Institutional Review Board for Baylor College of Medicine and Affiliated Hospitals

Protocol Number:    H-35071Status:                        ApprovedInitial Submit Date:  6/18/2014Approval Period:       5/5/2017 - 5/4/2018

Section Aa:  Title & PIA1.  Main Title

IDENTIFYING BIOMARKERS OF GI MORBIDITY IN ASD: LINKING MULTI-OMICS AND HUMAN BEHAVIOR

A2.  Principal InvestigatorName: RUTH ANN LUNA Phone: 832-824-1894Id: 044899 Fax: 832-825-1032Department: TCH PATHOLOGY Email: raluna@bcm.tmc.eduCenter: Mail Stn: BCM315

A3.  Administrative ContactName: MARLEN BANDA-RUIZ Phone: 832-824-3439Id: 179731 Fax:

Email: bandarui@bcm.tmc.eduMail Stn: BCM315

A3a.  Financial Conflict of Interest

Does any member of study personnel (Investigator (including investigator’s spouse and/or dependent children)) that are involved in the design, conduct, or reporting of the research have a Significant Financial Interest (SFI) that would reasonably appear to be affected by the research for which funding is sought and/or associated with an entity/business that would reasonably appear to be affected by the research?

     No

Section Ab:  General Information

A4.  Co-InvestigatorsName: JAMES VERSALOVIC Phone: 832-824-2213Id: 026810 Fax: 832-825-0164Department: TCH PATHOLOGY Email: jamesv@bcm.tmc.eduCenter: Mail Stn: BCM315

Name: CAROL ANN REDEL Phone: 832-822-1048Id: 030297 Fax: 832-825-3633Department: PEDIATRICS: GASTROENTEROLOGY Email: credel@bcm.tmc.eduCenter: Mail Stn: BCM320

Name: ROBIN PAGE KOCHEL Phone: 832-824-3390Id: 152719 Fax:Department: PEDIATRICS: PSYCHOLOGY Email: kochel@bcm.tmc.eduCenter: Mail Stn: BCM320

Name: JESSICA K RUNGE Phone: 832-824-3985Id: 162489 Fax:Department: TCH PATHOLOGY Email: runge@bcm.tmc.edu

Center: Mail Stn: BCM315

Name: MIRJANA MALETIC-SAVATIC Phone: 832-824-8807Id: 164647 Fax:Department: PEDIATRICS: NEUR NRI Email: maletics@bcm.tmc.eduCenter: Mail Stn: BCM320

Name: MICHELLE RUBIO-GONZALES Phone: 832-824-1716Id: 165042 Fax:Department: PATHOLOGY Email: rubiogon@bcm.tmc.eduCenter: Mail Stn: BCM315

Name: TOR SAVIDGE Phone: 832-824-2839Id: 179183 Fax:Department: PATHOLOGY Email: savidge@bcm.tmc.eduCenter: Mail Stn: BCM315

Name: COLIN D. MCGEE Phone: 832-824-4260Id: 181292 Fax:Department: TCH PATHOLOGY Email: cdmcgee@bcm.tmc.eduCenter: Mail Stn: BCM320

Name: ALAMELU VENKATACHALAM Phone: 832-824-3965Id: 184639 Fax:Department: TCH PATHOLOGY Email: venkatac@bcm.tmc.eduCenter: Mail Stn: BCM315

Name: MICHELLE KWOK Phone: 832-824-3943Id: 187113 Fax:Department: PATHOLOGY Email: mkwok@bcm.tmc.eduCenter: Mail Stn: BCM315

Name: JESSICA OROBIO Phone: 832-822-4759Id: 188388 Fax:Department: PEDIATRICS: PSYCHOLOGY Email: orobio@bcm.tmc.eduCenter: Mail Stn: BCM320

Name: KRISTIN ADKINS Phone:Id: Non-Baylor Fax:Institution: Texas Children's Hospital Email: kbadkins@texaschildrens.orgAddress: Fannin St

Name: LAUREN DOWELL Phone:Id: Non-Baylor Fax:Institution: Texas Children's Hospital Email: lrdowel1@texaschildrens.orgAddress: Fannin St

A5.  Funding Source:

Organization:  AUTISM SPEAKS

A6a.  Institution(s) where work will be performed:

BCM: Baylor College of MedicineTCH: Texas Children's Hospital

A6b.  Research conducted outside of the United States:

Country:  Facility/Institution:  Contact/Investigator:  Phone Number:  

If documentation of assurances has not been sent to the Office of Research, please explain:

A7.  Research Category:

A8. Therapeutic Intent

Does this trial have therapeutic intent?

No

Section B:  Exempt RequestB.  Exempt From IRB Review

Not Applicable

Section C:  Background InformationThe Microbiome-Gut-Brain Axis. It has been shown that initial colonization of the gut impacts brain development. More recent literature has highlighted the microbiome-gut-brain axis and reported that CNS neurotransmission can be significantly altered by the absence of a normal gut microbiome. There is a significant need for additional research involving the microbiome-gut-brain axis in autism spectrum disorders (ASDs), specifically in childhood when the establishment of the gut microbiome and manifestation of clinical symptoms of ASD occur concurrently. Few studies have focused on the gut microbiome in the pediatric ASD population, and those studies that do exist in the literature have multiple confounders and have produced conflicting results.

