NIH Human Microbiome Project - Core Microbiome Sampling Protocol A (HMP-A)

The collections of microbes that reside in the human body form complex communities and are found primarily in the oral cavity, on the skin, in the nasal cavity, in the gastrointestinal tract, and in the vagina (Paustian 2006; Tierno 2001). These microbial communities are intimately integrated with the human “self” and may lead to a new understanding of “self”. They vary with many factors, such as age, gender, environment, diet and disease state. How these variations are accomplished and the impact of these variations on human development, physiology, immunity, and nutrition is not understood. To pursue this understanding by taking advantage of recent technological advances, the NIH Roadmap has initiated the Human Microbiome Project (HMP), with the mission of generating resources to enable comprehensive characterization of the human microbiota and analysis of their role in human health and disease.

Traditional microbiology has focused on the study of individual species as isolated units. However many, if not most, have never successfully been isolated as viable specimens for analysis, presumably because their growth is dependent upon a specific microenvironment that has not, or cannot, be reproduced experimentally. Among those species that have been isolated, analyses of genetic makeup, gene expression patterns, and metabolic physiologies have rarely extended to inter-species interactions or microbe-host interactions. Advances in DNA sequencing technologies have created a new field of research, called metagenomics, allowing comprehensive examination of microbial communities, even those comprised of uncultivable organisms. Instead of examining the genome of an individual bacterial strain that has been grown in a laboratory, the metagenomic approach allows analysis of genetic material derived from complete microbial communities harvested from natural environments. In the HMP, this method will complement genetic analyses of known isolated strains, providing unprecedented information about the complexity of human microbial communities.

By leveraging both the metagenomic and traditional approaches to genomic DNA sequencing, the HMP will lay the foundation for further studies of human-associated microbial communities. Broadly, the project has set the following goals:

• Determining whether individuals share a core human microbiome
• Understanding whether changes in the human microbiome can be correlated with changes in human health
• Developing the new technological and bioinformatic tools needed to support these goals
• Addressing the ethical, legal and social implications raised by human microbiome research.

This first clinical study addresses the first HMP goal of determining whether individuals share a core human microbiome.

This study involves broad determination of the microbiota found in five anatomical sites: the oral cavity, skin, nasal cavity, gastrointestinal tract and vagina. This study will enroll healthy adults from two geographic regions of the US. The participation of healthy individuals will create a baseline for discovery of the core microbiota typically found in various areas of the human body. The information from this initial study can then be used to help assess the changes in the complement of microbiota found on or within diseased individuals. The specimens will be collected from approximately equal numbers of males and females. All subjects will be between the ages of 18 and 40. The age range of subjects has been established to minimize the influence of growth and developmental changes as well as those changes associated with the aging process.

The target for this multi-center study is approximately 300 healthy subjects between the ages of 18 and 40. Approximately half of the subjects will be female and approximately half will be male. A goal for minority participation will be at least 20% of the total number of subjects.

At Baylor College of Medicine (BCM), subjects will be recruited through the Vaccine Research Center under the direction of Wendy Keitel, MD. Dr. Keitel will serve as Clinical Principal Investigator for the study and will supervise the nursing team trained in clinical assessment (histories/physicals) and human sampling. Healthy adults will be recruited from the Texas Medical Center (TMC) and environs. The TMC represents over 42 member institutions, which includes 13 hospitals and 2 specialized patient facilities. Over 65,300 people were employed in the TMC in 2004. Eleven educational institutions are located in the TMC or are affiliated with the TMC; these enroll over 22,000 students. College students at Rice University (enrollment~5,000), the University of Houston (enrollment = 35,180), and other educational institutions also can be recruited for participation. Healthy adults also can be recruited from surrounding industrial populations such as oil companies, as has been done in the past. In addition, the BCM Vaccine Research Center maintains a registry of people who are interested in being contacted regarding upcoming studies, and the Office of Research at BCM maintains a Volunteer Line and registry that lists people who have consented to be contacted by an investigator from BCM for possible enrollment in an IRB approved protocol. Potential subjects will be recruited by posting fliers around the TMC, by sending email announcements via approved listserv distribution lists, and by contacting individuals who have given permission via email or telephone.

