A nationwide study of small intestinal histopathology and risk of autistic spectrum disorders

Autistic spectrum disorders (ASD)

ASD was defined as having a relevant ICD-9 or ICD-10 code (international classification of disease code in the Swedish Patient Register (Appendix)). The Swedish Patient Register started in 1964, becoming nationwide in 1987. Since 2001, it also includes hospital-based outpatient care. In Sweden, physicians examine all 4-year-old children as part of the national child health surveillance system. When an ASD is suspected at this mandatory screening, or earlier by parents or in infant care, the child is referred to a child psychiatrist specialist unit. The validity of the registry-based diagnosis is high. Two of the present authors (AR and CMH) recently conducted a medical record review by ascertainment of a random sample of cases (n=88) with ASD that appear in the Patient Registry and implemented a validation protocol developed by the Centers for Disease Control and Prevention (CDC).²¹ In 94.3% (83/88) could we substantiate the presence of DSM-IV autism according to medical records reviews, identical to findings from Denmark.²²

Celiac disease, inflammation, and normal mucosa

We searched computerized biopsy registers at Sweden’s 28 pathology departments and obtained data on personal identity number,²³ date of biopsy, and morphology (see Appendix for a list of histopathology codes according to the Swedish SnoMed system) in all individuals undergoing a duodenal or jejunal biopsy. CD was defined as having VA (equivalent to Marsh histopathology stage 3, see Supplementary Table 1).²⁴ We also collected data on individuals with a lesser degree of mucosal damage, such as inflammation without VA (equivalent to Marsh 1–2)²⁴ and normal mucosa (Marsh 0).²⁴ In total, we identified 287,586 unique individuals who had undergone a biopsy (29,148 with VA, 13,446 with inflammation, and 244,992 with normal mucosa)(Figure 1).²⁵ Data on normal biopsies performed at 10 university hospitals (n=121,952) were then sent for matching with CD serology at the 8 biochemistry departments responsible for the same catchment area as the university hospitals. We were thus able to identify 3,736 individuals with normal mucosa but positive IgA/IgG gliadin, endomysium, or tissue transglutaminase antibodies at time of biopsy (≤180 days before biopsy until ≤30 days after biopsy).²⁶ A detailed description of the data collection procedure has been published elsewhere.^{25, 26} We then excluded individuals with data irregularities (Figure 1), and those undergoing a biopsy (or entering the study) before 1 January 1987, since only then could a patient be diagnosed with ASD according to our definitions. In all, this study included 26,995 individuals with CD, 12,304 individuals with inflammation but no VA, and 3,719 individuals with normal mucosa but positive CD serology. (Of the latter 3,719 individuals, 359 had positive IgA endomysium or IgA tissue transglutaminase while 3,360 had positive IgA gliadin or positive IgG antibodies against endomysium, transglutaminase, or gliadin. See appendix for detailed information)

We did not require a positive CD serology for the diagnosis of CD, and data on positive CD serology were only collected in a sub-sample of individuals with CD (n=3,388 with positive serology) and inflammation (n=141). However, a patient chart validation found that 88% of individuals with available data on CD serology were positive at the time of biopsy.²⁵ This same evaluation found that the positive predictive value of VA for CD was 95% (n=108/114).²⁵

Controls

For each patient undergoing biopsy, the government agency Statistics Sweden (SCB, responsible for producing official statistics), identified up to five controls from the total population register, matched for sex, county, age, and calendar year. Controls were only selected from among individuals without a previous record of small intestinal biopsy. Finally, for arguments given above, we only kept controls entering the study in 1987 or later. In all, this study was based on 213,208 controls (CD controls: n=134,076, inflammation: n=60,654, and normal mucosa but positive CD serology: n=18,478). We did not have data on CD serology status in controls.

Analyses

In this paper we used two statistical approaches to examine the relationship between CD and ASD.

Ethics

The current study was approved by the Ethics review board in Stockholm, Sweden (2006/633-31/4; approved June 14^th, 2006). The board did not require individual informed consent because data were strictly register-based.

Background data

About half of the study participants underwent biopsy in 2000 or later (47.9% of CD individuals had been diagnosed since 2000). The majority were females (Supplementary Table 2). Some 40% of individuals with CD were diagnosed in childhood. Controls were matched on sex, age, and calendar period; thus, their distribution was identical to that of the individuals undergoing biopsy (Supplementary Tables 2 and 3).