Microbiome Characterization. Over the past 10 years, multiple bacterial organisms have been suspected of playing a role in the development of GI symptoms in patients with autism, and direct detection of these genera of interest has been performed via conventional methods such as culture and targeted real-time PCR. A recent thorough search of the literature produced only four previous studies utilizing next-generation sequencing (NGS) for the characterization of the gut microbiome in autism. NGS targeting the 16S rRNA gene was performed on intestinal biopsies from 15 ASD children with GI disease and 7 neurotypical children with GI disease. The subsequent analysis focused mainly on phylum-level differences, where decreased Bacteroidetes and increased Firmicutes and Proteobacteria were noted in the ASD group compared to the neurotypical group. Three additional studies in the ASD population involved microbiome characterization of stool samples via NGS and comparison with either unaffected siblings or unrelated healthy children. In 2010, Finegold, et al. recruited 33 ASD children, 7 unaffected siblings, and 8 healthy controls. The results suggested increased bacterial diversity in the ASD group with increased Bacteroidetes and decreased Firmicutes, findings that were contradictory to the study by Williams, et al., where a large sample size of 51 ASD children was compared to 53 unaffected siblings. In further contradiction to previous studies, no significant differences were noted between the groups, but critics have suggested that an unrelated healthy control group was needed to strengthen the study. The most recent study involved 20 ASD children and 20 unrelated healthy controls, and while Firmicutes and Bacteroidetes were found to be the most dominant phyla in the ASD group, no significant differences were noted in comparison to the healthy group, but a trend towards reduced abundance of fermenters was seen. When evaluating these four studies, common weaknesses include the absence of documented ASD assessment of the neurotypical controls (both siblings and unrelated children) and the lack of stratification by GI phenotype, which our preliminary pediatric IBS studies have demonstrated as significant. The studies also differed in terms of prior antibiotic use with exclusion criteria ranging from 15 days to 3 months. No study has compared ASD children with GI symptoms to other GI diseases with similar GI phenotypes, such as IBS. Metabolomics in Autism. There is a paucity of data regarding metabolomic analysis in the ASD population. Recent studies on urine specimens showed a decrease in several amino acids, decreased antioxidants, and significant alterations of gut bacterial metabolites in ASD children compared to healthy controls. The data suggest that metabolomic profiling is a promising avenue of research in this population, and recent optimization of metabolomic processing of stool specimens will further enable the necessary studies in pediatric ASD. No data currently exist from stool in this population, and this is a significant void if functional differences in the microbiome are to be investigated. With the addition of gut

metabolomics to gut microbiome data, a comprehensive data set in GI disease in ASDs could be established, as we have demonstrated in C.difficile-associated colitis. Our preliminary studies in IBS children indicate that metabolomic changes can also be identified where significant alterations in community 16S profiles have been hard to identify.

Section D:  Purpose and ObjectivesGI disease in the pediatric autism spectrum disorder (ASD) population ranges from constipation to diarrhea and is complicated by the non-verbal or limited communication abilities of these children. ASDs encompass a wide range of developmental brain disorders characterized by challenges in the areas of communication, social interaction, and behavior, with sensory difficulties and repetitive behaviors that make GI symptoms more challenging in these children. Prior studies have suggested that GI symptoms in ASD are associated with an altered gut microbiome, but no studies to date have assessed the functional importance of such microbial community shifts. This study will explore the functional microbiome, via a multi-omic analysis of microbiome, metabolome, and clinical data, of the GI tract in ASD children compared to healthy neurotypical children and children with functional GI disorders. In addition, behavioral characteristics of the ASD cohort will be compared to multi-omic signatures and association with abdominal pain. We hypothesize that the functional gut microbiome of ASD differs from that of healthy children and children with functional GI disorders.

Aim 1: Characterize and correlate the functional gut microbiome and metabolome in children with autism, neurotypical healthy children, and children with functional GI disorders. 1.1 Characterize the gut microbiome in children with ASD, unaffected siblings, and healthy children. We hypothesize that the gut microbiome of the ASD children will differ from their unaffected siblings as well as healthy children. We further hypothesize that microbiome differences will be seen based on GI phenotype. 1.2 Characterize the gut metabolome in children with ASD. We hypothesize that functional biochemical changes evident in metabolomic profiles are associated with microbial community shifts in ASD children. 1.3 Integrate gut microbiome, metabolome, and clinical data from ASD children with data from healthy and functional GI disorder pediatric cohorts. Utilizing a multi-omic analysis of clinical, microbial, and metabolomic data, we hypothesize that we will identify relevant functional pathways in ASD children with GI disease.

Section E:  Protocol Risks/SubjectsE1.  Risk Category

Category 1: Research not involving greater than minimum risk.

E2.  Subjects

Gender:Both

Age:Adolescent (13-17 yrs), Child (3-12 yrs), Infant/Toddler (0-36 mos)

Ethnicity:All Ethnicities

Primary Language:

English

Groups to be recruited will include:Both patients and healthy, non-patient, normals

Which if any of the following vulnerable populations will be recruited as subjects?

Children, Cognitively impaired

Vulnerable populations require special protections.  How will you obtain informed consent, protect subject confidentiality, and prevent undue coercion?

Consent will be obtained from the parent or guardian. Records will be kept in a secure area and confidentiality will be maintained within limits of the law. A number system will be used to identify the subject's data.

E3.  Pregnant woman/fetus

Will pregnant women and/or fetuses (as described in 45 CFR 46 Subpart B) be enrolled in the research?

No

E4.  Neonates

Will neonates of uncertain viability or nonviable neonates (as described in 45 CFR 46 Subpart B) be enrolled in the research?

