Although the major deficits in ASD are social and cognitive, many affected individuals with ASD also have substantial GI morbidity. Major findings in this study that may shed light on GI morbidity in ASD include the observations that: (1) levels of transcripts for disaccharidases and hexose transporters are reduced in AUT-GI children; (2) AUT-GI children have microbial dysbiosis in the mucoepithelium; and (3) dysbiosis is associated with deficiencies in host disacharidase and hexose transporter mRNA expression. Based on these findings, we propose a model whereby deficiencies in disaccharidases and hexose transporters alter the milieu of carbohydrates in the distal small intestine (ileum) and proximal large intestine (cecum), resulting in the supply of additional growth substrates for bacteria. These changes manifest in significant and specific compositional changes in the microbiota of AUT-GI children (see ).
A model for GI disease in children with autism.
A previous report on GI disturbances in ASD found low activities of at least one disaccharidase or glucoamylase in duodenum in 58% of children 
. In our study, 93.3% of AUT-GI children had decreased mRNA levels for at least one of the three disaccharidases (SI, MGAM, or LCT). In addition, we found decreased levels of mRNA for two important hexose transporters, SGLT1 and GLUT2. Congenital defects in these enzymes and transporters are extremely rare 
, and even the common variant for adult-type hypolactasia was not responsible for reduced LCT expression in AUT-GI children in this cohort. Therefore, it is unlikely that the combined deficiency of disaccharidases (maldigestion) and transporters (malabsorption) are indicative of a primary malabsorption resulting from multiple congenital or acquired defects in each of these genes. Transcripts for the enterocyte marker, villin, were not reduced in AUT-GI ilea and did not predict the expression levels of any of the disaccharidases or transporters in multiple regression models. This suggests that a general loss of enterocytes is unlikely. However, we cannot exclude the possibility that defects in the maturational status of enterocytes or enterocyte migration along the crypt-villus axis contribute to deficits in disaccharidase and transporter expression 
The ileal expression of CDX2, a master transcriptional regulator in the intestine, was a significant predictor of mRNA expression of all five disaccharidases and transporters in AUT-GI and Control-GI children, based on linear regression models. However, as ASD status remained a significant predictor of disaccharidase mRNA expression even after adjusting for CDX2 and villin, additional factors must also contribute. One potential factor is diet. Dietary intake of carbohydrates can regulate the mRNA expression of disaccharidases and hexose transporters in mice and rats 
. Several studies suggest that ASD children exhibit feeding selectivity and aberrant nutrient consumption 
. However, of the four studies reporting on carbohydrate intake, none found differences in total carbohydrate intake in ASD children 
. Furthermore, one study found no association between dietary intake of macronutrients (i.e., carbohydrates, proteins, or fats) and GI symptoms 
. Unfortunately, dietary diaries for the period immediately preceding biopsy were not available for the children evaluated in our study; hence, the extent to which dietary intake affected intestinal gene expression could not be determined.
Hormonal and growth factor regulation of some disaccharidases and hexose transporters have been reported in in vitro
studies and in animals 
. Inflammatory cytokines can regulate SI gene expression in human intestinal epithelial cells in vitro 
. Thus, immunological or hormonal imbalances reported in ASD children 
may also contribute to expression deficits. Additionally, intestinal microbes can influence the expression of disaccharidases and transporters 
through the influence of pathogen-associated molecular patterns (PAMPs) and butyrate (a byproduct of bacterial fermentation) on CDX2 expression and activity 
. In this regard, the observation that CDX2 was decreased in AUT-GI children with increased levels of Betaproteobacteria may be important.
Whatever the underlying mechanisms, reduced capacity for digestion and transport of carbohydrates can have profound effects. Within the intestine, malabsorbed carbohydrates can lead to osmotic diarrhea 
; non-absorbed sugars may also serve as substrates for intestinal microflora that produce fatty acids and gases (methane, hydrogen, and carbon dioxide), promoting additional GI symptoms such as bloating and flatulence 
. The deficiency of even a single gene in this important pathway can result in severe GI disease, as occurs with glucose-galactose malabsorption syndrome caused by SGLT1 deficiency, Fanconi-Bickel syndrome resulting from GLUT2 mutations, sucrase-isomaltase deficiency, and congenital lactase deficiency 
Changes in the type and quantity of dietary carbohydrates can influence composition and function of intestinal microflora 
; thus, we reasoned that carbohydrate maldigestion and malabsorption, resulting from deficient expression of disaccharidases and hexose transporters, might have similar effects in AUT-GI subjects. Pyrosequencing analysis of mucoepithelial bacteria revealed significant multicomponent dysbiosis in AUT-GI children, including decreased levels of Bacteroidetes, an increase in the Firmicute/Bacteroidete ratio, increased cumulative levels of Firmicutes and Proteobacteria, and increased levels of bacteria in the class Betaproteobacteria.