Conditional logistic regression: prior ASD

Having a prior diagnosis of ASD was not associated with CD (OR=0.93; 95% CI=0.51–1.68) or inflammation (OR=1.03; 95% CI=0.40–2.64) but with a highly increased risk of having a normal mucosa but positive CD serology (OR=4.57; 95% CI=1.58–13.22) (Table 1). The number of individuals with ASD is given in Supplementary Table 2. Six individuals with normal mucosa but positive CD serology had an earlier diagnosis of ASD. These individuals were positive for different antibodies (IgA Gliadin: n=3; IgG Gliadin: n=2; and IgA Endomysium: n=1) Adjusting for intrauterine growth retardation did not influence the risk estimate for CD (adjusted OR=1.04; 95% CI=0.56–1.95); nor did adjustment for education or country of birth (data not shown).

Sensitivity analyses showed similar ORs to the above (Table 1), with the exception of a slightly lower OR for normal mucosa and positive CD serology in individuals with at least two records of ASD (OR=3.14; 95% CI=0.94–10.51) and a slightly higher OR in the same patient group when we restricted study participants to those born in 1987 or later (OR=7.05; 95% CI=1.80–27.53) (Table 1).

No individual with CD who was diagnosed from 40 years of age or later had a record of earlier ASD. With this exception, and our finding of a non-significantly lower OR for CD in females with ASD, there were no differences in ORs based on age, sex, or calendar period (Supplementary Table 4).

In a posthoc analysis we estimated the OR for earlier ASD in patients with villous atrophy (here classified as CD) who also had positive CD serology. We found no association between serologically positive villous atrophy and earlier ASD (OR=1.47; 95%CI=0.52–4.15). None of the 141 individuals with inflammation and positive CD serology had an earlier diagnosis of ASD and hence no risk estimate could be calculated for that association.

In a second posthoc analysis we calculated the OR for earlier ASD in individuals who either had villous atrophy (here classified as CD) or positive IgA tissue transglutaminase/endomysium at time of biopsy. This combined group was not associated with earlier ASD (OR=1.21; 95%CI=0.74–1.97).

Cohort study: future ASD

Individuals with CD and inflammation were between 1.5 and 2 -fold increased risk of having a later diagnosis of ASD (Table 2), whereas the highest HRs for ASD were seen in individuals with normal mucosa but positive CD serology (Table 2).

Comparison with earlier literature

To our knowledge, the first large case series with autistic individuals undergoing investigation for CD was reported in 1973.²⁸ In that study 18 children with ASD were examined, of which 7 had a history of gastrointestinal symptoms.²⁸ Three children underwent small intestinal biopsy, but none of these children had VA.²⁸ In a second study eight autistic children with steatorrhea and alleged behavioral improvements on gluten restriction underwent biopsy but neither in this group did the researchers identify any patient with CD.²⁹

Two larger studies have investigated the association between CD and ASD. In the first Pavone et al evaluated 120 CD individuals from Catania, Italy.¹⁸ Parents were asked to answer 16 DSM-IIIR questions relating to their child's behavior.¹⁸ The researchers concluded that the prevalence of ASD was not elevated in CD. The same researchers tested 11/22 children with infantile autism in the same hospital for antigliadin and endomysium antibodies. Although two children were serologically positive, both had normal small intestinal mucosa. In a second study Batista et al examined 211 individuals with biopsy-proven CD for ASD.²⁰ Two of these 211 children had an ASD, resulting in a prevalence of ASD of 0.95% (95% CI=0.11–3.82%). Of 147 individuals with diagnosed ASD, 6 had positive antigliadin or transglutaminase antibodies, but all were negative for the more CD-specific endomysium antibodies. In contrast, Barcia et al reported that 5/150 individuals with ASD had both serological and histopathological findings of CD (p=0.014).¹⁷

Our data are consistent with earlier research in that we found no convincing evidence that CD is associated with ASD,^18–20 except for a small excess risk noted after CD diagnosis. A possible explanation for the excess risk of ASD after CD diagnosis is surveillance bias. We found a strong association (OR>l4) between positive serology (with normal mucosa) and later ASD. These individuals may suffer from non-celiac gluten sensitivity,¹² in which a gluten-free diet can be beneficial.³⁰ Markers of gluten sensitivity has been linked also to other neurological³¹ or psychiatric³² disorders such as schizophrenia.^{33, 34} Interestingly many antibody-positive patients are negative for HLA DQ2/8³⁵ suggesting that the response to gliadin in psychiatric and neurological disease³³ may be typical of non-celiac gluten sensitivity rather than CD. Sensitivity-related illnesses are increasing,³⁶ and effects from gluten may be mediated by a number of mechanisms.

Part of the positive association between positive serology and ASD could also be due to an increased likelihood of serological testing for CD in children with ASD but not altogether since with increased serological testing more individuals with true CD should also have been diagnosed. The association between serologically positive CD and earlier ASD was neutral although this analysis was limited in power.

