for the HLA-DR5–DQ7 haplotype (encoding the 
/
T-cell receptors, and cell densities were determined as previously described.14 The biopsy specimens were also stained for HLA class II molecules, and the expression of HLA-DR was considered to be enhanced when epithelial staining was strong or was confined to crypt cells.14 
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