Facilities at Baylor College of Medicine will enable all sampling procedures to occur at a single site. Oral assessment and sampling will be performed with the assistance of Catherine Flaitz, DDS, and her team of dental health care professionals including dental hygienists from the University of Texas Health Sciences Center – Dental Branch. Vaginal sampling will be performed with the assistance of Kjersti Aagard, MD, PhD, Amy Young, MD and the team of health care professionals from the Department of Obstetrics and Gynecology at Baylor College of Medicine. Joseph Petrosino, PhD will serve as co-investigator responsible for the DNA extraction laboratory. James Versalovic, MD, PhD will serve as Coordinating Investigator and Study Chair and will supervise a team of laboratory professionals from Baylor College of Medicine involved in specimen collection, processing, storage, and distribution.

At Washington University, Dr. Mark Watson is a member of the research faculty and as such, has access to the services offered by the Clinical Trials Unit. The Clinical Trials Unit will primarily recruit subjects through the Volunteer for Health (VFH) program. The Volunteer for Health program is sponsored by Washington University School of Medicine to support patient-oriented research at Washington University Medical Center. The overall purpose of VFH is to match interested volunteers with current clinical studies at the medical school. Currently, VFH has a database of 25,000+ prospective volunteers (persons who provided VFH with general health information and who gave their consent to be contacted about studies) that can be searched based on the initial inclusion/exclusion study criteria. Additionally they will place IRB-approved fliers, posters and advertisements in appropriate locations and media outlets in order to reach out to potential participants. Working with the VFH program will greatly facilitate tracking of subjects for resampling during this study and for participation in future studies related to the human microbiome.

Dr. Watson will serve as the Clinical Principal Investigator for the study and will oversee the collection of samples and generation of secondary resources. Dr. Watson is also Director, Siteman Cancer Center Tissue Procurement Core Facility. This entity will participate in purifying microbial nucleic acids as well as in storing and distributing the microbial nucleic acid samples. In this way, Dr. Watson will have complete oversight of the entire sample collection process. Sally Anderson, who serves as the Director of Clinical Research Services, will directly supervise the clinical nursing team collecting the samples. Dr. Mark Watson and Dr. Michael Dunne are leading a group of WU investigators who will provide ongoing support and oversight of this study. In addition to these professionals, Nathalia Garcia, DDS, will collect the oral samples and Tessa Madden, MD, will collect the vaginal samples.

A pre-screening questionnaire will be utilized prior to scheduling the Screening Visit in order to screen out potential ineligible subjects by history.

The Office for Human Research Protections (OHRP), Department of Health and Human Services (DHHS) considers unanticipated problems, in general, to include any incident, experience, or outcome that meets all of the following criteria:

• unexpected (in terms of nature, severity, or frequency) given (a) the research procedures that are described in the protocol-related documents, such as the IRB-approved research protocol and informed consent document; and (b) the characteristics of the subject population being studied;
• related or possibly related to participation in the research (possibly related means there is a reasonable possibility that the incident, experience, or outcome may have been caused by the procedures involved in the research); and
• suggests that the research places subjects or others at a greater risk of harm (including physical, psychological, economic, or social harm) than was previously known or recognized.

An incident, experience, or outcome that meets the three criteria above generally will warrant consideration of substantive changes in the research protocol or informed consent process/document or other corrective actions in order to protect the safety, welfare, or rights of subjects or others. Examples of corrective actions or substantive changes that might need to be considered in response to an unanticipated problem include:

• changes to the research protocol initiated by the investigator prior to obtaining IRB approval to eliminate apparent immediate hazards to subjects;
• modification of inclusion or exclusion criteria to mitigate the newly identified risks;
• implementation of additional procedures for monitoring subjects;
• suspension of enrollment of new subjects;
• suspension of research procedures in currently enrolled subjects;
• modification of informed consent documents to include a description of newly recognized risks; and
• provision of additional information about newly recognized risks to previously enrolled subjects.

