Notice Number: NOT-RM-12-028
Key Dates
Release Date: September 14, 2012
Response Date: October 5, 2012
Issued by
National Institutes of Health (NIH)
Purpose
This Request for Information (RFI) is intended to gauge interest in using stored biospecimens collected in the Genotype-Tissue Expression (GTEx) Project of the NIH Common Fund. The NIH is considering a Funding Opportunity Announcement (FOA) with set-aside funds to support future studies utilizing these biospecimens. Information obtained via this RFI will help the GTEx Working Group (WG) understand the potential demand for GTEx biospecimens, plan scientific reviews, and assess the need for other access mechanisms beyond an FOA.
The NIH is interested in making maximal use of this unique biospecimen resource, rich with clinical and genomic information. Except for the fibroblast and lymphoblastoid cell lines, biospecimens are of limited quantity and are non-renewable. Potential uses that are comprehensive (genomic vs single gene or small gene networks, proteomic vs single proteins or small networks of proteins, etc.) and complementary to existing gene expression and variation data, are preferred. Scientific questions that are equally well addressed using other sample sets will probably not be suitable, while those that take full advantage of the unique aspects of GTEx, such as the multiple tissues from each donor and the gene expression information, are particularly sought. In addition to scientific review, all proposals to use GTEx samples would also go through a Biospecimen Access Committee (currently being formed). GTEx data will be made available through dbGaP (http://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?study_id=phs000424), and eventually through other data portals, and is not the subject of this RFI.
Background
The primary goal of GTEx is to establish a resource database and associated tissue bank in which to study the relationship between genetic variation and gene expression and other molecular phenotypes in multiple reference tissues. An initial pilot phase resulted in biospecimen collection from over 175 post-mortem donors by the summer of 2012. Having met milestones, the project is scaling up to include approximately 900 post-mortem donors by the end of 2015. The GTEx project obtained authorization for broad future research use from the deceased donor’s next of kin.
The collection involves more than 30 tissues from each post-mortem donor (see Table 1). When consented, the cerebral cortex and cerebellum are sampled immediately, and the remaining whole brain is shipped to a brain bank, where 9 additional regions are sampled. Post-mortem donors of any racial and ethnic group and sex who are age 21-70 in whom biospecimen collection can start within 24 hours of death are eligible. Donors are expected to have a typical prevalence of chronic diseases because there are only a few exclusionary criteria, such as human immunodeficiency virus (HIV), viral hepatitis, and metastatic cancer. All donors are characterized for germline genetic variation through dense genotyping of a blood sample. All tissues of sufficient quality undergo gene expression analysis using deep RNA sequencing. This RFI deals with potential uses of biospecimens beyond the genotyping and gene expression analyses described above. More information about GTEx can be found at http://commonfund.nih.gov/GTEx/.
Biospecimen acquisition for GTEx is organized under the National Cancer Institute’s (NCI) cancer Human Biobank (caHUB) initiative (http://caHUB.cancer.gov), part of the Office of Biorepositories and Biospecimen Research (OBBR), together with SAIC-Frederick, Inc. (SAIC-F), the prime contractor to the NCI. Biospecimen collection is accomplished through Biospecimen Source Sites (BSS) and a Comprehensive Biospecimen Resource (CBR). Aliquots of tissue from each donor are processed by the GTEx Laboratory, Data Analysis and Coordinating Center (LDACC).
We expect to be able to make available to the scientific community the following sample types:
- PAXgene fixed, frozen tissue Tissue fixed using PAXgene Tissue (QIAGEN / BD, PreAnalytiX) and stored frozen (-80C) in PAXgene Tissue stabilizer. PAXgene Tissue is a proprietary solution containing alcohols (ethanol and methanol), acetic acid, and a soluble organic compound; it fixes primarily by protein precipitation.
