Ignite Creation Date:
2024-05-06 @ 4:07 AM
Last Modification Date:
2024-10-26 @ 11:44 AM
Study NCT ID:
NCT02457104
Status:
UNKNOWN
Last Update Posted:
2021-02-18
First Post:
2015-05-27
Brief Title:
The Effect of PPI Therapy on Weight Gut Microbiome and Expression of GPR41 and GPR43
Sponsor:
University of Colorado Denver
Organization:
University of Colorado Denver
Study Overview
Official Title:
The Effect of PPI Therapy on Weight Gut Microbiome and Expression of GPR41 and GPR43
Status:
UNKNOWN
Status Verified Date:
2021-02
Last Known Status:
RECRUITING
Delayed Posting:
No
If Stopped, Why?:
Not Stopped
Has Expanded Access:
False
If Expanded Access, NCT#:
N/A
Has Expanded Access, NCT# Status:
N/A
Acronym:
None
Brief Summary:
The investigators long-term goal is to understand how PPIs influence energy balance in both obese and normal-weight individuals The overall goal of this study is to determine whether PPI use causes detrimental changes in the composition and functional properties of the gut microbiome and whether any such effects are mediated by altered responses of human fatty acid receptors eg GPR4143
Detailed Description:
Research Plan
Patients without gastrointestinal symptoms scheduled for a screening or surveillance colonoscopy for colorectal cancer at the UCH endoscopy lab will be screened for the study based on the inclusion and exclusion criteria Eligible patients who consent to the study will have height weight and demographic information measured and recorded They will complete a weightdiet history questionnaire Patients will undergo the screeningsurveillance colonoscopy per routine clinical care Residual stool is standardly aspirated through the colonoscope to ensure full visualization of the mucosa to enhance polyp detection We will take stool aspirates from three separate locations sigmoid colon cecum and terminal ileum Evidence suggests that a bowel preparation prior to colonoscopy does not significantly change the composition of the intestinal microbiota for the majority of subjects We will also take four small pinch biopsies at each of these three sites Stool aspirates and biopsy specimens will be snap-frozen in liquid N2 and frozen at -80C until required for their assays
We will enroll a total of 48 subjects divided equally between four groups OBPPI OBnon-PPI NWPPI and NWnon-PPI Approximately 40-50 patients per week meeting the above inclusionexclusion criteria undergo a screeningsurveillance colonoscopy at UCH making recruitment of 2-3 patientsweek over 6-9 months realistic to reach targeted enrollment
Aim 1 To examine the effects of PPI use on the gut microbiota in the colon and terminal ileum of normal-weight NW and obese OB patients Intestinal microbiomes will be profiled by 16S rRNA sequencing to identify PPI-associated alterations in gut biodiversity
Separate sterile specimen collection containers will be used for the stool aspirates and mucosal biopsies from the sigmoid colon cecum and terminal ileum High-throughput DNA sequencing for microbiome analysis will be used
Microbiome 16S rRNA Profiling Total community genomic DNA will be prepared from intestinal specimens and 16S rRNA genes PCR amplified as previously described Paired-end multiplexed sequencing will be performed 10-20 million DNA sequences will be generated in a single run and 100000 rRNA reads generated per sample to ensure 99 sequence coverage Demultiplexing quality filtering chimera-removal and sequence classification using SINASilva will follow previous publications Similar sequences are grouped into operational taxonomic units OTUs based on taxonomy
Microbiome Data Analysis The main outcome is the percent relative abundance PRA of microbial groups and we hypothesize this will differ between PPI users and non-users of acid suppression medications Specific microbial groups speciesgeneraphyla that differ between experimental groups will be identified using a non-parametric two-part statistic40 Statistical analyses will be assessed at 005 and p-values corrected for false discovery rate FDR41 Results will be validated by species-specific QPCR
Aim 2 To determine whether PPI use alters the relative amount of gut bacterial RNA dedicated to the fermentation cycle in NW and OB individuals We will perform shotgun sequencing of bulk RNA prepared from intestinal microbiomes to identify microbial metabolic pathways differentially expressed in patients on PPIs Stool aspirates from the terminal ileum cecum and sigmoid colon will be obtained as detailed above
RNA Isolation and Sequencing Microbial RNA will be extracted from 50 mg of fecal aspirates using the RiboPure Bacteria extraction kit Life Technologies Inc USA For this pilot we will assay specimens from a total of 48 subjects divided equally between four groups OBPPI OBnon-PPI NWPPI and NWnon-PPI Host and microbial rRNA will be removed using the Ribo-Zero bacteria and Ribo-Zero mouse kits Epicentre Inc USA RNA samples will be prepared for sequencing using the ScriptSeq v2 RNA-Seq bacteria kits Epicentre Inc USA Sequencing will be performed on an Illumina HiSeq2000 platform UC-Denver Microarray and Genomics Core which provides high depth of coverage For quality assurance of reproducibility two mRNA samples of each specimen will be pooled and sequenced In this pilot experiment we will generate 20x106 single-end 150-base reads per sample for each microbial meta-transcriptome dataset