No

E5.  Children

Will children be enrolled in the research?Yes

Section F:  Design/ProcedureF1.  Design

Select one category that most adequately describes your research:

c) Pilot

Discuss the research design including but not limited to such issues as: probability of group assignment, potential for subject to be randomized to placebo group, use of control subjects, etc.

Access to a large pediatric cohort will enable recruitment of participants with ASD and/or functional GI disorders (FGIDs) from Texas Children’s Hospital. Parents of children may also search the study via website or flyer and contact the coordinator for enrollment. The study will include recruitment of 5 groups: 1. children with ASD and FGIDs, 2. children with ASD and without FGIDs, 3. unaffected siblings (non-ASD) without FGIDs, 4. neurotypical children with FGIDs, and 5. neurotypical children without FGIDs.

Inclusion Criteria:1. Age 2-17 years old. 2. For the ASD cohort, a confirmed ASD diagnosis with the Autism Diagnostic Observation Schedule (ADOS or ADOS-2) administered by a qualified individual/center and reviewed by co-investigators at Texas Children's Hospital. 3. For the non-ASD cohort (unaffected siblings and neurotypical children), Social Responsiveness Scale (SRS-2) screens will be used to rule out ASD (SRS-2 score < 55). The SRS-2 will also be administered to the ASD cohort for comparison. 4. For the FGIDs cohorts, presence of constipation and/or abdominal pain for ≥2 months not due to a known physiological or organic cause for the GI symptoms. All children with GI symptoms will undergo an evaluation by a pediatric GI specialist for an organic cause of their symptoms prior to being enrolled into the study. 5. For the non-FGIDs cohorts, no history of GI complaints or disorders. All participants will be screened with the Parent report version of the Rome III Diagnostic Questionnaire for the Pediatric Functional GI Disorders to ensure there is no history of FGIDs in this cohort, a strategy used in previous studies of FGIDs in children with autism.

Note: For participating families not local to the Houston area or otherwise unable to be present for an ADOS evaluation, other diagnostic tools will be considered on a case-by-case basis (ex. the Autism Diagnostic Interview-R, ADI-R, is another widely accepted method of obtaining an accurate ASD diagnosis).

Exclusion Criteria:Exclusion criteria include: past bowel surgery; a serious chronic medical condition (e.g., diabetes); weight and/or height < 2 SD for age; anti-inflammatory use; history of inflammatory bowel disease, Celiac disease, H. pylori infection, or eosinophilic disorders (e.g., eosinophilic esophagitis).

F2.  Procedure

Recruitment Potential participants will be identified during clinical visits to the Autism Center or GI clinic. In addition, interested families will contact the research coordinator directly. The Autism Center will also recruit through prior research studies by contacting families that have given permission to be recontacted for the purpose of learning about new studies. Inclusion and exclusion criteria will be evaluated in each case, and if criteria are met, informed consent will be obtained. Potential participants will also be recruited at Nationwide Children's Hospital (Columbus, OH) and UT-Southwestern (Dallas, TX), with all specimens, surveys, and data transferred to TCH. IRB approval has been obtained and is attached in Section S.

Clinical Metadata and Behavioral Assessments: GI symptoms will be assessed using the modified 6-item GI Severity Index (6-GSI) previously employed in the autism population. Additional clinical data related to diet, prior antibiotics/antifungals/antivirals, supplements, current/recent medications, and general clinical history will be obtained from medical records and caregiver interview. Extensive clinical data will be obtained on each participant, including detailed diet history, 6-Item GI severity index scores, other medical co-morbidities, and genetic and behavioral characteristics. Behavioral assessments for all children to be included in the multi-omic analysis will be the Sensory Profile, the Repetitive Behavior Scale-Revised, Aberrant Behaviors Checklist, and the Child Behavioral Checklist. Each participant will also be screened by the Social Responsiveness Scale-2, and should a child with no history of an autism diagnosis receive a score suggesting a possible diagnosis of autism, the research coordinators and study investigators will refer the family for a comprehensive evaluation through the Texas Children's Autism Center. In the ASD groups, autism diagnoses will be verified with an Autism Diagnostic Observation Schedule (ADOS) evaluation if one had not previously been done or if it was deemed invalid. For participating families not local to the Houston area or otherwise unable to be present for an ADOS evaluation, other diagnostic tools will be considered on a case-by-case basis (ex. the Autism Diagnostic Interview-R, ADI-R, is another widely accepted method of obtaining an accurate ASD diagnosis). ROME III Diagnostic questionnaire: The Rome Foundation has developed diagnostic questionnaires in coordination with the Rome III committees to assist physicians, health care professionals and researchers in identifying individuals who have one or more functional gastrointestinal disorders (FGIDs). The questionnaire uses 5 point scales to measure frequency, severity, and duration of symptoms. In addition, it may be scored to assess whether a patient meets the criteria for each of the individual functional gastrointestinal disorders. The questionnaire is followed by a coding system that identifies provisional diagnoses from the responses to the questions. The QPGS-RIII cannot substitute for the medical evaluation and clinical judgment required for an accurate diagnosis. The questionnaire will be administered to both subjects and their parent/guardian during the clinic visit. Time to completion is approximately 45 minutes.

