It was hypothesised that primary gastrointestinal (GI) pathologies and intestinal barrier defects could play an important role in the triggering and clinical expression of some childhood developmental disorders, including autism (1–4) (5) (6,7) (8,9)
Ileocolonic lymphoid nodular hyperplasia and mild acute and chronic inflammation of the colon-rectum, small bowel, and stomach have been reported in children with ASD. Although some of the reported findings are still controversial (3,10–12) (13) intestinal permeability (IPT) has also been reported (14) (4)
Although the possible mechanisms are unknown, it has long been suggested that some intestinal lesions that increase IPT to exogenous peptides of dietary origin may lead to the disruption of neuroregulatory mechanisms and normal brain development (enterocolonic encephalopathy) (15) (10)
The question then arises as to whether it is possible to identify subgroups—that is, either gut inflammation or abnormal IPT, or both of them, in children with ASD as the first steps towards the design of efficacious therapeutical interventions. IPT, as measured by the urinary excretion of metabolically inert sugars after oral dosing, is a surrogate marker of mucosal integrity (16) (17–20) (14) (21) (5)
The presence of a genetic predisposing factor for the leaky gut condition could be inferred through the study of IPT in first-degree relatives of subjects with ASD, just as it has already been done in inflammatory bowel disease (IBD) and other GI conditions (18–20)
GI inflammation can be investigated by measuring faecal calprotectin (FC), a protein produced by intestinal granulocytes (22)
The aim of the present research is to verify whether the GI barrier is actually impaired and whether gut inflammation is present in a large cohort of patients with ASD and in their first-degree relatives, using noninvasive tools.
PATIENTS AND METHODS
To carry out the present research we investigated 90 children with ASD and 146 of their first-degree relatives; data were compared with 64 children and 146 adult normal subjects as control groups. All of the subjects were given an IPT test (lactulose [LA]/mannitol [MA]), and all of the patients and their first-degree relatives underwent an FC determination. Blood tests for coeliac disease markers—serum anti-tissue transglutaminase antibodies (anti-tTG), anti-gliadin antibodies (AGA IgG and AGA IgA), anti-endomysium antibodies (EMA), and human leucocyte antigen (HLA) DQ2-DQ8 (DQ2/8)—were performed in all of the patients with ASD as well as in all of the first-degree relatives and controls who showed high values of IPT and/or FC. Informed consent was obtained from the parents of each patient and control subject before starting any procedure.
Subjects
Ninety subjects with ASD were recruited from either the outpatient or inpatient services of the Child and Adolescent Neuropsychiatry Unit at the Second University of Naples, Italy. The sample included 81 boys and 9 girls (mean age ± SD 7.4 ± 5.1 years). There were 4 couples of twins. All of the children were administered the Autism Diagnostic Interview-Revised version (23) (24) (25) Diagnostic and Statistical Manual of Mental Disorders-IV criteria for AD.
One hundred forty-six first-degree relatives were included (mean age ± SD 40.2 ± 8.7; female = 72, male = 74; siblings = 8, female = 4, male = 4, mean age ± SD 12.4 ± 5.0). A total of 85 families took part in the study, including 2 children whose (foster) parents did not take part in the study. An expert clinical review was conducted of their social functioning and participation history to exclude the presence of symptoms related to pervasive developmental disorders.
One hundred forty-six healthy subjects (age range 19–66 years, mean age ± SD 31.8 ± 12.3; female = 98, male = 48) were recruited from among the staff members of the department and their families, together with 64 healthy children (mean age ± SD 7.1 ± 3.1; female = 30, male = 34). None of these subjects claimed any recent GI symptoms, nor were they affected by any major GI diseases.
A carefully detailed GI anamnesis was collected for each subject, with special regard to type of special diet if any (eg, gluten-free or gluten-casein–free diet [GCFD]), reported food intolerances, and recently determined aspartase aminotransferase and alanine aminotransferase values to assess the general state of health of the liver.
