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Original Articles: Gastroenterology

Increased Prevalence of Anti-gliadin Antibodies and Anti-tissue Transglutaminase Antibodies in Children With Cerebral Palsy

Stenberg, Reidun*; Dahle, Charlotte; Lindberg, Eva*; Schollin, Jens*

Author Information
Journal of Pediatric Gastroenterology and Nutrition: October 2009 - Volume 49 - Issue 4 - p 424-429
doi: 10.1097/MPG.0b013e31819a4e52
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Abstract

Children with severe cerebral palsy (CP) are often malnourished and have a retarded development of weight and height. Long-lasting starvation in early childhood is known to have a negative impact on motor and brain development (1,2). Some of the factors leading to malnourishment in children with CP are well known, and these children are nowadays receiving more support and help concerning their medical and habilitational needs (3,4).

Nourishing food that promotes good growth is a prerequisite for achieving optimal habilitation of children with CP. However, there are still a number of unsolved questions regarding nutrition and growth in these children. One of the questions that has not been thoroughly addressed concerns the intestinal uptake of nutrients.

A small pilot study recently indicated that children with CP have increased levels of anti-gliadin antibodies (AGA) (5). This finding raises the question of whether among children with CP there is an increased prevalence of celiac disease (CD) that may contribute to subnormal growth and development. So far, we know that there is an increased prevalence of childhood CD in Down syndrome, Turner syndrome, and diabetes mellitus (6). Furthermore, both neurological symptoms and white matter lesions have been reported in people with CD, both children and adults (7–10).

We, therefore, conducted the present study in a relatively large population of children with CP, to elucidate the questions of a possible association between CP and CD. The study protocol was approved by the human research ethics committees in Örebro and Uppsala.

PATIENTS AND METHODS

The study comprised children with CP living in the Swedish counties of Värmland and Närke, with a total number of inhabitants of 547,467. All of the children with a diagnosis of CP were registered and are well known at 2 large habilitation centers, 1 in each county. Among a total of 275 children registered as having CP, 91 children or their parents agreed to the children's participation in the study. One child was excluded after the enrollment because the CP diagnosis was not correct. Information about the type of CP was collected from medical records. The diagnoses and subtyping of CP were made in all cases by a pediatric neurologist. The nomenclature of CP and the subgroups were based on studies by Hagberg (11). During the study a functional assessment of each child was made on the basis of the Gross Motor Function Classification System (GMFCS) (12). The classification is graded I–V in which V is the most severe. The total load of disability was assessed from a combination of the classification of CP and GMFCS.

At the time of enrollment, the ages of the included children varied from 18 months to 18 years (median age 9 years). Forty-two were girls and 48 were boys. None of the children had any comorbidity for CD such as diabetes mellitus or dermatitis herpetiformis.

Body weight and height were recorded and blood samples were collected on inclusion. Sera were analyzed with regard to IgG and IgA antibodies against gliadin (AGA), endomysium (EMA), and tissue transglutaminase (tTG). The levels of total IgA were determined in 86 sera. All children with elevated serum levels of 1 or more of the antibodies tested were offered small-bowel biopsies. At the same time as the biopsy, blood samples for total IgG were collected. Total IgG was measured to find out whether there were elevated levels indicating a polyclonal hypergammaglobulinemic reaction.

Laboratory Analyses

Anti-gliadin Antibodies

Anti-gliadin antibodies of both the IgG and IgA class were determined by ELISA as previously described. The cutoff levels used for IgG-AGA (20 U/mL) and IgA-AGA (30 U/mL) were based on investigations of healthy blood donors (n = 1866) and children (n = 181, 1.5–17 years of age) and set at the 92.6th and 91.3rd percentile, respectively (13,14).

Anti-tissue Transglutaminase Antibodies

IgA and IgG antibodies against tTG were measured by ELISA, using human recombinant tTG (Biosystems Electrabox, Barcelona, Spain) in accordance with the manufacturer's instructions. For IgA-tTG the recommended cutoff level of 8 U/mL and a gray zone value of 6–8 U/mL were used. The IgG-tTG value was based on a qualitative analysis in accordance with the manufacturer's instructions. The results of IgG-tTG are expressed as a ratio, calculated as absorbance of the sample/absorbance of the cutoff control and were considered positive if the ratio was at least 1 (15,16).

