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Comparison of Fecal Microbiota in Children with Autism Spectrum Disorders and Neurotypical Siblings in the Simons Simplex Collection
Open accessAbstract
In order to assess potential associations between autism spectrum disorder (ASD) phenotype, functional GI disorders and fecal microbiota, we recruited simplex families, which had only a single ASD proband and neurotypical (NT) siblings, through the Simons Simplex Community at the Interactive Autism Network (SSC@IAN). Fecal samples and metadata related to functional GI disorders and diet were collected from ASD probands and NT siblings of ASD probands (age 7-14). Functional gastrointestinal disorders (FGID) were assessed using the parent-completed ROME III questionnaire for pediatric FGIDs, and problem behaviors were assessed using the Child Behavior Check List (CBCL). Targeted quantitative polymerase chain reaction (qPCR) assays were conducted on selected taxa implicated in ASD, including Sutterella spp., Bacteroidetes spp. and Prevotella spp. Illumina sequencing of the V1V2 and the V1V3 regions of the bacterial 16S rRNA genes from fecal DNA was performed to an average depth of 208,000 and 107,000 high-quality reads respectively. Twenty-five of 59 ASD children and 13 of 44 NT siblings met ROME III criteria for at least one FGID. Functional constipation was more prevalent in ASD (17 of 59) compared to NT siblings (6 of 44, P = 0.035). The mean CBCL scores in NT siblings with FGID, ASD children with FGID and ASD without FGID were comparably higher (58-62 vs. 44, P < 0.0001) when compared to NT children without FGID. There was no significant difference in macronutrient intake between ASD and NT siblings. There was no significant difference in ASD severity scores between ASD children with and without FGID. No significant difference in diversity or overall microbial composition was detected between ASD children with NT siblings. Exploratory analysis of the 16S rRNA sequencing data, however, identified several low abundance taxa binned at the genus level that were associated with ASD and/or first order ASD*FGID interactions (FDR <0.1).
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TL;DR
In simplex families from SSC@IAN, ASD children and their neurotypical siblings showed no significant difference in overall fecal microbial diversity or composition, but functional constipation was more common in ASD children. Exploratory 16S analyses found only low-abundance genus-level taxa associated with ASD and/or ASD*FGID interactions.
Key findings
- Among 59 ASD children and 44 NT siblings from 66 families, 25/59 ASD children (42%) and 13/44 NT siblings (30%) met ROME III criteria for at least one FGID; functional constipation was more prevalent in ASD children (17/59) than NT siblings (6/44, P = 0.035).
- Mean CBCL scores in NT siblings with FGID, ASD children with FGID, and ASD children without FGID were comparably higher than NT children without FGID (58–62 vs. 44, P < 0.0001); no significant difference in macronutrient intake was found between ASD and NT siblings, and ADOS-CSS did not differ significantly between ASD children with and without FGID.
- No significant difference in α-diversity, β-diversity, or overall microbial composition was detected between ASD children and NT siblings; targeted qPCR showed no significant differences for Sutterella, Prevotella, Bacteroidetes, C. coccoides-E. rectales, Faecalibacterium prausnitzii, or Escherichia coli.
- Exploratory 16S rRNA sequencing identified several low-abundance genus-level taxa associated with ASD and/or first-order ASD*FGID interactions at FDR < 0.1, including Cyanobacteria/Chloroplast, which was increased in the ASD-with-FGID group and appeared linked to chia seed consumption in two ASD children with functional constipation.
Methods
- Families were recruited through the Simons Simplex Community at the Interactive Autism Network (SSC@IAN); of 245 potentially eligible families with 339 children, 107 families with 145 children consented and 66 families with 103 children completed the study. Participants were ASD probands and NT siblings aged 7–14, excluding those with GI disease, serious medical problems, or antibiotics within one month of stool collection.
- Parents completed the ROME III questionnaire for pediatric FGIDs, the CBCL/6-18, and a one-week food diary; fecal samples underwent targeted qPCR for selected bacterial subgroups and Illumina sequencing of the V1V2 and V1V3 regions of bacterial 16S rRNA genes.
- Analyses included Fisher exact tests, mixed-effect models with ASD, FGID, and ASD*FGID as fixed effects and family as random effect, α-diversity indices, PERMANOVA for β-diversity, and negative binomial/zero-inflated negative binomial models for individual OTUs with Benjamini-Hochberg FDR adjustment.
Limitations
- The paper states that although this is one of the larger studies of its kind, the sample size is still modest given the number of variables included, and that additional ASD children, NT siblings (preferably family matched), and unrelated controls using the same phenotyping tools are needed to confirm the findings.
- The paper states that it may be necessary to restrict dietary intake of plant-derived supplements such as chia seeds to determine whether the Cyanobacteria/Chloroplast signal is biologically significant for ASD pathophysiology.
- Evident from the text: the study lacked unrelated NT controls, so it cannot distinguish whether NT siblings of ASD children have altered microbiomes relative to the general population; the significant microbial findings are exploratory, low-abundance, and not consistently replicated across V1V2 and V1V3 datasets.