Metatranscriptome Data Analysis Annotation and enumeration of bacterial transcripts will be performed at the peptide level using BLASTX searches against the approximately 1800 bacterial genome sequences currently available in GenBank as well as the NCBI non-redundant protein database using an E-value cutoff of 10-544 Broad-level functional differences between samples will be assessed using annotations generated through the COG47 KEGG48 and RASTSEED tools49 50 Statistical analysis of enteric microbial transcriptomes will focus on the identification of metabolic pathways that are differentially expressed between groups44 Genes with high-quality annotations E10-5 will be used to construct an NxM matrix of genes M by subjects N recording the number of sequence reads annotated for a given gene and subject A similar NxM matrix of gene categories M by subjects N will be constructed to tabulate the distribution of sequence reads assigned to functional categories Genes or gene categories that differ in prevalence or abundance between treatment groups will be identified by the Fisher exact test or Kruskal-Wallis test respectively applied to each genecategory in the matrix Significance will be assessed at 005 following correction for FDR41 P-values will be used to prioritize a candidate gene list for follow-up validation using QPCR to screen for gene expression across the entire patient cohort
Aim 3 To determine the extent to which PPI use alters expression of GPR41 GPR43 and their effector genes in the colon and terminal ileum of NW and OB patients Biopsy specimens will be subjected to a panel of RT-QPCR assays to monitor expression of target genes impacted by microbial SCFA production Biopsies from the terminal ileum cecum and sigmoid colon will be obtained as detailed above
Quantitative reverse transcriptase-PCR qRT-PCR analysis Total RNA will be extracted using the RNeasy Mini Kit Qiagen and ISOGEN WAKO Complementary DNAs will be transcribed using RNAs as templates with Moloney murine leukaemia virus reverse transcriptase Invitrogen cDNAs will be amplified by PCR with Taq DNA polymerase TaKaRa using the appropriate primers qRT-PCR analyses will be performed using DNA Engine Opticon-2 MJ Research The expression will be quantified in duplicate
Sample Size and Power Analysis
We will enroll 24 NW and 24 obese OB participants Half of the NW and OB groups will be PPI users and half will be non-users of acid suppressing medications The primary outcome for Aim 1 is the difference in the PRA of Firmicutes and Bacteroidetes A two-way ANOVA factors of BMI category and PPI use will be used Based on our preliminary data we will have 94 power to detect a difference in the PRA of Firmicutes 72 vs 52 with SD of 20 and 95 power to detect differences in the PRA of Bacteroidetes 16 vs 5 with SD of 5 between PPI users and non-users The primary outcome for Aim 2 is the difference in bacterial RNA dedicated to the fermentation cycle while the outcome for Aim 3 is the difference in expression of GPR 41 and GPR 43 Based on the expected differences in the gut microbiota profile and subsequent SCFA production we expect a 25 difference between PPI users and non-users for both outcomes giving us 99 power for both Aims 2 and 3 We will also perform subgroup analyses within the NW and OB groups
Patients without gastrointestinal symptoms scheduled for a screening or surveillance colonoscopy for colorectal cancer at the UCH endoscopy lab will be screened for the study based on the inclusion and exclusion criteria Eligible patients who consent to the study will have height weight and demographic information measured and recorded They will complete a weightdiet history questionnaire Patients will undergo the screeningsurveillance colonoscopy per routine clinical care Residual stool is standardly aspirated through the colonoscope to ensure full visualization of the mucosa to enhance polyp detection We will take stool aspirates from three separate locations sigmoid colon cecum and terminal ileum Evidence suggests that a bowel preparation prior to colonoscopy does not significantly change the composition of the intestinal microbiota for the majority of subjects We will also take four small pinch biopsies at each of these three sites Stool aspirates and biopsy specimens will be snap-frozen in liquid N2 and frozen at -80C until required for their assays
We will enroll a total of 48 subjects divided equally between four groups OBPPI OBnon-PPI NWPPI and NWnon-PPI Approximately 40-50 patients per week meeting the above inclusionexclusion criteria undergo a screeningsurveillance colonoscopy at UCH making recruitment of 2-3 patientsweek over 6-9 months realistic to reach targeted enrollment
Aim 1 To examine the effects of PPI use on the gut microbiota in the colon and terminal ileum of normal-weight NW and obese OB patients Intestinal microbiomes will be profiled by 16S rRNA sequencing to identify PPI-associated alterations in gut biodiversity
Separate sterile specimen collection containers will be used for the stool aspirates and mucosal biopsies from the sigmoid colon cecum and terminal ileum High-throughput DNA sequencing for microbiome analysis will be used
Microbiome 16S rRNA Profiling Total community genomic DNA will be prepared from intestinal specimens and 16S rRNA genes PCR amplified as previously described Paired-end multiplexed sequencing will be performed 10-20 million DNA sequences will be generated in a single run and 100000 rRNA reads generated per sample to ensure 99 sequence coverage Demultiplexing quality filtering chimera-removal and sequence classification using SINASilva will follow previous publications Similar sequences are grouped into operational taxonomic units OTUs based on taxonomy