Endomysium Antibodies

IgA- and IgG-EMA were assessed by indirect immunofluorescence microscopy. For detection of IgA-EMA, in-house cryosections of monkey esophagus were used, and for detection of IgG-EMA commercial slides with fixed sections of monkey esophagus (SciMedX, Denville, NJ) were used. Briefly, sera diluted 1/10 in phosphate-buffered saline (PBS, pH 7.2–7.4) with 1% BSA (bovine serum albumin fraction V from ICN cat. no. 810034) were applied to antigen-coated (monkey esophagus) slides and incubated for 30 minutes in a humid chamber at room temperature. After washing with PBS, the sections were covered with α- or γ-chain specific fluorescein isothiocyanate conjugated rabbit Fab2 antihuman IgA/IgG (DAKO Copenhagen, Denmark) and incubated for another 30 minutes in a humid chamber at room temperature. Finally, the slides were washed with PBS and mounted with PBS-glycerin on slides for fluorescence microscopy (UV microscope Zeiss, HBO 50 W). The result was considered positive if the presence of a reticulin-like pattern was recorded at a dilution of at least 1/10 (17,18).

Small-bowel Biopsy

Gastroscopes used were Pentax 2540, 2940, or 1840 (diameter 8.33, 9.67, and 6 mm, respectively). Biopsy specimens were taken from at least 2 parts of the duodenum and were then immersed in formaldehyde solution. The specimens were examined histologically at the Department of Pathology of the University Hospital in Örebro and were classified as follows: normal; duodenal intraepithelial lymphocytes, that is, mucosa with normal villi and thickness but with >30 intraepithelial lymphocytes per 100 epithelial cells; CD3 staining was done in most cases; partially villous, that is, the villi are reduced in length but the length is greater than the breadth and there is an increased number of intraepithelial lymphocytes; and subtotal villous atrophy; the breadth of the villi is greater than the length, or there is total villous atrophy (flat mucosa) with intraepithelial lymphocytes (19).

Statistical Analyses

Differences between groups were evaluated for statistical significance with Pearson χ2 test in cross-tabulations. An independent t test was used for body mass index (BMI) (standard deviation, SD), height (SD), and weight (SD), using 2-tailed significance. The level of significance was set at 0.05.

RESULTS

Thirty-nine (43%) of the 90 included children had elevated serum levels of 1 or more of the antibodies tested. None of the children had IgA deficiency (<0.07 g/L). Children with elevated antibodies had a lower body weight (P = 0.049), height (P = 0.041), and BMI (P = 0.014). One of 86 tested had increased total IgA. One of 19 tested had increased total IgG.

AGA

Thirty-two (36%) of the children with CP showed elevated levels of IgG-AGA, of which 4 of these levels were high (>200 U/mL). Nine (10%) tested positive for IgA-AGA, 1 of whom had high levels (>70 U/mL). Twenty-eight of the children showed an increase in IgG-AGA alone, 5 in IgA-AGA alone, and 4 in both (Fig. 1).

FIG. 1
FIG. 1:
Percentage and distribution of the elevated antibodies (n = 39) tested in 90 children with cerebral palsy.

Anti-tTG

Twenty-five (30%) of 85 (reagents were missing in 5) children tested for IgG-tTG had elevated levels and 6 of 90 children tested for IgA-tTG (7%) (Fig. 1). Twenty-one showed an increase in IgG-tTG alone, 1 in IgA-tTG alone, and 4 in both.

EMA

Neither IgG- nor IgA-EMA could be detected in any of the subjects.

Sex

No statistically significant difference was found between girls and boys regarding elevation of serum levels of AGA and/or anti-tTG (P = 0.172).