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A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“No significant difference (p < 0.05) was noted in β-diversity between ASD and NT sibling bacterial communities analyzed at the phyla, family or genus level and using Bray-Curtis, Jaccard or Morisita-Horn indices as dissimilarity measurements.”
§ Comparison of Fecal Microbiota in Children with Autism Spectrum Disorders and Neurotypical Siblings in the Simons Simplex Collection > Results > 16S rRNA sequence analysis of ASD children and NT siblings
Load-bearingStrength:plain· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passes- Rests on an assumption: The study had adequate statistical power to detect differences in the microbial outcomes measured between ASD and NT siblings.
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“there was no significant change in the prevalence of the GI symptoms occurred over time.”
- Also stated in: § Demographics and functional GI disorders (FGID) in ASD and NT siblings · plain
“There was no significant change in the prevalence of any of the seven problematic GI symptoms in the ASD children at the two time points.”
§ Comparison of Fecal Microbiota in Children with Autism Spectrum Disorders and Neurotypical Siblings in the Simons Simplex Collection > Results > Demographics and functional GI disorders (FGID) in ASD and NT siblings
Strength:plain· not yet calibratedn = 59Tests✓ Asserted: passes✓ Checkable: passes? Load-bearing: unclear✓ Based: passes- Also stated in: § Demographics and functional GI disorders (FGID) in ASD and NT siblings · plain
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“The mean relative abundance of this genus was increased in the ASD-with FGID group compared to those of the other three categories (ASD without FGID, NT siblings with FGID and NT siblings without FGID, see S4 Table for mean relative abundances).”
§ Comparison of Fecal Microbiota in Children with Autism Spectrum Disorders and Neurotypical Siblings in the Simons Simplex Collection > Results > 16S rRNA sequence analysis of ASD children and NT siblings
Tests✓ Asserted: passes✓ Checkable: passes? Load-bearing: unclear✓ Based: passesA result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“No significant associations were detected between the relative abundance of the Sutterella or Prevotella genera and ASD, FGID or ASD*FGID first order interactions (see Table 6) in either the V1V2 or V1V3 datasets.”
§ Comparison of Fecal Microbiota in Children with Autism Spectrum Disorders and Neurotypical Siblings in the Simons Simplex Collection > Results > 16S rRNA sequence analysis of ASD children and NT siblings
Strength:plain· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes? Load-bearing: unclear✓ Based: passesA result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Inspection of the daily dietary logs of the ASD children with high counts of Chloroplast sequences revealed that these particular children were fed shakes containing chia seeds.”
§ Comparison of Fecal Microbiota in Children with Autism Spectrum Disorders and Neurotypical Siblings in the Simons Simplex Collection > Discussion
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesA result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Exploratory analysis of the 16S rRNA sequencing data, however, identified several low abundance taxa binned at the genus level that were associated with ASD and/or first order ASD*FGID interactions (FDR <0.1).”
§ Comparison of Fecal Microbiota in Children with Autism Spectrum Disorders and Neurotypical Siblings in the Simons Simplex Collection > Abstract
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesA result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Functional constipation was more prevalent in ASD (17 of 59) compared to NT siblings (6 of 44, P = 0.035).”
- Also stated in: § Demographics and functional GI disorders (FGID) in ASD and NT siblings · plain
“ASD children exhibited a higher prevalence of functional constipation in compared to NT siblings (P = 0.035) when both matched and unmatched individuals were included in the analysis.”
§ Comparison of Fecal Microbiota in Children with Autism Spectrum Disorders and Neurotypical Siblings in the Simons Simplex Collection > Abstract
Strength:plain· not yet calibratedCausal:correlational· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes? Load-bearing: unclear✓ Based: passes- Also stated in: § Demographics and functional GI disorders (FGID) in ASD and NT siblings · plain
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Permutational Hotelling T test revealed no significant difference in the overall microbial composition when binned at the phyla level, between ASD children and NT siblings.”
§ Comparison of Fecal Microbiota in Children with Autism Spectrum Disorders and Neurotypical Siblings in the Simons Simplex Collection > Results > 16S rRNA sequence analysis of ASD children and NT siblings
Strength:plain· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes? Load-bearing: unclear✓ Based: passesA result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“No significant difference was observed for any of these bacterial subgroups in the 37 ASD/NT sibling pairs (see Methods).”
§ Comparison of Fecal Microbiota in Children with Autism Spectrum Disorders and Neurotypical Siblings in the Simons Simplex Collection > Results > No associations (FDR < 0.1) detected in targeted bacterial subgroups between ASD, FGID and/or first order ASD*FGID by QPCR
Load-bearingStrength:plain· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passes- Rests on an assumption: The study had adequate statistical power to detect differences in the microbial outcomes measured between ASD and NT siblings.
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“We also detected no significant difference in the prevalence of specific FGIDs such as functional constipation in the family matched ASD/NT sibling pairs.”