Diet, Abdominal Pain, and Stool Diaries: Subjects will be instructed on keeping daily diet, pain, and stool diaries over a two week period. The two-week Food Record will capture the types of foods and quantity of each food eaten for each day. The pain diaries have been validated and successfully used in numerous studies within our GI section. This will allow for clinical phenotyping of pain (frequency, severity, timing, and location), interference of activities, and bowel movements (frequency, and form). Abdominal pain ratings are made 3 times per day (awakening, after lunch, and evening) during the 2-week period. Children will rate their pain using a 0 to 10 scale with 0 being “no pain at all’’ and 10 representing the “worst pain you can imagine.” This pain scale has been validated for measuring abdominal pain in children. Pain frequency was recorded during the same 2-week period. When possible, stools passed during the 2 week period will be collected. They will be characterized by the children using the modified pediatric Bristol stool form chart used in our GI section. Verbal children with ASD will attempt to keep these diaries, and parents will assess these items if possible in limited verbal and non-verbal children.

Specimen Collection and Processing: All participants will provide stool specimens for both microbiome and metabolome characterization. Stool specimens will be collected with the assistance of a caregiver and processed by the Texas Children’s Microbiome Center (TCMC). Stool specimens will be self-collected at home by each subject. The stool will be collected as is done routinely in the clinical setting to obtain stool samples for testing for blood, parasites, etc. Specifically, a plastic stool collection frame is placed over the toilet seat and a collection bowl is placed in the frame. The subject stools normally, and the stool is captured in the bowl. The bowl is then removed, sealed with a lid, and placed in a plastic bag and into the freezer until transport to Texas Children’s Hospital. Upon receipt, specimens will be stored at -80 degrees within the Texas Children’s Microbiome Center (TCMC) until processing. Bacterial DNA will be extracted using a commercial DNA extraction kit, the MoBio Powersoil DNA kit, with minor modifications implemented by the Human Microbiome Consortium. The resulting microbial DNA will be quantitated by the Nanodrop-1000 and Qubit methodologies to ensure adequate quality and quantity. For metabolomic analysis, an aliquot of stool will be placed in a pre-weighed tube and submitted for metabolomic characterization. Buccal swabs will also be collected by swabbing the inside of the cheek with a swab. These swabs will only be stored, and subjects may be contacted for participation and consent for additional studies. The specimens and associated data will be banked indefinitely. Gut Microbiome Characterization:

Microbiome characterization will be performed by sequencing the 16S rRNA gene and ITS genes followed by extensive bioinformatics analysis related to microbial composition, diversity, and community structure. Microbial DNA extraction, 16S rRNA gene-based next-generation sequencing (using the Illumina MiSeq), quality filtering, and microbiome-based analysis will be performed within the Texas Children's Microbiome Center. Each sample will be amplified using a barcoded primer, which yields a unique sequence identifier tagged onto each individual sample library. Illumina-based sequencing will yield > 5,000 reads per sample. All generated sequences will be quality filtered to remove mismatched sequences (to primer or molecular identifier), short reads, and low quality sequences (including ambiguous base calls). The remaining quality-checked sequence set will be analyzed via several bioinformatics approaches, including but not limited to QIIME and PiCRUST. Gut Metabolome Characterization: Metabolome characterization of stool aliquots will be completed using LC- and GC-mass spectrometry to identify biomarkers of interest in GI morbidity, as well as microbial-produced neurotransmitters. In addition, a novel biomarker panel associated with functional GI disorders (FGIDs) will be evaluated in the ASD cohort. Biochemical changes will be compared between groups, between GI phenotypes, and parallel with behavioral data.

Section G:  Sample Size/Data AnalysisG1.  Sample Size

How many subjects (or specimens, or charts) will be used in this study?

Local: 500              Worldwide: 500

Please indicate why you chose the sample size proposed:This is a pilot study. Detailed power calculations will be performed on the initial data obtained, and amendments to the IRB will be made if an increase in study numbers is indicated and feasible. As currently designed, this study would be the largest of its kind based on published research. Note: Potential sample size has been increased from 375 to 500 in the hopes that more participants can be recruited throughout the course of the study to increase our statistical power across the 5 groups.

G2.  Data Analysis

Provide a description of your plan for data analysis. State the types of comparisons you plan (e.g. comparison of means, comparison of proportions, regressions, analysis of variance). Which is the PRIMARY comparison/analysis? How will the analyses proposed relate to the primary purposes of your study?

Multiple analyses will be performed on the 16S rRNA gene sequencing data, including determination of bacterial diversity, evenness, richness, and relative abundance of the bacteria identified in each sample. QIIME and supervised machine learning will be employed to characterize changes in relative abundances of specific microbial taxa and overall bacterial richness/diversity. Network analysis strategies will allow for the evaluation of the underlying community structure of the gut microbiome in all groups. If available, the ITS1 target will also be sequenced to characterize the fungal components of the microbiome. For metabolomic data, biochemical changes will be compared between groups, between GI phenotypes, and in parallel with behavioral data. Complete multi-omic analysis of all five groups, including microbiome, metabolome, and clinical metadata, will allow the identification of correlative profiles between pediatric GI disorders and specific GI phenotypes in ASD, including associated behavioral characteristics.

Section H:  Potential Risks/DiscomfortsH1.  Potential Risks/Discomforts

Describe and assess any potential risks/discomforts; (physical, psychological, social, legal, or other) and assess the likelihood and seriousness of such risks:

There are no known risks associated with collection of stool, answering the questionnaires, or keeping the diaries. The investigators acknowledge the potential for the loss of confidentiality during the study. All efforts will be made to maintain anonymity of the specimens and associated data, specimens and data will be assigned only to a unique identifier with no personal identifiers associated with any data or specimens. Only the PI and research coordinator will have access to medical records and all data will be kept in a locked office and/or secure server.

H2.  Data and safety monitoring plan

     Do the study activities impart greater than minimal risk to subjects?