Small-bowel Biopsies

In 25 of 39 children with elevated serum levels of 1 or more of the antibodies tested, small-bowel biopsy was performed during the study. Another 2 patients had previously undergone small-bowel biopsy because of clinical suspicion of CD. One of these biopsies was performed before the study and confirmed CD, but this child had not since been on a strict gluten-free diet (GFD). The other child underwent biopsy earlier, with a normal histological result. No major difference in the distribution of subtypes and functional grades of CP between children who did or did not undergo a duodenal biopsy was seen, with reservation for the small number of children in the dropout group for biopsy (n = 12).

Intraepithelial lymphocytosis was found in 2 of the 25 biopsied children during the study. One of these 2 children tested positive for IgG-tTG (ratio 3.31) and negative for IgA-tTG; the IgA-AGA level was 79 U/mL and the IgG-AGA level 369 U/mL. The other child had raised levels of IgG-AGA (94 U/mL) and IgG-tTG (ratio 1.09), but was negative for IgA-AGA and IgA-tTG. In the other 23 children, the mucosal biopsy results were normal.

In conclusion, small-bowel biopsy was performed in 27 of 39 children. Celiac disease had been confirmed earlier in 1 child and intraepithelial lymphocytosis in another 2 children.

Relation to Clinical Findings

Elevated levels of 1 or more of the tested antibodies were significantly more often found in children with a more severe type of CP (tetraplegia or dyskinesia) compared with other CP types (P = 0.045) (Fig. 2A). The same pattern was found when individual GMFCS classes were compared with each other, however, not statistically significant (Fig. 2B). Children with the most severe handicap (GMFCS V) had significantly higher levels of tested antibodies compared with children with less severe functional classes (GMFCS I–IV) (P = 0.008).

FIG. 2
FIG. 2:
Distribution of children with different subgroups of cerebral palsy and Gross Motor Function Classification System (GMFCS) and its relation to elevated anti-gliadin antibodies and/or antibodies to transglutaminase. (A) Subgroups of cerebral palsy. (B) GMFCS: functional classification, in which V is the most severe disability.

Children with elevated levels of at least 1 of the tested antibodies had a significantly lower body weight (P = 0.019), height (P = 0.026), and BMI (P = 0.004) (Fig. 3).

FIG. 3
FIG. 3:
Distribution of weight, height, and body mass index (BMI) in standard deviations among children with cerebral palsy in relation to elevated (n = 39) and normal (n = 51) levels of anti-gliadin antibodies and/or antibodies to transglutaminase.

One child with high levels of IgG-AGA and a slightly elevated ratio for IgG-tTG had gastrointestinal problems with diarrhea. He had previously undergone a liver transplantation because of sclerosing cholangitis and biopsy was not performed. He was put on GFD and the diarrhea subsequently decreased.

The biopsy samples from 2 children showed intraepithelial lymphocytosis. One of these children had clearly elevated IgG-tTG (ratio 3.31) and high levels of IgA- (79 U/mL) and IgG-AGA (370 U/mL). This child had gastrointestinal problems with constipation and vomiting and was put on GFD. His parents reported that he became more alert and communicative after the introduction of GFD. The other child had a high level of IgG-AGA (93.9 U/mL) and a slightly elevated level of IgG-tTG (1.09). After GFD had been introduced, he gained weight and became more alert.

Eleven of the children had a gastrostomy and 8 of these had elevated levels of 1 or more of the antibodies tested. In at least half of these children, the gastrostomy had been performed early in life (<4 years of age). The majority of these children were taking some gluten-containing food orally.

DISCUSSION

The main finding in this study was that among the studied children with CP, a high proportion had antibodies to gliadin and a somewhat lower proportion to tTG. The explanation for this is not clear, but several questions arise regarding an etiopathogenic association with CD and a possible link between the 2 diseases.

