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Original Articles: Gastroenterology

Serologic and Genetic Markers of Celiac Disease: A Sequential Study in the Screening of First Degree Relatives

Bonamico, Margherita*; Ferri, Mirella*; Mariani, Paolo*; Nenna, Raffaella*; Thanasi, Enina*; Luparia, Rita P. L*; Picarelli, Antonio; Magliocca, Fabio M; Mora, Barbara; Bardella, Maria Teresa§; Verrienti, Antonella; Fiore, Benedetta; Uccini, Stefania; Megiorni, Francesca; Mazzilli, Maria Cristina; Tiberti, Claudio

Author Information
Journal of Pediatric Gastroenterology and Nutrition: February 2006 - Volume 42 - Issue 2 - p 150-154
doi: 10.1097/01.mpg.0000189337.08139.83
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Celiac disease (CD) is a chronic, gluten-dependent enteropathy occurring in genetically predisposed individuals characterized by villous atrophy and crypt hyperplasia of the small bowel mucosa. In addition to cancer (1), the complications of the disease include those related to malabsorption, such as iron-deficient anemia (2) and osteopathy (3), and those associated with the autoimmune process underlying the disease, such as type 1 diabetes mellitus (4) and thyropathy (5). In addition, infertility (6), low-weight birth infants (7), dilated cardiomyopathy, and other forms of heart failure (8) have been observed in CD patients.

Several studies reported an increasing prevalence of CD in the general population, in many European countries as well as in the United States (9-12). In Italy, the prevalence of the disease in school children is 0.55%, with a ratio between symptomatic and asymptomatic patients of 1:7 (11). The increased recognition of the disease is related to the availability of serologic markers, which in positive cases permit us to select candidates for the intestinal biopsy, an invasive examination. In fact, at present, the detection of typical histologic lesions of the intestinal mucosa is mandatory for the diagnosis of CD (13). These tests permit the investigation of both general population and CD high-risk groups, such as Down syndrome patients (14), Turner syndrome girls (15), and relatives of CD patients (12,16-19).

The prevalence of biopsy-proven CD among first degree relatives varies between 5.5% (18) and 8.3% (19), reaching a value of 18% in siblings (17). In the course of time, serologic CD screening has been performed, first using antigliadin antibodies (AGA) and antireticulin (16) and then antiendomysium antibodies (EMA) (18,19). The identification of tissue transglutaminase (tTG) as the main self-antigen recognized by EMA (20) and the important role for this enzyme in the pathogenesis of CD (21) led to the development of immunoenzymatic assays for anti-tTG antibody detection using guinea pig liver tTG (22,23). The availability of human recombinant tTG has improved the performance of this analysis (24,25), and a novel radioimmunologic assay using human recombinant tTG appears to be a very sensitive method (26-28).

CD develops mostly in subjects carrying the DQA1*05-DQB1*02 or DQA1*03-DQB1*0302 alleles coding for the human leukocyte antigen (HLA)-DQ2 and -DQ8 high-risk heterodimers. In Italy, we have previously reported that 91% of patients and 26% of controls carried the DQ2 or DQ8 dimers (29).

We studied a large series of CD relatives with the following aims:

  • Identifying a useful serologic strategy to select CD relatives as candidates for the intestinal biopsy;
  • Evaluating the possible role of HLA typing in the diagnostic algorithm for CD relatives.


Study Population and Study Design

All first degree relatives (parents and siblings) of the 181 CD children who attended the Pediatrics Department of the University of Rome “La Sapienza” were invited to participate in the study. The screening for CD was also offered to offspring of 27 CD parents attending the Department of Clinical Science of the University of Rome “La Sapienza.” Four hundred forty-one first degree relatives older than 2 years (223 males) were enrolled in this study.

