(A–C) Increases or decreases in AUT-GI children in both gene expression (A) and bacterial parameters (B and C) were determined for each individual based on the levels of each parameter in the Control-GI group. (A) The gene expression levels in the AUT-GI children that exceeded the 75^th percentile of Control-GI values and were at least 2-fold increased relative to the Control-GI mean (red arrow) or below the 25^th percentile of Control-GI values and at least 2-fold decreased relative to the Control-GI mean (green arrow) were scored as an increase or decrease, respectively. Values above the 90^th or below the 10^th percentiles of Control-GI children are indicated by double arrows. (B and C) Bacterial parameters in AUT-GI children that exceeded the 75^th percentile of Control-GI values (red arrows) or were below the 25^th percentile of Control-GI values (green arrows) were scored as an increase or decrease, respectively. Values above the 90^th or below the 10^th percentiles of Control-GI children are indicated by double arrows. Results are shown for data obtained by real-time PCR (RT), where performed, and pyrosequencing (454). (n.c. = no change relative to defined cut-off values for Control-GI children).

(A–B) Firmicute/Bacteroidete ratio from pyrosequencing reads obtained from ileal (A; Mann-Whitney, p = 0.026) and cecal (B; Mann-Whitney, p = 0.032) biopsies. (C–D) Firmicute/Bacteroidete ratios obtained by real-time PCR for ilea (C; Mann-Whitney, p = 0.0006) and ceca (D; Mann-Whitney, p = 0.022). (E–F) Cumulative abundance of Firmicutes and Proteobacteria from ileal (E; Mann-Whitney, p = 0.015) and cecal (F; Mann-Whitney, p = 0.007) biopsies. (G–H) Cumulative levels of members of the families Lachnospiraceae and Ruminococcaceae in ileal (G; Mann-Whitney; p = 0.062) and cecal (H; Mann-Whitney; p = 0.098) biopsies. (I–J) Family-level abundance distributions of the class Clostridia in ileum (I) and cecum (J): bottom row displays cumulative levels of all family members by patient; gray cells indicate where no sequences were observed for a given taxa. *, p<0.05; **, p<0.01; ***, p<0.001; †, p<0.1 (trend).

(A–B) Abundance of Clostridiales from ileal (A) and cecal (B) biopsies from AUT-GI and Control-GI patients (n = 7), with AUT-GI stratified by whether the onset of GI symptoms occurred after (n = 5) the onset of autism symptoms (GI-After) or before and at the same time (n = 10) as autism symptoms (GI-Before/Same). [A: AUT (GI-After) vs. AUT (GI-Before/Same), Mann-Whitney, p = 0.028; AUT (GI-Before/Same) vs. Control-GI, Mann-Whitney, p = 0.015; AUT (GI-After) vs. Control-GI, Mann-Whitney, p = 0.935] [B: AUT (GI-After) vs. AUT (GI-Before/Same), Mann-Whitney, p = 0.037; AUT (GI-Before/Same) vs. Control-GI, Mann-Whitney, p = 0.019; AUT (GI-After) vs. Control-GI, Mann-Whitney, p = 0.935]. (C–D) Cumulative abundance of Lachnospiraceae and Ruminococcaceae from ileal (C) and cecal (D) biopsies from AUT-GI and Control-GI patients (n = 7), with AUT-GI stratified by whether the onset of GI symptoms occurred after (n = 5) the onset of autism symptoms or before and at the same time (n = 10) as autism symptoms [C: AUT (GI-After) vs. AUT (GI-Before/Same), Mann-Whitney, p = 0.028; AUT (GI-Before/Same) vs. Control-GI, Mann-Whitney, p = 0.015; AUT (GI-After) vs. Control-GI, Mann-Whitney, p = 0.808] [D: AUT (GI-After) vs. AUT (GI-Before/Same), Mann-Whitney, p = 0.020; AUT (GI-Before/Same) vs. Control-GI, Mann-Whitney, p = 0.011; AUT (GI-After) vs. Control-GI, Mann-Whitney, p = 0.685]. (E) Age at GI onset (in months) for AUT-GI and Control-GI patients, with AUT-GI stratified by whether GI onset occurred after (n = 5) the onset of autism symptoms or before and at the same time (n = 10) as autism symptoms [E: AUT (GI-After) vs. AUT (GI-Before/Same), Mann-Whitney, tied p = 0.007; AUT (GI-Before/Same) vs. Control-GI, Mann-Whitney, tied p = 0.757; AUT (GI-After) vs. Control-GI, Mann-Whitney, tied p = 0.027]. *, p<0.05; **, p<0.01; n.s., not significant.

Box-and-whisker plots displaying (A) SI (Mann-Whitney; p = 0.001), (B) MGAM (Mann-Whitney; p = 0.003), (C) LCT (Mann-Whitney; p = 0.032), (D) SGLT1 (Mann-Whitney; p = 0.008), (E) GLUT2 (Mann-Whitney; p = 0.010), (F) Villin (Mann-Whitney; p = 0.307), and (G) CDX2 (Mann-Whitney; p = 0.192) mRNA expression normalized to GAPDH mRNA in ileal biopsies from AUT-GI (AUT) and Control-GI (Control) patients. *, p<0.05; **, p<0.01; n.s., not significant.

(A–B) Phylum-level comparison of the average relative abundance of bacterial taxa in ileal (A) and cecal (B) biopsies. (C–D) Bacteroidete abundance, obtained from pyrosequencing for ileal (C; Mann-Whitney, p = 0.012) and cecal (D; Mann-Whitney, p = 0.008) biopsies. (E–F) Bacteroidete-specific quantitative real-time PCR analysis of ileal (E; Mann-Whitney, p = 0.003) and cecal (F; Mann-Whitney, p = 0.022) biopsies; copy number values are normalized relative to total bacteria copy numbers. *, p<0.05; **, p<0.01.

(A–B) Phyla-level abundance of Proteobacteria members in ileal (A; Mann-Whitney, p = 0.549) and cecal (B; Mann-Whitney, p = 0.072) biopsies obtained by pyrosequencing. (C–D) Class-level abundance of Betaproteobacteria members in ileal (C; Mann-Whitney, p = 0.072) and cecal (D; Mann-Whitney, p = 0.038) biopsies. (E–F) Family-level abundance distributions of bacteria within the classes Alpha-, Beta-, and Gammaproteobacteria in ileal (E) and cecal (F) biopsies: bottom row of each heatmap displays the cumulative levels of family members in each class of Proteobacteria by patient; gray cells indicate where no sequences were identified for a given taxa. *, p<0.05; †, p<0.1 (trend); n.s., not significant.

A) Schematic representation of enterocyte-mediated digestion of disaccharides and transport of monosaccharides in the small intestine. Disaccharidases (SI, MGAM, and LCT) in the enterocyte brush border break down disaccharides into their component monosaccharides. The monosaccharides, glucose and galactose, are transported from the small intestinal lumen into enterocytes by the sodium-dependent transporter SGLT1. On the basolateral enterocyte membrane, GLUT2, transports glucose, galactose, and fructose out of enterocytes and into the circulation. The expression levels of disaccharidases and hexose transporters may be controlled, in part, by the transcription factor CDX2. B) In the normal small intestine, where expression of disaccharidases and hexose transporters are high, nearly all disaccharides are efficiently digested and monosaccharides are absorbed from the lumen. C) In the AUT-GI intestine, where expression of disaccharidases and hexose transporters are deficient, mono- and disaccharides accumulate in the lumen of the ileum and cecum resulting in dysbiosis, diarrhea, bloating, and flatulence.