In our study, the children with positive antibody tests had a significantly lower weight, height, and BMI, and were more severely disabled compared with those with negative tests. From earlier studies, it is known that the majority of children with dyskinesia have a normal birth weight (20). We found that the patients with CP with dyskinesia or tetraplegia had significantly higher frequencies of positive AGA and/or anti-tTG. Among the children in the present study, 1 had CD, which is an expected finding considering the Swedish CD prevalence rate of 1 to 2.5/100 (21). In addition, 2 of the 30 children who underwent duodenal biopsy had intraepithelial lymphocytosis, which is a nonspecific sign of inflammation, but an underlying gluten-induced reaction cannot be ruled out.

Our most notable finding was an increased prevalence of IgG-AGA in patients as compared with that in healthy children (14), but only 5% of the patients exhibited high antibody levels; the clinical implication of this finding is indecisive. In view of its low disease specificity, IgG-AGA is not regarded as a reliable diagnostic marker of CD, but IgG-class antibodies to tTG and EMA are valuable diagnostic CD markers in patients with IgA deficiency (22,23).

Concerning antibodies of the IgA class, AGA is still considered to be the most sensitive CD marker in children younger than 2 years, but not at older ages, as was the case in the majority of the patients in the present study (24). We did not find any patient with a positive EMA test, which is a highly specific marker for untreated CD. However, it is known that untreated patients with CD, with or without severe intestinal damage, can be EMA negative (25). In contrast to IgA-EMA, IgA anti-tTG was found in a substantial number of our patients, although usually at low levels (Fig. 1).

IgA-tTG is considered to have a high specificity for CD (16,17). Recently, however, several reports have been published regarding occurrence of IgA-tTG in primary biliary cirrhosis, end-stage heart failure, and in acute coronary syndrome without concomitant CD (26–28).

The terms “early developed CD” and “latent CD” have not been clearly defined, although latent CD is often diagnosed retrospectively and can be provoked by gliadin (29,30). Whether or not AGA/EMA/anti-tTG can be seen before the occurrence of villous atrophy is unclear. Bearing this in mind, we decided to offer a duodenal biopsy to all children with a positive test in any of the analyses performed in this study.

Hadjivassiliou et al (31) suggested that the occurrence of IgG-AGA in adults may be due to increased sensitivity to gliadin without the presence of an intestinal affection and that IgG-AGA may cross-react with Purkinje cells in the cerebellum, leading to cerebellar ataxia, that is, gluten ataxia. From these possibilities, it may be speculated whether gliadin and cerebellar Purkinje cells share common epitopes. It has also been proposed that AGA may cross-react with vascular endothelial cells in the brain (32), although the specificity of these reactions can be questioned. It is tempting to speculate that antigenic cross-reactions may also concern other areas of the central nervous system, which hypothetically could be relevant to the present study. Immunoreactivity to gliadin has recently been reported to result in cross-reactivity with neuronal antigens; for example, binding of AGA to synapsin I has been observed in patients with ataxia, seizures, neuropathy, and neurobehavioral symptoms with and without affection on the gut (33).

In a study by Kieslich et al (10) it was found that 20% of children with CD had magnetic resonance–verified lesions of the brain, which could represent an extraintestinal manifestation of CD (9,34,35).

In our study, positive IgG-AGA and IgG-anti-tTG tests were not accompanied by histological damage to the gut mucosa in children with CP. The whole group of children with CP had lower weight, height, and BMI compared with healthy children. The children who had elevated antibodies of AGA and/or tTG were significantly more affected in these parameters. The reason for this is not clear. These children may have an affection of the gut that is too early to be recognized by our present methods. One can also speculate whether these children have an unspecific mucosal inflammation that contributes to the retarded weight and height. However, this does not exclude the possibility of a deficient mucosal barrier, for example, after exposure to gliadin with subsequent immunization. CD does not seem to be the reason for the retarded development of weight and height in these children.

In conclusion, we found elevated levels of antibodies to gliadin and/or to tTG among 39 of 90 children with CP without evidence of an increased prevalence of CD. The clinical implications of our findings are unclear, but call for extended studies.

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Keywords:

Antibodies to tissue transglutaminase; Antigliadin IgG and IgA antibodies; Celiac disease; Cerebral palsy; Small-bowel biopsy

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