- PAXgene Fixed, Paraffin Embedded (PFPE) Tissue Aliquots, similar in size and adjacent to those described above, that are fixed in PAXgene Tissue fixative and paraffin embedded. The result of histopathological review of an H&E stained section from each tissue will be available.
- Flash frozen brain samples subregions of the brain are sampled and flash frozen in liquid nitrogen at the brain bank, including the cerebral cortex and cerebellum that were already sampled and fixed in PAXgene reagents at the BSS.
- Genomic DNA - routinely extracted from a 20 mg aliquot of all PAXgene Tissue fixed tissues using the PAXgene tissue DNA kit (Qiagen - Catalog # 767134). Quantification and quality assessment (agarose gel) are performed, demonstrating high molecular weight DNA in the vast majority of samples. DNA from blood is obtained using acid citrate dextrose (ACD) collection tubes and is used for genotyping.
- mRNA and microRNA a 10 mg portion of the original PAXgene Tissue fixed aliquot is used to extract RNA using PAXgene Tissue miRNA Kit (Qiagen - Catalog # 766134); stored frozen at -80C. RNA samples undergo quality assessment (Agilent Bioanalyzer), and those of sufficient quality are analyzed for gene expression. RNA from blood is obtained from a sample collected in PAXgene Blood RNA tubes.
- Lymphoblastoid and fibroblast cell lines. Skin and blood biospecimens are sent directly to the LDACC for establishment of fibroblast and EBV-transformed lymphoblastoid cell cultures; stored frozen in liquid nitrogen. Lymphoblastoid cell lines are successfully established for ~ 60% of donors (23 as of July 2012) while fibroblast lines are successfully established for >90% of donors (39 as of July 2012).
In addition to biospecimens, the following data will be available (potential uses of the data are not the subject of this RFI):
Openly available (data portal under development):
- Molecular data that does not provide direct genetic variation information (e.g., expression arrays, summary sequence-based gene expression estimates (rpkm) stripped of variant information; RNA Integrity Number (RIN) values; eQTL results;
- Standard Operating Procedures (SOPs), available at http://biospecimens.cancer.gov/resources/sops/default.asp;
- Histopathological interpretations;
- Laboratory processing variables (e.g., cDNA library preparation methods, processing batch variables);
- Sex and age at death (in 5- or 10-year intervals) for each donor;
Available only through controlled access (dbGaP):
- Medical and social history information - This information is collected from family members and/or the medical record, when available;
- Molecular results that contain direct genetic variation information (e.g., SNP genotyping files, RNA-Seq BAM files). Whole-genome single-nucleotide polymorphism (SNP) and CNV genotyping are performed using a high-density genotyping array (Illumina 5M) and an exon SNP array (Affymetrix GeneChip Human Exon 1.0 ST) on peripheral blood biospecimen DNA. The primary gene expression method is RNA-seq using the Illumina Hiseq 2000 platform, but some biospecimens have also undergone gene expression array analysis using Affymetrix Gene 1.0ST or 1.5ST platform.
See table 1 for a summary of sample and tissue types collected in GTEx. Collection and analyses are ongoing and therefore results will change over time. A searchable database of available samples is under development.
Table 1: Summary of approximate numbers of tissues collected and amount of sample remaining based on the first 150 donors.