Institutions engaged in human subjects research conducted or supported by DHHS must have written procedures for ensuring prompt reporting to the IRB, appropriate institutional officials, and any supporting department or agency head of any unanticipated problem involving risks to subjects or others (45 CFR 46.103(b)(5)). Furthermore, for research covered by an assurance approved for federalwide use by OHRP, DHHS regulations at 45 CFR 46.103(a) require that institutions promptly report any unanticipated problems to OHRP.

Incidents or events that meet the OHRP criteria for unanticipated problems require the completion of an unanticipated problem report form. OHRP recommends that investigators include the following information when reporting an adverse event, or any other incident, experience, or outcome as an unanticipated problem to the IRB:

• appropriate identifying information for the research protocol, such as the title, investigator’s name, and the IRB project number;
• a detailed description of the adverse event, incident, experience, or outcome;
• an explanation of the basis for determining that the adverse event, incident, experience, or outcome represents an unanticipated problem;
• a description of any changes to the protocol or other corrective actions that have been taken or are proposed in response to the unanticipated problem.

For multicenter research protocols, if a local investigator at one institution engaged in the research independently proposes changes to the protocol or informed consent document in response to an unanticipated problem, the investigator should consult with the study sponsor or coordinating center regarding the proposed changes because changes at one site could have significant implications for the entire research study.

Forms describing unanticipated problems will be submitted to the CDCC and the IRB. The CDCC will notify the NIH study representative of the unanticipated problem. Unanticipated problems will be reported immediately to the NIH Clinical Studies Oversight Committee (CSOC). The CSOC may convene an ad hoc meeting for review of the unanticipated problem and consideration of corrective actions. Other supporting documentation of the problem may be requested by the CSOC and should be provided as soon as possible. A summary of the CSOC meeting and proposed actions will be provided to the study Steering Committee. The study PIs will then submit the CSOC review and any protocol changes to the IRBs.

Investigators will be responsible for reporting unanticipated problems from the time that the first subject is enrolled until one year after the final subject is enrolled.

The OHRP, DHHS defines an Adverse Event (AE) as any untoward or unfavorable medical occurrence in a human subject, including any abnormal sign (for example, abnormal physical exam or laboratory finding), symptom, or disease, temporally associated with the subject’s participation in the research, whether or not considered related to the subject’s participation in the research (modified from the definition of adverse events in the 1996 International Conference on Harmonisation E6 Guidelines for Good Clinical Practice). A Serious Adverse Event/Experience (SAE) is any adverse event/experience that meets any of the following criteria:

• Results in death
• Is life-threatening (places the subject at immediate risk of death from the event as it occurred)
• Requires inpatient hospitalization or prolongation of existing hospitalization
• Results in a persistent or significant disability or incapacity
• Results in congenital anomaly/birth defect
• Any other adverse event that, based upon appropriate medical judgment, may jeopardize the subject and may require medical or surgical intervention to prevent one of the other outcomes listed in this definition.

For those events meeting the definition of Serious Adverse Events, the completion of an SAE form is required.

OHRP considers adverse events that are unexpected, related or possibly related to participation in research, and serious to be the most important subset of adverse events representing unanticipated problems, because such events always suggest that the research places subjects or others at a greater risk of physical or psychological harm than was previously known or recognized, and routinely warrant consideration of substantive changes in the research protocol or informed consent process/document or other corrective actions in order to protect the safety, welfare, or rights of subjects. Furthermore, OHRP notes that IRBs have authority to suspend or terminate approval of research that, among other things, has been associated with unexpected serious harm to subjects (45 CFR 46.113). In order for IRBs to exercise this important authority in a timely manner, they must be informed promptly of those adverse events that are unexpected, related or possibly related to participation in the research, and serious (45 CFR 46.103(b)(5)).