     No

H3.  Coordination of information among sites for multi-site research

Is the BCM Principal Investigator acting as the SPONSOR-INVESTIGATOR for this multi-site research?

     No or Not Applicable

Is BCM the COORDINATING CENTER for this multi-site research?     No or Not Applicable

Section I:  Potential BenefitsDescribe potential benefit(s) to be gained by the individual subject as a result of participating in the planned work.

Subjects will not benefit personally from providing the specimens because this research will take time to produce medically useful results.

Describe potential benefit(s) to society of the planned work.The participants will be part of an innovative study that will help researchers around the world and the general public gain knowledge and understand more about the human microbiome and how it relates to health and disease. It will also contribute to knowledge about microbial and metabolic differences in ASD and healthy children and the association with GI symptoms.

Do anticipated benefits outweigh potential risks? Discuss the risk-to-benefit ratio.The benefits of learning more about the human microbiome and how it relates to GI symptoms in children with ASD and neurotypical children far outweigh the current and potential future minimal risks. Therefore, the risk-to-benefit ratio is favorable.

Section J:  Consent ProceduresJ1.  Waiver of Consent

Will any portion of this research require a waiver of consent and authorization?

No

J1a.  Waiver of requirement for written documentation of Consent

Will this research require a waiver of the requirement for written documentation of informed consent?

No

J2.  Consent Procedures

Who will recruit subjects for this study?PIPI's staff

Describe how research population will be identified, recruitment procedures, any waiting period between informing the prospective participant and obtaining consent, steps taken to minimize the possibility of coercion or undue influence and consent procedures in detail.

The study will be open to all persons who satisfy the study entry criteria, and assessment of each potential participant based on inclusion and exclusion criteria will be done by the research coordinator with review by the PI or designee. Informed consent will be obtained from the parent or guardian of all eligible participants. Children will give assent as appropriate, specifically children who are not cognitively impaired will give assent.

Some children will be recruited through the Texas Children's Hospital Pediatric Gastroenterology, Hepatology,

and Nutrition Service or through the TCH Autism Center. Unaffected siblings will also be recruited through the TCH Autism Center at the time the child with ASD is consented for the study. Potential participants will be made aware of the study and provided with a flyer (that includes contact information) or directed to an institutional website. The recruiting physician or the research coordinator can also perform the screening questionnaire at this time.

Some children with be recruited through advertisements (e.g. www.clinicaltrials.org website) or via flyer in pediatric offices and will contact the coordinator for screening. These families may not have a child that is a patient at TCPA or TCH and therefore will give consent to view the child's medical records and will consult with the GI physician or Autism Center if needed.

When each potential participant has been deemed eligible by the screening questionnaire, then an in-person visit will be scheduled with the research coordinator. The coordinator will explain the study, including the types of diaries required, behavioral surveys to be completed, and specimens to be collected. Participation is voluntary, and informed consent will be obtained following explanation of the complete protocol and resolution of any questions by the potential participants and parents. Assent will be obtained from children who have no cognitive impairment. Additionally, videoconferencing with the family will be an option available for those not local to the Houston area.

Are foreign language consent forms required for this protocol?

No

J3.  Privacy and Intrusiveness

Will the research involve observation or intrusion in situations where the subjects would normally have an expectation of privacy?

No

J4.  Children

Will children be enrolled in the research?Yes

J5.  Neonates

Will non-viable neonates or neonates of uncertain viability be involved in research?

No

J6.  Consent Capacity - Adults who lack capacity

Will Adult subjects who lack the capacity to give informed consent be enrolled in the research?

No

J7.  Prisoners

Will Prisoners be enrolled in the research?

No

Section K:  Research Related Health Information and ConfidentialityWill research data include identifiable subject information?

Yes

Information from health records such as diagnoses, progress notes, medications, lab or radiology findings, etc.

Yes

Specific information concerning alcohol abuse:

No

Specific information concerning drug abuse:

No

Specific information concerning sickle cell anemia:

No

Specific information concerning HIV:

No

Specific information concerning psychiatry notes:

No

Demographic information (name, D.O.B., age, gender, race, etc.):

Yes

Full Social Security #:No

Partial Social Security # (Last four digits):

No

Billing or financial records:

No

Photographs, videotapes, and/or audiotapes of you:

No

Other:No

At what institution will the physical research data be kept?The physical research data will kept on the 9th floor of the Feigin Center within the Texas Children's Microbiome Center.

How will such physical research data be secured?All physical research data will be stored in locked cabinets inside a locked office within the Texas Children's Microbiome Center.

At what institution will the electronic research data be kept?Electronic research data will be stored on the BCM network within Texas Children's Hospital (Feigin Center). All data will reside on local computers within the Texas Children's Microbiome Center or on BCM servers.

Such electronic research data will be secured via BCM IT Services- provided secured network storage of electronic

research data (Non-Portable devices only):Yes

Such electronic research data will be secured via Other:

No

Will there be anyone besides the PI, the study staff, the IRB and the sponsor, who will have access to identifiable research data?

No

Please describe the methods of transmission of any research data (including PHI, sensitive, and non-sensitive data) to sponsors and/or collaborators.

Any transfer of study data between our lab, the sponsors, or collaborators will be done via secure/encrypted email only.

Will you obtain a Certificate of Confidentiality for this study?

No

Please further discuss any potential confidentiality issues related to this study.No patient identifiers will be reported in any published version of this study. The PHI will not be reused or disclosed to (shared with) any other person or entity, except as required by law, for authorized oversight of the research study, or for other research for which the use or disclosure of the PHI would be permitted under the Privacy Rule.