Family members included 257 parents, (127 fathers and 130 mothers, aged 28-63 years, median age 40 years), 146 siblings (76 brothers and 70 sisters, aged 2-25 years, median age 10 years), and 38 offspring (20 males and 18 females, aged 2-16 years, median age 5 years). There were two sets of twins among siblings and one among offspring. All family members were on a gluten-containing diet when serologic tests were performed.

The intestinal biopsy was suggested to all EMA or anti-tTG antibody positive subjects. Some relatives, negative to serology, underwent intestinal biopsy because of gastrointestinal complaints. Subjects with subtotal or partial villous atrophy and crypt hyperplasia (2, 3 a-c degree, according to the Oberhuber classification) (30) were diagnosed as being affected by CD, and a gluten-free diet was prescribed. To characterize the clinical form of the disease, a standardized questionnaire, including gastrointestinal signs and symptoms (classic form), symptoms not related to the gastrointestinal tract (atypical form), and absence of symptoms (silent form), was completed. For the nonceliac relatives who carried the HLA heterodimers related to an increased risk for CD (DQ2 and/or DQ8), a serologic follow-up was suggested.

Serologic Markers

All subjects were previously tested for serum immunoglobulin (Ig)A presence. The serologic screening included: (1) IgA EMA, tested by an indirect immunofluorescence method (Eurospital, Trieste, Italy), using as a substrate sections from the distal portion of monkey's esophagus and fluorescein-labeled goat anti-human IgA antibody as secondary antibody. The patient's serum was diluted 1:5 in phosphate buffer at pH 7.4, and the presence of a brilliant green network pattern under fluorescence microscope was considered positive (31); (2) IgA antitransglutaminase autoantibodies (TGAA), evaluated with a quantitative radioimmunoprecipitation assay (RIA), using human recombinant 35S-methionine labeled tTG. Autoantibody levels were expressed as an index defined as (unknown sample cpm - negative control cpm)/(positive control cpm - negative control cpm). A receiver operator characteristic analysis was used to identify the optimal threshold value for sensitivity and specificity of the TGAA RIA method (28).

A TGAA index of 0.050 was considered as the limit of positivity of this assay. The subjects initially found IgA deficient were investigated for IgG EMA and IgG anti-tTG antibody presence (32). IgG anti-tTG were tested using the same procedure used for TGAA detection but with replacement of the anti-human IgA agarose with 50% protein A Sepharose. Autoantibody levels and the optimal threshold value for sensitivity and specificity of the IgG anti-tTG method were calculated as for TGAA assay. An IgG anti-tTG antibody index of 0.065 was considered as the limit of positivity of this assay.

HLA Typing

Three hundred sixty-four subjects were typed for HLA-DRB1, -DQA1, and -DQB1 genes by polymerase chain reaction sequence specific primers using commercial kits (Dynal, Bromborough, UK).

Small Bowel Histology

Multiple duodenal biopsies were obtained during upper endoscopy. Particularly in children, one sample from the bulb and four samples from the distal duodenum were taken (33). In adults, three samples of the distal duodenum were obtained. Histologic evaluation was performed by two independent experienced observers, one for children and adolescents (FMM) and one for adults (SU). Intestinal biopsies were evaluated according to the Oberhuber classification (30).

Ethical Issues

The study was performed according to the Declaration of Helsinki. All subjects were informed about the objectives of the study and the eventual necessity of small intestinal biopsy. Informed consent was obtained from adults and from the parents for their children.


High levels of TGAA were present in 46 of 439 (10.5%) CD relatives, whereas IgA EMA were positive in 38 of 439 (8.6%). The ages of the four subjects who were IgA-TGAA positive and IgA-EMA negative with villous atrophy were 12, 15, 40, and 63 years. The intestinal biopsy was offered to all positive subjects. However, three relatives (1 daughter EMA and TGAA positive, DQ2/DQ8 negative, 1 mother and 1 father, both DQ2 and TGAA positive) did not receive the examination. In addition, 21 CD relatives with a negative serology underwent intestinal biopsy because of gastrointestinal symptoms. Table 1 shows the results of serologic findings and intestinal biopsy in these subjects.