§ Comparison of Fecal Microbiota in Children with Autism Spectrum Disorders and Neurotypical Siblings in the Simons Simplex Collection > Results > Demographics and functional GI disorders (FGID) in ASD and NT siblings
Strength:plain· not yet calibratedn = 37Tests✓ Asserted: passes✓ Checkable: passes? Load-bearing: unclear✓ Based: passes
- It may state two separate claims as one. 11
- Probably not a substantial claim. 23
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Comparisons of the relative abundances of the Bacteroidetes phylum, and the Sutterella and Prevotella genera reveal high correlations between MiSeq V1V2 and V1V3 (ρ≈0.9) and small proportional biases (S5 Table and S1 Fig).”
§ Comparison of Fecal Microbiota in Children with Autism Spectrum Disorders and Neurotypical Siblings in the Simons Simplex Collection > Results > Comparison of MiSeq V1V2, V1V3 and qPCR measurements of selected bacterial categories
Strength:plain· not yet calibratedCausal:correlational· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes? Load-bearing: unclear✓ Based: passesIt may state two separate claims as one.
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“No significant effect of ASD, FGID or ASD*FGID was observed on OTU complexity or richness, two measures of α-diversity, Shannon diversity and Chao 1 (see Table 5for mean values).”
§ Comparison of Fecal Microbiota in Children with Autism Spectrum Disorders and Neurotypical Siblings in the Simons Simplex Collection > Results > 16S rRNA sequence analysis of ASD children and NT siblings
Load-bearingStrength:plain· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesIt may state two separate claims as one.
- Rests on an assumption: The study had adequate statistical power to detect differences in the microbial outcomes measured between ASD and NT siblings.
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Fifteen ASD children and 13 NT siblings in 37 families met ROME III criteria for at least one FGID, with concordance for FGIDs in 6 (18%) ASD/NT sibling pairs.”
§ Comparison of Fecal Microbiota in Children with Autism Spectrum Disorders and Neurotypical Siblings in the Simons Simplex Collection > Results > Demographics and functional GI disorders (FGID) in ASD and NT siblings
Strength:plain· not yet calibratedn = 37Tests✓ Asserted: passes✓ Checkable: passes? Load-bearing: unclear✓ Based: passesIt may state two separate claims as one.
The paper states this without a source or data behind it here, so there is nothing to check it against. The quote shows it is in the paper.
In the paper
“None of the other subjects consumed chia seeds except one ASD child who ingested a serving of mixed grain bread that included chia grain.”
§ Comparison of Fecal Microbiota in Children with Autism Spectrum Disorders and Neurotypical Siblings in the Simons Simplex Collection > Results > 16S rRNA sequence analysis of ASD children and NT siblings
Strength:plain· not yet calibratedSingle caseTests✓ Asserted: passes✓ Checkable: passes✗ Load-bearing: fails✓ Based: passesProbably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Mean total, internalizing, externalizing and subscale CBCL scores were significantly increased (p < 0.05) in the ASD children in both the ASD/NT sibling pairs and in comparison of all the ASD with NT children (matched and unmatched).”
§ Comparison of Fecal Microbiota in Children with Autism Spectrum Disorders and Neurotypical Siblings in the Simons Simplex Collection > Results > Comparison of CBCL and macronutrient intake
Strength:plain· not yet calibratedCausal:correlational· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes? Load-bearing: unclear✓ Based: passesIt may state two separate claims as one.
The paper states this without a source or data behind it here, so there is nothing to check it against. The quote shows it is in the paper.
In the paper
“While there were generally positively correlated measurements between the MiSeq and qPCR platforms, we observed large systematic biases particularly with the two relative low abundance genera, Sutterella and Provetella.”
§ Comparison of Fecal Microbiota in Children with Autism Spectrum Disorders and Neurotypical Siblings in the Simons Simplex Collection > Results > Comparison of MiSeq V1V2, V1V3 and qPCR measurements of selected bacterial categories
Strength:plain· not yet calibratedCausal:correlational· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes? Load-bearing: unclear✓ Based: passesIt may state two separate claims as one.
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Our study also detected no differences in either α or β diversity between ASD and NT siblings.”
§ Comparison of Fecal Microbiota in Children with Autism Spectrum Disorders and Neurotypical Siblings in the Simons Simplex Collection > Discussion
Load-bearingStrength:plain· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesIt may state two separate claims as one.
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Phyla of lower abundance, including RF3, Tenericutes, Cyanobacteria, Verrucomicrobia, Lentisphaerae, and Fusobacterium were also detected (see S3 Table).”
§ Comparison of Fecal Microbiota in Children with Autism Spectrum Disorders and Neurotypical Siblings in the Simons Simplex Collection > Results > 16S rRNA sequence analysis of ASD children and NT siblings
Strength:plain· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes✗ Load-bearing: fails✓ Based: passesProbably not a substantial claim. The paper’s conclusions may not depend on it.
The paper states this without a source or data behind it here, so there is nothing to check it against. The quote shows it is in the paper.
In the paper
“The Silva database used in the current study, however, binned the Melainabacteria sequences in a taxon that was distinct from the Chloroplast taxon.”