Inclusion criteria for control subjects and first-degree relatives were as follows. All of the subjects were requested not to smoke, drink alcohol, or take any kind of nonsteroidal anti-inflammatory drugs (or other anti-inflammatory drugs) for at least 3 days before the test. The exclusion criteria were pregnancy and/or the concomitant condition of known coeliac disease and/or other major diseases of the intestinal tract, such as IBD and hepatic disorders, as well as known and serologically proven food intolerances. In all of the subjects with ASD, serum anti-tTG, AGA IgG and AGA IgA, EMA, and HLA DQ2-DQ8 were determined. The above parameters were also investigated in all of the first-degree relatives and controls who showed high values of IPT and/or FC.
Informed consent was obtained from each selected subject and from the parents of children. The study was approved by the ethics committee of our department and was carried out in accordance with the Helsinki Declaration of 1975.
Intestinal Permeability IPT is regarded as a valuable and noninvasive test for monitoring mucosal damage of the small intestine. The procedure is based on the simultaneous oral administration of 2 sugar probes of different molecular sizes and absorption routes and the estimation of urinary recovery of each molecule. In the present study, an LA/MA test was administered, as previously described (26,27) (26) (26,27)
Faecal Calprotectin
FC was determined from a stool sample from each subject to investigate intestinal inflammation. FC was detected by means of enzyme-linked immunosorbent assay (ELISA) (Calprest, Eurospital, Italy). Briefly, this method is based on the use of a polyclonal antibody against calprotectin in an ELISA system, with the addition of a final coloured product. Normal values were estimated to be <100 μg/g stool (adult and children) on the basis of previous reports (28,29)
Anti-gliadin Antibodies
The determination of specific IgA and IgG antibodies against α-gliadin in serum was achieved by means of an ELISA (Eurospital, Italy). Normal values were set as <8 U/mL and <50 U/mL, respectively.
Anti-tissue Transglutaminase Antibodies
The determination of specific IgA antibodies against tissue transglutaminase in serum was achieved by means of an ELISA (Eurospital, Italy). Normal values were <8 U/mL.
Anti-endomysium Antibodies
The detection of class IgA EMA was achieved by indirect immunofluorescence on sections of human umbilical cord (Eurospital, Italy).
Human Leucocyte Antigen
The Eurospital Eu-DQ kit was used for the determination of HLA II, DQ2, and DQ8 haplotypes in human whole blood, respectively coded by alleles DQA1*05-DQA1*0201/DQB1*02 and DQB1*0302. It requires the preliminary DNA extraction and purification from whole blood, subsequent DNA amplification in PCR, and detection of amplificates on agarose gel.
Statistical Analyses
Statistical significance was assessed at a level of 0.05. Variables were summarised either as mean ± SD and/or as frequency and percentage. The Student t test, the Mann-Whitney test, and analysis of variance (ANOVA) with Bonferroni correction were used to evaluate the differences among means. The dependence between pairs of parameters was evaluated as a simple linear correlation (r ) with the Spearman test. Data handling and analysis were performed through Graph Pad Prism 5 (GraphPad Software Inc, La Jolla, CA).
RESULTS
Among patients with ASD, 33 of 90 (36.7%) showed abnormal IPT compared with 31 of 146 (21.2%) first-degree relatives, 7 of 146 (4.8%) adult controls, and none of the child controls. The percentage of abnormal values of patients with ASD and that of their relatives were significantly different from those of the respective control groups (Fisher exact test, P < 0.0001) (Table 1 ).
TABLE 1: Demographic characteristics and permeability data of the investigated subjects
IPT mean value results were significantly different among the 4 investigated groups: ASD, 0.041 ± 0.08; relatives, 0.028 ± 0.050; adult controls, 0.013 ± 0.01; and child controls, 0.023 ± 0.01 as shown in Table 1 and Figure 1 . The 1-way ANOVA with Bartlett correction gave the results P < 0.0001. The difference between mean values from relatives and adult controls was significant, with P = 0.019 (Mann-Whitney test).