All serious adverse events will be:

• recorded on the appropriate serious adverse event case report form;
• followed until satisfactory resolution or until the Principal Investigator or Subinvestigator deems the event to be chronic or the patient to be stable;
• reported to the IRB and to the CDCC;
• entered in the CDCC data management system.

Any adverse event considered serious by the Principal Investigator or Sub-investigator or which meets the aforementioned criteria must be submitted on an SAE form to the CDCC. Investigators will be responsible for reporting SAEs that occur during sampling or within 48 hours of a study visit.

The study clinician will complete a Serious Adverse Event Form within the following timelines:

• All deaths and immediately life-threatening events, whether related or unrelated, will be recorded on the Serious Adverse Event Form and sent by fax within 24 hours of site awareness.
• Serious adverse events other than death and immediately life-threatening events, regardless of relationship, will be reported via fax within 72 hours of site awareness.

The CDCC will notify the NIH study representative of the serious adverse event within one business day. SAEs that are related to study participation will be reported immediately to the NIH CSOC. The CSOC may convene an ad hoc meeting for review of the SAE and consideration of corrective actions. Other supporting documentation of the event may be requested by the CSOC and should be provided as soon as possible. A summary of the CSOC meeting and proposed actions will be provided to the study Steering Committee. The study PIs will then submit the CSOC review and any protocol changes to the IRBs.

Serious adverse events that are considered unrelated to study participation will be provided to the CSOC in a line listing to be reviewed at regularly scheduled meetings.

This study aims to discover the microbial diversity found within specimens collected from different individuals, but from the same regions of the body. It will also contribute to knowledge about microbial diversity within individuals over time. The amount of microbial diversity will be measured by the identification of said microbes found in the nucleic acids isolated from the body site specimens. It should be noted, however, that it may not be possible in all cases to identify all microbes contributing to the diversity of the subject.

The study is exploratory in nature and the number of samples planned for it is pragmatically derived. Originally, the study aimed to enroll approximately 250 subjects. Based on preliminary analysis of data, the total enrollment goal was increased to 300 subjects, to provide more robust data on the composition of the microbiome at multiple body sites. Preliminary database searches at the collection sites found a significant number of candidate subjects available for this study.

This study will consent and enroll approximately 300 subjects. Subjects who are sampled at two visits could be enrolled in the study for up to 1 year. Approximately 100 subjects (50 at each site) who are sampled a third time could be enrolled in the study for up to 18 months. It is anticipated that subject retention will rely on the interest of the subjects in the study as well as on reimbursement for their time and effort.

Analysis of microbial sequence data will be conducted by the study chair and others as needed. These efforts will include analysis of 16S rRNA sampling data used to define the microbial “species” isolated in each specimen. Once identified, these sequences can be used to assess the diversity found both within specimens isolated from the same individual and across specimens isolated from the same body regions from multiple individuals. All sequence data collected will be made available for analysis. Spurious data will be isolated and removed as DNA sequence assessment tools allow for identification of said sequences. Missing data are not significant because the sample size was pragmatic. As such, any sequence information gained from any of the specimens will be found to contribute to the overall understanding of the microbial diversity found in the human population. Genotyping or sequencing of human DNA isolated from the blood of subjects who provide clinical specimens may be used to examine the relationship of host genotype to the microbiota present on/in an individual.

This study will be conducted according to the principles of respect for persons, beneficence, and justice as stated in the Belmont Report. This study will also embrace the principles set forth in the Declaration of Helsinki. Investigators will comply with the provisions for the protection of the rights and welfare of human research subjects set forth in the U.S. Code of Federal Regulations, Title 45 Part 46, and in compliance with determinations of all Institutional Review Boards (IRBs) overseeing the research. All institutions participating in the protocol will have in place a Federal Wide Assurance (FWA) with the DHHS Office of Human Subjects Research Protections. The assurance documents the institution’s commitment to the human subjects regulations.