Section L:  Cost/PaymentDelineate clinical procedures from research procedures. Will subject's insurance (or subject) be responsible for research related costs? If so state for which items subject's insurance (or subject) will be responsible (surgery, device, drugs, etc). If appropriate, discuss the availability of financial counseling.

The patient will not be billed for any of the testing proposed in this study.

If subjects will be paid (money, gift certificates, coupons, etc.) to participate in this research project, please note the total dollar amount (or dollar value amount) and distribution plan (one payment, pro-rated payment, paid upon completion, etc) of the payment.

Dollar Amount:50

Distribution Plan:Subjects will be compensated $50 when they have completed all aspects of the study.

Section M:  GeneticsHow would you classify your genetic study?

Discuss the potential for psychological, social, and/or physical harm subsequent to participation in this research. Please discuss, considering the following areas: risks to privacy, confidentiality, insurability, employability, immigration status, paternity status, educational opportunities, or social stigma.

NA

Will subjects be offered any type of genetic education or counseling, and if so, who will provide the education or counseling and under what conditions will it be provided? If there is the possibility that a family's pedigree will be presented or published, please describe how you will protect family member's confidentiality?

NA

Section N:  Sample CollectionSAMPLE:     Other: Stool

What is the purpose of the sample collection?Collect material for microbial DNA sequencing and metabolomic analysis

For blood draws, specify the amount drawn, in teaspoons, at each visit and across the course of the subjects entire participation time.

Collect at least 3 inches (more than 10 grams) of stool from all children.

Is there the possibility that cell lines will be developed with this sample?No

Sample will be obtained from:Other: From subject

Will the sample be stripped of identifiers?

No

If sample will be released outside the hospital:

Will sample be released to anyone not listed as an investigator on the protocol? Will the information be identifiable, coded or de-identified?

Stool coded with an anonymous identifier will be released to an outside lab for metabolomic testing.

Will sample material be sold or transferred to any third parties? Will the information be de-identified?Stool aliquots with an anonymous identifier will be transferred to Metabolon for metabolomic testing.

If sample will be banked for future use:

Where will the sample be banked and for how long?The remaining stool material and bacterial DNA will be banked within the Texas Children's Microbiome Center indefinitely.

Does the banking institution have an approved policy for the distribution of samples?

Specimens will not be distributed outside of the research group.

If the entire sample will NOT be used during the course of this research study:

Will the remaining tissue be discarded? If not what will be done with the remaining sample after study completion and how long will the sample be kept?

Stool specimens, isolated DNA, and nucleic acid sequence data will be stored indefinitely for potential follow-up studies by the investigators on this protocol.

Will samples be made available to the research subject (or his/her medical doctor) for other testing?

No

If a subject withdraws from the study:

Will subject have the option to get the remaining portion of their sample back?

No

Will samples be destroyed? If not, will they be kept anonymously? What will happen to the sample if the subject revokes authorization?

Remaining coded samples will be destroyed.

Will data obtained from their sample be deleted? What will happen to the sample if the subject revokes authorization?

If the subject revokes authorization, the data will be deleted.

Will study data or test results be recorded in the subject's medical records?

No

Will results of specific tests and/or results of the overall study be revealed to the research subject and or his/her doctor?

Neither the subject nor the subjects' physicians will receive test results. Results of overall study will be revealed upon request.

Please identify all third parties, including the subject's physician, to receive the test results.

Neither the subject nor the subjects' physicians will receive test results.

SAMPLE:     Buccal Brushing

What is the purpose of the sample collection?Buccal specimens will be collected and stored for potential follow-up studies that would require additional consent.

For blood draws, specify the amount drawn, in teaspoons, at each visit and across the course of the subjects entire participation time.

A single swab will be collected.

Is there the possibility that cell lines will be developed with this sample?No

Sample will be obtained from:Other: From subject

Will the sample be stripped of identifiers?

No

If sample will be released outside the hospital:

Will sample be released to anyone not listed as an investigator on the protocol? Will the information be identifiable, coded or de-identified?

No.

Will sample material be sold or transferred to any third parties? Will the information be de-identified?

Sample material will not be sold or transferred to any third parties without additional consent.

If sample will be banked for future use:

Where will the sample be banked and for how long?The buccal swab will be banked within the Texas Children's Microbiome Center indefinitely.

Does the banking institution have an approved policy for the distribution of samples?The swab will not be distributed outside of the research group without additional consent.

If the entire sample will NOT be used during the course of this research study:

Will the remaining tissue be discarded? If not what will be done with the remaining sample after study completion and how long will the sample be kept?

Buccal swab samples will be stored indefinitely for potential follow-up studies by the investigators on this protocol.

Will samples be made available to the research subject (or his/her medical doctor) for other testing?

No

If a subject withdraws from the study:

Will subject have the option to get the remaining portion of their sample back?

No

Will samples be destroyed? If not, will they be kept anonymously? What will happen to the sample if the subject revokes authorization?

Remaining coded samples will be destroyed.

Will data obtained from their sample be deleted? What will happen to the sample if the subject revokes authorization?

If the subject revokes authorization, the data will be deleted.

Will study data or test results be recorded in the subject's medical records?

No

Will results of specific tests and/or results of the overall study be revealed to the research subject and or his/her doctor?

Neither the subject nor the subjects' physicians will receive test results. Results of overall study will be revealed upon request.

Please identify all third parties, including the subject's physician, to receive the test results.

Neither the subject nor the subjects' physicians will receive test results.

