Assess Genetic Susceptibility to Manganese Neurotoxicity

NOTICE OF INTENT TO ISSUE A PURCHASE ORDER. The Centers for Disease Control and Prevention, National Institute for Occupational Safety and Health (NIOSH), Cincinnati, Ohio hereby announces its intent to issue a purchase order to The University of Cincinnati for the following: Analyses to Assess Genetic Susceptibility to Manganese Neurotoxicity. Background and Objectives Manganese is an essential nutrient with roles in normal human physiology; however, chronic exposure to high doses of inhaled manganese dust or fumes can result in a well-described Parkinson-like syndrome known as "manganism." Effects include tremors, weakness, reduced hand-eye coordination and psychological impairments. There is also substantial evidence from human studies that chronic low doses of inhaled manganese can produce deleterious effects. Common workplace exposures include arc welding and steel production. Recent reports of cognitive deficits in chronically exposed workers indicate current OELS, including the NIOSH REL, may not provide sufficient protection against manganese-induced neurotoxicity. The goal of the proposed project is to improve risk assessment for inhaled Mn by incorporating the most recent information on genetic susceptibility and human genetic diversity into models currently in use. Traditional genotyping methods are available to identify potential biomarkers of susceptibility using known DNA polymorphisms. In addition, new methods of DNA analysis offer promise as novel, early biomarkers of exposure, susceptibility and disease prior to the development of irreversible adverse health effects. We propose to obtain data from both traditional and novel methods to better assess genetic susceptibility to neurotoxicity following chronic, low-level occupational exposure to inhaled manganese Only one study has focused on genetic susceptibility to neurological deficits following occupational manganese exposure. That study identified a common CYP2D6 allele as a risk factor for manganism in a Chinese worker population. Interestingly, CYP2D6 genotype may also play a role in the development of Parkinson's Disease. However, there is widely recognized phenotypic variation in CYP2D6 between Chinese and Caucasian populations. Therefore, the role of CYP2D6 polymorphisms in the development of neurological deficits following occupational manganese exposure must be confirmed in a non-Chinese population. We will obtain data using traditional genotyping methodology to compare phenotypic variation in a non-Chinese, manganese-exposed population. A secondary goal is to obtain data which can be used to develop new biomarkers of exposure, illness, and susceptibility. The latency to develop neurological symptoms following occupational manganese exposure can stretch over many years; therefore, the development of an early biomarker is an important research need. Blood Mn levels do not accurately reflect chronic exposures due to different pharmacokinetics in blood and other target tissues. The specific focus will be on the genes most relevant to its absorption, distribution, metabolism and excretion: (SLC11A2/DMT1 (Divalent metal transporter 1) TF (Transferrin) TFRC (Transferrin receptor) SLC39A4 (ZIP4) SLC39A8 (ZIP 8) SLC 39A14 (ZIP 14) HFE (hereditary hemochromatosis factor). The endpoint of greatest interest is neurotoxicity, because neurological impairment is often irreversible and has high social and economic costs. Genes which might serve as biomarkers of disease include: GABRA2, GAD1, HOXA1, NEUROD1, NEUROD2, PGR, STK11, SYK Data will be obtained using blood previously collected from a manganese-exposed cohort. A detailed description of the work to be completed is described in the Statement of Work. Statement of Work Task 1. RFLP Genotyping Data (CYP2D6) and Mn Data Summary Allele-specific PCR-RFLP analysis will be done for 80 Mn-exposed adults, using the methods of Zheng et al.1,2 to determine each subject's CYP2D6 genotype. All data will be provided without subject names, personal identifiers or any coding that could be used to link samples with individual names. Contractor researchers will maintain full responsibility for privacy, confidentiality, and security of computers where personal identifiers and codes are stored. Reports to CDC-NIOSH will include the following information: age, sex, CYP2D6 genotype, environmental Mn exposure, blood Mn level, hair Mn level, results from postural sway testing, results from postural hand tremor testing. Task 2: DNA Methylation Array Data and Analysis With the assistance of the NIOSH project officer, 8 individuals within the larger cohort will be classified as "most affected" and 8 as "least affected" based on exposure and outcome data provided in Task 1. Using the NimbleGen array technology and the manufacturer's protocols, DNA methylation patterns will be compared between the 8 most highly affected and the 8 least affected Mn-exposed adults. Genomic DNA will be sonicated to produce fragments ~200-700 bp, then immunoprecipitated with an antibody against 5-methyl cytidine. Enrichment of MeDIP DNA will be validated before labeling of experimental and total DNA and co-hybridization on a NimbleGen Promoter plus CpG island array. Array images will be used for data extraction, comparing the intensity ratio of immunoprecipitated DNA to total DNA, thus identifying methylated regions across the genome. A known methylated region will