The study will also be conducted under Good Clinical Practice (GCP) as laid out in the International Conference on Harmonisation (ICH) E6 GCP Consolidated Guidance (ICH 1996). Investigator responsibilities are set out in Section 4 of the E6 Guideline (as published in the Federal Register May 1997). Sponsor responsibilities are set out in Section 5 of the E6 ICH Guideline (as published in the Federal Register May 1997).

Only those IRB members who are independent of the investigator should provide opinion on a study-related matter.

This protocol (unaltered and in its entirety) and any other documents that the IRB may need to fulfill its responsibility, including subject-recruitment procedures, and information about payment and compensation available to subjects, will be submitted to an appropriate IRB by the investigator or designate, and their written unconditional approval should be in the possession of the investigator and the sponsor before commencement of the study.

No deviations from, or changes to, the protocol should be initiated without prior written IRB approval/favorable opinion of an appropriate amendment, except when necessary to eliminate immediate hazards to the subjects or when the change(s) involves only logistical or administrative aspects of the study. Protocol administrative changes are submitted to the IRB for information only. However, written verification that a memo describing the protocol administrative change was submitted should be obtained.

The IRB must be informed by the investigator (or designate) of the following:

• all subsequent protocol amendments, informed consent changes or revisions of other documents originally submitted for review - requires IRB approval;
• all subsequent protocol administrative changes - for information;
• new information that may affect adversely the safety of subjects or the conduct of the study;
• an annual update and/or request for re-approval - Continuing Review - requires IRB approval;
• when the study has been completed.

Each institution will use a separate version of the study informed consent, developed from a template provided by the NIH. All subjects screened must document informed consent by signing and dating the study consent form. Those subjects found ineligible by the screening process for enrollment will not be scheduled for the baseline specimen collection visit. Subjects who agree to be sampled a third time will sign an informed consent document that includes information about the third specimen collection. Subjects may be contacted in the future and be invited to participate in future sampling studies.

The written consent document embodies the elements of informed consent as described in the Declaration of Helsinki and in the U.S. Code of Federal Regulations, Title 45 Part 46, and will adhere to the ICH Harmonized Tripartite guidelines for Good Clinical Practice. Informed consent should be implemented before any protocol-specific procedures are carried out. Information will be presented orally and in written form.

An investigator or designate will describe the protocol to potential subjects face to face. The Subject Information and Consent Form may be read to the subjects, but, in any event, the investigator will give the subjects ample opportunity to inquire about details of the study and ask any questions before dating and signing the consent form. All illiterate individuals will have the Subject Information and Consent Form explained to them point by point by the interviewer in the presence of an impartial witness. The witness should personally sign and date the consent form.

Subjects must be informed that the study involves research, and must be given information about the purpose of the study, expected benefits, possible risks, the expected duration of the subject’s participation in the study, the subject’s responsibilities, the approximate number of subjects involved in the study, and the procedures associated with the research study. Subjects will be informed that they will be notified in a timely manner if information becomes available that may be relevant to their willingness to continue participation in the study. Subjects must receive an explanation as to whether compensation and any medical treatments are available if injury occurs and, if so, what that consists of, or whether further information may be obtained. Subjects must be informed of the anticipated financial expenses, if any, to the subject for participating in the study. They must be informed whom to contact for answers to any questions relating to the research project (e.g., the investigator), relating to the research subject’s rights, and relating to a research-related injury (e.g., the IRB, an ombudsman, an ethics committee, or other informed administrative body). The subjects must be informed that participation is voluntary and that they are free to withdraw from the study for any reason at any time, without penalty or loss of benefits to which the subject is otherwise entitled. Neither the investigator nor the study staff should coerce or unduly influence a subject to participate or continue to participate in the study. The extent of confidentiality of subject records must be defined, and subjects must be informed that applicable data protection legislation will be complied with. Subjects must be informed that the monitor(s), auditor(s), IRB, NIH, and the regulatory authorities will be granted direct access to the subject’s original medical records for verification of clinical study procedures and/or data, without violating the confidentiality of that subject, to the extent permitted by the applicable laws and/or regulations, and that by signing a written consent form, the subject is authorizing such access. Subjects must be informed that records identifying the subject will be kept confidential, and to the extent permitted by the applicable laws and/or regulations, will not be made publicly available and, if the results of the study are published, the subject’s identity will remain confidential.