FIGURE 1: IPT is measured in the 4 investigated subgroups and expressed as LA/MA values (mean ± SD): patients with autism spectrum disorders (N = 90), relatives (N = 146), adult controls (N = 146), child controls (N = 64). One-way ANOVA with Bartlett correction was P < 0.0001. The difference between mean values from relatives to adult controls was significant, with P = 0.019 (Mann-Whitney test).
Mean values of MA% and LA% recovered in the various groups are reported in Table 1 . The increase in LA/MA values, in both patients with ASD and their relatives, is mainly due to the 2- to 3-fold increase in LA compared with MA recovery.
Most of the recruited children with ASD had received a final diagnosis in the 2 years before entering the study and only a small percentage of them (23/90; 25.6%; mean age ± SD 9.1 ± 4.7) were on a reported GCFD. As shown in Figure 2 , changes in feeding regimens affected IPT because in children on GCFD the LA/MA mean value was 0.017 ± 0.012, lower than that of children on an unrestricted diet (0.055 ± 0.097) (P = 0.034, Mann-Whitney test). IPT mean values of the children on GCFD showed significantly different results from those of the control children (P = 0.039, Student t test) and the children on an unrestricted diet (P = 0.014, Student t test).
FIGURE 2: Small intestine barrier function is more deregulated in the children with autism spectrum disorders with regular eating habits than in those who are on a gluten-casein–free diet (GCFD) (P = 0.034, Mann-Whitney test). Intestinal permeability , expressed as LA/MA values (mean ± SD), in the 2 groups and child controls is reported. The differences between the 2 groups (GCFD and free diet) vs controls are both significant (P = 0.039 and P = 0.014, respectively; Student t test).
To examine any possible correlation between abnormal IPT values and the presence of GI symptoms, as well as other specific characteristics of the patients (eg, sex, age, HLA DQ2/DQ8) children with ASD were divided into 2 groups, abnormal or normal IPT. As shown in Table 2 , there was no correlation between the presence of GI symptoms and abnormality of IPT values. GI symptoms were referred by the parents and were present in 42 of 90 children (46.7%), namely constipation in 20 of 44 (45.5%), diarrhoea in 15 of 44 (34.1%), and others (eg, alternating diarrhoea/constipation, abdominal pain) in 7 of 44 (15.9%). Sex and age distribution were comparable between the 2 subgroups and HLA DQ2/DQ8 were present, as is normally found in the general population.
TABLE 2: IPT values of patients with ASD (N = 90)
An ANOVA conducted to compare individual IPT values with various scores from the Autism Diagnostic Interview-Revised, Autism Diagnostic Observation Schedule, and Childhood Autism Rating Scale questionnaires showed no significant correlations (F = 1.708; F = 0.595; F = 1.464).
The values of FC were abnormal in 22 of 90 (24.6%; range 102.5–387.4 μg/g) of patients with ASD and 17 of 146 (11.7%; range 108.7–375.7 μg/g) of relatives; that is, these subjects showed FC values higher than normal (ie, FC >100 μg/g). In Table 3 the means of the pathological values in the 2 groups are given to show how their entity could account for a condition of mild inflammation (30)
TABLE 3: FC in patients with ASD and their first-degree relatives
There was no correlation between the IPT and FC values; the linear correlation analysis gave the results r = 0.09 (ASD) and r = 0.23 (relatives) (Spearman test).
Serological parameters to exclude coeliac disease are reported in Table 4 . The investigated patients with ASD were negative for tTG, EMA, and AGA IgA, whereas 2 of them showed high AGA IgG values. These 2 high values (both >100 U/mL) were associated with high FC values (200 and 248 μg/g) and normal IPT (0.012 and 0.008). Familial and personal anamneses were both free from any indication of food intolerances.
TABLE 4: Serum parameters (mean ± SD) to evaluate presence of concomitant coeliac disease and/or hepatic involvement
First-degree relatives who showed abnormal IPT were first screened for the presence of HLA DQ2/DQ8 alleles, which gave positive results in 10 of 31 (32.3%) cases; 3 had DQ8 and 7 had DQ2. FC was abnormal in only 2 of them. Although no other serological signs of coeliac disease were found (Table 4 ), the subjects were invited to undergo an upper GI endoscopy.