be used as a positive control and a known unmethylated region as the negative control. Four technical replicates will be performed to determine platform variability. This work will be conducted at the contractor's facility and will include a statistical analysis of the array results by GML bioinformatics staff. The report to NIOSH will include a list of all differentially methylated genes, a recommendation of the ten best candidate genes for verification, images used for data analysis, and the statistical interpretation of the array data. Task 3: Validation of Genes Using Bisulfite Sequencing Following consultation with, and approval of, the NIOSH project officer, differential DNA methylation patterns on five candidate genes identified in Task 2 will be verified using bisulfite sequencing and specific DNA methylation sites on the genes of interest will be determined. A total of 50 DNA samples will be used in Task 3. Delivery, Review, and Payment The data and final reports will be delivered to the NIOSH project officer in three segments as described in the "Deliverables Schedule." For each deliverable, the following system will be used: 1.) Deliver a draft report in the specified file format (See "Format Requirements") to the NIOSH Project Officer one month prior to the completion date specified in the "Deliverables Schedule." 2.) The NIOSH Project Officer will return comments and requests for clarifications and revisions within 7 business days of receipt of the draft report. 3.) Deliver a final, revised report in the specified file format (See "Format Requirements") to the NIOSH Project Officer by the date specified in "Deliverables Schedule." Format Requirements for Reports, Data Files, and Computer Programs All reports will be delivered in both paper and electronic form, in CDC approved word processing software (Microsoft Word 2003 or earlier), formatted for 8 1/2" x 11" paper, with 1-inch margins and numbered pages. The cover page will include the project/award number, title, author(s), affiliation(s), and date completed. Data files will be delivered in the format specified by NIOSH: Microsoft Excel compatible with Office 2003 for data summaries and JPEG files (600 dpi minimum) for array image files. All files must be PC-compatible. Each report should consist of: - A cover page including the project/award number, title, author(s), affiliation(s), and date completed (Word 2003 or earlier) - A summary of the work completed and data delivered along with a brief description of all files and records included in the report for that task (Word 2003 or earlier). - Data summarized in an Excel (2003 or earlier) spreadsheet The report for Task 1 (RFLP Genotyping and Mn Data) should also include: - a JPEG image of a representative genotyping gel - a unique sample code linking RFLP data with Mn exposure and health effects data (This code cannot be the same code used by UC researchers analyzing the Marietta cohort data.) The report for Task 2 (DNA Methylation Arrays) should also include: - JPEG images of DNA methylation array data - Statistical analysis by GML bioinformatics staff summarized in Microsoft Excel (2003 or earlier) Deliverables Schedule (final reports) Task Description Due Date* 1 RFLP Genotyping Data and Mn Data Summary 4 Months after award of order. 2 DNA Methylation Array Data and Analysis 6 Months after award of order. 3 Validation of Genes by Bisulfite Sequencing 12 Months after award of order. * Months after award/purchase order date. References Cited 1. Zheng, Y.X., et al., Polymorphism of metabolic genes and susceptibility to occupational chronic manganism. Biomarkers, 2002. 7(4): p. 337-346. 2. Heim, M. and Meyer, U.A., Genotyping of poor metabolisers of debrisoquine by allele-specific PCR amplication. The Lancet, 1990. 336: p. 529-532 The University of Cincinnati has blood samples and informed consent for genetic testing in a Mn-exposed adult cohort. The University of Cincinnati has multiple measures of Mn internal dose (i.e. blood and hair Mn levels) and has completed neurological testing on the cohort. This project is supported by justification for other than full and open competition to The University of Cincinnati under authority of 41 U.S.C.253(c)(1). Responsible sources that believe they possess the expertise and capabilities identified above are may submit to the Contracting Officer within 15 days from the posting date of this notice, their written capabilities statement and pricing information in the format they choose, not to exceed 10 pages. Please forward the capability statement and pricing information to Jennifer Bayer, Purchasing Agent, Reference #000HCCEE-2008-59423, CDC/NIOSH, 4676 Columbia Parkway, Cincinnati, OH 45226, ATTN: Mail Stop C-4. All vendors must be registered in the Central Contractor Registry (CCR) prior to an award of a federal contract. The website is: www.ccr.gov. The Government will review any/all capabilities statements and pricing information submitted and determine if other qualified sources do exist that could provide this requirement. Information received in response to this announcement will be used solely for the purpose of determining whether to conduct a competitive procurement. If no affirmative responses are received within 15 days, in accordance with FAR 13.106-1(b)(1), negotiations will be conducted with The University of Cincinnati as the only source and a purchase order will be issued without any additional notices being posted.