A recent pyrosequencing study reported an increase in Bacteroidetes in fecal samples of ASD subjects 
. Although these findings may appear to be incongruent with those reported here, our data were obtained using biopsies rather than free fecal material. Others have reported differences in the composition of fecal versus mucosal microflora 
. Only about 50% of cells in feces are viable, with dead and injured cells making up the remaining fractions 
. The loss of Bacteroidetes from the mucoepithelium as a result of death, injury, or competition for binding in the mucosal space can result in increased wash out of Bacteroidete cells into the fecal stream. Thus, higher levels of Bacteroidetes in feces could be indicative of an inability to thrive in the mucosal microbiome rather than an indication that Bacteroidetes are found at higher levels in the microbiome.
The trend toward increased Firmicutes was largely attributable to Clostridia with Ruminococcaceae
as major contributors. Several Ruminococcaceae
are known butyrate producers and may thus influence short-chain fatty acid (SCFA) levels 
. SCFA influence colonic pH, and some Bacteroides sp.
are sensitive to acidic pH 
. Three previous reports indicated differences in Clostridia species in children with ASD, including greater abundance of Clostridium
clusters I, II, XI and C. bolteae 
. Stratification of AUT-GI children based on the timing of GI symptom development relative to autism onset revealed that the levels of Clostridiales and cumulative levels of Lachnospiraceae
were significantly higher in AUT-GI children for whom GI symptoms developed before or at the same time as the onset of autism symptoms compared to AUT-GI children for whom GI symptoms developed after the onset of autism and compared to Control-GI children. However, we cannot discern whether changes in Clostridiales occurred before the onset of autism in this subgroup. We can only conclude that increased levels of Clostridiales members in biopsies taken after the development of both GI symptoms and autism are associated with the timing of GI onset relative to autism onset in this cohort. Although the reason for this association remains unclear, this finding may suggest that the timing of GI onset relative to autism is an important variable to consider in the design of future prospective studies investigating the microbiota of children with autism.
Although we found only a trend for increased Firmicutes in AUT-GI children, the cumulative levels of Firmicutes and Proteobacteria were significantly higher. These results suggest that, in some patients, a decrease in Bacteroidetes is associated with an increase in Firmicutes, whereas, in others, increases in Proteobacteria are associated with a reduced abundance of Bacteroidetes. Three AUT-GI patients had high levels of Alpha-, Beta-, or Gammaproteobacteria. In addition, the AUT-GI group had elevated levels of Betaproteobacteria compared to the Control-GI group, primarily reflecting the presence of Alcaligenaceae. Alcaligenaceae sequences were not detected in any tissues from Control-GI children.
Deficient digestion and absorption of di- and monosaccharides in the small intestine may alter the balance of growth substrates, thus eliminating the growth advantages that Bacteroidetes enjoy in the healthy intestine and enabling competitive growth of bacterial phylotypes better suited for growth on undigested and unabsorbed carbohydrates. In support of this hypothesis, multiple linear regression models demonstrated that levels of ileal SGLT1 and SI mRNA were associated with levels of Bacteroidetes in ileum and cecum, or cecum alone, respectively. Levels of ileal SI, MGAM, and GLUT2 mRNA were associated with levels of cecal Firmicutes, although the magnitude of the effects of MGAM and GLUT2 differed between AUT-GI and Control-GI children. Significant associations were also observed between levels of SI and MGAM mRNA and of Proteobacteria in ileum and cecum, and of Betaproteobacteria in cecum. Although deficiencies in disaccharidase and transporter expression appear to at least partially contribute to these alterations in the AUT-GI microbiota, diagnostic status remained a significant predictor of Bacteroidete and cecal Firmicute abundance even after adjusting for gene expression.
Metabolic interactions between intestinal microflora and their hosts are only beginning to be understood. Nonetheless, there is already abundant evidence that microflora can have system-wide effects 
and influence immune responses, brain development and behavior 
. We acknowledge that this is a small study comprising 22 subjects. The small sample size evaluated in this study is a limitation arising from the difficulty in obtaining biopsies from young children undergoing invasive endoscopic examination. This caveat notwithstanding, our data show that at least some children with autism have a distinct intestinal profile that is linked to deficient expression of disaccharidases and hexose transporters, potentially promoting maldigestion, malabsorption, and multicomponent, compositional dysbiosis. Although the underlying cause of these changes and the extra-intestinal effects these changes may elicit remain speculative, the identification of specific molecular and microbial signatures that define GI pathophysiology in AUT-GI children sets the stage for further research aimed at defining the epidemiology, diagnosis, and informed treatment of GI symptoms in autism.