From a familial point of view, when a relative had abnormal IPT, a child with ASD also showed this condition in 15 of 31 cases (48.4%). In 5 cases (5/85 families, 5.6%), we found 3 or more members of the same family with abnormal IPT. The clinical concordance for ASD between the investigated twins was 100%; that is, all of the investigated couples of twins were both affected. Conversely, the concordance of IPT values was 75%.
DISCUSSION
In the present study, evidence is provided that supports the view that a genetically determined abnormal IPT is present in ASD (4,14)
As a matter of fact, IPT was abnormal in a large percentage of subjects with ASD; subjects with ASD were reported to benefit from a gluten-free diet (1) (31–35) (36–38)
A recovered barrier function would eventually prevent digestion products of natural food from entering the blood through the leaky mucosa and inducing antigenic responses as well as reducing the interference with the central nervous system. Some recent studies have found a close relation between dietary change and the onset of symptoms in patients with ASD (6,39–42) (15,43) (9) (15)
Another possibility is that 1 or more enzymatic defects affecting the digestion of such proteins are present; they could produce “aberrant” peptides, which would cross a weak barrier—a weakened intestinal barrier still is a necessary condition—thus reaching peripheral areas of the body causing damage (4)
The detection by D'Eufemia et al (14) Table 2 ). Again, a possible subgroup stratification evolves in which “GI symptomatic” does not always overlap to “barrier function deficit.” In many cases, however, the perception remains that intestinal symptoms are expected in children with developmental disorders, reflecting the effect rather than a primary GI dysfunction. A recent article (21)
Many studies were conducted in the first-degree relatives of patients with IBD in the attempt to prove the existence of a genetic defect (18–20)
Abnormal IPT was found in asymptomatic subjects, including relatives and adult controls, suggesting that a weakened intestinal barrier function could be present and underestimated in a part of the general population. The identification of increased IPT in a subject is not a diagnostic endpoint, but does indicate the need for additional detailed investigation.
Coeliac disease was excluded in all of the investigated subjects, based on the clinical and serological markers, thus confirming the previous findings (44) (45)
In the present study, the increased IPT is mainly due to the 2- to 3-fold increase in passive LA absorption. The MA, being a monosaccharide and relatively small, is absorbed via a transcellular route (reflecting absorptive capacity), and LA, a disaccharide, is absorbed via an inter- or paracellular route (reflecting barrier function). An increase in LA urinary recovery compared with that of MA indicates a fall in the intestinal barrier function (16,46) (27)
IPT was reported to be often high in the presence of intestinal inflammation, as in Crohn disease and coeliac disease (18–20,47) (22) (48) (49) (50)
FC is a protein typically produced by granulocytes, and its quantity in stools is directly correlated to the degree of inflammation. In IBD, increased permeability is the consequence of inflammation; in ASD, IPT is most likely a primary defect independent of inflammation related to functional changes that can be secondary to specific genetic predisposition. IPT alteration in ASD is not directly correlated with inflammation: A massive intervention of immune system cells is secondary to a prolonged and/or disruptive breakdown of the intestinal barrier. The mean pathological value of FC found in these patients (159.7 ± 74.0 μg/g; Table 3 ), although the results should be confirmed by repeating the measurement (48) (29,30)
To summarise, our results show that IPT is abnormal in a subgroup of children affected by ASD. This result may be extremely important in that IPT determination could be used as a biomarker for a subgroup of children who can benefit from treatments targeting specifically the leaky gut. The presence of a genetic factor influencing the intestinal barrier is suggested through the finding that a large number of first-degree relatives showed IPT impairment as well. The influence of environment (ie, “toxic” gluten) (35,51)
Acknowledgments
This research would have been impossible without the support of the “Oltre Il Muro” association as well as the active and compliant participation of many parents of children with ASD. The authors also wish to thank Eurospital for their technical support; Prof Alessio Fasano for scientific support and unconditional encouragement; and Mrs Luciana Polidoro and Ms Julia Colacino for linguistic assistance.
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