Preserved Tissue (PAXgene - not paraffin embedded, unless otherwise specified) |
Stored RNA |
Stored DNA |
||||
Tissue Type |
N (N with RIN>6) |
Median Weight (mg) |
Median Total Amt (ug) |
Median Conc. (ng/uL) |
Median Total Amt (ug) |
Median Conc. (ng/uL) |
Adipose, Subcutaneous |
144 (108) |
360 |
1.8 |
29.1 |
0.8 |
7.6 |
Adipose, Visceral (Omentum) |
17 (10) |
299 |
2.4 |
36.3 |
0.9 |
9.7 |
Adrenal Gland |
107 (62) |
315 |
13.5 |
314.9 |
6.0 |
55.6 |
Artery, Aorta (Ascending or Thoracic) |
133 (83) |
254 |
2.2 |
64.3 |
1.1 |
10.2 |
Artery, Coronary |
97 (65) |
110 |
2.9 |
87.5 |
1.8 |
17.4 |
Artery, Tibial |
142 (115) |
95 |
1.7 |
62.6 |
1.7 |
18.1 |
Bladder, Urinary*^ |
121 (75) |
420 |
4.8 |
144.8 |
2.9 |
30.2 |
Brain, Cerebellum |
36 (24) |
209 |
8.1 |
262.6 |
20.9 |
145.8 |
Brain, Cortex |
35 (22) |
203 |
5.4 |
192.4 |
1.8 |
15.8 |
Brain, Amygdala@ |
34 (21) |
130 |
5.3 |
188.4 |
1.0 |
19.4 |
Brain, Anterior cingulate cortex (BA24)@ |
35 (16) |
144 |
6.9 |
212.8 |
1.0 |
18.0 |
Brain, Caudate (basal ganglia)@ |
35 (28) |
136 |
5.9 |
195.6 |
0.9 |
15.6 |
Brain, Cerebellar Hemisphere@ |
35 (21) |
200 |
8.1 |
266.1 |
7.8 |
136.1 |
Brain, Frontal Cortex (BA9)@ |
35 (22) |
210 |
5.9 |
188.5 |
1.0 |
19.9 |
Brain, Hippocampus@ |
34 (24) |
164 |
5.4 |
202.0 |
0.9 |
20 |
Brain, Hypothalamus@ |
34 (19) |
123 |
4.3 |
165.8 |
1.0 |
23.7 |
Brain, Nucleus accumbens (basal ganglia)@ |
35 (28) |
115 |
5.6 |
200.2 |
0.9 |
16.3 |
Brain, Putamen (basal ganglia)@ |
33 (18) |
118 |
5.7 |
196.0 |
1.2 |
21.7 |
Brain, Spinal cord (cervical c-1)@ |
24 (17) |
136 |
2.6 |
86.6 |
0.6 |
13.4 |
Brain, Substantia nigra@ |
33 (20) |
120 |
4.8 |
146.1 |
0.6 |
14.5 |
Breast, Mammary Tissue |
87 (56) |
375 |
2.2 |
32.1 |
1.1 |
11.6 |
Cervix, Ectocervix* |
32 (24) |
303 |
3.6 |
111.7 |
3.6 |
33.2 |
Cervix, Endocervix* |
34 (22) |
311 |
5.0 |
150.8 |
6.3 |
47.6 |
Colon, Transverse |
144 (84) |
288 |
8.2 |
235.3 |
6.2 |
58.6 |
Colon, Sigmoid |
15 (9) |
317 |
6.9 |
200.1 |
5.1 |
53.3 |
Esophagus, Gastroesophageal Junction |
16 (10) |
489 |
3.7 |
106.3 |
3.6 |
38.1 |
Esophagus, Mucosa |
139 (111) |
159 |
14.4 |
332.7 |
5.5 |
49.2 |
Esophagus, Muscularis |
140 (100) |
345 |
5.5 |
166.4 |
2.9 |
28.3 |
Fallopian Tube* |
32 (26) |
183 |
7.5 |
239.6 |
6.9 |
62.8 |
Heart, Atrial Appendage |
12 (11) |
329 |
4.0 |
132.8 |
2.0 |
21.5 |
Heart, Left Ventricle |
116 (79) |
312 |
4.4 |
185.5 |
1.6 |
14.8 |
Kidney, Cortex |
73 (10) |
318 |
8.9 |
265.5 |
9.6 |
80.0 |
Kidney, Medulla* |
41 (10) |
322 |
8.2 |
240.1 |
9.0 |
84.9 |
Liver |
81 (43) |
367 |
23.8 |
361.3 |
4.8 |
48.0 |
Lung |
129 (108) |
303 |
4.9 |
204.2 |
5.6 |
54.4 |
Muscle, Skeletal |
146 (133) |
350 |
2.9 |
114.1 |
1.4 |
12.0 |
Nerve, Tibial |
139 (97) |
212 |
1.2 |
48.0 |
2.1 |
19.8 |
Ovary |
45 (35) |
400 |
10.4 |
283.9 |
14.6 |
124.9 |
Pancreas^ |
130 (67) |
375 |
50.7 |
363.6 |
10.1 |
93.5 |
Pituitary Gland |
33 (16) |
119 |
17.7 |
335.5 |
14.1 |
153.5 |
Prostate |
83 (39) |
345 |
7.9 |
236.2 |
6.0 |
60.5 |
Skin, Sun exposed (Lower Leg) |
147 (94) |
300 |
2.6 |
98.2 |
2.6 |
24.4 |
