Grantee Research Project Results
2016 Progress Report: Identifying In Utero Exposures that are Risk Factors for Childhood Leukemia
EPA Grant Number: R836159C002Subproject: this is subproject number 002 , established and managed by the Center Director under grant R836159
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
Center: Center for Integrative Research on Childhood Leukemia and the Environment - 2015
Center Director: Metayer, Catherine
Title: Identifying In Utero Exposures that are Risk Factors for Childhood Leukemia
Investigators: Rappaport, Stephen M. , Hubbard, Alan , Whitehead, Todd
Institution: University of California - Berkeley
EPA Project Officer: Callan, Richard
Project Period: September 1, 2015 through August 31, 2019 (Extended to August 31, 2020)
Project Period Covered by this Report: September 1, 2015 through August 31,2016
RFA: Children's Environmental Health and Disease Prevention Research Centers (2014) RFA Text | Recipients Lists
Research Category: Human Health , Children's Health
Objective:
Aim 1: Finalize methods for profiling small molecules and Cys34 adducts in archive newborn specimens (ANBS).
Aim 2: Measure and annotate omic features in ANBS extracts from ALL cases and matched controls.
Aim 3: Process and compare data from cases and controls to find discriminating omic features.
Aim 4: Perform semi-targeted analyses of small molecules chosen a priori as possible biomarkers of childhood ALL.
Aim 5: Measure and compare omic features between pairs of ANBS and maternal blood samples collected during pregnancy from 200 mothers of childhood ALL cases and 400 mothers of control children.
Progress Summary:
Aim 1: We developed methods for profiling small molecules and Cys34 adducts of human serum albumin (HSA) from ANBS and then measuring these chemicals via LC-HRMS. Each set of untargeted analyses is performed with a 3-mm or 4.7-mm punch from an ANBS. In the prior funding period, the method for extraction and detection of Cys34 adducts was reported. In Year 1 of current funding, we continued work started in the first cycle and conducted a study to validate the untargeted analysis of small molecules. Punches (4.7-mm) from 10 ANBS from control children and adjacent filter paper from the same Guthrie cards (IRB-approved from previous cycle of CIRCLE) and were extracted with 80% acetonitrile and aliquots were injected into a UPLC-HRMS platform (1290 UPLC and 6550 Q-TOF, Agilent) in both positive and negative modes using a C-18 reverse phase column. The method included measurement of potassium in the extract to facilitate normalization of analyte levels for hematocrit content, which is highly correlated with potassium levels. Data were processed using in-house software written in R. About 82,000 small molecule features were detected. After filtering to remove features with fold changes (FC) (blood-derived/filter material) greater than 2, coefficients of variation (CV) < 30%, and FDR-adjusted P-values < 0.05, over 10,000 features were available for analysis. Tandem MS/MS spectra for over 150 features matched those in on-line databases for blood-derived molecules. Annotated small molecules included fatty acids, lipids, monosaccharides, microbial metabolites, steroids & hormones, nucleotides and glutathione conjugates.
Aim 2: Extracts from 4.7-mm punches from 97 pairs of ANBS from CL cases and matched controls (IRB-approved from previous cycle of CIRCLE) were analyzed using the same UPLC-HRMS platform described above. Matching was based on gender, date of birth and the child’s ethnicity. More than 150,000 small-molecule features were detected in the combined negative- and positive-mode analyses. Using strict filtering criteria (FC > 5, CV < 20%, clustering of highly correlated features) 1,722 testable clusters were obtained.
Aim 3: Several strategies were applied for statistical analysis of small-molecule clusters from the 97 case/ control pairs (Aim 2). After stratification by gender, four clusters (three in females and one in males) were found to be present at either higher or lower concentrations in CL cases than controls with significance levels at or near a FDR-adjusted P-value of 0.05. Two of these discriminating features have been identified through matching of MS/MS spectra and comparisons with reference standards.
Aim 4: In parallel with Aim 2 we detected several targeted molecules that had been selected as putative causes of CL (metabolites of benzene, biomarkers of coffee and alcohol consumption, smoking). However, none of these targeted molecules were detected at significantly higher levels in CL cases. Aim 5. This aim was not pursued in Year 1.
Future Activities:
Aim 1: In Year 2, the LC-HRMS method for small-molecule omics will be expanded to include HILIC chromatography (for polar metabolites) as well as reverse-phase chromatography. The method will also be simplified by employing separate ANBS punches for small molecule omics and Cys34 adductomics. This will increase precision and sample throughput. Steps of bioinformatic processing will be finalized with R programming for data-acquisition, quality control, peak-picking, batch adjustment & clustering. Aim 2: In Year 2, additional batches of 100 CL cases & controls will be analyzed for small molecules and Cys34 adducts. Replication of findings from the first cycle of CIRCLE will be important to assess robustness of the method used. MS/MS spectra will be used for annotations based on comparisons with our in-house libraries and with online databases.
Aim 2: In Year 2, additional batches of 100 CL cases & controls will be analyzed for small molecules and Cys34 adducts. MS/MS spectra will be used for annotations based on comparisons with our in-house libraries and with online databases.
Aim 3: In Year 2, statistical tests will be performed with batches of samples from Aim 2 to compare levels of small molecules and Cys34 adducts between CL cases and controls. Tests will be performed batch-wise to validate results and with data combined from all batches to increase power.
Aim 4: In Year 2, targeted analytes representing possible CL risk factors (metabolites of benzene, biomarkers of coffee and alcohol consumption, smoking & oxidative stress) will be quantitated in each batch of samples. Aim 5: This aim will not be pursued in Year 2.
Journal Articles:
No journal articles submitted with this report: View all 4 publications for this subprojectProgress and Final Reports:
Original AbstractMain Center Abstract and Reports:
R836159 Center for Integrative Research on Childhood Leukemia and the Environment - 2015 Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
R836159C001 In Utero Chemical Exposures, Immune Status, and Childhood Leukemia
R836159C002 Identifying In Utero Exposures that are Risk Factors for Childhood Leukemia
R836159C003 Prenatal Exposures, Constitutive Genetics, DNA Methylation & Childhood Leukemia
The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.
Project Research Results
2 journal articles for this subproject
Main Center: R836159
37 publications for this center
35 journal articles for this center