§ Comparison of Fecal Microbiota in Children with Autism Spectrum Disorders and Neurotypical Siblings in the Simons Simplex Collection > Results > 16S rRNA sequence analysis of ASD children and NT siblings
Strength:plain· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes✗ Load-bearing: fails✓ Based: passesProbably not a substantial claim. The paper’s conclusions may not depend on it.
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“At the genus level, 359 operational taxonomic units (OTU) were detected for the V1V2 dataset and 189 OTUs for the V1-V3 datasets, respectively.”
§ Comparison of Fecal Microbiota in Children with Autism Spectrum Disorders and Neurotypical Siblings in the Simons Simplex Collection > Results > 16S rRNA sequence analysis of ASD children and NT siblings
Strength:plain· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes✗ Load-bearing: fails✓ Based: passesProbably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.
In the paper
“The CBCL/6-18 is a parent report form to screen for emotional, behavioral, and social problems in children ages 6–18 [33].”
§ Comparison of Fecal Microbiota in Children with Autism Spectrum Disorders and Neurotypical Siblings in the Simons Simplex Collection > Materials and Methods > Child Behavior Checklist (CBCL)
What the sources say
- Not checked yetcites [33] TM Achenbach, 2001
Strength:plain· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes✗ Load-bearing: fails✓ Based: passesProbably not a substantial claim. The paper’s conclusions may not depend on it.
A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“This may reflect the smaller number of subjects enrolled in the previous studies, possibly resulting in Type 1 errors.”
§ Comparison of Fecal Microbiota in Children with Autism Spectrum Disorders and Neurotypical Siblings in the Simons Simplex Collection > Discussion
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesProbably not a substantial claim.
In the paper
“The p-values for each OTU were adjusted for multiple comparisons to calculate a false discovery rate (FDR) using the Benjamini-Hochberg procedure [59].”
§ Comparison of Fecal Microbiota in Children with Autism Spectrum Disorders and Neurotypical Siblings in the Simons Simplex Collection > Materials and Methods > Statistical analysis
What the sources say
- Not checked yetcites [59] Y Benjamini, 1995, Controlling the false discovery rate: a practical and powerful approach to multiple testing, J R Statist Soc B source
Load-bearingStrength:plain· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesProbably not a substantial claim.
In the paper
“Studies tracking siblings of ASD children have detected vulnerabilities in some neurocognitive domains in the absence of an ASD diagnosis [72] suggesting that some of the NT siblings may exhibit a broader subclinical ASD phenotype.”
§ Comparison of Fecal Microbiota in Children with Autism Spectrum Disorders and Neurotypical Siblings in the Simons Simplex Collection > Discussion
What the sources say
- Not checked yetcites [72] M Wade, 2015, Using sibling designs to understand neurodevelopmental disorders: from genes and environments to prevention programming, Biomed Res Int source
Strength:hedged· not yet calibratedCausal:correlational· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes? Load-bearing: unclear✓ Based: passesIt may state two separate claims as one.
In the paper
“An alternative explanation for why a study comparing ASD children with NT siblings did not replicate differences reported between ASD children with unrelated controls, is that NT siblings of ASD children have altered microbiomes compared to that of unrelated children [24].”
§ Comparison of Fecal Microbiota in Children with Autism Spectrum Disorders and Neurotypical Siblings in the Simons Simplex Collection > Discussion
What the sources say
- Not checked yetcites [24] R Krajmalnik-Brown, 2015, Gut bacteria in children with autism spectrum disorders: challenges and promise of studying how a complex community influences a complex disease, Microb Ecol Health Dis
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesProbably not a substantial claim.
What the paper did — it cites no source for this, so there is none to check. The quote shows it is in the paper.
In the paper
“Plasmid quantification standards were prepared from representative clones of the target organisms to insure that the assays were conducted within the linear range and with similar slopes.”
§ Comparison of Fecal Microbiota in Children with Autism Spectrum Disorders and Neurotypical Siblings in the Simons Simplex Collection > Materials and Methods > Quantitative PCR (qPCR) for targeted bacterial subgroups
Strength:plain· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes✗ Load-bearing: fails✓ Based: passesProbably not a substantial claim. The paper’s conclusions may not depend on it.
In the paper
“which is the current international standard by which functional GI disorders (FGID) are diagnosed and classified [24].”
§ Comparison of Fecal Microbiota in Children with Autism Spectrum Disorders and Neurotypical Siblings in the Simons Simplex Collection > Introduction
What the sources say
- Not checked yetcites [24] R Krajmalnik-Brown, 2015, Gut bacteria in children with autism spectrum disorders: challenges and promise of studying how a complex community influences a complex disease, Microb Ecol Health Dis
Load-bearingStrength:plain· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesProbably not a substantial claim.
What the paper did — it cites no source for this, so there is none to check. The quote shows it is in the paper.
In the paper
“All assays were carried out in triplicate.”