The consent forms will be formatted according to Institutional requirements using a model provided by NIH. Consent forms will be in a language fully comprehensible to the prospective subjects. Informed consent shall be documented by the use of a written consent form approved by the IRB and signed and dated by the subject, and by the person who conducted the informed consent discussion. The signature confirms that the consent is based on information that has been provided to the subject and that there has been opportunity to clarify this information. Each subject’s signed consent form must be kept on file by the investigator for possible inspection by the Regulatory Authorities and/or NIH. The subject should receive a copy of the signed and dated written consent form and any other written information provided to the subjects, and should receive copies of any signed and dated consent form updates and any amendments to the written information provided to subjects.

Children under 18 are excluded from this study because an adult representative population is being sought in order to establish a baseline representative catalogue of the microbiome.

Protected Health Information (PHI) is health information that identifies a subject. PHI is protected by federal law under the Health Insurance Portability and Accountability Act (HIPAA). To take part in this research, subjects must give the research team permission to use and disclose (share) their PHI for the study explained in the consent form for this protocol.

A separate Certificate of Confidentiality (CoC) from the US Department of Health and Human Services will be sought by each Institution recruiting, consenting and sampling subjects. This will help to further protect information that may identify subjects. The CoC prevents the investigator from being forced to disclose identifying information for use in court. The investigator may not even be forced by court subpoena. Courts that may be prevented from getting subjects’ information include any federal, state, local, civil, criminal, administrative, legislative, or other court proceeding. Subjects will be informed that a CoC does not prevent them or a member of their family from voluntarily releasing information about themselves or their involvement in this research. The investigator may not withhold information if subjects give their insurer or employer permission to receive information about their participation in this research. This means that they and their family must also actively protect the subjects’ privacy. The CoC does not prevent the researchers from taking steps, including reporting to authorities, to prevent serious harm to the subject or others.

The consent form documents that the research team may share a subject’s information with:

• The Department of Health and Human Services (DHHS) to complete federal responsibilities for audit or evaluation of this study;
• Public health agencies to complete public health reporting requirements;
• Hospital or University representatives, to complete Hospital or University responsibilities for oversight of this study;
• Primary care physician if a medical condition that needs urgent attention is discovered;
• Appropriate authorities to the extent necessary to prevent serious harm to the subject or others.

Subject confidentiality is strictly held in trust by the participating investigators, their staff, and the sponsor(s) and their agents. The microbial sequence data will be placed in an open access database available on the internet, but reasonable efforts will be made to remove all traces of human sequence data before these data are posted, so it will be virtually impossible for anyone to identify a subject from looking at these data. The subjects’ medical data, and any data generated from the sequencing of the subjects’ blood specimens, will be placed in a controlled access database available on the internet. An NIH Data Access Committee will control access to these data, to ensure that the data are made available to qualified researchers, consistent with the terms of the informed consent. No information concerning the study or the data will be released to any unauthorized third party without prior written approval of the sponsor. The study monitor or other authorized representatives of the sponsor may inspect all documents and records required to be maintained by the Investigator, including but not limited to, medical records (office, clinic or hospital) and pharmacy records for the subjects in this study. The clinical study site will permit access to such records.