Section O:  Drug StudiesDoes the research involve the use of ANY drug* or biologic? (*A drug is defined as any substance that is used to elicit a pharmacologic or physiologic response whether it is for treatment or diagnostic purposes)

No

Does the research involve the use of ANY gene transfer agent for human gene transfer

research?No

O1.  Current Drugs

Is this study placebo-controlled?

No

Will the research involve a radioactive drug that is not approved by the FDA?

No

Section P:  Device Studies     Does this research study involve the use of ANY device?

     No

Section Q:     Consent Form(s) Autism GI

Section R:  AdvertisementsMode of Advertising:  Internet

Exact language of Advertisement:Identifying Biomarkers of GI Morbidity in Autism Spectrum Disorders: Linking Multi-omics and Human Behavior

We are seeking participants for a research study of abdominal pain in children with autism spectrum disorders. Eligible participants include children 4-12 years of age, specifically children with a diagnosis of autism spectrum disorder, children with abdominal pain, and children with no abdominal pain. Siblings of children with autism with no abdominal pain are also eligible. The study will encompass behavioral, abdominal pain, and the bacteria (microbiome) and small biological molecules (metabolome) in the gastrointestinal tract. Participation is easy, there is no pain involved, and your time will be compensated.

For more information on participating in this study, please contact Ruth Ann Luna, PhD (raluna@bcm.edu). This study is being conducted by Baylor College of Medicine in conjunction with Texas Children's Hospital, and this research is funded by Autism Speaks.

CONSENT FORMInstitutional Review Board for Baylor College of Medicine and Affiliated Hospitals

Autism GI

H-35071 - IDENTIFYING BIOMARKERS OF GI MORBIDITY IN ASD: LINKING MULTI-OMICS AND HUMAN BEHAVIOR

BackgroundWe now know that the bacteria in our intestines can be important to our health. However, we do not understand very well what types of bacteria keep us healthy and what types may lead to health problems. We believe that differences in the bacteria in our intestines and inflammation may help to explain why some children experience frequent stomach (abdominal) pain and others do not. We also believe that in the stomach pain may be linked to behavior in children with autism.

Healthy children, children with autism spectrum disorder, and children with functional GI disorders will be asked to provide one stool specimen and one cheek swab specimen as well as complete several surveys and diaries over a two week period. The stool specimen will be used to determine differences in bacteria and metabolites between children with and without abdominal pain. Behavioral traits will also be compared to the results of the bacteria and metabolites analyses.

In order to participate, your child must not have taken antibiotics, antifungals, or antivirals by mouth (oral) or given in the vein in the previous 3 months prior to enrollment. In this document, the word “you” refers to "your child or you (the parent)”.This research study is funded by Autism SpeaksPurposeWe want to compare the types of bacteria found in children with frequent abdominal (stomach) pain to the types of bacteria found in children without frequent abdominal pain. We also will see if there is any relationship between the type of bacteria and how often a child has pain or a change in their bowel habits (diarrhea or constipation). We will compare these differences in children with and without autism as well as assess the relationship with changes in behavior.ProceduresThe research will be conducted at the following location(s): Baylor College of Medicine and TCH: Texas Children's Hospital.We are studying healthy children without stomach (abdominal) pain as well as children with functional abdominal pain. A research coordinator will contact you by phone or email at a time that is convenient to you and will schedule an initial visit that will take about 30 minutes. You and your child will be asked about your child’s general health history, how your child is feeling and about any stomach or intestinal tract problems or other diseases he or she may have had. The coordinator will ask you and your child what he or she ate for 24 hours show you how to keep a diet record on the day the stool is collected. In total, you will be asked for a 2 week diet history. The coordinators will explain how to collect stool samples and cheek swab samples (one of each will be collected for the study). Stool will be collected at home during typical bathroom trips using the materials provided by the research coordinator, and they will be kept on ice or

in the freezer until transported to Texas Children’s Hospital. Cheek swabs will be collected by a gentle brushing of the inside of the cheek, and the swab will then be stored on ice or in the refrigerator until transport to Texas Children's Hospital. The stool collection will look for certain bacteria and metabolites (small biological molecules) that occur naturally in their intestines. You and/or your child will also receive instructions on how to complete a two-week diary to record any abdominal (stomach) pain and information about their stooling habits. The total length of the study will be 2 weeks.

All children and/or parents will also complete several surveys and assessments to collect information on behavior that might be related to abdominal pain. These surveys should take approximately 45 minutes in total to complete and can be completed at home during the 2 weeks of the study. The research coordinator will be available for any questions you may have. All children will be screened using the Social Responsiveness Scale-2 (SRS-2), and should a child with no history of autism diagnosis receive a score suggesting a possible diagnosis of autism, the research coordinators and study investigators will refer the family for a comprehensive evaluation through the Texas Children’s Autism Center. If an ADOS (Autism Diagnostic Observation Schedule) was not performed previously or was deemed not valid in a child with a diagnosis of autism, then the Autism Center will confirm the diagnosis by performing an ADOS evaluation.

If you participate, you voluntarily agree to have Dr. Luna or her designee access your child's pediatric medical records once during the course of the study to obtain any information related to autism or abdominal pain. All information will be kept anonymous and recorded by study number only with no identifiable information. In addition, every effort will be made to protect your privacy and keep all data confidential, including keeping all samples and information generated in secure places like locked offices and strictly secured servers at Baylor College of Medicine.