The role of gluten and a gluten-free diet in individuals with ASD is under debate. One study reported that a gluten-free diet in 15 children with ASD had no effect; however, after the trial, parents of 9 of the children wanted to continue with the diet because they thought their autistic children had improved.^{37, 38} A Cochrane review found only small effect of gluten- and casein-free diets on ASD,³⁹ whereas a later randomized single-blind study reported that dietary intervention with a gluten- and casein-free diet had a beneficial effect in some children with ASD.⁴⁰

The current paper has some limitations, including the fact that we did not have data on symptoms in individuals with ASD. If these individuals have more gastrointestinal symptoms because of other disorders than CD, that could result in surveillance bias. Awareness of the potential effect of dietary interventions in ASD may also have led to more testing for CD. However, as stated previously, this should have resulted in a higher OR for all three pathology groups and not just for having a positive serology with normal mucosa.

At the same time, impaired communication in autistic individuals may lead to difficulties in communicating gastrointestinal symptoms, but could also make physicians hesitant to perform a biopsy before CD diagnosis in individuals with ASD. A major strength of our paper is therefore our use of several patient groups in which the high risk for positive serology in those with normal mucosa shows that the lack of association with CD is unlikely to be due to physicians refusing to perform small intestinal biopsies.

We used nationwide registers to ascertain ASD. Although the prevalence of ASD in our study is lower than in smaller data sets in which individuals are screened for ASD, our prevalence of ASD (either before or after study entry) in our reference population (controls) was nevertheless 2.8/1000 (606 divided by 213,208), which is consistent with American register-based data on ASD prevalence (0.6–4.6/1000).⁴¹

Another shortcoming is that we lacked data on gluten-free diet. A gluten-free diet usually results in negative CD serology and a lower probability of undergoing small intestinal biopsy. Hence, if a large number of autistic individuals were on a gluten-free diet already when being investigated for CD that could underestimate the association with CD. However, the strong positive association between ASD and positive serology makes such an explanation unlikely. Antibody levels will usually decrease before the mucosa heals in persons on a gluten-free diet.⁴²

At the same time, we cannot rule out that the moderate excess risk for ASD seen in individuals with diagnosed CD (HR=1.49) may be restricted to CD individuals with poor compliance, although we believe surveillance bias is a more likely explanation. In a validation of a random subset of individuals with CD 17% had evidence of poor dietary adherence.²⁵

Another limitation is that we did not have access to data on family history and could not adjust for parental autoimmunity. We have previously shown that parental autoimmunity is a risk factor for ASD. However, different patterns of parental autoimmunity are unlikely to explain the highly increased risk for positive serology and normal mucosa, as well as a largely neutral risk for CD. Finally we did not have data on serology in all individuals with CD and inflammation.

Apart from the different comparison groups, the main strength of our paper is its statistical power. This study included more cases of individuals with both CD and ASD than all previous papers taken together (12 before CD diagnosis and 127 after, n=139). The large power allowed us to calculate narrow confidence intervals and to test the association with positive CD serology in various sensitivity analyses. We also had data on a number of potential confounders, including country of birth, education, and, in a subset of individuals, intrauterine growth retardation. Adjustment for these variables had little effect on the risk estimates.

Other advantages include the high sensitivity and specificity of VA as proof of diagnosed CD. More than 95% of Swedish gastroenterologist and pediatricians perform a biopsy before celiac diagnosis,²⁵ and Swedish pathologists classify 90% of all slides with VA correctly.²⁵

Another strength is our long follow-up. This study was based on data on ASD recorded in the Swedish National Patient Register over a period of more than 20 years.

Potential mechanisms

Given that ASD is linked to early brain development, the reason for the positive association with positive CD serology is not self-evident. Because most individuals with positive CD serology are likely to have the same HLA as CD individuals with VA (where little association with ASD was seen), shared genetics is unlikely to explain our findings. Instead, we speculate that the positive association between ASD and positive celiac serology observed in this study may be due to increased mucosal permeability noted in some individuals with early CD^{43, 44} or individuals with elevated levels of more non-specific antigliadin antibodies.⁴⁵

A recent Italian study found a significantly higher intestinal permeability in patients with ASD and their first-degree relatives compared to healthy controls.⁴⁶ However, this study also showed that patients with ASD who avoided gluten and casein had lower intestinal permeability compared to those consuming gluten and casein.⁴⁶ A high intestinal permeability may allow an increased absorption of short peptides that trigger the immune system leading to ASD. We cannot, however, explain the different pattern in individuals with CD vs. those with normal mucosa but positive CD serology. Unfortunately we had no information on antibodies to other food antigens that could perhaps shed light on whether this is a broad sensitization.

In conclusion, we found weak evidence of a link between ASD and CD but a strong association between ASD and positive CD serology in individuals with normal mucosa.