§ Comparison of Fecal Microbiota in Children with Autism Spectrum Disorders and Neurotypical Siblings in the Simons Simplex Collection > Materials and Methods > Quantitative PCR (qPCR) for targeted bacterial subgroups
Strength:plain· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes✗ Load-bearing: fails✓ Based: passesProbably not a substantial claim. The paper’s conclusions may not depend on it.
None of the sources cited for this claim have been checked yet.
In the paper
“Previous epidemiological studies using the ROME III process to survey the prevalence of a child having at least one FGID in general pediatric clinics reported a prevalence of 2% and 16%, respectively [73,74]”
§ Comparison of Fecal Microbiota in Children with Autism Spectrum Disorders and Neurotypical Siblings in the Simons Simplex Collection > Discussion
What the sources say
- Not checked yetcites [73] E Miele, 2004, Functional gastrointestinal disorders in children: an Italian prospective survey, Pediatrics source
- Not checked yetcites [74] GS Karabulut, 2013, The incidence of irritable bowel syndrome in children using the Rome III criteria and the effect of trimebutine treatment, J Neurogastroenterol Motil source
Load-bearingStrength:plain· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesProbably not a substantial claim.
In the paper
“One study used intestinal mucosal [10] rather than fecal samples.”
§ Comparison of Fecal Microbiota in Children with Autism Spectrum Disorders and Neurotypical Siblings in the Simons Simplex Collection > Discussion
What the sources say
- Not checked yetcites [10] BL Williams, 2012, Application of novel PCR-based methods for detection, quantitation, and phylogenetic characterization of Sutterella species in intestinal biopsy samples from children with autism and gastrointestinal disturbances, MBio source
Strength:plain· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes✗ Load-bearing: fails✓ Based: passesProbably not a substantial claim. The paper’s conclusions may not depend on it.
None of the sources cited for this claim have been checked yet.
In the paper
“Review of previous studies suggest that GI symptoms are more common in ASD children than in NT children, but these studies suffer from methodological differences in assessing GI symptoms [28–31].”
§ Comparison of Fecal Microbiota in Children with Autism Spectrum Disorders and Neurotypical Siblings in the Simons Simplex Collection > Discussion
What the sources say
- Not checked yetcites [28] BO McElhanon, 2014, Gastrointestinal symptoms in autism spectrum disorder: a meta-analysis, Pediatrics source
- Not checked yetcites [29] EY Hsiao, 2014, Gastrointestinal issues in autism spectrum disorder, Harv Rev Psychiatry source
- Not checked yetcites [30] GT Furuta, 2012, Management of constipation in children and adolescents with autism spectrum disorders, Pediatrics source
- Not checked yetcites [31] P Gorrindo, 2012, Gastrointestinal dysfunction in autism: parental report, clinical evaluation, and associated factors, Autism Res source
Strength:hedged· not yet calibratedCausal:correlational· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes✗ Load-bearing: fails✓ Based: passesProbably not a substantial claim. The paper’s conclusions may not depend on it.
What the paper did — it cites no source for this, so there is none to check. The quote shows it is in the paper.
In the paper
“Our assessment of GI symptoms differs from previous studies in that we used the international standard for assessing FGIDs in the general pediatric population.”
§ Comparison of Fecal Microbiota in Children with Autism Spectrum Disorders and Neurotypical Siblings in the Simons Simplex Collection > Discussion
Strength:plain· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes✗ Load-bearing: fails✓ Based: passesProbably not a substantial claim. The paper’s conclusions may not depend on it.
What the paper did — it cites no source for this, so there is none to check. The quote shows it is in the paper.
In the paper
“In this study, 16S rRNA sequencing using MiSeq was carried out to a greater depth than previous studies utilizing pyrosequencing.”
§ Comparison of Fecal Microbiota in Children with Autism Spectrum Disorders and Neurotypical Siblings in the Simons Simplex Collection > Discussion
Strength:plain· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes✗ Load-bearing: fails✗ Based: failsProbably not a substantial claim. The paper’s conclusions may not depend on it. The paper may give no reason why it holds.
What the paper did — it cites no source for this, so there is none to check. The quote shows it is in the paper.
In the paper
“Of the 44 NT siblings, 37 were family matched to the ASD children in the study and the remaining seven NT children were siblings of ASD children who were not in the current study, in some cases because the ASD child was older than 14 years of age.”
§ Comparison of Fecal Microbiota in Children with Autism Spectrum Disorders and Neurotypical Siblings in the Simons Simplex Collection > Discussion
Strength:plain· not yet calibratedn = 37Tests✓ Asserted: passes✓ Checkable: passes✗ Load-bearing: fails✓ Based: passesProbably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.
What the paper did — it cites no source for this, so there is none to check. The quote shows it is in the paper.
In the paper
“Although this is one of the larger studies comparing ASD, FGID and the gut microbiota in ASD children and NT siblings, our sample size is still modest given the number of variables included.”