Samples will be analyzed at Baylor College of Medicine and Texas Children's Hospital, and some samples may be analyzed by our collaborators at an outside facility/laboratory, specifically stool specimens may be sent to Metabolon for more in-depth metabolite (small biological molecule) testing. The entire sample may not be used during the study, and remaining sample material will be kept and stored securely for an indefinite amount of time. If you choose to withdraw or are withdrawn from the study, your samples and any associated data will be discarded. Samples will not be sold to any third party.

You may qualify for additional studies based on the outcome of this study or future planned studies. If you participate in this study, you voluntarily agree to be contacted if you qualify for these additional studies, and your participation is your choice.Research Related Health InformationAuthorization to Use or Disclose (Release) Health Information that Identifies You for a Research Study

If you sign this document, you give permission to people who give medical care and

ensure quality from Baylor College of Medicine and TCH: Texas Children's Hospital to use or disclose (release) your health information that identifies you for the research study described in this document.

The health information that we may use or disclose (release) for this research includes:

Information from health records such as diagnoses, progress notes, medications, lab or radiology findings, etc.

Demographic information (name, D.O.B., age, gender, race, etc.)

The health information listed above may be used by and or disclosed (released) to researchers, their staff and their collaborators on this research project, the Institutional Review Board, Baylor College of Medicine, TCH: Texas Children's Hospital, and AUTISM SPEAKS and their representatives.

Use or Disclosure Required by Law

Your health information will be used or disclosed when required by law.

Your health information may be shared with a public health authority that is authorized by law to collect or receive such information for the purpose of preventing or controlling disease, injury, or disability and conducting public health surveillance, investigations or interventions.

Baylor College of Medicine and TCH: Texas Children's Hospital are required by law to protect your health information. By signing this document, you authorize Baylor College of Medicine and TCH: Texas Children's Hospital to use and/or disclose (release) your health information for this research. Those persons who receive your health information may not be required by Federal privacy laws (such as the Privacy rule) to protect it and may share your information with others without your permission, if permitted by laws governing them.

Please note that the research does not involve treatment. Baylor College of Medicine and TCH: Texas Children's Hospital may not condition (withhold or refuse) treating you on whether you sign this Authorization.

Please note that you may change your mind and revoke (take back) this Authorization at any time. Even if you revoke this Authorization, researchers, their staff and their collaborators on this research project, the Institutional Review Board, AUTISM SPEAKS and their representatives, regulatory agencies such as the U.S. Department of Health and Human Services, Baylor College of Medicine, and TCH: Texas Children's Hospital may still use or disclose health information they already have obtained about you as necessary to maintain the integrity or reliability of the current research. If you revoke this Authorization, you may no longer be allowed to participate in the research

described in this Authorization.

To revoke this Authorization, you must write to: Ruth Ann Luna Feigin Center 830 Texas Children's Hospital 1102 Bates Avenue Houston, TX 77030

This authorization does not have an expiration date. If all information that does or can identify you is removed from your health information, the remaining information will no longer be subject to this authorization and may be used or disclosed for other purposes.

No publication or public presentation about the research described above will reveal your identity without another authorization from you.Potential Risks and DiscomfortsThere are no known risks to the stool collection, cheek swab collection, questionnaires, or diary.There is the potential for loss of confidentiality during the study, but this risk is minimal and every effort will be made to maintain confidentiality including giving all specimens, surveys, and diaries unique and anonymous codes and keeping all data in locked offices and on highly secure computers.Study staff will update you in a timely way on any new information that may affect your decision to stay in the study. There is a small risk for the loss of confidentiality. However, the study personnel will make every effort to minimize these risks.Potential BenefitsYou will receive no direct benefit from your participation in this study. However, your participation may help the investigators better understand the human intestinal microbiome (bacteria in our intestines) in children and how it relates to abdominal pain in children with and without autism..AlternativesYou may choose to not participate in this study.Investigator Withdrawal of Subject from a StudyThe investigator or sponsor may decide to stop you from taking part in this study at any time. You could be removed from the study for reasons related only to you (for example, if you move to another city or if you do not keep the diaries as requested) or because the entire study is stopped. The investigator or Institutional Review Board may stop the study at any time.Subject Costs and PaymentsYou will not be asked to pay any costs related to this research.At the completion of the study, subjects will be compensated $50.Subject's RightsYour signature on this consent form means that you have received the information about this study and that you agree to volunteer for this research study.

You will be given a copy of this signed form to keep. You are not giving up any of your

rights by signing this form. Even after you have signed this form, you may change your mind at any time. Please contact the study staff if you decide to stop taking part in this study.

If you choose not to take part in the research or if you decide to stop taking part later, your benefits and services will stay the same as before this study was discussed with you. You will not lose these benefits, services, or rights.

The investigator, RUTH ANN LUNA, and/or someone he/she appoints in his/her place will try to answer all of your questions. If you have questions or concerns at any time, or if you need to report an injury related to the research, you may speak with a member of the study staff: Ruth Ann Luna, Ph.D. (832-824-1894) or Research Coordinator, Michelle Rubio-Gonzales (832-824-3342)

Members of the Institutional Review Board for Baylor College of Medicine and Affiliated Hospitals (IRB) can also answer your questions and concerns about your rights as a research subject. The IRB office number is (713) 798-6970. Call the IRB office if you would like to speak to a person independent of the investigator and research staff for complaints about the research, if you cannot reach the research staff, or if you wish to talk to someone other than the research staff.If your child is the one invited to take part in this study you are signing to give your permission. Each child may agree to take part in a study at his or her own level of understanding. When you sign this you also note that your child understands and agrees to take part in this study according to his or her understanding.

Please print your child's name here __________________________