Microbiome Data Analysis The main outcome is the percent relative abundance PRA of microbial groups and we hypothesize this will differ between PPI users and non-users of acid suppression medications Specific microbial groups speciesgeneraphyla that differ between experimental groups will be identified using a non-parametric two-part statistic40 Statistical analyses will be assessed at 005 and p-values corrected for false discovery rate FDR41 Results will be validated by species-specific QPCR
Aim 2 To determine whether PPI use alters the relative amount of gut bacterial RNA dedicated to the fermentation cycle in NW and OB individuals We will perform shotgun sequencing of bulk RNA prepared from intestinal microbiomes to identify microbial metabolic pathways differentially expressed in patients on PPIs Stool aspirates from the terminal ileum cecum and sigmoid colon will be obtained as detailed above
RNA Isolation and Sequencing Microbial RNA will be extracted from 50 mg of fecal aspirates using the RiboPure Bacteria extraction kit Life Technologies Inc USA For this pilot we will assay specimens from a total of 48 subjects divided equally between four groups OBPPI OBnon-PPI NWPPI and NWnon-PPI Host and microbial rRNA will be removed using the Ribo-Zero bacteria and Ribo-Zero mouse kits Epicentre Inc USA RNA samples will be prepared for sequencing using the ScriptSeq v2 RNA-Seq bacteria kits Epicentre Inc USA Sequencing will be performed on an Illumina HiSeq2000 platform UC-Denver Microarray and Genomics Core which provides high depth of coverage For quality assurance of reproducibility two mRNA samples of each specimen will be pooled and sequenced In this pilot experiment we will generate 20x106 single-end 150-base reads per sample for each microbial meta-transcriptome dataset
Metatranscriptome Data Analysis Annotation and enumeration of bacterial transcripts will be performed at the peptide level using BLASTX searches against the approximately 1800 bacterial genome sequences currently available in GenBank as well as the NCBI non-redundant protein database using an E-value cutoff of 10-544 Broad-level functional differences between samples will be assessed using annotations generated through the COG47 KEGG48 and RASTSEED tools49 50 Statistical analysis of enteric microbial transcriptomes will focus on the identification of metabolic pathways that are differentially expressed between groups44 Genes with high-quality annotations E10-5 will be used to construct an NxM matrix of genes M by subjects N recording the number of sequence reads annotated for a given gene and subject A similar NxM matrix of gene categories M by subjects N will be constructed to tabulate the distribution of sequence reads assigned to functional categories Genes or gene categories that differ in prevalence or abundance between treatment groups will be identified by the Fisher exact test or Kruskal-Wallis test respectively applied to each genecategory in the matrix Significance will be assessed at 005 following correction for FDR41 P-values will be used to prioritize a candidate gene list for follow-up validation using QPCR to screen for gene expression across the entire patient cohort
Aim 3 To determine the extent to which PPI use alters expression of GPR41 GPR43 and their effector genes in the colon and terminal ileum of NW and OB patients Biopsy specimens will be subjected to a panel of RT-QPCR assays to monitor expression of target genes impacted by microbial SCFA production Biopsies from the terminal ileum cecum and sigmoid colon will be obtained as detailed above
Quantitative reverse transcriptase-PCR qRT-PCR analysis Total RNA will be extracted using the RNeasy Mini Kit Qiagen and ISOGEN WAKO Complementary DNAs will be transcribed using RNAs as templates with Moloney murine leukaemia virus reverse transcriptase Invitrogen cDNAs will be amplified by PCR with Taq DNA polymerase TaKaRa using the appropriate primers qRT-PCR analyses will be performed using DNA Engine Opticon-2 MJ Research The expression will be quantified in duplicate
Sample Size and Power Analysis
We will enroll 24 NW and 24 obese OB participants Half of the NW and OB groups will be PPI users and half will be non-users of acid suppressing medications The primary outcome for Aim 1 is the difference in the PRA of Firmicutes and Bacteroidetes A two-way ANOVA factors of BMI category and PPI use will be used Based on our preliminary data we will have 94 power to detect a difference in the PRA of Firmicutes 72 vs 52 with SD of 20 and 95 power to detect differences in the PRA of Bacteroidetes 16 vs 5 with SD of 5 between PPI users and non-users The primary outcome for Aim 2 is the difference in bacterial RNA dedicated to the fermentation cycle while the outcome for Aim 3 is the difference in expression of GPR 41 and GPR 43 Based on the expected differences in the gut microbiota profile and subsequent SCFA production we expect a 25 difference between PPI users and non-users for both outcomes giving us 99 power for both Aims 2 and 3 We will also perform subgroup analyses within the NW and OB groups
Study Oversight
Has Oversight DMC:
None
Is a FDA Regulated Drug?:
None
Is a FDA Regulated Device?:
None
Is an Unapproved Device?:
None
Is a PPSD?:
None
Is a US Export?:
None
Is an FDA AA801 Violation?:
None