§ Comparison of Fecal Microbiota in Children with Autism Spectrum Disorders and Neurotypical Siblings in the Simons Simplex Collection > Discussion
Strength:plain· not yet calibratedTests✓ Asserted: passes✗ Checkable: fails✗ Load-bearing: fails✓ Based: passesProbably not a substantial claim. It may be too vague to check against evidence. The paper’s conclusions may not depend on it.
In the paper
“The Actinobacteria/Mobiluncus genus has previously been associated with bacterial vaginosis [71].”
§ Comparison of Fecal Microbiota in Children with Autism Spectrum Disorders and Neurotypical Siblings in the Simons Simplex Collection > Results > 16S rRNA sequence analysis of ASD children and NT siblings
What the sources say
- Not checked yetcites [71] RF Lamont, 2011, The vaginal microbiome: new information about genital tract flora using molecular based techniques, BJOG source
Strength:plain· not yet calibratedCausal:correlational· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes✗ Load-bearing: fails✓ Based: passesProbably not a substantial claim. The paper’s conclusions may not depend on it.
In the paper
“The Firmicutes/Asteroleplasma genus appears to represent a group of uncultured wall-less mycobacterium like organisms within the class Erysipelotrichia [69].”
§ Comparison of Fecal Microbiota in Children with Autism Spectrum Disorders and Neurotypical Siblings in the Simons Simplex Collection > Results > 16S rRNA sequence analysis of ASD children and NT siblings
What the sources say
- Not checked yetcites [69] JJ Davis, 2013, Genomes of the class Erysipelotrichia clarify the firmicute origin of the class Mollicutes, Int J Syst Evol Microbiol source
Strength:hedged· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes✗ Load-bearing: fails✓ Based: passesProbably not a substantial claim. The paper’s conclusions may not depend on it.
In the paper
“The Proteobacteria/Thalassospira genus appears to represent alpha-proteobacteria present in bacterioplankton and a number of uncultivated organisms [70].”
§ Comparison of Fecal Microbiota in Children with Autism Spectrum Disorders and Neurotypical Siblings in the Simons Simplex Collection > Results > 16S rRNA sequence analysis of ASD children and NT siblings
What the sources say
- Not checked yetcites [70] Q Lai, 2014, Multilocus sequence analysis for assessment of phylogenetic diversity and biogeography in Thalassospira bacteria from diverse marine environments, PLoS One source
Strength:hedged· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes✗ Load-bearing: fails✓ Based: passesProbably not a substantial claim. The paper’s conclusions may not depend on it.
None of the sources cited for this claim have been checked yet.
In the paper
“Constipation has been previously recognized as a significant comorbidity in ASD children [28–31].”
§ Comparison of Fecal Microbiota in Children with Autism Spectrum Disorders and Neurotypical Siblings in the Simons Simplex Collection > Discussion
What the sources say
- Not checked yetcites [28] BO McElhanon, 2014, Gastrointestinal symptoms in autism spectrum disorder: a meta-analysis, Pediatrics source
- Not checked yetcites [29] EY Hsiao, 2014, Gastrointestinal issues in autism spectrum disorder, Harv Rev Psychiatry source
- Not checked yetcites [30] GT Furuta, 2012, Management of constipation in children and adolescents with autism spectrum disorders, Pediatrics source
- Not checked yetcites [31] P Gorrindo, 2012, Gastrointestinal dysfunction in autism: parental report, clinical evaluation, and associated factors, Autism Res source
Strength:plain· not yet calibratedCausal:correlational· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes✗ Load-bearing: fails✓ Based: passesProbably not a substantial claim. The paper’s conclusions may not depend on it.
None of the sources cited for this claim have been checked yet.
In the paper
“Metagenomic studies have detected a novel nonphotosynthetic taxa termed “Melainabacteria” present in human gut flora that represents either a sibling phylum or a class within the Cyanobacteria phylum [65,66].”
§ Comparison of Fecal Microbiota in Children with Autism Spectrum Disorders and Neurotypical Siblings in the Simons Simplex Collection > Results > 16S rRNA sequence analysis of ASD children and NT siblings
What the sources say
- Not checked yetcites [65] SC Di Rienzi, 2013, The human gut and groundwater harbor non-photosynthetic bacteria belonging to a new candidate phylum sibling to Cyanobacteria, Elife source
- Not checked yetcites [66] RM Soo, 2014, An expanded genomic representation of the phylum cyanobacteria, Genome Biol Evol source
Strength:plain· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes✗ Load-bearing: fails✓ Based: passesProbably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.
The paper relies on this without support.
The 7-day parent-recorded food diary accurately estimates children's daily macronutrient intake.
“The parents were asked to record the daily dietary intake of ASD and NT siblings on the Stony Brook University Medical Center Department of Pediatrics Food Diary/Calorie Count sheet for 7 days prior to the collection of the stool sample.”
The paper relies on this without support.
The study had adequate statistical power to detect differences in the microbial outcomes measured between ASD and NT siblings.
“No significant difference in diversity or overall microbial composition was detected between ASD children with NT siblings.”
“No significant difference was observed for any of these bacterial subgroups in the 37 ASD/NT sibling pairs (see Methods).”