Results of IgA EMA and anti-tTG antibodies in immunocompetent relatives of celiac patients together with the results of intestinal biopsy

Two fathers, one with IgA deficiency, IgG EMA, IgG anti-tTG, and DQ2 positive, and the other on steroid therapy, IgG EMA negative, IgG anti-tTG weakly positive, and DQ8 positive, both showing 3a type lesions of the duodenal mucosa, were not included in the evaluation of IgA serologic tests. Intestinal lesions related to CD were observed in 40 patients and in the two immunodeficient fathers; consequently, the strict prevalence of CD in this group is 9.5% (42/441). This prevalence could increase to 10.2% if we take into account the three relatives positive to the serology who did not perform the biopsy and also to 10.9.% including one EMA and TGAA low positive and two TGAA medium-high positive relatives showing a normal small bowel mucosa, “enlarged prevalence.” All 21 subjects serologically negative who performed intestinal biopsy for clinical reasons showed a normal mucosa (Table 1).

Clinical Characteristics of CD Relatives Affected by CD

CD was diagnosed in 42 subjects, 26 females and 16 males (F/M = 1.6:1); among this series, there were 15 parents, 19 siblings, and 8 offspring. The highest percentage was observed among daughters (38.9%), followed by sisters (17.1%), brothers (9.2%), fathers (6.3%), mothers (5.4%), and sons (5.0%). Typical, atypical, and silent forms of CD were recognized in 42.8%, 11.9%, and 45.2% of cases, respectively; offspring particularly showed only typical forms. Five of these 42 CD patients (11.9%, 2 fathers and 3 adolescent siblings) presented an associated autoimmune pathology: Hashimoto thyroiditis (2 cases), hypothyroidism, psoriasis, and nephritis. Among them, two sisters showed a classic form of CD, whereas the other three were clinically silent.

Genetic Study

Table 2 shows the HLA typing of 364 relatives divided on the basis of the disease status and kinship degree. The HLA-DQ2 and -DQ8 high-risk molecules are present in 95% (38/40) of celiac and in 59.5% (193/324) of nonceliac relatives. The two CD relatives, negative for the DQ2 and DQ8 heterodimers, carried the HLA-DRB1*07-DQA1*0201-DQB1*02 haplotype.

Human leukocyte antigen typing of 364 relatives divided on the basis of the disease status and kinship degree

Among the subjects studied, three parents, carrying HLA DQ2 heterodimer, previously EMA negative, during the follow-up lasting 13, 25, and 20 years, respectively, one became EMA and TGAA positive, the other two only TGAA positive. Two of them were asymptomatic, one was affected by herpetiform dermatitis. Intestinal biopsy showed 3a type lesions of the duodenal mucosa (30) in the two fathers, whereas the other parent, a mother of two celiac offspring, has not yet performed endoscopy because she is undergoing chemotherapy for a mammary neoplasm.


The prevalence of CD among relatives observed in our series is higher than previously recognized in other recent studies, ranging from 5.5%, detected by Farrè et al. (18) using IgA EMA and IgA AGA, to 8.3% in the study of Högberg et al. (19), who evaluated AGA, EMA, and TGAA using a commercial enzyme-linked immunoabsorbent assay kit. In the large series investigated by Fasano et al. (12) in the United States, the prevalence of CD in first degree relatives was 4.5%; the criteria for the diagnosis were either EMA positivity with an intestinal biopsy consistent with CD or EMA positivity and HLA haplotypes compatible with CD when a biopsy was not performed (in 145/205 EMA positive first degree relatives).

