PROGRAM DESCRIPTION

Overview:
The AGRE research program is located in the Autism Speaks office in Los Angeles, CA. In collaboration with the AGRE Steering Committee, the AGRE clinical staff oversees protocols for phlebotomy, ADI-R and ADOS evaluations, data collection and storage, pedigree production and the distribution of quality controlled DNA, cell line and serum samples. Biomaterial production and storage is located at the Rutgers University Cell Repository (RUCDR) directed by Dr. Jay Tischfield and under contract to AGRE.

Scientific Oversight:
The AGRE Steering Committee is composed of advisors with backgrounds in genetics and neurological diseases who volunteer their time to provide oversight and direction for this program. The Steering Committee comes together yearly to review program progress and provide direction. Throughout the course of the year, the committee is routinely queried when issues arise or advice is needed. John Constantino, MD, Chair of the Steering Committee, is a Professor of Psychiatry and Pediatrics at Washington University School of Medicine in St. Louis. Dr. Constantino's research interests include disorders of social development in children and psychiatric complications of neurodevelopmental disorders. Dan Geschwind, M.D., Ph.D., a neurologist and neurogeneticist at UCLA, is the Chief Scientific Advisor of the AGRE program. The Steering Committee provides continual scientific oversight for the program. Three subcommittees are in place, the Family Assessment Committee, the Collaborations Committee and the Researcher Review Committee. The Family Assessment Committee reviews initial pedigrees and prioritizes them for blood collection. For example, families who have participated in other genetic studies or families with affected monozygotic twins will be given a lower priority. The Collaborations Committee helps the AGRE staff and management to choose the collaborations which are embarked upon with outside institutions. The Researcher Review Committee evaluates applications to use the resource.

Family Enrollment: (see Family Recruitment Flowchart)
Enrollment: AGRE strives to be as inclusive as possible in initially enrolling families affected by the autism spectrum disorders. Families with more than one member who has autism, PDD or Asperger’s syndrome may enroll in the AGRE program. Families are found through the CAN mailing list and web site, at conferences around the country and through posters placed in physicians’ offices and schools. Many AGRE recruiters are the parents of autistic children themselves. At initial contact, AGRE provides the family a packet of information on the program and a preliminary enrollment form. Families can access a Family Recruiter, using the AGRE 800 number, to answer any questions or assist them in completing the enrollment form. It is AGRE’s experience that when families learn that they can make a very real and meaningful contribution to genetic research in autism by participating in this program, and that this is an open resource that takes no proprietary interest in their DNA, they are eager to enroll.

On-line Questionnaires:
Online Questionnaire: Because there may be a substantial wait before a phlebotomist or psychometrician is able to visit the family in their home, AGRE participants are able to fill out some of the study questionnaires on-line prior to the initial home visit and during subsequent phases of the research project. This is made possible through AGRE’s online data acquisition system, referred to as the Online System for Clinical Research (OSCR). OSCR is updated periodically with new assessments and questionnaires for subjects to complete, allowing AGRE to continue data collection and add new questionnaires even after in-home data collection has been completed. OSCR also allows users to view summaries of how the user community as a whole has responded to specific questions, thus providing feedback to families and keeping them even more connected to the research project.

Family Diagnosis and Characterization:
Current Characterization: On the family enrollment form, families identify the diagnosis they received from their physician or specialist. Autism, PDD and Asperger's syndrome are accepted. However, before a family is scheduled for blood draw, AGRE requires that the family have an Autism Diagnostic Interview (ADI) conducted to confirm the diagnosis and characterize the family in relation to the autism spectrum disorders.

Currently, only the diagnosis of "autism" is backed by the empirically defined algorithms of the ADI. The catalogue makes use of the two alternate diagnosis of PDD and Aspergers as indicated by differential ADI ratings. These reflect accepted DSM-IV/ICD-10 criteria, but the authors of the ADI wish it to be made clear that these are not yet considered by all in the field to be empirically definable diagnosis.

AGRE uses trained ADI raters who travel to the family home to conduct the interview. All AGRE raters are trained under the supervision of Catherine Lord, Ph.D., at the University of Chicago. These raters are initially checked for reliability and are subsequently checked on a regular basis by an experienced ADI trainer. The family is required to sign a consent form that allows for the ADI interview and the transfer of data to the AGRE program. The interview, with parental permission, is audio taped. The ADI rater is periodically videotaped conducting the interview as a quality control performance check. Once the interview is completed, the ADI rater sends a hard copy of the interview, the audiotape and the videotape to the AGRE office for data entry and secured storage. Only families whose diagnosis is confirmed with autism spectrum disorder are considered for blood collection.

A full in-home clinical exam, The Autism Diagnostic Observation Scale (ADOS), and various other measures will be conducted by specially trained pediatric neurologists. These AGRE clinicians receive mentoring from various experts in the field of autism. Training on the ADOS is provided by certified ADOS trainers supervised by Catherine Lord, Ph.D., at the University of Michigan. All data collected from these visits is entered into the online AGRE phenotypic database.

AGRE Databases

AGRE Internal Demographic Database (AGRE ID):

When a potential family is identified at a conference, or through a mailing, they are assigned a unique identifier code and entered into the AGRE demographic database at the AGRE offices. The AGRE demographic database is the only location where identifying information is stored. A family pedigree is drawn and only the code numbers (no identifiers) accompany that pedigree assuring family confidentiality as data and biomaterials are shared or distributed.