The paper relies on this without support.
The study had adequate statistical power to detect differences in macronutrient intake between ASD and NT siblings.
“There was no significant difference in macronutrient intake between ASD and NT siblings.”
Claims other papers make about this one
Each is checked against this paper’s own text: does it say what the citing paper says it does?
In the citing paper
“Two studies showed increases [31, 33] and six studies showed no significant changes in alpha diversity [36, 42, 43, 48, 50, 54].”
From Gut microbiota changes in children with autism spectrum disorder: a systematic review (2020) · cites this paper as [48]
In the citing paper
“The rest of the studies observed either a decrease in Bacteroidetes percentage [50, 54] or no significant differences between cases and controls [31, 36, 48].”
From Gut microbiota changes in children with autism spectrum disorder: a systematic review (2020) · cites this paper as [48]
In the citing paper
“Among the seven [31, 36, 40–42, 48, 50] papers that reported on beta diversity, four [31, 40, 41, 50] papers showed significant differences in beta diversity between ASD and SIB/HC while the rest did not (Additional file 1: Table S2).”
From Gut microbiota changes in children with autism spectrum disorder: a systematic review (2020) · cites this paper as [48]
In the citing paper
“At the phylum level, none of the six studies showed an increase in absolute or relative abundance [31, 33, 36, 48, 51, 54].”
From Gut microbiota changes in children with autism spectrum disorder: a systematic review (2020) · cites this paper as [48]
In the citing paper
“The two studies on absolute abundance did not reveal any differences between ASD, SIB, and HC [48, 51].”
From Gut microbiota changes in children with autism spectrum disorder: a systematic review (2020) · cites this paper as [48]
In the citing paper
“Instead, these seven studies showed either a non-significant result in relative abundance [41, 48, 50, 52] or a lower relative abundance in children classified as ASD versus SIB or HC [31, 39, 42].”
From Gut microbiota changes in children with autism spectrum disorder: a systematic review (2020) · cites this paper as [48]
In the citing paper
“Cluster XIVa and XIVb, however, were quantitatively similar in percentage [52] and counts [45, 48, 49] in the available four studies.”
From Gut microbiota changes in children with autism spectrum disorder: a systematic review (2020) · cites this paper as [48]
In the citing paper
“For relative abundance in both total and metabolically active Firmicutes, the studies demonstrated either a decrease [31, 33, 36] or no significant differences between children with ASD, SIB, and HC [48, 51, 54].”
From Gut microbiota changes in children with autism spectrum disorder: a systematic review (2020) · cites this paper as [48]
In the citing paper
“Actinobacteria as a phylum did not reveal any compelling results [33, 36, 48];”
From Gut microbiota changes in children with autism spectrum disorder: a systematic review (2020) · cites this paper as [48]
In the citing paper
“Despite speculation that Sutterella was increased in children with ASD [53, 55], this was not true in every study. There were three studies suggesting no significant differences [38, 41, 48] and one suggesting a decrease [44], but this particular study included adolescents in their cohort.”
From Gut microbiota changes in children with autism spectrum disorder: a systematic review (2020) · cites this paper as [48]
In the citing paper
“The general trend, however, supports no alterations in E. coli for children in ASD in five studies [30, 35, 42, 48, 52] vs one study [31] documenting a decrease in E. coli percentage among other microbiota.”
From Gut microbiota changes in children with autism spectrum disorder: a systematic review (2020) · cites this paper as [48]
In the citing paper
“Fusobacteria phylum suggested a lower relative abundance in children with ASD vs unaffected siblings, although one study [48] demonstrated the opposite in terms of Fusobacterium absolute numbers.”
From Gut microbiota changes in children with autism spectrum disorder: a systematic review (2020) · cites this paper as [48]
In the citing paper
“No specific findings were identified in the Verrucomicrobia phylum [31, 33, 36, 39, 48] and in the Verrucomicrobiaceae family [31, 37, 39, 41, 52].”
From Gut microbiota changes in children with autism spectrum disorder: a systematic review (2020) · cites this paper as [48]
In the citing paper
“In this review, only a few studies have involved developmental specialists and psychologists who are apt in monitoring changes in ASD symptoms [32, 48].”
From Gut microbiota changes in children with autism spectrum disorder: a systematic review (2020) · cites this paper as [48]
In the citing paper
“One study suggests that children with ASD may have an increased intake of chia seeds in smoothies, which is associated with specific microbiome findings [48].”
From Gut microbiota changes in children with autism spectrum disorder: a systematic review (2020) · cites this paper as [48]
In the citing paper
“but more recent studies failed to reach a consensus as to whether an ASD-specific microbial signature exists.24, 51”
From Distinct Microbiome-Neuroimmune Signatures Correlate With Functional Abdominal Pain in Children With Autism Spectrum Disorder (2017) · cites this paper as [51]
In the citing paper
“In addition, Son et al.21 conducted further nutritional analysis of one-week food diary, no significant difference between two group with respect to daily intake of macronutrients (calories, protein, fats, carbohydrates, sugars or dietary fiber) were noted.”