It is anticipated that the original clinical specimens collected from the oral cavity, skin, nasal cavity, and vagina will be completely consumed in the DNA extraction process. It is anticipated that the original stool specimen will be in excess of the amount needed for DNA extraction. The primary focus of this study is extraction and sequencing of microbial DNA. If the volume of clinical specimen allows, it is possible that RNA extraction may be undertaken from the same specimens for RNA sequencing and gene expression studies. Any body site specimens remaining after completion of nucleic acid extraction and sequencing will be destroyed. Specimens will be stored temporarily to enable sufficient quality assessment (amount, purity) of extracted nucleic acid prior to sequencing. Nucleic acid may be re-extracted from the same clinical specimen(s) if sufficient material remains intact after the initial extraction. Any residual microbial DNA or RNA remaining after sequencing will be stored at the clinical research sites. Coded human DNA and (possibly) lymphoblastoid cell lines from the blood specimens will be maintained after the study is completed. Coded serum specimens will also be maintained after study completion.

The human DNA, the lymphoblastoid cell lines, the serum, and the microbial nucleic acids obtained under this project may be used for future approved studies related to the human microbiome. The repositories will require all researchers who apply to use the human DNA, the lymphoblastoid cell lines and the microbial nucleic acids to submit a written description of the research, which will be reviewed to make sure that the researcher requesting the specimens plans to use them in a way that is consistent with the subject consent (i.e., related to human microbiome research). It is anticipated that researchers from universities, hospitals, non-profit groups, companies, and government research laboratories in the United States and in other countries will study the specimens and the information from them.

Coded serum specimens will be stored in repositories at the clinical research sites and may be distributed to other researchers for future studies related to the human microbiome, and with the permission of the subject, to other researchers for future studies not necessarily related to the human microbiome. The clinical site repositories will require all researchers who apply to use the serum specimens to submit a written description of the research intent and obtain approval for use of the serum.

If a subject chooses to withdraw consent for future use of stored specimens, specimens will be destroyed and every attempt will be made to exclude data from that subject in future releases of DNA sequence data. However, if data have been released to researchers prior to the subject’s withdrawal of consent, data cannot be retracted.

Precautions are in place to ensure accurate record keeping procedures and to maintain subject confidentiality. Following clinical specimens collection, all of the specimens and information will be processed according to the procedures in the Manual of Procedures.

The responsibilities of data management will be handled by the clinical site, the study CDCC, and, for the blood specimens, the Coriell Institute. The specific responsibilities of the clinical site include accurate collection and storage of all information pertaining to the specimens obtained for this project. The clinical site will code each specimen and maintain this information in a secure location, as described in the Manual of Procedures.

All source documents and laboratory reports will be reviewed by the clinical team and data entry staff, who will ensure that they are accurate and complete. Serious adverse events must be assessed for causality and reviewed by the site Principal Investigator or designee. Data collection will be the responsibility of the study staff at the site under the supervision of the site Principal Investigator. During the study, the Investigator will maintain complete and accurate documentation for the study.

The laboratories isolating nucleic acids will have standard lab practices for record keeping and take steps to make sure specimen integrity is maintained. The laboratories will also maintain and distribute specimen specific metrics used for specimen evaluation.

Responsibilities for the CDCC include cumulative record keeping of all information associated with the project including coded subject information collected at the time of consent, specimen specific metrics, DNA sequence information and the resulting analysis information. At the end of the study, a copy of all datasets will be provided to the NIH. The CDCC will send a copy of all datasets to the NIH electronically.

Only coded information will be transferred between the entities involved in this study. This coded information will be shared using a secure file transfer protocol. Only the study investigators at the clinical sites will have access to the study identification code list.

Data capture methods used for this study include the following: paper copies that will be kept centrally and processed on an ongoing basis, electronic spreadsheets that will be distributed and processed on an ongoing basis, and databases that will be kept centrally as well as distributed and updated on an ongoing basis.

Clinical data (including SAEs and concomitant medications) will be entered into a 21 CFR Part 11-compliant internet data entry system (IDES) provided by The EMMES Corporation. The data system includes password protection and internal quality checks, such as automatic range checks, to identify data that appear inconsistent, incomplete, or inaccurate. Clinical data will be entered directly from the source documents.