The confirmation that CD relatives are a population at high risk of developing a gluten intolerance suggests the need for an extensive screening policy in these subjects. Otherwise, the high prevalence of CD and consequently the high number of relatives who must be screened suggest researching a useful screening algorithm. It implies a strategy that includes accuracy, low invasiveness, and possibly low cost. This is true if we consider that a subject can become celiac at any time during his life (34,35), as has also been shown in our series. Therefore, the screening must be periodically repeated. Although the gold standard for CD diagnosis is the intestinal biopsy, its invasiveness and cost preclude the submission of all CD relatives to this examination. For this reason, serologic tests are used to select subjects who need intestinal biopsy.

In the 1990s, EMA detection proved to be a very sensitive and specific test (31,36-38). However, the lack of absolute sensitivity (28,39), as well as some other methodologic problems (semiquantitative test, operator-dependent results), led to other markers being recognized.

The identification of human tTG as the putative antigen of the EMA permitted us to set up new specific and sensitive assays able to evaluate immunoreactivity in CD. Among these methods, the detection of TGAA with a fluid-phase RIA proved to be a powerful method for CD screening (26-28). In this study, once again, this method was able to evaluate CD-related immunoreactivity with higher sensitivity in comparison with EMA detection (100% and 90%, respectively). Nevertheless, we found three TGAA positive subjects (1 of 3 also EMA positive) with a normal intestinal mucosa; two of them carried the DQ2 heterodimer, one the DRB1*07-DQA1*0201-DQB1*02 haplotype present in a small percentage of celiac patients (40). These subjects could be considered to have potential forms of CD. We have not evaluated the specificity of TGAA because, for ethical reasons, we could not suggest the intestinal biopsy to asymptomatic subjects with negative serology. In addition, it is important to consider that previous studies performed with our RIA method demonstrated a very good specificity (26,28).

The presence of one IgA deficient father among our celiac relatives is not surprising and confirms the importance of assaying IgA levels. In this patient, IgG EMA and IgG anti-tTG antibodies were present, indicating that intestinal biopsy should be performed.

According to the results of the present study, a screening strategy based on the determination of HLA status as a first step would exclude approximately one third of relatives; however, two DQ2/DQ8 negative cases would be missed. Moreover, the prevalence of DQ2/DQ8 negative celiacs (5%) in our series agrees with that already observed by others (40) and us (41). As reported in other studies (34,35), we also noted among our relatives three parents who became serologically positive during a follow-up lasting more than 10 years because of the presence of the DQ2 heterodimer. So, we propose the expensive genetic study as a second step in previously TGAA negative subjects to limit the population for the serologic follow-up. Therefore, one algorithm could be proposed for relatives more than 2 years old. First, evaluation of RIA TGAA and total IgA (in the IgA deficient relatives, determination of RIA IgG anti-tTG) could be performed, followed by intestinal biopsy in positive subjects. To all TGAA negative relatives, the genetic study and a second antibody evaluation 2 to 3 years later are offered. The HLA-DQ2 or -DQ8 positive subjects, negative to the serology, should continue the serologic follow-up, whereas a clinical follow-up is sufficient for the relatives who do not carry the at risk heterodimers.

In our series, almost half of the celiac relatives were clinically silent, whereas a minority presented atypical symptoms and the remaining a classic form of CD; the percentage of classic form was 100%, 31.6%, and 36.4% in offspring, siblings, and parents, respectively. It is possible that, after a first diagnosis of CD, the family became more alert in recognizing mild symptoms related to the disease, particularly with children. The observation that three of five patients affected by autoimmune pathologies presented a silent form of CD corroborates the importance of screening all CD first degree relatives.

In conclusion, the present carefully conducted study, based on a large number of first degree relatives of CD patients and on the use of a very sensitive RIA method, confirms the high prevalence of the disease in this series, which is frequently silent and can be identified only with a serologic screening. A diagnostic algorithm, including sequentially RIA TGAA evaluation and HLA typing, may be useful for the diagnosis and the follow-up of CD relatives.


The authors thank Mrs. Patricia Byrne for help with English style.


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Celiac disease; Relatives; Serology; HLA

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