From Altered composition and function of intestinal microbiota in autism spectrum disorders: A systematic review (2019) · cites this paper as [21]
In the citing paper
“Besides, Son et al.21, De Angelis et al.23 and Gondalia et al.26 recruited healthy siblings as control, whereas other studies recruited normal control from the whole population.”
From Altered composition and function of intestinal microbiota in autism spectrum disorders: A systematic review (2019) · cites this paper as [21]
In the citing paper
“Whereas, in the study of Son et al.21, there was no difference of functional modules between ASD patients and HCs, which was accordance with the result of gut microbiota composition.”
From Altered composition and function of intestinal microbiota in autism spectrum disorders: A systematic review (2019) · cites this paper as [21]
In the citing paper
“It can be an alternative explanation that why studies21,26 using NT sibling controls did not find any significant difference, while other studies using non-sibling controls did.”
From Altered composition and function of intestinal microbiota in autism spectrum disorders: A systematic review (2019) · cites this paper as [21]
Self-check
Percentages, sums and reported differences re-computed from the paper’s own numbers.
Each contradiction between two passages listed here was confirmed by an independent check.
Completed-study group counts conflict
MajorThe methods say 66 ASD children and 37 NT siblings completed the study, while the results say 59 ASD children and 44 NT siblings completed it. Both descriptions total 103 children but assign seven children to different groups.
“66 families with 103 children (66 ASD probands and 37 NT siblings) completed the study.”
“A total of 59 ASD children and 44 NT siblings from 66 families completed the study (Table 1).”
Diet-restriction total disagrees with Table 3
MinorThe four groups in Table 3 list 1, 3, 0, and 1 children on a gluten- or casein-free diet, totaling five rather than four.
Reported 4 · recomputed 5
“Only 4 children were reported to adhere to a gluten and/or casein free diet.”
FGID concordance percentage uses the wrong denominator
MinorSix of the 37 matched sibling pairs is 16.2%, not the reported 18%.
Reported 18 · recomputed 16.216216
“with concordance for FGIDs in 6 (18%) ASD/NT sibling pairs.”
Matched NT sibling FGID count differs from Table 1
MinorThe results text says 13 matched NT siblings had an FGID, but Table 1 reports 12.
“Fifteen ASD children and 13 NT siblings in 37 families met ROME III criteria for at least one FGID”
“| FGID present (family matched) | 15 (41%) | 12 (32%) | 0.31 |”
H1b and H1c diagnoses are reversed in Table 1
MinorThe results text identifies H1b as cyclic vomiting syndrome and H1c as aerophagia, while Table 1 assigns those labels the other way around.
“H1b) Cyclic vomiting syndrome; H1c) Aerophagia.”
“| H1b. Aerophagia | 2 | 2 | 0.44 | | H1c. Cyclic Vomiting Syndrome | 0 | 1 | 0.33 |”
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Metadata
- Authors
- Son, J. S., Zheng, L. J., Rowehl, L. M., Tian, X., Zhang, Y., Zhu, W., Litcher-Kelly, L., Gadow, K. D., Gathungu, G., Robertson, C. E., Ir, D., Frank, D. N., & Li, E.
- Affiliations
- Stony Brook University (US)
- University of Colorado Anschutz (US)
- Corresponding author
- Li, Ellen
- Venue
- PLOS ONE
- Publisher
- Public Library of Science (PLoS)
- Published
- 2015-10-01
- Volume
- 10
- Issue
- 10
- Pages
- e0137725
- ISSN
- 1932-6203 (ISSN-L)
- PMC
- PMC4591364
- OpenAlex
- W1860290282
- Cited by
- 229
- References
- 73
- Citations by year
- 2026: 9 · 2025: 17 · 2024: 23 · 2023: 17 · 2022: 18 · 2021: 29 · 2020: 29 · 2019: 33 · 2018: 24 · 2017: 13 · 2016: 14 · 2015: 1
- FWCI
- 7.52
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- PLoS ONE (publishedVersion, cc-by)
- Europe PMC (PubMed Central) (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- Academic Commons (Stony Brook University) (submittedVersion, cc-by)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Figshare (submittedVersion, cc-by)
- Funders
- Simons Foundation
- Simons Foundation Autism Research Initiative
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Gut microbiota and health
- Life Sciences › Neuroscience › Cognitive Neuroscience › Autism Spectrum Disorder Research
- Health Sciences › Medicine › Gastroenterology › Gastrointestinal motility and disorders
- Keywords
- autism spectrum disorder, fecal microbiota, functional gastrointestinal disorders, constipation, siblings, gut microbiota, Sutterella, Bacteroidetes, Prevotella, child behavior problems, macronutrient intake, ASD severity, microbial taxa
- MeSH terms
- Autism Spectrum Disorder, Adolescent, Bacteria, Child, Diet, Feces, Female, Humans, Male, Phenotype, RNA, Ribosomal, 16S, Case-Control Studies, Siblings, Gastrointestinal Tract, Microbiota
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