AGRE Genotypic and Phenotypic Databases at www.agre.org

Genotypic Data-
Columbia University has conducted a genome scan on the AGRE family collection using the James Weber version 6 microsatellite marker collection with additional markers selected from the Marshfield Database to fill in gaps. This data, coded for confidentiality, is currently available on the website for download by AGRE approved researchers.

The genome scan has been extended to 345 AGRE families and additional
fine mapping markers have been analyzed in suggestive regions.

Additional candidate gene and candidate loci genotyping is available from researchers who have shared their raw data with AGRE, including microsatellite genotyping of GABRB3, chromosomes 2q, 5, and 15, and SNP genotyping of 7 candidate genes.

Finally, whole genome association data is available on both the Affymetrix 5.0 chips (751 families) and the Illumina Hap550 (943 families).

Phenotypic Data-ISAAC
A phenotypic database is currently available on the website for AGRE approved researchers. The ISAAC (Internet System for Assessing Autistic Children) database contains:

-Autism Diagnostic Interviews - Revised (ADI-R)

-Autism Diagnostic Observation Schedule (ADOS)

-Birth and Medical History

-Language Questionnaire

-Physical and Neurological Exam

-Parental Family and Medical History

-Peabody Picture Vocabulary Test (PPVT)

-Ravens Colored Progressive Matrices

-Repetitive Behavior Scale

-Social Responsiveness Scale

-Stanford Binet Intelligence Scales (SB-5)

-Vineland Adaptive Behavior Scales

All data is double entered and the entries compared for accuracy. This data is accessible to researchers approved by the AGRE Researcher Review Committee. Data may be accessed at: www.agre.org. Data is made available as it is validated. Therefore, phenotype data will often precede the availability of biomaterial samples.

Family Recruitment

Types of Families Recruited: Currently, AGRE is most interested in recruiting multiplex families where two or more siblings are affected by the autism spectrum disorder. Additionally, trios (two parents and an affected sibling) are being considered for recruitment through an exchange with other autism research groups. These families will be essential for further candidate gene studies, positional cloning and linkage disequilibrium studies. No family is actually entered into the collection and the repository until it meets the AGRE diagnostic criteria and is approved for recruitment by the Family Assessment Committee.

Sharing Researcher Collections: AGRE is establishing a sharing researcher program which allows other researchers in the field to contribute their multiplex, simplex or cousin family samples to the gene bank and be given an equal number of samples in exchange, free of cost.

Family Consent for Diagnosis and Blood Collection: AGRE consent forms have been developed with review and input from the AGRE Steering Committee. In the design of the AGRE consent form, AGRE has consulted with Bartha Knoppers, Ph.D., L.L.D., Chair of the Bioethics Committee of the Human Genome Project.

Autism research has particularly difficult consent issues because the subjects are often children, and the research and diagnostic issues require video recording. Families are given a multi-level consent allowing them to grant or withhold consent for each stage of diagnosis and blood collection. The consent form informs the family that the samples will be used by more than one research group studying neuropsychiatric research. Researchers have no direct access to these families but AGRE is able to recontact families as needed.

Institutional Review Board (IRB) Approval: AGRE has IRB approval from Western Institutional Review Board, Inc (www.wirb.com) and UCLA. AGRE also requires all approved users to obtain IRB approval through their own institutions.

Family Blood Collection
Once the diagnosis is confirmed and the Family Assessment Committee has prioritized the family for blood collection, AGRE schedules the family for a blood draw. AGRE arranges for a carefully screened phlebotomist, who is proficient in working with developmentally delayed children, to visit the family home and collect blood on all participating members. The phlebotomist is supplied with a custom-designed blood collection kit that includes all appropriate tubes, instructions for blood draw and consent forms. When the blood draw is complete, the phlebotomist ships the bloods to the Rutgers repository by overnight air courier.

Repository Protocol
We collect three (3) tubes of blood from each individual and ship them to the Rutgers University Cell and DNA Repository.

In the past, one tube was used to extract DNA from whole blood and two tubes were used to immortalize cel lines. DNA extracted from whole blood yields approximately 1 mg. When extracted from immortalized cell lines, the yield will be closer to 2 mg.

Currently, we do not extract any DNA from whole blood. Instead we use all 3 tubes to immortalize cell lines. Some of the blood is saved as a back-up in the event that the EBV transformation process fails.

Quality control testing of DNA is performed. This testing includes measurement of OD 260/280 ratio, DNA concentration, agarose gel electrophoresis and digestibility by two endonucleoses.

To establish immortalized cell lines, EBV transformation is performed on isolated lymphocytes. Frozen stock of immortalized cell lines are stored in liquid nitrogen. Microbiological and mycoplasma quality control screening is performed on these cell lines.

Previously, sera was collected for analysis. This has been discontinued. Serum was collected from centrifuged blood. The serum was aliquotted, frozen and stored for distribution.

The AGRE staff tracks the receipt of family samples at the repository. Biomaterial production is monitored by the AGRE staff. If cell line production is unsuccessful, the AGRE recruiting staff contacts the family to arrange for another blood draw. The percentage of rebleeds in this collection is approximately 5%.

Sample Distribution
AGRE has a subcommittee in place, the Researcher Review Committee, whose responsibility it is to review and approve researcher access applications. Once approved, a researcher is provided with a login and password which are used to access the website data and order biomaterials from AGRE. Orders for biomaterials taken by the AGRE office are forwarded to the Rutgers Cell Repository where samples are shipped to the researcher within a 2-4 week period.
All available data can be downloaded from the AGRE website by approved researchers. The login and password are used on the website to restrict access to all data to AGRE approved researchers only.

For assistance please contact the researcher liaison by phone (323-931-6577 x731) or by email (research@agre.org).