The following types of coded data will be collected:

• Subject’s health history;
• Specimen specific metrics such as DNA quantity and quality;
• DNA sequence from each specimen.

As described in the Manual of Procedures, subject ID codes will be assigned to subjects sequentially as they are screened. The Subject ID will be entered on all specimens and source documents and will be used to identify all subject data records.

A Steering Committee (see Manual of Procedures) will hold regular discussions via conference calls on the overall strategy and progress of the project. The information discussed on these calls will be used to generate reports used to document the overall progress of the study. The Steering Committee will review the progress of specimen collection, DNA isolation, and analysis of the sequence data.

Sequencing metrics will also be reported to NHGRI on a quarterly basis. Information such as the number of subjects, sequence pass rates and total bases of sequencing information will be included in these documents. Information from these reports will also be provided to the Steering Committee for their review.

The collection sites are responsible for preparing clinical study reports and providing them to the appropriate regulatory authorities, according to the applicable regulatory requirements.

The Clinical Principal Investigator at each site must maintain adequate and accurate records as specified in Essential Documents for the Conduct of a Clinical Trial (Section 8 of the ICH E6 Guideline) to enable the conduct of the study to be fully documented and the study data to be subsequently verified. These documents should be classified into two separate categories: (1) Investigator's study file and (2) Subject clinical source documents. Essential documents should be retained at least seven years after the grant is officially closed or until the NIH authorizes transfer or destruction of study records. No study records will be destroyed without prior authorization from the NIH.

Repeat specimen collection will be performed if additional nucleic acid extraction is necessary and the clinical specimen(s) is exhausted. Additional nucleic acid extraction might be necessary because of compromised clinical specimens (inappropriate storage at home, in the lab, etc.), the presence of amplification/sequencing inhibitors in particular specimens, insufficient nucleic acid yields or DNA/RNA of poor quality. Technical errors during nucleic acid extraction may also result in yields of low amount or quality.

If DNA amount and purity are questionable, as detected by quality assessment procedures in the clinical laboratories, and the source clinical specimen is exhausted, the clinical staff at the site will be notified as soon as possible. In this situation, one of two options may apply: 1) the same subject could be resampled, or 2) new subjects could be screened and enrolled in the study. If subjects are contacted for repeat body site sampling, the specimen collection will be targeted so that only the body sites that yielded inadequate nucleic acid preparations will be re-sampled.

Additional individuals may need to be recruited if enrolled subjects that are re-contacted are no longer available or refuse to provide additional specimens. Subjects may be unavailable for different reasons including health reasons (exclusion criteria) or geographic relocation. In these cases, new individuals will be screened and considered for enrollment in the study. These subjects would be newly consented for complete body sampling including the sites of interest that yielded inadequate nucleic acid preparations. The study Steering Committee, in consultation with the local IRB, will make final decisions on whether the total numbers of subjects and specimens need to be reconsidered due to specific issues with DNA collection or funding constraints.

A protocol deviation is any noncompliance with the clinical trial protocol, Good Clinical Practice (GCP), or Manual of Procedures requirements. The noncompliance may be on the part of the subject, the investigator, or the study site staff. As a result of deviations, corrective actions are to be developed by the site and implemented promptly.

These practices are consistent with Good Clinical Practice:

It is the responsibility of the site to use continuous vigilance to identify and report deviations within 5 working days of identification of the protocol deviation, or within 5 working days of the scheduled protocol-required activity. All deviations must be promptly reported to NIH, via the EMMES Corporation’s IDES.

All deviations from the protocol must be addressed in study source documents. A completed copy of the NIH Protocol Deviation (PD) Form must be maintained in the Regulatory File. Protocol deviations must be submitted to the local IRB/IEC per their guidelines. The site PI/study staff is responsible for knowing and adhering to their IRB requirements.