Skin, Not sun exposed (Suprapubic) |
17 (11) |
359 |
3.4 |
118.8 |
3.3 |
35.4 |
Spleen^ |
137 (33) |
417 |
15.4 |
346.8 |
13.9 |
133.2 |
Stomach^ |
144 (82) |
338 |
12.3 |
299.6 |
6.7 |
57.7 |
Terminal Ileum (Peyer’s patch) |
15 (9) |
288 |
7.5 |
229.2 |
9.8 |
104.8 |
Testis |
92 (64) |
339 |
13.5 |
328.1 |
7.4 |
62.2 |
Thyroid |
126 (94) |
341 |
4.9 |
213.9 |
8.2 |
81.6 |
Uterus |
41 (31) |
364 |
7.9 |
226.8 |
9.8 |
73.9 |
Vagina |
48 (34) |
387 |
4.8 |
141.2 |
4.5 |
43.1 |
Whole Blood& |
149 (144) |
- |
6.6 |
102.8 |
19.4 |
115.1 |
* Tissues collected only during the pilot phase (i.e., the final numbers will not be significantly higher); @ Fresh Frozen Tissue; ^ 15-30% of samples show severe autolysis; & Blood for RNA collected in PAXgene Blood RNA tubes, Blood for DNA collected in acid citrate dextrose (ACD) tubes. Protocols for collecting minor salivary glands are under review.
Information Requested
The NIH Common Fund GTEx Working Group is interested in knowing the level of interest in using GTEx biospecimens, the types of analyses being considered, the level of funding support that would be required, and how well suited the existing sample types are for the proposed analyses.
Please provide the following types of information for assays or ideas that could be accomplished with GTEx biospecimens.
- An indication of how likely you are to request GTEx samples.
- The name or type of assay and the research question being addressed.
- An indication of whether the assay is known to work on PAXgene (alcohol-based) fixed or other types of biospecimens GTEx stores or whether a technical feasibility study to demonstrate suitability of GTEx samples and/or additional processing would be required (e.g. tissue microarray).
- An estimate of the number of donors needed and the number, types, and amounts of samples needed.
- An indication of whether financial support would be required to perform the assays and analysis, or whether you have funds from existing peer-reviewed grants or other sources.
Responses
To assure consideration, your responses must be sent to [email protected] and received by midnight on October 5, 2012.
Responses to this RFI are voluntary. The comments collected will be analyzed and considered in planning and development of future initiatives. We do not intend to publish a summary of responses received or any other information provided, except very broad characterizations, such as the number of responses received and the relative demand of various tissue types. Despite this, proprietary, classified or confidential information should not be included in your response. This RFI is for planning purposes only and is not a solicitation for applications or an obligation on the part of the United States (U.S.) Government to provide support for any ideas identified in response to it. Please note that the U.S. Government will not pay for the preparation of any comment submitted or for its use of that comment.
Inquiries
For further information contact:
Simona Volpi, Ph.D.
Email: [email protected]
and Human Services (HHS)
